Research ArticleDecoction and Fermentation of Selected Medicinal HerbsPromote Hair Regrowth by Inducing Hair Follicle Growth inConjunction with Wnts Signaling

Su Kil Jang1 Seung Tae Kim1 Do Ik Lee2 Jun Sub Park1 Bo Ram Jo1 Jung Youl Park3

Jong Heo4 and Seong Soo Joo1

1College of Life Science Gangneung-Wonju National University 120 Gangneung Daehangno GangneungGangwon 210-702 Republic of Korea2College of Pharmacy Chung-Ang University 221 Heukseok-dong Dongjak-gu Seoul 156-756 Republic of Korea3Industry-Academic Cooperation Foundation Hanbat National University Daejeon 305-719 Republic of Korea4Heo-jong Oriental Medicine Clinic Mia-dong Gangbuk-gu 62-3 Seoul 01205 Republic of Korea

Correspondence should be addressed to Seong Soo Joo larryjoohanmailnet

Received 26 November 2015 Revised 25 January 2016 Accepted 7 February 2016

Academic Editor Jian-Li Gao

Copyright copy 2016 Su Kil Jang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

It is well recognized that regulating the hair follicle cycle in association withWnt signaling is one of the most interesting targets forpromoting hair regrowth In this study we examined whether selected herbal medicines processed by decoction and fermentationpromote hair growth by upregulating the number and size of hair follicles andWnt signaling including activation of 120573-catenin andAkt in telogen-synchronized C57BL6NmiceThe results revealed that the fermented extract after decoction (FDE)more effectivelypromoted hair growth than that of a nonfermented extract (DE) Notably FDE effectively enhanced formation of hair follicleswith clearer differentiation between the inner and outer root sheath which is observed during the anagen phase Mechanisticevidence was found for increased 120573-catenin and Akt phosphorylation levels in dorsal skin tissue along with elevated expressionof hair regrowth-related genes such as Wnt310a10b Lef1 and fibroblast growth factor 7 In conclusion our findings suggest thatFDE plays an important role in regulating the hair cycle by increasing expression of hair regrowth-related genes and activatingdownstreamWnt signaling targets

1 Introduction

Hair is a defining property of mammals that plays importantroles in keeping the body warm and dry and protectingagainst harmful environments Thus new hair is requiredconstantly throughout life In general new hair is suppliedby existing follicles through the anagen (growing phaseof follicular epithelium) catagen (apoptosis and regressionphase) and telogen phases (resting phase for the epithelium)A hair follicle is a skin organ that generates hair and has beenthe focus of stem cell studies [1 2]

Hair follicles may be self-renewed by keratinocyte stemcells (KSCs) located at the bulge region which containsundifferentiated cells [3 4] During the transition from

the telogen to the anagen phases Wnt signaling which isrequired to establish the hair follicle and is upregulatedonly at the end of the telogen phase to promote entry intoanagen plays a key role in activating bulge stem cells toprogress toward hair formation and these signals are relayedin association with 120573-catenin and lymphoid enhancer factor1 (Lef1) [5] Wnt ligand expression is not detected in telogenphase follicles and Wnt10a and 10b are only expressed at theonset of the anagen phase in the dermal papilla and secondaryhair germ cells respectively [6] This is supported by theobservation that 120573-catenin is generally confined to the mem-branes of bulge cells through most of the telogen phase andthat nuclear 120573-catenin only becomes apparent in hair germcells just before the follicle enters the anagen phase [5 7]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 4541580 10 pageshttpdxdoiorg10115520164541580

2 Evidence-Based Complementary and Alternative Medicine

To date comprehensively effective highly safe candi-date compounds prepared from herbal extracts have beenexamined for their hair regrowth activities Among thesewe selected eight herbs and derived an optimal extractthrough decoction and fermentation processes In this studywe examined the hair regrowth activity of the extract inC57BL6N mice and its prospective mechanism in conjunc-tion with Wnt signaling

2 Materials and Methods

21 Preparation of the Study Sample Eight selectedmedicinalherbs such as Cynanchum wilfordii Mori Fructus Schisan-drae Fructus Perillae Herba Houttuyniae Herba LigustriFructus Longanae Arillus and Polygonati Rhizoma werepurchased from Saerom Pharmaceutical Co Ltd (KyunggiRepublic of Korea)The study samples were prepared accord-ing to the method developed in our laboratory In briefto prepare the decoction evenly weighed herbs (1 1) werefinely ground and primarily immersed in autoclaved distilledwater (1 10 wv) for 30min followed by boiling on anelectric heater for 2 h The decoction was filtered usingWhatman Grade No 1 Filter Paper (Whatman InternationalLtd Maidstone UK) and centrifuged at 5000 rpm for 15minThe supernatant was collected lyophilized and stored at 4∘Cbefore use For fermentation Bacillus subtilis was propagatedtwice in 50mL MRS broth (Difco Detroit MI USA) at 37∘Covernight for fermentation Then 107 CFUmL of B subtiliswas inoculated into the decocted extracts and fermented at37∘C for 48 h (FDE) Nonfermented decoction extracts (DE)were used as a normal control Both samples were seriallyfiltered with a 60120583m nylon net filter and a 022120583m syringefilter (Millipore Bedford MA USA) precipitated overnightlyophilized (supernatant) and stored in desiccators at roomtemperature before use During fermentation (24 and 48 h)samples of the liquid culture were examined under phase-contrast microscopy to visualize basic cell characteristics

22 Animals Healthy male C57BL6N mice (7 weeks old)were obtained from Central Lab Animal Inc (Seoul Repub-lic of Korea) and were adapted to laboratory conditions (tem-perature 20 plusmn 2∘C relative humidity 50 and lightdarkcycle 12 h) for 1 week The animals (119899 = 3group) were main-tained at a constant temperature (23 plusmn 2∘C) relative humidity(55plusmn 10) and a 12 h lightdark cycle and fed standard rodentchow and purified water ad libitum Two days before theexperiments all mice (8 weeks of age) were shaved usinganimal clippers Telogen-synchronized C57BL6Nmice weredivided into six groups each containing three male miceThe FDE and DE extracts as well as finasteride (FIN Sigma-Aldrich St Louis MO USA) were administered daily for20 consecutive days The FDE and DE groups were given 64or 128mgkg and 140 or 280mgkg respectively consideringthe vaporized solid parts of FDE (39 g100mL) and DE(87 gmL) FIN (1mgkg) was administered as the positiveat the same time points All mice were sacrificed on day21 and the dorsal hair growth patterns were photographedon days 0 3 6 9 12 15 18 and 20 The back area of each

mousewas photographedwith a digital camera and the imagewas inputted to a computer for measurement according tothe following formula [ Hair regrowth = hairy black areadivide hair removal area] to quantitatively compare the hairregrowth patternsThe animal experiments were approved bythe Gangneung-Wonju National University Animal Care andUse Committee (Approval number GWNU-2014-27) and allprocedures were conducted in accordance with the Guide forCare and Use of Laboratory Animals published by the USNational Institutes of Health

23 Cell Culture Human mesenchymal stem cells (hMSCs)used in this study were provided by Professor Kim (Chung-buk National University Republic of Korea) [8 9] Cells werecultured at a density of 5 times 103 cellscm2 in complete mediumand Dulbeccorsquos modified Eaglersquos medium (DMEM low glu-cose) was supplemented with 10 fetal bovine serum (FBSHyclone Logan UT USA) 25 ngmL hFGF2 100UmLpenicillin and 100 120583gmL streptomycin (Invitrogen Carls-bad CA USA) Complete medium was changed every 2-3days and hMSCs were subcultured when they reach 1 times 104cellscm2 Cultures were maintained under 5 CO

2at 37∘C

in tissue culture flasks

24 Cellular Cytotoxicity (Lactate Dehydrogenase LDH)Assay The cytotoxicity induced by the FDE and DE wasquantified by measuring LDH release LDH content wasdetermined using a commercial nonradioactive LDH assaykit CytoTox 96 (Promega Madison WI USA) which isbased on a coupled enzymatic reaction that results in theconversion of a tetrazolium salt into a red formazan productThe increase in the amount of formazan produced in theculture supernatant directly correlates with the increase inthe number of lysed cells The formazan was quantified spec-trophotometrically by measuring its absorbance at 490 nm(Spectra Max 340 Molecular Devices Sunnyvale CA USA)Cytotoxicity in experimental samples was determined as LDH release compared with that in cells treated with 1Triton X-100

25 Quantitative Real-Time Polymerase Chain Reaction (PCR)Assay Total RNA from C57BL6N dorsal skin tissues orhMSCs was prepared using the TRIZOL method (Invitro-gen) cDNAs were synthesized from RNA by reverse tran-scription of 1 120583g total RNA using the ImProm-II reverse tran-scription system (Promega) and oligo dT primers in a totalvolume of 20 120583L PCR amplification was performed usingthe primers described in Table 1 (Bioneer Daejeon Republicof Korea) Quantitative real-time PCR (qPCR) reactionswere run on a Rotor-Gene 6000 (Corbett Research SydneyAustralia) using SYBR Green PCR Master Mix (QiagenValencia CA USA) in 20120583L reaction mixtures Each real-time PCRmaster mix contained 10 120583L 2x enzymeMastermix70 120583L RNase free water 1 120583L of each primer (10 pmole each)and 1 120583L diluted template PCR was performed with an initialpreincubation step for 10min at 95∘C followed by 45 cyclesof 95∘C for 15 s annealing at 52∘C for 15 s and extension at72∘C for 10 s A melting curve analysis was used to confirm

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primer sequences used for the real-time polymerase chain reaction analysis

Gene Primer Amino acid sequences Product size (bp) Accession number

Mouse

FGF7 51015840 primer 51015840-TGCTTCCACCTCGTCTGTCT 212 NM 00800831015840 primer 51015840-GAGGCAAAGTGAAAGGGACC

Wnt3 51015840 primer 51015840-AGAGACGGGCTCCTTTGGTA 123 NM 00952131015840 primer 51015840-TTCTCCTTCCGTTTCTCCGT

Wnt10a 51015840 primer 51015840-GTGCGCTCTGGGTAAACTGA 232 NM 00951831015840 primer 51015840-AGAGAAGCGTTCTCCGAAGC

Wnt10b 51015840 primer 51015840-TCTTGGCTTTGTTCAGTCGG 124 NM 01171831015840 primer 51015840-CCCAGCTGTCGCTTACTCAG

120573-catenin 51015840 primer 51015840-AGGCTTTTCCCAGTCCTTCA 122 M9036431015840 primer 51015840-TCTGCATGCCCTCATCTAGC

Lef1 51015840 primer 51015840-CGTCCTCTCAGGAGCCCTAC 169 X5863631015840 primer 51015840-GGAGAAAGGGACCCATTTGA

120573-actin 51015840 primer 51015840-TACAGCTTCACCACCACAGC 187 NM 00739331015840 primer 51015840-AAGGAAGGCTGGAAAAGAGC

Human

Wnt3 51015840 primer 51015840-CACATGCACCTCAAATGCAA 132 AB06762831015840 primer 51015840-CGAGGCGCTGTCATACTTGT

Wnt10a 51015840 primer 51015840-TTCCACTGGTGCTGCGTAGT 107 AB05957031015840 primer 51015840-CTGCGCGAAGTCAGTCTAGC

Wnt10b 51015840 primer 51015840-CATACAGGGCATCCAGATCG 148 AB05956931015840 primer 51015840-AAAAGCGCTCTCTCGGAAAC

GAPDH 51015840 primer 51015840-GGAGCCAAAAGGGTCATCAT 203 AK 02652531015840 primer 51015840-GTGATGGCATGGACTGTGGT

formation of the expected PCR product and products fromall assays were tested by 12 agarose gel electrophoresis toconfirm the correct lengths An interrun calibrator was usedand a standard curve was created for each gene to obtainPCR efficiencies Relative sample expression levels werecalculated using Rotor-Gene 6000 Series Software 17 andwere expressed relative to glyceraldehyde 3-phosphate dehy-drogenase and corrected for between-run variability Data areexpressed as a percentage of the internal control gene

26 Western Blot Analysis C57BL6N dorsal skin tissueswere homogenized and lysed in 1 RIPA buffer containingprotease and phosphatase inhibitors (Roche MannheimGermany) and total proteins were separated on 10 SDS-PAGE After electrophoresis the proteins were transferredto polyvinylidene fluoride membranes and the membraneswere blocked with 5 skim milk in Tris-buffered salinesolution containing 01 Tween-20 The membranes wereimmunoblotted with primary antibodies including anti-120573-catenin anti-phospo-Akt anti-Akt and anti-actin (SantaCruz Biotechnology Santa Cruz CA USA) followed byincubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Stressgen SanDiego CA USA) The blots were developed using anenhanced chemiluminescent solution (Thermo Rockford ILUSA)

27 Histological Examination and Hair Follicle Count Rect-angular pieces of central dorsal skin were collected parallel to

the vertebral line and fixed in 10 neutral buffered formalin(4 g sodium phosphate monobasic 65 g sodium phosphatedibasic 100mL of 37 formalin and 900mL distilled water)for 5 days The tissues were then embedded in paraffincut into sections (5120583m) and stained with a hematoxylin-eosin (HampE) solution The sections were deparaffinized withxylene hydrated in a descending graded ethanol series andstainedwith hematoxylin for 2min followed by 2minwashesand eosin staining for 5 s All tissue samples were examinedand imaged in a blinded fashion Hair follicle counts wereperformed by using a digital photomicrograph and all of theimages were cropped in a fixed area (300 pixels in width)Data were evaluated from representative areas at a fixedmagnification of 100x Images were captured using a NikonEclips Ti-S invertedmicroscope (Nikon Tokyo Japan) at 40xmagnification

28 Statistical Analysis Statistical comparisons betweengroups were performed using one-way analysis of variancewith Dunnettrsquos post hoc test and SPSS v 17 software (SPSSInc Chicago IL USA) A 119875 lt 005 was considered signifi-cant

3 Results

31 Hair Growth-Promoting Action in Male C57BL6N MiceHair growth patterns in the shaved area were comparedin all groups To evaluate the hair growth effect FIN(1mgkg) FDE (low 64mgkg high 128mgkg) and DE

4 Evidence-Based Complementary and Alternative Medicine

(Day

)

0

3

6

9

12

15

18

20

Ctrl FIN High Low High LowFDE DE

(a)

CtrlFINF-low

F-highD-lowD-high

0

20

40

60

80

100

Relat

ive a

rea (

)

3 6 90 15 18 2012

Days

(b)

Figure 1 Hair growth-promoting effect in C57BL6N mice The dorsal skin of male C57BL6N mice was shaved after the mice were orallyadministered the fermented herbal extract after decoction (FDE) the nonfermented herbal extract after decoction (DE) or finasteride for 20days (a) The shaved dorsal skin was photographed at 0 3 6 9 12 15 18 and 20 days (b) The area of hair regrowth was measured by imagesoftware on the indicated day FIN finasteride F FDE D DE

Evidence-Based Complementary and Alternative Medicine 5

Ctrl FIN

FDE-high FDE-low

DE-high DE-low

ML

HF

DP IRS

ORS

(a)

Ctrl FIN High Low High LowFDE DE

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast lowastlowastlowast

0

10

20

30

40

Hai

r fol

licle

coun

ts

(b)

Figure 2 Comparison of hair follicle growth in C57BL6N mice (a) Hematoxylin-eosin staining of dorsal skin from mice administered thefermented herbal extract after decoction (FDE) the nonfermented herbal extract after decoction (DE) or finasteride for 20 days was analyzedArrows are muscle layer (ML) dermal papilla (DP) outer root sheath (ORS) and inner root sheath (IRS) (b) The number of hair follicles indeep subcutis Values are mean plusmn standard deviations lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrl control group

(low 140mgkg high 280mgkg) were administered andpatterns of dorsal hair growth were examined on days0 3 6 9 12 15 18 and 20 (Figure 1(a)) As shown inFigure 1(a) the FDE and DE caused a gray hair color on day12 after induction and the hair shafts were visible on day15 whereas hair in the control group remained unpigmenteduntil day 15 Interestingly regrowth of hair in FDE-treatedmice was as much as that seen in the FIN positive controlwhich is a representative oral hair loss drug used worldwide(Figure 1(b))

32 Effects of FDEDE on Hair Follicle Structure Hair folliclegrowth (anagen) between groups was compared in accor-dance with accepted morphological guidelines [10] Ourresults revealed that FDE and DE increased the number andsize of hair follicles which are markers for transition of folli-cles from the telogen to anagen phase of hair growth whereashair in the control group was in the early anagen phasein which enlarged dermal papilla and the bulb located inthe dermis are distinct characteristics (Figure 2(a)) NotablyHampE staining of FDE and DE mice well demonstrated that

6 Evidence-Based Complementary and Alternative Medicine

Ctrl FIN Low High Low High00

05

10

15

20

25

FDE DE

Rela

tive c

once

ntra

tion

(Wnt

3 m

RNA

)no

rmal

ized

to120573

-act

in

lowastlowast

lowastlowastlowast

(a)

Rela

tive c

once

ntra

tion

(Wnt

10a m

RNA

)no

rmal

ized

to120573

-act

in

0

1

2

3

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(b)

Rela

tive c

once

ntra

tion

(Wnt

10b

mRN

A)

norm

aliz

ed to

120573-a

ctin

0

2

4

6

8

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(c)

Rela

tive c

once

ntra

tion

(Lef

1 m

RNA

)no

rmal

ized

to120573

-act

in

0

2

4

6

8

10

Ctrl FIN Low High Low HighFDE DE

lowastlowast

lowastlowast

lowastlowastlowast

(d)

Relat

ive c

once

ntra

tion

(FG

F7 m

RNA

)no

rmal

ized

to120573

-act

in

00

05

10

15

20

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)

Figure 3 Comparative analysis of the expression of hair regrowth-related genes at the mRNA level in dorsal skin tissue The tissues werecollected on day 21 andmRNAswere harvested using TRIZOLWnt310a10b Lef1 and fibroblast growth factor (FGF) 7 geneswere comparedby real-time quantitative polymerase chain reaction Results are expressed as means plusmn standard deviations lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

the hair follicles were at least in the anagen IIIc-IV phaserepresenting thinner dermal papilla maximal hair bulb sizeand volume and newly formed hair shafts The number ofhair follicles in the longitudinal sections of the FDE-treatedmice increased similar to that observed in the positive FINcontrol (Figure 2(b))

33 Expression of Wnt Fibroblast Growth Factor (FGF) 7 andLef1 Genes in C57BL6N Dorsal Skin Primary Wnt (Wnt3)

and secondary Wnts (Wnt10a and Wnt10b) are essential forhair follicle initiation morphogenesis and development [11]and were examined to determine whether FDE and DEincreased expression of dermal Wnt genes implicated inthe first signal essential for inducing hair follicles Figures3(a)ndash3(c) indicate that oral administration of FDE and DEcontributed largely to differentiation of the hair medullaand regulation of matrixprecortex cells by stimulating Wntgenes (3 10a and 10b) at the higher concentration Moreover

Evidence-Based Complementary and Alternative Medicine 7

Ctrl FINFDE DE

120573-cateninPhospho-Akt

AktActin

Low High Low High

(a)

0

50

100

150

120573-c

aten

ina

ctin

()

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

0

100

200

300

p-A

ktA

kt (

)Ctrl FIN Low High Low High

FDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(c)

Figure 4 Western blot analysis of 120573-catenin and Akt phosphorylation (a) Immunoblotting analysis of 120573-catenin and phospho-Akt proteinlevels was conducted in C57BL6N dorsal skin tissue sampled on day 21 after treatment (b) A bar graph shows the quantification of 120573-cateninactin and (c) phospho-AktAkt ratio Finasteride (FIN) was used as the positive control lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrlcontrol group

FDE and DE upregulated expression of Lef1 which is anessential regulatory gene in the Wnt signaling pathway thatcontrols cell growth and differentiation through a signalingcascade from Wnts to Lef1 (Figure 3(d)) The FGF7 genewas also overexpressed in the FDE- and DE-treated groups(Figure 3(e)) suggesting a prolonged anagen phase anddelayed progression into the catagen phase in dermal papillacells [12]

34 Activation of 120573-Catenin and Akt Signaling by FDE andDE Cytosolic 120573-catenin an essential molecule in the Wntsignaling pathway translocates into the nucleus where itinduces transcription of target genes Thus we investigatedwhether FDE or DE upregulates 120573-catenin levels in dorsalskin tissues 120573-catenin level increases during the initial stagesof hair regeneration We also compared the protein levelof phosphorylated Akt a downstream target of phospho-inositide 3-kinase with that in the FIN group 120573-cateninexpression increased in response to the high doses of FDEand DE to levels comparable to those in the FIN groupMoreover Akt activation which promotes hair regrowth byregulating dermal papilla cell proliferation in the hair folliclewas upregulated in the presence of FDE and DE with moreupregulation in the FDE than that in the DE (Figures 4(a)ndash4(c))

35 Cytotoxicity and Profiles of Wnts mRNAs in hMSCs Thecytotoxicity induced by the FDE and DE was quantified bymeasuring LDH release at varying ranges of concentration

(1ndash1000120583gmL) Incubating hMSCs with each of the studysamples did not result in cell cytotoxicity except at the highestdose (1000 120583gmL) Figures 5(a) and 5(b) show that the FDEand DE did not significantly increase LDH release for 1ndash100 120583gmL exposure for up to 24 h but a higher concen-tration (1000 120583gmL) induced a significant increase in LDHrelease Figures 5(c)ndash5(e) clearly show that FDE significantlyincreased Wnt3 Wnt10a and Wnt10b mRNA in hMSCs Asexpected results of this in vitro study coincidewith the in vivodorsal skin results

4 Discussion

The hair follicle is a mammalian skin organ that produceshair which is repeatedly renewed through canonical sig-naling for proper growth and patterning Otherwise thehair cycle is disrupted and hair is lost from the head orbody which is called alopecia To date two representativetypes of drugs have been prescribed for pattern baldnessFIN which is a 5 alpha-reductase inhibitor and minoxi-dil which is an antihypertensive vasodilator are the mostfrequently prescribed medications for alopecia The selectedherbal medicines used in this study have been reportedto have diverse pharmacological activities such as antihy-percholesterolemia immunoregulation antiallergy antibac-teria antioxidation anticancer hepatoprotection and anti-insomnia [13ndash18]

As C57BL6N mice have been shown to be in a syn-chronized telogen stage of the hair cycle at 8 weeks of age

8 Evidence-Based Complementary and Alternative Medicine

1 10 100 1000

Cyto

toxi

city

()

hMSCs

0

20

40

60

80

100ve

rsus

Ctr

l

lowastlowastlowast

lowastlowastlowast

H2O2FDE (120583gmL)

(a)

Cyto

toxi

city

()

hMSCs

20

40

60

80

100

vers

us C

trl

lowastlowastlowast

lowastlowastlowast

1 10 100 10000

H2O2DE (120583gmL)

(b)

Ctrl PMA FDE DE

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

00

05

10

15

20

(Wnt

3 m

RNA

)

lowastlowastlowast

lowastlowast

(c)

hMSCs

Rela

tive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

(Wnt

10a m

RNA

)

Ctrl PMA FDE DE00

lowastlowast

(d)

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

25

(Wnt

10b

mRN

A)

Ctrl PMA FDE DE00

lowast

lowastlowast

(e)

Figure 5 Effect on Wnts (Wnt3 10a and 10b) gene expression in hMSCs (a-b) Cytotoxicity assays were performed in a 96-well plate for24 h and the results were expressed as the percent cytotoxicity for identical treatments of HDE and DE (1ndash1000 120583gmL) For gene expressionanalysis cells were seeded on a 6-well plate and treated with the HDE (100120583gmL) or DE (100120583gmL) in the presence or absence of 50120583Mphorbolmyristic acetate (PMA) for 24 h (cndashe)Wnt3Wnt10a andWnt10bmRNAwere quantified by fold units using the real-time polymerasechain reaction Results are expressed as means plusmn standard deviations from three separate experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

their hair follicle regeneration and regrowth have been welldescribed The high dose of FDE promoted hair regrowthduring the 20 consecutive days of administration that wasequal to that seen in the positive FIN controlWnt signaling isrequired to establish the hair follicle [19] and plays a key roleactivating bulge stem cells to progress toward hair formationand this signal is relayed by 120573-catenin and Lef1 [5]

In recent studies a hierarchy of Wnts that control hairfollicle development has been introduced According tothe prior reports among 19 Wnts Wnts 3 4 and 6 wereconsidered to mediate hair follicle initiation and Wnts 2 7b10a and 10b were considered to depend on the epidermalactivation [6 10 20] In line with thisWnt secretionmediateshair follicle development by acting as signalingmolecules and

Evidence-Based Complementary and Alternative Medicine 9

they can be classified as primary Wnts (Wnts 3 4 and 6)and secondary Wnts (Wnts 2 7b 10a and 10b) which areessential for initiating hair follicle growth and are involvedin development of the hair follicle respectively [10 21]In particular matrix and precortex cells express Wnt3 andWnt10ab which send a signaling cascade to Lef1 [22 23]Importantly120573-catenin which is a key regulator of hair folliclegrowth is involved in inducing the transition from telogento anagen [23] Thus it is important that a hair regrowthcandidate should be associated with Wnt signaling HampEstaining demonstrated that FDE and DE actively promotedhair follicle regrowth by increasing the number and size ofhair follicles which is an indicator for the transition of hairgrowth from the telogen to anagen phases at particular timesThe hair of FDE-treated mice was more prone to transitioninto the late anagen phase (ie IIIc) in which the dermalpapilla reaches the deepest position and rests close to themuscle layer along with the distinguishable inner and outerroot sheaths [10]

The increase in Wnt310a10b and Lef1 gene expressionin the dorsal skin strongly indicates that hair follicles wereactively developing because these genes are predominantlyexpressed in the hair placode epithelium during induction ofthe hair follicle [24ndash26] Coincident data were obtained fromqPCR analysis in hMSCs which are known to express all ofthe common stem cell markers and can be differentiated intovarious types of specialized cells under appropriate growthconditions [27] Taken together FDE could play an essentialrole in the initiation and development of hair follicles asshown by theHampE staining results Furthermore the increasein FGF7 mRNA helps understand how FDE prolongs theanagen phase and delays progression into the catagen phase

5 Conclusion

Our data clearly demonstrate that FDE effectively promoteshair regrowth by enhancing Wnt signaling and activatingAkt together with overexpression of hair regrowth-relatedprimary and secondaryWnts (Wnt310a10b) Lef1 and FGF7Although more in-depth chemical screening studies arerequired it is certain that the extract from eight selectedherbal medicines could be a promising hair regrowth candi-date when fermented after decoction

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Su Kil Jang and Seung Tae Kim equally contributed to thispaper

Acknowledgment

This study was supported by a grant from the Korea Health-care Technology RampD Project Ministry for Health Welfareand Family Affairs Republic of Korea (A091121)

References

[1] R Paus andGCotsarelis ldquoThebiology of hair folliclesrdquoTheNewEngland Journal of Medicine vol 341 no 7 pp 491ndash497 1999

[2] E Fuchs B J Merrill C Jamora and R Dasgupta ldquoAt the rootsof a never-ending cyclerdquo Developmental Cell vol 1 no 1 pp13ndash25 2001

[3] S Claudinot M Nicolas H Oshima A Rochat and Y Bar-randon ldquoLong-term renewal of hair follicles from clonogenicmultipotent stem cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 41 pp14677ndash14682 2005

[4] M Ohyama A Terunuma C L Tock et al ldquoCharacterizationand isolation of stem cell-enriched human hair follicle bulgecellsrdquoTheJournal of Clinical Investigation vol 116 no 1 pp 249ndash260 2006

[5] W E Lowry C Blanpain J A Nowak G Guasch L Lewis andE Fuchs ldquoDefining the impact of 120573-cateninTcf transactivationon epithelial stem cellsrdquo Genes amp Development vol 19 no 13pp 1596ndash1611 2005

[6] S Reddy T Andl A Bagasra et al ldquoCharacterization of Wntgene expression in developing and postnatal hair follicles andidentification of Wnt5a as a target of Sonic hedgehog in hairfollicle morphogenesisrdquo Mechanisms of Development vol 107no 1-2 pp 69ndash82 2001

[7] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[8] J C Ra I S Shin S H Kim et al ldquoSafety of intravenousinfusion of human adipose tissue-derived mesenchymal stemcells in animals and humansrdquo Stem Cells and Development vol20 no 8 pp 1297ndash1308 2011

[9] D Park G Yang D K Bae et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve cognitive functionand physical activity in ageing micerdquo Journal of NeuroscienceResearch vol 91 no 5 pp 660ndash670 2013

[10] S Muller-Rover B Handjiski C van der Veen et al ldquoA com-prehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stagesrdquoThe Journal of InvestigativeDermatology vol 117 no 1 pp 3ndash15 2001

[11] T Andl S T Reddy T Gaddapara and S E Millar ldquoWNT sig-nals are required for the initiation of hair follicle developmentrdquoDevelopmental Cell vol 2 no 5 pp 643ndash653 2002

[12] DM Danilenko B D Ring D Yanagihara et al ldquoKeratinocytegrowth factor is an important endogenousmediator of hair folli-cle growth development and differentiation normalization ofthe nunu follicular differentiation defect and amelioration ofchemotherapy-induced alopeciardquo American Journal of Pathol-ogy vol 147 no 1 pp 145ndash154 1995

[13] G S KimDHKim J J Lim et al ldquoBiological and antibacterialactivities of the natural herb Houttuynia cordata water extractagainst the intracellular bacterial pathogen Salmonella withinthe RAW 2647 macrophagerdquo Biological amp PharmaceuticalBulletin vol 31 no 11 pp 2012ndash2017 2008

[14] X-Y Yang G-S Park M H Lee et al ldquoToll-like receptor 4-mediated immunoregulation by the aqueous extract of MoriFructusrdquo Phytotherapy Research vol 23 no 12 pp 1713ndash17202009

[15] H-S Lee J-H Choi Y-E Kim I-H Kim B-M Kim and C-H Lee ldquoEffects of the cynanchum wilfordii ethanol extract onthe serum lipid profile in hypercholesterolemic ratsrdquo PreventiveNutrition and Food Science vol 18 no 3 pp 157ndash162 2013

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

To date comprehensively effective highly safe candi-date compounds prepared from herbal extracts have beenexamined for their hair regrowth activities Among thesewe selected eight herbs and derived an optimal extractthrough decoction and fermentation processes In this studywe examined the hair regrowth activity of the extract inC57BL6N mice and its prospective mechanism in conjunc-tion with Wnt signaling

2 Materials and Methods

21 Preparation of the Study Sample Eight selectedmedicinalherbs such as Cynanchum wilfordii Mori Fructus Schisan-drae Fructus Perillae Herba Houttuyniae Herba LigustriFructus Longanae Arillus and Polygonati Rhizoma werepurchased from Saerom Pharmaceutical Co Ltd (KyunggiRepublic of Korea)The study samples were prepared accord-ing to the method developed in our laboratory In briefto prepare the decoction evenly weighed herbs (1 1) werefinely ground and primarily immersed in autoclaved distilledwater (1 10 wv) for 30min followed by boiling on anelectric heater for 2 h The decoction was filtered usingWhatman Grade No 1 Filter Paper (Whatman InternationalLtd Maidstone UK) and centrifuged at 5000 rpm for 15minThe supernatant was collected lyophilized and stored at 4∘Cbefore use For fermentation Bacillus subtilis was propagatedtwice in 50mL MRS broth (Difco Detroit MI USA) at 37∘Covernight for fermentation Then 107 CFUmL of B subtiliswas inoculated into the decocted extracts and fermented at37∘C for 48 h (FDE) Nonfermented decoction extracts (DE)were used as a normal control Both samples were seriallyfiltered with a 60120583m nylon net filter and a 022120583m syringefilter (Millipore Bedford MA USA) precipitated overnightlyophilized (supernatant) and stored in desiccators at roomtemperature before use During fermentation (24 and 48 h)samples of the liquid culture were examined under phase-contrast microscopy to visualize basic cell characteristics

22 Animals Healthy male C57BL6N mice (7 weeks old)were obtained from Central Lab Animal Inc (Seoul Repub-lic of Korea) and were adapted to laboratory conditions (tem-perature 20 plusmn 2∘C relative humidity 50 and lightdarkcycle 12 h) for 1 week The animals (119899 = 3group) were main-tained at a constant temperature (23 plusmn 2∘C) relative humidity(55plusmn 10) and a 12 h lightdark cycle and fed standard rodentchow and purified water ad libitum Two days before theexperiments all mice (8 weeks of age) were shaved usinganimal clippers Telogen-synchronized C57BL6Nmice weredivided into six groups each containing three male miceThe FDE and DE extracts as well as finasteride (FIN Sigma-Aldrich St Louis MO USA) were administered daily for20 consecutive days The FDE and DE groups were given 64or 128mgkg and 140 or 280mgkg respectively consideringthe vaporized solid parts of FDE (39 g100mL) and DE(87 gmL) FIN (1mgkg) was administered as the positiveat the same time points All mice were sacrificed on day21 and the dorsal hair growth patterns were photographedon days 0 3 6 9 12 15 18 and 20 The back area of each

mousewas photographedwith a digital camera and the imagewas inputted to a computer for measurement according tothe following formula [ Hair regrowth = hairy black areadivide hair removal area] to quantitatively compare the hairregrowth patternsThe animal experiments were approved bythe Gangneung-Wonju National University Animal Care andUse Committee (Approval number GWNU-2014-27) and allprocedures were conducted in accordance with the Guide forCare and Use of Laboratory Animals published by the USNational Institutes of Health

23 Cell Culture Human mesenchymal stem cells (hMSCs)used in this study were provided by Professor Kim (Chung-buk National University Republic of Korea) [8 9] Cells werecultured at a density of 5 times 103 cellscm2 in complete mediumand Dulbeccorsquos modified Eaglersquos medium (DMEM low glu-cose) was supplemented with 10 fetal bovine serum (FBSHyclone Logan UT USA) 25 ngmL hFGF2 100UmLpenicillin and 100 120583gmL streptomycin (Invitrogen Carls-bad CA USA) Complete medium was changed every 2-3days and hMSCs were subcultured when they reach 1 times 104cellscm2 Cultures were maintained under 5 CO

2at 37∘C

in tissue culture flasks

24 Cellular Cytotoxicity (Lactate Dehydrogenase LDH)Assay The cytotoxicity induced by the FDE and DE wasquantified by measuring LDH release LDH content wasdetermined using a commercial nonradioactive LDH assaykit CytoTox 96 (Promega Madison WI USA) which isbased on a coupled enzymatic reaction that results in theconversion of a tetrazolium salt into a red formazan productThe increase in the amount of formazan produced in theculture supernatant directly correlates with the increase inthe number of lysed cells The formazan was quantified spec-trophotometrically by measuring its absorbance at 490 nm(Spectra Max 340 Molecular Devices Sunnyvale CA USA)Cytotoxicity in experimental samples was determined as LDH release compared with that in cells treated with 1Triton X-100

25 Quantitative Real-Time Polymerase Chain Reaction (PCR)Assay Total RNA from C57BL6N dorsal skin tissues orhMSCs was prepared using the TRIZOL method (Invitro-gen) cDNAs were synthesized from RNA by reverse tran-scription of 1 120583g total RNA using the ImProm-II reverse tran-scription system (Promega) and oligo dT primers in a totalvolume of 20 120583L PCR amplification was performed usingthe primers described in Table 1 (Bioneer Daejeon Republicof Korea) Quantitative real-time PCR (qPCR) reactionswere run on a Rotor-Gene 6000 (Corbett Research SydneyAustralia) using SYBR Green PCR Master Mix (QiagenValencia CA USA) in 20120583L reaction mixtures Each real-time PCRmaster mix contained 10 120583L 2x enzymeMastermix70 120583L RNase free water 1 120583L of each primer (10 pmole each)and 1 120583L diluted template PCR was performed with an initialpreincubation step for 10min at 95∘C followed by 45 cyclesof 95∘C for 15 s annealing at 52∘C for 15 s and extension at72∘C for 10 s A melting curve analysis was used to confirm

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primer sequences used for the real-time polymerase chain reaction analysis

Gene Primer Amino acid sequences Product size (bp) Accession number

Mouse

FGF7 51015840 primer 51015840-TGCTTCCACCTCGTCTGTCT 212 NM 00800831015840 primer 51015840-GAGGCAAAGTGAAAGGGACC

Wnt3 51015840 primer 51015840-AGAGACGGGCTCCTTTGGTA 123 NM 00952131015840 primer 51015840-TTCTCCTTCCGTTTCTCCGT

Wnt10a 51015840 primer 51015840-GTGCGCTCTGGGTAAACTGA 232 NM 00951831015840 primer 51015840-AGAGAAGCGTTCTCCGAAGC

Wnt10b 51015840 primer 51015840-TCTTGGCTTTGTTCAGTCGG 124 NM 01171831015840 primer 51015840-CCCAGCTGTCGCTTACTCAG

120573-catenin 51015840 primer 51015840-AGGCTTTTCCCAGTCCTTCA 122 M9036431015840 primer 51015840-TCTGCATGCCCTCATCTAGC

Lef1 51015840 primer 51015840-CGTCCTCTCAGGAGCCCTAC 169 X5863631015840 primer 51015840-GGAGAAAGGGACCCATTTGA

120573-actin 51015840 primer 51015840-TACAGCTTCACCACCACAGC 187 NM 00739331015840 primer 51015840-AAGGAAGGCTGGAAAAGAGC

Human

Wnt3 51015840 primer 51015840-CACATGCACCTCAAATGCAA 132 AB06762831015840 primer 51015840-CGAGGCGCTGTCATACTTGT

Wnt10a 51015840 primer 51015840-TTCCACTGGTGCTGCGTAGT 107 AB05957031015840 primer 51015840-CTGCGCGAAGTCAGTCTAGC

Wnt10b 51015840 primer 51015840-CATACAGGGCATCCAGATCG 148 AB05956931015840 primer 51015840-AAAAGCGCTCTCTCGGAAAC

GAPDH 51015840 primer 51015840-GGAGCCAAAAGGGTCATCAT 203 AK 02652531015840 primer 51015840-GTGATGGCATGGACTGTGGT

formation of the expected PCR product and products fromall assays were tested by 12 agarose gel electrophoresis toconfirm the correct lengths An interrun calibrator was usedand a standard curve was created for each gene to obtainPCR efficiencies Relative sample expression levels werecalculated using Rotor-Gene 6000 Series Software 17 andwere expressed relative to glyceraldehyde 3-phosphate dehy-drogenase and corrected for between-run variability Data areexpressed as a percentage of the internal control gene

26 Western Blot Analysis C57BL6N dorsal skin tissueswere homogenized and lysed in 1 RIPA buffer containingprotease and phosphatase inhibitors (Roche MannheimGermany) and total proteins were separated on 10 SDS-PAGE After electrophoresis the proteins were transferredto polyvinylidene fluoride membranes and the membraneswere blocked with 5 skim milk in Tris-buffered salinesolution containing 01 Tween-20 The membranes wereimmunoblotted with primary antibodies including anti-120573-catenin anti-phospo-Akt anti-Akt and anti-actin (SantaCruz Biotechnology Santa Cruz CA USA) followed byincubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Stressgen SanDiego CA USA) The blots were developed using anenhanced chemiluminescent solution (Thermo Rockford ILUSA)

27 Histological Examination and Hair Follicle Count Rect-angular pieces of central dorsal skin were collected parallel to

the vertebral line and fixed in 10 neutral buffered formalin(4 g sodium phosphate monobasic 65 g sodium phosphatedibasic 100mL of 37 formalin and 900mL distilled water)for 5 days The tissues were then embedded in paraffincut into sections (5120583m) and stained with a hematoxylin-eosin (HampE) solution The sections were deparaffinized withxylene hydrated in a descending graded ethanol series andstainedwith hematoxylin for 2min followed by 2minwashesand eosin staining for 5 s All tissue samples were examinedand imaged in a blinded fashion Hair follicle counts wereperformed by using a digital photomicrograph and all of theimages were cropped in a fixed area (300 pixels in width)Data were evaluated from representative areas at a fixedmagnification of 100x Images were captured using a NikonEclips Ti-S invertedmicroscope (Nikon Tokyo Japan) at 40xmagnification

28 Statistical Analysis Statistical comparisons betweengroups were performed using one-way analysis of variancewith Dunnettrsquos post hoc test and SPSS v 17 software (SPSSInc Chicago IL USA) A 119875 lt 005 was considered signifi-cant

3 Results

31 Hair Growth-Promoting Action in Male C57BL6N MiceHair growth patterns in the shaved area were comparedin all groups To evaluate the hair growth effect FIN(1mgkg) FDE (low 64mgkg high 128mgkg) and DE

4 Evidence-Based Complementary and Alternative Medicine

(Day

)

0

3

6

9

12

15

18

20

Ctrl FIN High Low High LowFDE DE

(a)

CtrlFINF-low

F-highD-lowD-high

0

20

40

60

80

100

Relat

ive a

rea (

)

3 6 90 15 18 2012

Days

(b)

Figure 1 Hair growth-promoting effect in C57BL6N mice The dorsal skin of male C57BL6N mice was shaved after the mice were orallyadministered the fermented herbal extract after decoction (FDE) the nonfermented herbal extract after decoction (DE) or finasteride for 20days (a) The shaved dorsal skin was photographed at 0 3 6 9 12 15 18 and 20 days (b) The area of hair regrowth was measured by imagesoftware on the indicated day FIN finasteride F FDE D DE

Evidence-Based Complementary and Alternative Medicine 5

Ctrl FIN

FDE-high FDE-low

DE-high DE-low

ML

HF

DP IRS

ORS

(a)

Ctrl FIN High Low High LowFDE DE

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast lowastlowastlowast

0

10

20

30

40

Hai

r fol

licle

coun

ts

(b)

Figure 2 Comparison of hair follicle growth in C57BL6N mice (a) Hematoxylin-eosin staining of dorsal skin from mice administered thefermented herbal extract after decoction (FDE) the nonfermented herbal extract after decoction (DE) or finasteride for 20 days was analyzedArrows are muscle layer (ML) dermal papilla (DP) outer root sheath (ORS) and inner root sheath (IRS) (b) The number of hair follicles indeep subcutis Values are mean plusmn standard deviations lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrl control group

(low 140mgkg high 280mgkg) were administered andpatterns of dorsal hair growth were examined on days0 3 6 9 12 15 18 and 20 (Figure 1(a)) As shown inFigure 1(a) the FDE and DE caused a gray hair color on day12 after induction and the hair shafts were visible on day15 whereas hair in the control group remained unpigmenteduntil day 15 Interestingly regrowth of hair in FDE-treatedmice was as much as that seen in the FIN positive controlwhich is a representative oral hair loss drug used worldwide(Figure 1(b))

32 Effects of FDEDE on Hair Follicle Structure Hair folliclegrowth (anagen) between groups was compared in accor-dance with accepted morphological guidelines [10] Ourresults revealed that FDE and DE increased the number andsize of hair follicles which are markers for transition of folli-cles from the telogen to anagen phase of hair growth whereashair in the control group was in the early anagen phasein which enlarged dermal papilla and the bulb located inthe dermis are distinct characteristics (Figure 2(a)) NotablyHampE staining of FDE and DE mice well demonstrated that

6 Evidence-Based Complementary and Alternative Medicine

Ctrl FIN Low High Low High00

05

10

15

20

25

FDE DE

Rela

tive c

once

ntra

tion

(Wnt

3 m

RNA

)no

rmal

ized

to120573

-act

in

lowastlowast

lowastlowastlowast

(a)

Rela

tive c

once

ntra

tion

(Wnt

10a m

RNA

)no

rmal

ized

to120573

-act

in

0

1

2

3

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(b)

Rela

tive c

once

ntra

tion

(Wnt

10b

mRN

A)

norm

aliz

ed to

120573-a

ctin

0

2

4

6

8

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(c)

Rela

tive c

once

ntra

tion

(Lef

1 m

RNA

)no

rmal

ized

to120573

-act

in

0

2

4

6

8

10

Ctrl FIN Low High Low HighFDE DE

lowastlowast

lowastlowast

lowastlowastlowast

(d)

Relat

ive c

once

ntra

tion

(FG

F7 m

RNA

)no

rmal

ized

to120573

-act

in

00

05

10

15

20

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)

Figure 3 Comparative analysis of the expression of hair regrowth-related genes at the mRNA level in dorsal skin tissue The tissues werecollected on day 21 andmRNAswere harvested using TRIZOLWnt310a10b Lef1 and fibroblast growth factor (FGF) 7 geneswere comparedby real-time quantitative polymerase chain reaction Results are expressed as means plusmn standard deviations lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

the hair follicles were at least in the anagen IIIc-IV phaserepresenting thinner dermal papilla maximal hair bulb sizeand volume and newly formed hair shafts The number ofhair follicles in the longitudinal sections of the FDE-treatedmice increased similar to that observed in the positive FINcontrol (Figure 2(b))

33 Expression of Wnt Fibroblast Growth Factor (FGF) 7 andLef1 Genes in C57BL6N Dorsal Skin Primary Wnt (Wnt3)

and secondary Wnts (Wnt10a and Wnt10b) are essential forhair follicle initiation morphogenesis and development [11]and were examined to determine whether FDE and DEincreased expression of dermal Wnt genes implicated inthe first signal essential for inducing hair follicles Figures3(a)ndash3(c) indicate that oral administration of FDE and DEcontributed largely to differentiation of the hair medullaand regulation of matrixprecortex cells by stimulating Wntgenes (3 10a and 10b) at the higher concentration Moreover

Evidence-Based Complementary and Alternative Medicine 7

Ctrl FINFDE DE

120573-cateninPhospho-Akt

AktActin

Low High Low High

(a)

0

50

100

150

120573-c

aten

ina

ctin

()

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

0

100

200

300

p-A

ktA

kt (

)Ctrl FIN Low High Low High

FDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(c)

Figure 4 Western blot analysis of 120573-catenin and Akt phosphorylation (a) Immunoblotting analysis of 120573-catenin and phospho-Akt proteinlevels was conducted in C57BL6N dorsal skin tissue sampled on day 21 after treatment (b) A bar graph shows the quantification of 120573-cateninactin and (c) phospho-AktAkt ratio Finasteride (FIN) was used as the positive control lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrlcontrol group

FDE and DE upregulated expression of Lef1 which is anessential regulatory gene in the Wnt signaling pathway thatcontrols cell growth and differentiation through a signalingcascade from Wnts to Lef1 (Figure 3(d)) The FGF7 genewas also overexpressed in the FDE- and DE-treated groups(Figure 3(e)) suggesting a prolonged anagen phase anddelayed progression into the catagen phase in dermal papillacells [12]

34 Activation of 120573-Catenin and Akt Signaling by FDE andDE Cytosolic 120573-catenin an essential molecule in the Wntsignaling pathway translocates into the nucleus where itinduces transcription of target genes Thus we investigatedwhether FDE or DE upregulates 120573-catenin levels in dorsalskin tissues 120573-catenin level increases during the initial stagesof hair regeneration We also compared the protein levelof phosphorylated Akt a downstream target of phospho-inositide 3-kinase with that in the FIN group 120573-cateninexpression increased in response to the high doses of FDEand DE to levels comparable to those in the FIN groupMoreover Akt activation which promotes hair regrowth byregulating dermal papilla cell proliferation in the hair folliclewas upregulated in the presence of FDE and DE with moreupregulation in the FDE than that in the DE (Figures 4(a)ndash4(c))

35 Cytotoxicity and Profiles of Wnts mRNAs in hMSCs Thecytotoxicity induced by the FDE and DE was quantified bymeasuring LDH release at varying ranges of concentration

(1ndash1000120583gmL) Incubating hMSCs with each of the studysamples did not result in cell cytotoxicity except at the highestdose (1000 120583gmL) Figures 5(a) and 5(b) show that the FDEand DE did not significantly increase LDH release for 1ndash100 120583gmL exposure for up to 24 h but a higher concen-tration (1000 120583gmL) induced a significant increase in LDHrelease Figures 5(c)ndash5(e) clearly show that FDE significantlyincreased Wnt3 Wnt10a and Wnt10b mRNA in hMSCs Asexpected results of this in vitro study coincidewith the in vivodorsal skin results

4 Discussion

The hair follicle is a mammalian skin organ that produceshair which is repeatedly renewed through canonical sig-naling for proper growth and patterning Otherwise thehair cycle is disrupted and hair is lost from the head orbody which is called alopecia To date two representativetypes of drugs have been prescribed for pattern baldnessFIN which is a 5 alpha-reductase inhibitor and minoxi-dil which is an antihypertensive vasodilator are the mostfrequently prescribed medications for alopecia The selectedherbal medicines used in this study have been reportedto have diverse pharmacological activities such as antihy-percholesterolemia immunoregulation antiallergy antibac-teria antioxidation anticancer hepatoprotection and anti-insomnia [13ndash18]

As C57BL6N mice have been shown to be in a syn-chronized telogen stage of the hair cycle at 8 weeks of age

8 Evidence-Based Complementary and Alternative Medicine

1 10 100 1000

Cyto

toxi

city

()

hMSCs

0

20

40

60

80

100ve

rsus

Ctr

l

lowastlowastlowast

lowastlowastlowast

H2O2FDE (120583gmL)

(a)

Cyto

toxi

city

()

hMSCs

20

40

60

80

100

vers

us C

trl

lowastlowastlowast

lowastlowastlowast

1 10 100 10000

H2O2DE (120583gmL)

(b)

Ctrl PMA FDE DE

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

00

05

10

15

20

(Wnt

3 m

RNA

)

lowastlowastlowast

lowastlowast

(c)

hMSCs

Rela

tive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

(Wnt

10a m

RNA

)

Ctrl PMA FDE DE00

lowastlowast

(d)

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

25

(Wnt

10b

mRN

A)

Ctrl PMA FDE DE00

lowast

lowastlowast

(e)

Figure 5 Effect on Wnts (Wnt3 10a and 10b) gene expression in hMSCs (a-b) Cytotoxicity assays were performed in a 96-well plate for24 h and the results were expressed as the percent cytotoxicity for identical treatments of HDE and DE (1ndash1000 120583gmL) For gene expressionanalysis cells were seeded on a 6-well plate and treated with the HDE (100120583gmL) or DE (100120583gmL) in the presence or absence of 50120583Mphorbolmyristic acetate (PMA) for 24 h (cndashe)Wnt3Wnt10a andWnt10bmRNAwere quantified by fold units using the real-time polymerasechain reaction Results are expressed as means plusmn standard deviations from three separate experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

their hair follicle regeneration and regrowth have been welldescribed The high dose of FDE promoted hair regrowthduring the 20 consecutive days of administration that wasequal to that seen in the positive FIN controlWnt signaling isrequired to establish the hair follicle [19] and plays a key roleactivating bulge stem cells to progress toward hair formationand this signal is relayed by 120573-catenin and Lef1 [5]

In recent studies a hierarchy of Wnts that control hairfollicle development has been introduced According tothe prior reports among 19 Wnts Wnts 3 4 and 6 wereconsidered to mediate hair follicle initiation and Wnts 2 7b10a and 10b were considered to depend on the epidermalactivation [6 10 20] In line with thisWnt secretionmediateshair follicle development by acting as signalingmolecules and

Evidence-Based Complementary and Alternative Medicine 9

they can be classified as primary Wnts (Wnts 3 4 and 6)and secondary Wnts (Wnts 2 7b 10a and 10b) which areessential for initiating hair follicle growth and are involvedin development of the hair follicle respectively [10 21]In particular matrix and precortex cells express Wnt3 andWnt10ab which send a signaling cascade to Lef1 [22 23]Importantly120573-catenin which is a key regulator of hair folliclegrowth is involved in inducing the transition from telogento anagen [23] Thus it is important that a hair regrowthcandidate should be associated with Wnt signaling HampEstaining demonstrated that FDE and DE actively promotedhair follicle regrowth by increasing the number and size ofhair follicles which is an indicator for the transition of hairgrowth from the telogen to anagen phases at particular timesThe hair of FDE-treated mice was more prone to transitioninto the late anagen phase (ie IIIc) in which the dermalpapilla reaches the deepest position and rests close to themuscle layer along with the distinguishable inner and outerroot sheaths [10]

The increase in Wnt310a10b and Lef1 gene expressionin the dorsal skin strongly indicates that hair follicles wereactively developing because these genes are predominantlyexpressed in the hair placode epithelium during induction ofthe hair follicle [24ndash26] Coincident data were obtained fromqPCR analysis in hMSCs which are known to express all ofthe common stem cell markers and can be differentiated intovarious types of specialized cells under appropriate growthconditions [27] Taken together FDE could play an essentialrole in the initiation and development of hair follicles asshown by theHampE staining results Furthermore the increasein FGF7 mRNA helps understand how FDE prolongs theanagen phase and delays progression into the catagen phase

5 Conclusion

Our data clearly demonstrate that FDE effectively promoteshair regrowth by enhancing Wnt signaling and activatingAkt together with overexpression of hair regrowth-relatedprimary and secondaryWnts (Wnt310a10b) Lef1 and FGF7Although more in-depth chemical screening studies arerequired it is certain that the extract from eight selectedherbal medicines could be a promising hair regrowth candi-date when fermented after decoction

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Su Kil Jang and Seung Tae Kim equally contributed to thispaper

Acknowledgment

This study was supported by a grant from the Korea Health-care Technology RampD Project Ministry for Health Welfareand Family Affairs Republic of Korea (A091121)

References

[1] R Paus andGCotsarelis ldquoThebiology of hair folliclesrdquoTheNewEngland Journal of Medicine vol 341 no 7 pp 491ndash497 1999

[2] E Fuchs B J Merrill C Jamora and R Dasgupta ldquoAt the rootsof a never-ending cyclerdquo Developmental Cell vol 1 no 1 pp13ndash25 2001

[3] S Claudinot M Nicolas H Oshima A Rochat and Y Bar-randon ldquoLong-term renewal of hair follicles from clonogenicmultipotent stem cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 41 pp14677ndash14682 2005

[4] M Ohyama A Terunuma C L Tock et al ldquoCharacterizationand isolation of stem cell-enriched human hair follicle bulgecellsrdquoTheJournal of Clinical Investigation vol 116 no 1 pp 249ndash260 2006

[5] W E Lowry C Blanpain J A Nowak G Guasch L Lewis andE Fuchs ldquoDefining the impact of 120573-cateninTcf transactivationon epithelial stem cellsrdquo Genes amp Development vol 19 no 13pp 1596ndash1611 2005

[6] S Reddy T Andl A Bagasra et al ldquoCharacterization of Wntgene expression in developing and postnatal hair follicles andidentification of Wnt5a as a target of Sonic hedgehog in hairfollicle morphogenesisrdquo Mechanisms of Development vol 107no 1-2 pp 69ndash82 2001

[7] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[8] J C Ra I S Shin S H Kim et al ldquoSafety of intravenousinfusion of human adipose tissue-derived mesenchymal stemcells in animals and humansrdquo Stem Cells and Development vol20 no 8 pp 1297ndash1308 2011

[9] D Park G Yang D K Bae et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve cognitive functionand physical activity in ageing micerdquo Journal of NeuroscienceResearch vol 91 no 5 pp 660ndash670 2013

[10] S Muller-Rover B Handjiski C van der Veen et al ldquoA com-prehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stagesrdquoThe Journal of InvestigativeDermatology vol 117 no 1 pp 3ndash15 2001

[11] T Andl S T Reddy T Gaddapara and S E Millar ldquoWNT sig-nals are required for the initiation of hair follicle developmentrdquoDevelopmental Cell vol 2 no 5 pp 643ndash653 2002

[12] DM Danilenko B D Ring D Yanagihara et al ldquoKeratinocytegrowth factor is an important endogenousmediator of hair folli-cle growth development and differentiation normalization ofthe nunu follicular differentiation defect and amelioration ofchemotherapy-induced alopeciardquo American Journal of Pathol-ogy vol 147 no 1 pp 145ndash154 1995

[13] G S KimDHKim J J Lim et al ldquoBiological and antibacterialactivities of the natural herb Houttuynia cordata water extractagainst the intracellular bacterial pathogen Salmonella withinthe RAW 2647 macrophagerdquo Biological amp PharmaceuticalBulletin vol 31 no 11 pp 2012ndash2017 2008

[14] X-Y Yang G-S Park M H Lee et al ldquoToll-like receptor 4-mediated immunoregulation by the aqueous extract of MoriFructusrdquo Phytotherapy Research vol 23 no 12 pp 1713ndash17202009

[15] H-S Lee J-H Choi Y-E Kim I-H Kim B-M Kim and C-H Lee ldquoEffects of the cynanchum wilfordii ethanol extract onthe serum lipid profile in hypercholesterolemic ratsrdquo PreventiveNutrition and Food Science vol 18 no 3 pp 157ndash162 2013

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primer sequences used for the real-time polymerase chain reaction analysis

Gene Primer Amino acid sequences Product size (bp) Accession number

Mouse

FGF7 51015840 primer 51015840-TGCTTCCACCTCGTCTGTCT 212 NM 00800831015840 primer 51015840-GAGGCAAAGTGAAAGGGACC

Wnt3 51015840 primer 51015840-AGAGACGGGCTCCTTTGGTA 123 NM 00952131015840 primer 51015840-TTCTCCTTCCGTTTCTCCGT

Wnt10a 51015840 primer 51015840-GTGCGCTCTGGGTAAACTGA 232 NM 00951831015840 primer 51015840-AGAGAAGCGTTCTCCGAAGC

Wnt10b 51015840 primer 51015840-TCTTGGCTTTGTTCAGTCGG 124 NM 01171831015840 primer 51015840-CCCAGCTGTCGCTTACTCAG

120573-catenin 51015840 primer 51015840-AGGCTTTTCCCAGTCCTTCA 122 M9036431015840 primer 51015840-TCTGCATGCCCTCATCTAGC

Lef1 51015840 primer 51015840-CGTCCTCTCAGGAGCCCTAC 169 X5863631015840 primer 51015840-GGAGAAAGGGACCCATTTGA

120573-actin 51015840 primer 51015840-TACAGCTTCACCACCACAGC 187 NM 00739331015840 primer 51015840-AAGGAAGGCTGGAAAAGAGC

Human

Wnt3 51015840 primer 51015840-CACATGCACCTCAAATGCAA 132 AB06762831015840 primer 51015840-CGAGGCGCTGTCATACTTGT

Wnt10a 51015840 primer 51015840-TTCCACTGGTGCTGCGTAGT 107 AB05957031015840 primer 51015840-CTGCGCGAAGTCAGTCTAGC

Wnt10b 51015840 primer 51015840-CATACAGGGCATCCAGATCG 148 AB05956931015840 primer 51015840-AAAAGCGCTCTCTCGGAAAC

GAPDH 51015840 primer 51015840-GGAGCCAAAAGGGTCATCAT 203 AK 02652531015840 primer 51015840-GTGATGGCATGGACTGTGGT

formation of the expected PCR product and products fromall assays were tested by 12 agarose gel electrophoresis toconfirm the correct lengths An interrun calibrator was usedand a standard curve was created for each gene to obtainPCR efficiencies Relative sample expression levels werecalculated using Rotor-Gene 6000 Series Software 17 andwere expressed relative to glyceraldehyde 3-phosphate dehy-drogenase and corrected for between-run variability Data areexpressed as a percentage of the internal control gene

26 Western Blot Analysis C57BL6N dorsal skin tissueswere homogenized and lysed in 1 RIPA buffer containingprotease and phosphatase inhibitors (Roche MannheimGermany) and total proteins were separated on 10 SDS-PAGE After electrophoresis the proteins were transferredto polyvinylidene fluoride membranes and the membraneswere blocked with 5 skim milk in Tris-buffered salinesolution containing 01 Tween-20 The membranes wereimmunoblotted with primary antibodies including anti-120573-catenin anti-phospo-Akt anti-Akt and anti-actin (SantaCruz Biotechnology Santa Cruz CA USA) followed byincubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Stressgen SanDiego CA USA) The blots were developed using anenhanced chemiluminescent solution (Thermo Rockford ILUSA)

27 Histological Examination and Hair Follicle Count Rect-angular pieces of central dorsal skin were collected parallel to

the vertebral line and fixed in 10 neutral buffered formalin(4 g sodium phosphate monobasic 65 g sodium phosphatedibasic 100mL of 37 formalin and 900mL distilled water)for 5 days The tissues were then embedded in paraffincut into sections (5120583m) and stained with a hematoxylin-eosin (HampE) solution The sections were deparaffinized withxylene hydrated in a descending graded ethanol series andstainedwith hematoxylin for 2min followed by 2minwashesand eosin staining for 5 s All tissue samples were examinedand imaged in a blinded fashion Hair follicle counts wereperformed by using a digital photomicrograph and all of theimages were cropped in a fixed area (300 pixels in width)Data were evaluated from representative areas at a fixedmagnification of 100x Images were captured using a NikonEclips Ti-S invertedmicroscope (Nikon Tokyo Japan) at 40xmagnification

28 Statistical Analysis Statistical comparisons betweengroups were performed using one-way analysis of variancewith Dunnettrsquos post hoc test and SPSS v 17 software (SPSSInc Chicago IL USA) A 119875 lt 005 was considered signifi-cant

3 Results

31 Hair Growth-Promoting Action in Male C57BL6N MiceHair growth patterns in the shaved area were comparedin all groups To evaluate the hair growth effect FIN(1mgkg) FDE (low 64mgkg high 128mgkg) and DE

4 Evidence-Based Complementary and Alternative Medicine

(Day

)

0

3

6

9

12

15

18

20

Ctrl FIN High Low High LowFDE DE

(a)

CtrlFINF-low

F-highD-lowD-high

0

20

40

60

80

100

Relat

ive a

rea (

)

3 6 90 15 18 2012

Days

(b)

Figure 1 Hair growth-promoting effect in C57BL6N mice The dorsal skin of male C57BL6N mice was shaved after the mice were orallyadministered the fermented herbal extract after decoction (FDE) the nonfermented herbal extract after decoction (DE) or finasteride for 20days (a) The shaved dorsal skin was photographed at 0 3 6 9 12 15 18 and 20 days (b) The area of hair regrowth was measured by imagesoftware on the indicated day FIN finasteride F FDE D DE

Evidence-Based Complementary and Alternative Medicine 5

Ctrl FIN

FDE-high FDE-low

DE-high DE-low

ML

HF

DP IRS

ORS

(a)

Ctrl FIN High Low High LowFDE DE

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast lowastlowastlowast

0

10

20

30

40

Hai

r fol

licle

coun

ts

(b)

Figure 2 Comparison of hair follicle growth in C57BL6N mice (a) Hematoxylin-eosin staining of dorsal skin from mice administered thefermented herbal extract after decoction (FDE) the nonfermented herbal extract after decoction (DE) or finasteride for 20 days was analyzedArrows are muscle layer (ML) dermal papilla (DP) outer root sheath (ORS) and inner root sheath (IRS) (b) The number of hair follicles indeep subcutis Values are mean plusmn standard deviations lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrl control group

(low 140mgkg high 280mgkg) were administered andpatterns of dorsal hair growth were examined on days0 3 6 9 12 15 18 and 20 (Figure 1(a)) As shown inFigure 1(a) the FDE and DE caused a gray hair color on day12 after induction and the hair shafts were visible on day15 whereas hair in the control group remained unpigmenteduntil day 15 Interestingly regrowth of hair in FDE-treatedmice was as much as that seen in the FIN positive controlwhich is a representative oral hair loss drug used worldwide(Figure 1(b))

32 Effects of FDEDE on Hair Follicle Structure Hair folliclegrowth (anagen) between groups was compared in accor-dance with accepted morphological guidelines [10] Ourresults revealed that FDE and DE increased the number andsize of hair follicles which are markers for transition of folli-cles from the telogen to anagen phase of hair growth whereashair in the control group was in the early anagen phasein which enlarged dermal papilla and the bulb located inthe dermis are distinct characteristics (Figure 2(a)) NotablyHampE staining of FDE and DE mice well demonstrated that

6 Evidence-Based Complementary and Alternative Medicine

Ctrl FIN Low High Low High00

05

10

15

20

25

FDE DE

Rela

tive c

once

ntra

tion

(Wnt

3 m

RNA

)no

rmal

ized

to120573

-act

in

lowastlowast

lowastlowastlowast

(a)

Rela

tive c

once

ntra

tion

(Wnt

10a m

RNA

)no

rmal

ized

to120573

-act

in

0

1

2

3

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(b)

Rela

tive c

once

ntra

tion

(Wnt

10b

mRN

A)

norm

aliz

ed to

120573-a

ctin

0

2

4

6

8

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(c)

Rela

tive c

once

ntra

tion

(Lef

1 m

RNA

)no

rmal

ized

to120573

-act

in

0

2

4

6

8

10

Ctrl FIN Low High Low HighFDE DE

lowastlowast

lowastlowast

lowastlowastlowast

(d)

Relat

ive c

once

ntra

tion

(FG

F7 m

RNA

)no

rmal

ized

to120573

-act

in

00

05

10

15

20

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)

Figure 3 Comparative analysis of the expression of hair regrowth-related genes at the mRNA level in dorsal skin tissue The tissues werecollected on day 21 andmRNAswere harvested using TRIZOLWnt310a10b Lef1 and fibroblast growth factor (FGF) 7 geneswere comparedby real-time quantitative polymerase chain reaction Results are expressed as means plusmn standard deviations lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

the hair follicles were at least in the anagen IIIc-IV phaserepresenting thinner dermal papilla maximal hair bulb sizeand volume and newly formed hair shafts The number ofhair follicles in the longitudinal sections of the FDE-treatedmice increased similar to that observed in the positive FINcontrol (Figure 2(b))

33 Expression of Wnt Fibroblast Growth Factor (FGF) 7 andLef1 Genes in C57BL6N Dorsal Skin Primary Wnt (Wnt3)

and secondary Wnts (Wnt10a and Wnt10b) are essential forhair follicle initiation morphogenesis and development [11]and were examined to determine whether FDE and DEincreased expression of dermal Wnt genes implicated inthe first signal essential for inducing hair follicles Figures3(a)ndash3(c) indicate that oral administration of FDE and DEcontributed largely to differentiation of the hair medullaand regulation of matrixprecortex cells by stimulating Wntgenes (3 10a and 10b) at the higher concentration Moreover

Evidence-Based Complementary and Alternative Medicine 7

Ctrl FINFDE DE

120573-cateninPhospho-Akt

AktActin

Low High Low High

(a)

0

50

100

150

120573-c

aten

ina

ctin

()

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

0

100

200

300

p-A

ktA

kt (

)Ctrl FIN Low High Low High

FDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(c)

Figure 4 Western blot analysis of 120573-catenin and Akt phosphorylation (a) Immunoblotting analysis of 120573-catenin and phospho-Akt proteinlevels was conducted in C57BL6N dorsal skin tissue sampled on day 21 after treatment (b) A bar graph shows the quantification of 120573-cateninactin and (c) phospho-AktAkt ratio Finasteride (FIN) was used as the positive control lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrlcontrol group

FDE and DE upregulated expression of Lef1 which is anessential regulatory gene in the Wnt signaling pathway thatcontrols cell growth and differentiation through a signalingcascade from Wnts to Lef1 (Figure 3(d)) The FGF7 genewas also overexpressed in the FDE- and DE-treated groups(Figure 3(e)) suggesting a prolonged anagen phase anddelayed progression into the catagen phase in dermal papillacells [12]

34 Activation of 120573-Catenin and Akt Signaling by FDE andDE Cytosolic 120573-catenin an essential molecule in the Wntsignaling pathway translocates into the nucleus where itinduces transcription of target genes Thus we investigatedwhether FDE or DE upregulates 120573-catenin levels in dorsalskin tissues 120573-catenin level increases during the initial stagesof hair regeneration We also compared the protein levelof phosphorylated Akt a downstream target of phospho-inositide 3-kinase with that in the FIN group 120573-cateninexpression increased in response to the high doses of FDEand DE to levels comparable to those in the FIN groupMoreover Akt activation which promotes hair regrowth byregulating dermal papilla cell proliferation in the hair folliclewas upregulated in the presence of FDE and DE with moreupregulation in the FDE than that in the DE (Figures 4(a)ndash4(c))

35 Cytotoxicity and Profiles of Wnts mRNAs in hMSCs Thecytotoxicity induced by the FDE and DE was quantified bymeasuring LDH release at varying ranges of concentration

(1ndash1000120583gmL) Incubating hMSCs with each of the studysamples did not result in cell cytotoxicity except at the highestdose (1000 120583gmL) Figures 5(a) and 5(b) show that the FDEand DE did not significantly increase LDH release for 1ndash100 120583gmL exposure for up to 24 h but a higher concen-tration (1000 120583gmL) induced a significant increase in LDHrelease Figures 5(c)ndash5(e) clearly show that FDE significantlyincreased Wnt3 Wnt10a and Wnt10b mRNA in hMSCs Asexpected results of this in vitro study coincidewith the in vivodorsal skin results

4 Discussion

The hair follicle is a mammalian skin organ that produceshair which is repeatedly renewed through canonical sig-naling for proper growth and patterning Otherwise thehair cycle is disrupted and hair is lost from the head orbody which is called alopecia To date two representativetypes of drugs have been prescribed for pattern baldnessFIN which is a 5 alpha-reductase inhibitor and minoxi-dil which is an antihypertensive vasodilator are the mostfrequently prescribed medications for alopecia The selectedherbal medicines used in this study have been reportedto have diverse pharmacological activities such as antihy-percholesterolemia immunoregulation antiallergy antibac-teria antioxidation anticancer hepatoprotection and anti-insomnia [13ndash18]

As C57BL6N mice have been shown to be in a syn-chronized telogen stage of the hair cycle at 8 weeks of age

8 Evidence-Based Complementary and Alternative Medicine

1 10 100 1000

Cyto

toxi

city

()

hMSCs

0

20

40

60

80

100ve

rsus

Ctr

l

lowastlowastlowast

lowastlowastlowast

H2O2FDE (120583gmL)

(a)

Cyto

toxi

city

()

hMSCs

20

40

60

80

100

vers

us C

trl

lowastlowastlowast

lowastlowastlowast

1 10 100 10000

H2O2DE (120583gmL)

(b)

Ctrl PMA FDE DE

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

00

05

10

15

20

(Wnt

3 m

RNA

)

lowastlowastlowast

lowastlowast

(c)

hMSCs

Rela

tive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

(Wnt

10a m

RNA

)

Ctrl PMA FDE DE00

lowastlowast

(d)

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

25

(Wnt

10b

mRN

A)

Ctrl PMA FDE DE00

lowast

lowastlowast

(e)

Figure 5 Effect on Wnts (Wnt3 10a and 10b) gene expression in hMSCs (a-b) Cytotoxicity assays were performed in a 96-well plate for24 h and the results were expressed as the percent cytotoxicity for identical treatments of HDE and DE (1ndash1000 120583gmL) For gene expressionanalysis cells were seeded on a 6-well plate and treated with the HDE (100120583gmL) or DE (100120583gmL) in the presence or absence of 50120583Mphorbolmyristic acetate (PMA) for 24 h (cndashe)Wnt3Wnt10a andWnt10bmRNAwere quantified by fold units using the real-time polymerasechain reaction Results are expressed as means plusmn standard deviations from three separate experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

their hair follicle regeneration and regrowth have been welldescribed The high dose of FDE promoted hair regrowthduring the 20 consecutive days of administration that wasequal to that seen in the positive FIN controlWnt signaling isrequired to establish the hair follicle [19] and plays a key roleactivating bulge stem cells to progress toward hair formationand this signal is relayed by 120573-catenin and Lef1 [5]

In recent studies a hierarchy of Wnts that control hairfollicle development has been introduced According tothe prior reports among 19 Wnts Wnts 3 4 and 6 wereconsidered to mediate hair follicle initiation and Wnts 2 7b10a and 10b were considered to depend on the epidermalactivation [6 10 20] In line with thisWnt secretionmediateshair follicle development by acting as signalingmolecules and

Evidence-Based Complementary and Alternative Medicine 9

they can be classified as primary Wnts (Wnts 3 4 and 6)and secondary Wnts (Wnts 2 7b 10a and 10b) which areessential for initiating hair follicle growth and are involvedin development of the hair follicle respectively [10 21]In particular matrix and precortex cells express Wnt3 andWnt10ab which send a signaling cascade to Lef1 [22 23]Importantly120573-catenin which is a key regulator of hair folliclegrowth is involved in inducing the transition from telogento anagen [23] Thus it is important that a hair regrowthcandidate should be associated with Wnt signaling HampEstaining demonstrated that FDE and DE actively promotedhair follicle regrowth by increasing the number and size ofhair follicles which is an indicator for the transition of hairgrowth from the telogen to anagen phases at particular timesThe hair of FDE-treated mice was more prone to transitioninto the late anagen phase (ie IIIc) in which the dermalpapilla reaches the deepest position and rests close to themuscle layer along with the distinguishable inner and outerroot sheaths [10]

The increase in Wnt310a10b and Lef1 gene expressionin the dorsal skin strongly indicates that hair follicles wereactively developing because these genes are predominantlyexpressed in the hair placode epithelium during induction ofthe hair follicle [24ndash26] Coincident data were obtained fromqPCR analysis in hMSCs which are known to express all ofthe common stem cell markers and can be differentiated intovarious types of specialized cells under appropriate growthconditions [27] Taken together FDE could play an essentialrole in the initiation and development of hair follicles asshown by theHampE staining results Furthermore the increasein FGF7 mRNA helps understand how FDE prolongs theanagen phase and delays progression into the catagen phase

5 Conclusion

Our data clearly demonstrate that FDE effectively promoteshair regrowth by enhancing Wnt signaling and activatingAkt together with overexpression of hair regrowth-relatedprimary and secondaryWnts (Wnt310a10b) Lef1 and FGF7Although more in-depth chemical screening studies arerequired it is certain that the extract from eight selectedherbal medicines could be a promising hair regrowth candi-date when fermented after decoction

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Su Kil Jang and Seung Tae Kim equally contributed to thispaper

Acknowledgment

This study was supported by a grant from the Korea Health-care Technology RampD Project Ministry for Health Welfareand Family Affairs Republic of Korea (A091121)

References

[1] R Paus andGCotsarelis ldquoThebiology of hair folliclesrdquoTheNewEngland Journal of Medicine vol 341 no 7 pp 491ndash497 1999

[2] E Fuchs B J Merrill C Jamora and R Dasgupta ldquoAt the rootsof a never-ending cyclerdquo Developmental Cell vol 1 no 1 pp13ndash25 2001

[3] S Claudinot M Nicolas H Oshima A Rochat and Y Bar-randon ldquoLong-term renewal of hair follicles from clonogenicmultipotent stem cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 41 pp14677ndash14682 2005

[4] M Ohyama A Terunuma C L Tock et al ldquoCharacterizationand isolation of stem cell-enriched human hair follicle bulgecellsrdquoTheJournal of Clinical Investigation vol 116 no 1 pp 249ndash260 2006

[5] W E Lowry C Blanpain J A Nowak G Guasch L Lewis andE Fuchs ldquoDefining the impact of 120573-cateninTcf transactivationon epithelial stem cellsrdquo Genes amp Development vol 19 no 13pp 1596ndash1611 2005

[6] S Reddy T Andl A Bagasra et al ldquoCharacterization of Wntgene expression in developing and postnatal hair follicles andidentification of Wnt5a as a target of Sonic hedgehog in hairfollicle morphogenesisrdquo Mechanisms of Development vol 107no 1-2 pp 69ndash82 2001

[7] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[8] J C Ra I S Shin S H Kim et al ldquoSafety of intravenousinfusion of human adipose tissue-derived mesenchymal stemcells in animals and humansrdquo Stem Cells and Development vol20 no 8 pp 1297ndash1308 2011

[9] D Park G Yang D K Bae et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve cognitive functionand physical activity in ageing micerdquo Journal of NeuroscienceResearch vol 91 no 5 pp 660ndash670 2013

[10] S Muller-Rover B Handjiski C van der Veen et al ldquoA com-prehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stagesrdquoThe Journal of InvestigativeDermatology vol 117 no 1 pp 3ndash15 2001

[11] T Andl S T Reddy T Gaddapara and S E Millar ldquoWNT sig-nals are required for the initiation of hair follicle developmentrdquoDevelopmental Cell vol 2 no 5 pp 643ndash653 2002

[12] DM Danilenko B D Ring D Yanagihara et al ldquoKeratinocytegrowth factor is an important endogenousmediator of hair folli-cle growth development and differentiation normalization ofthe nunu follicular differentiation defect and amelioration ofchemotherapy-induced alopeciardquo American Journal of Pathol-ogy vol 147 no 1 pp 145ndash154 1995

[13] G S KimDHKim J J Lim et al ldquoBiological and antibacterialactivities of the natural herb Houttuynia cordata water extractagainst the intracellular bacterial pathogen Salmonella withinthe RAW 2647 macrophagerdquo Biological amp PharmaceuticalBulletin vol 31 no 11 pp 2012ndash2017 2008

[14] X-Y Yang G-S Park M H Lee et al ldquoToll-like receptor 4-mediated immunoregulation by the aqueous extract of MoriFructusrdquo Phytotherapy Research vol 23 no 12 pp 1713ndash17202009

[15] H-S Lee J-H Choi Y-E Kim I-H Kim B-M Kim and C-H Lee ldquoEffects of the cynanchum wilfordii ethanol extract onthe serum lipid profile in hypercholesterolemic ratsrdquo PreventiveNutrition and Food Science vol 18 no 3 pp 157ndash162 2013

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

(Day

)

0

3

6

9

12

15

18

20

Ctrl FIN High Low High LowFDE DE

(a)

CtrlFINF-low

F-highD-lowD-high

0

20

40

60

80

100

Relat

ive a

rea (

)

3 6 90 15 18 2012

Days

(b)

Figure 1 Hair growth-promoting effect in C57BL6N mice The dorsal skin of male C57BL6N mice was shaved after the mice were orallyadministered the fermented herbal extract after decoction (FDE) the nonfermented herbal extract after decoction (DE) or finasteride for 20days (a) The shaved dorsal skin was photographed at 0 3 6 9 12 15 18 and 20 days (b) The area of hair regrowth was measured by imagesoftware on the indicated day FIN finasteride F FDE D DE

Evidence-Based Complementary and Alternative Medicine 5

Ctrl FIN

FDE-high FDE-low

DE-high DE-low

ML

HF

DP IRS

ORS

(a)

Ctrl FIN High Low High LowFDE DE

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast lowastlowastlowast

0

10

20

30

40

Hai

r fol

licle

coun

ts

(b)

Figure 2 Comparison of hair follicle growth in C57BL6N mice (a) Hematoxylin-eosin staining of dorsal skin from mice administered thefermented herbal extract after decoction (FDE) the nonfermented herbal extract after decoction (DE) or finasteride for 20 days was analyzedArrows are muscle layer (ML) dermal papilla (DP) outer root sheath (ORS) and inner root sheath (IRS) (b) The number of hair follicles indeep subcutis Values are mean plusmn standard deviations lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrl control group

(low 140mgkg high 280mgkg) were administered andpatterns of dorsal hair growth were examined on days0 3 6 9 12 15 18 and 20 (Figure 1(a)) As shown inFigure 1(a) the FDE and DE caused a gray hair color on day12 after induction and the hair shafts were visible on day15 whereas hair in the control group remained unpigmenteduntil day 15 Interestingly regrowth of hair in FDE-treatedmice was as much as that seen in the FIN positive controlwhich is a representative oral hair loss drug used worldwide(Figure 1(b))

32 Effects of FDEDE on Hair Follicle Structure Hair folliclegrowth (anagen) between groups was compared in accor-dance with accepted morphological guidelines [10] Ourresults revealed that FDE and DE increased the number andsize of hair follicles which are markers for transition of folli-cles from the telogen to anagen phase of hair growth whereashair in the control group was in the early anagen phasein which enlarged dermal papilla and the bulb located inthe dermis are distinct characteristics (Figure 2(a)) NotablyHampE staining of FDE and DE mice well demonstrated that

6 Evidence-Based Complementary and Alternative Medicine

Ctrl FIN Low High Low High00

05

10

15

20

25

FDE DE

Rela

tive c

once

ntra

tion

(Wnt

3 m

RNA

)no

rmal

ized

to120573

-act

in

lowastlowast

lowastlowastlowast

(a)

Rela

tive c

once

ntra

tion

(Wnt

10a m

RNA

)no

rmal

ized

to120573

-act

in

0

1

2

3

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(b)

Rela

tive c

once

ntra

tion

(Wnt

10b

mRN

A)

norm

aliz

ed to

120573-a

ctin

0

2

4

6

8

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(c)

Rela

tive c

once

ntra

tion

(Lef

1 m

RNA

)no

rmal

ized

to120573

-act

in

0

2

4

6

8

10

Ctrl FIN Low High Low HighFDE DE

lowastlowast

lowastlowast

lowastlowastlowast

(d)

Relat

ive c

once

ntra

tion

(FG

F7 m

RNA

)no

rmal

ized

to120573

-act

in

00

05

10

15

20

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)

Figure 3 Comparative analysis of the expression of hair regrowth-related genes at the mRNA level in dorsal skin tissue The tissues werecollected on day 21 andmRNAswere harvested using TRIZOLWnt310a10b Lef1 and fibroblast growth factor (FGF) 7 geneswere comparedby real-time quantitative polymerase chain reaction Results are expressed as means plusmn standard deviations lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

the hair follicles were at least in the anagen IIIc-IV phaserepresenting thinner dermal papilla maximal hair bulb sizeand volume and newly formed hair shafts The number ofhair follicles in the longitudinal sections of the FDE-treatedmice increased similar to that observed in the positive FINcontrol (Figure 2(b))

33 Expression of Wnt Fibroblast Growth Factor (FGF) 7 andLef1 Genes in C57BL6N Dorsal Skin Primary Wnt (Wnt3)

and secondary Wnts (Wnt10a and Wnt10b) are essential forhair follicle initiation morphogenesis and development [11]and were examined to determine whether FDE and DEincreased expression of dermal Wnt genes implicated inthe first signal essential for inducing hair follicles Figures3(a)ndash3(c) indicate that oral administration of FDE and DEcontributed largely to differentiation of the hair medullaand regulation of matrixprecortex cells by stimulating Wntgenes (3 10a and 10b) at the higher concentration Moreover

Evidence-Based Complementary and Alternative Medicine 7

Ctrl FINFDE DE

120573-cateninPhospho-Akt

AktActin

Low High Low High

(a)

0

50

100

150

120573-c

aten

ina

ctin

()

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

0

100

200

300

p-A

ktA

kt (

)Ctrl FIN Low High Low High

FDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(c)

Figure 4 Western blot analysis of 120573-catenin and Akt phosphorylation (a) Immunoblotting analysis of 120573-catenin and phospho-Akt proteinlevels was conducted in C57BL6N dorsal skin tissue sampled on day 21 after treatment (b) A bar graph shows the quantification of 120573-cateninactin and (c) phospho-AktAkt ratio Finasteride (FIN) was used as the positive control lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrlcontrol group

FDE and DE upregulated expression of Lef1 which is anessential regulatory gene in the Wnt signaling pathway thatcontrols cell growth and differentiation through a signalingcascade from Wnts to Lef1 (Figure 3(d)) The FGF7 genewas also overexpressed in the FDE- and DE-treated groups(Figure 3(e)) suggesting a prolonged anagen phase anddelayed progression into the catagen phase in dermal papillacells [12]

34 Activation of 120573-Catenin and Akt Signaling by FDE andDE Cytosolic 120573-catenin an essential molecule in the Wntsignaling pathway translocates into the nucleus where itinduces transcription of target genes Thus we investigatedwhether FDE or DE upregulates 120573-catenin levels in dorsalskin tissues 120573-catenin level increases during the initial stagesof hair regeneration We also compared the protein levelof phosphorylated Akt a downstream target of phospho-inositide 3-kinase with that in the FIN group 120573-cateninexpression increased in response to the high doses of FDEand DE to levels comparable to those in the FIN groupMoreover Akt activation which promotes hair regrowth byregulating dermal papilla cell proliferation in the hair folliclewas upregulated in the presence of FDE and DE with moreupregulation in the FDE than that in the DE (Figures 4(a)ndash4(c))

35 Cytotoxicity and Profiles of Wnts mRNAs in hMSCs Thecytotoxicity induced by the FDE and DE was quantified bymeasuring LDH release at varying ranges of concentration

(1ndash1000120583gmL) Incubating hMSCs with each of the studysamples did not result in cell cytotoxicity except at the highestdose (1000 120583gmL) Figures 5(a) and 5(b) show that the FDEand DE did not significantly increase LDH release for 1ndash100 120583gmL exposure for up to 24 h but a higher concen-tration (1000 120583gmL) induced a significant increase in LDHrelease Figures 5(c)ndash5(e) clearly show that FDE significantlyincreased Wnt3 Wnt10a and Wnt10b mRNA in hMSCs Asexpected results of this in vitro study coincidewith the in vivodorsal skin results

4 Discussion

The hair follicle is a mammalian skin organ that produceshair which is repeatedly renewed through canonical sig-naling for proper growth and patterning Otherwise thehair cycle is disrupted and hair is lost from the head orbody which is called alopecia To date two representativetypes of drugs have been prescribed for pattern baldnessFIN which is a 5 alpha-reductase inhibitor and minoxi-dil which is an antihypertensive vasodilator are the mostfrequently prescribed medications for alopecia The selectedherbal medicines used in this study have been reportedto have diverse pharmacological activities such as antihy-percholesterolemia immunoregulation antiallergy antibac-teria antioxidation anticancer hepatoprotection and anti-insomnia [13ndash18]

As C57BL6N mice have been shown to be in a syn-chronized telogen stage of the hair cycle at 8 weeks of age

8 Evidence-Based Complementary and Alternative Medicine

1 10 100 1000

Cyto

toxi

city

()

hMSCs

0

20

40

60

80

100ve

rsus

Ctr

l

lowastlowastlowast

lowastlowastlowast

H2O2FDE (120583gmL)

(a)

Cyto

toxi

city

()

hMSCs

20

40

60

80

100

vers

us C

trl

lowastlowastlowast

lowastlowastlowast

1 10 100 10000

H2O2DE (120583gmL)

(b)

Ctrl PMA FDE DE

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

00

05

10

15

20

(Wnt

3 m

RNA

)

lowastlowastlowast

lowastlowast

(c)

hMSCs

Rela

tive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

(Wnt

10a m

RNA

)

Ctrl PMA FDE DE00

lowastlowast

(d)

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

25

(Wnt

10b

mRN

A)

Ctrl PMA FDE DE00

lowast

lowastlowast

(e)

Figure 5 Effect on Wnts (Wnt3 10a and 10b) gene expression in hMSCs (a-b) Cytotoxicity assays were performed in a 96-well plate for24 h and the results were expressed as the percent cytotoxicity for identical treatments of HDE and DE (1ndash1000 120583gmL) For gene expressionanalysis cells were seeded on a 6-well plate and treated with the HDE (100120583gmL) or DE (100120583gmL) in the presence or absence of 50120583Mphorbolmyristic acetate (PMA) for 24 h (cndashe)Wnt3Wnt10a andWnt10bmRNAwere quantified by fold units using the real-time polymerasechain reaction Results are expressed as means plusmn standard deviations from three separate experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

their hair follicle regeneration and regrowth have been welldescribed The high dose of FDE promoted hair regrowthduring the 20 consecutive days of administration that wasequal to that seen in the positive FIN controlWnt signaling isrequired to establish the hair follicle [19] and plays a key roleactivating bulge stem cells to progress toward hair formationand this signal is relayed by 120573-catenin and Lef1 [5]

In recent studies a hierarchy of Wnts that control hairfollicle development has been introduced According tothe prior reports among 19 Wnts Wnts 3 4 and 6 wereconsidered to mediate hair follicle initiation and Wnts 2 7b10a and 10b were considered to depend on the epidermalactivation [6 10 20] In line with thisWnt secretionmediateshair follicle development by acting as signalingmolecules and

Evidence-Based Complementary and Alternative Medicine 9

they can be classified as primary Wnts (Wnts 3 4 and 6)and secondary Wnts (Wnts 2 7b 10a and 10b) which areessential for initiating hair follicle growth and are involvedin development of the hair follicle respectively [10 21]In particular matrix and precortex cells express Wnt3 andWnt10ab which send a signaling cascade to Lef1 [22 23]Importantly120573-catenin which is a key regulator of hair folliclegrowth is involved in inducing the transition from telogento anagen [23] Thus it is important that a hair regrowthcandidate should be associated with Wnt signaling HampEstaining demonstrated that FDE and DE actively promotedhair follicle regrowth by increasing the number and size ofhair follicles which is an indicator for the transition of hairgrowth from the telogen to anagen phases at particular timesThe hair of FDE-treated mice was more prone to transitioninto the late anagen phase (ie IIIc) in which the dermalpapilla reaches the deepest position and rests close to themuscle layer along with the distinguishable inner and outerroot sheaths [10]

The increase in Wnt310a10b and Lef1 gene expressionin the dorsal skin strongly indicates that hair follicles wereactively developing because these genes are predominantlyexpressed in the hair placode epithelium during induction ofthe hair follicle [24ndash26] Coincident data were obtained fromqPCR analysis in hMSCs which are known to express all ofthe common stem cell markers and can be differentiated intovarious types of specialized cells under appropriate growthconditions [27] Taken together FDE could play an essentialrole in the initiation and development of hair follicles asshown by theHampE staining results Furthermore the increasein FGF7 mRNA helps understand how FDE prolongs theanagen phase and delays progression into the catagen phase

5 Conclusion

Our data clearly demonstrate that FDE effectively promoteshair regrowth by enhancing Wnt signaling and activatingAkt together with overexpression of hair regrowth-relatedprimary and secondaryWnts (Wnt310a10b) Lef1 and FGF7Although more in-depth chemical screening studies arerequired it is certain that the extract from eight selectedherbal medicines could be a promising hair regrowth candi-date when fermented after decoction

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Su Kil Jang and Seung Tae Kim equally contributed to thispaper

Acknowledgment

This study was supported by a grant from the Korea Health-care Technology RampD Project Ministry for Health Welfareand Family Affairs Republic of Korea (A091121)

References

[1] R Paus andGCotsarelis ldquoThebiology of hair folliclesrdquoTheNewEngland Journal of Medicine vol 341 no 7 pp 491ndash497 1999

[2] E Fuchs B J Merrill C Jamora and R Dasgupta ldquoAt the rootsof a never-ending cyclerdquo Developmental Cell vol 1 no 1 pp13ndash25 2001

[3] S Claudinot M Nicolas H Oshima A Rochat and Y Bar-randon ldquoLong-term renewal of hair follicles from clonogenicmultipotent stem cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 41 pp14677ndash14682 2005

[4] M Ohyama A Terunuma C L Tock et al ldquoCharacterizationand isolation of stem cell-enriched human hair follicle bulgecellsrdquoTheJournal of Clinical Investigation vol 116 no 1 pp 249ndash260 2006

[5] W E Lowry C Blanpain J A Nowak G Guasch L Lewis andE Fuchs ldquoDefining the impact of 120573-cateninTcf transactivationon epithelial stem cellsrdquo Genes amp Development vol 19 no 13pp 1596ndash1611 2005

[6] S Reddy T Andl A Bagasra et al ldquoCharacterization of Wntgene expression in developing and postnatal hair follicles andidentification of Wnt5a as a target of Sonic hedgehog in hairfollicle morphogenesisrdquo Mechanisms of Development vol 107no 1-2 pp 69ndash82 2001

[7] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[8] J C Ra I S Shin S H Kim et al ldquoSafety of intravenousinfusion of human adipose tissue-derived mesenchymal stemcells in animals and humansrdquo Stem Cells and Development vol20 no 8 pp 1297ndash1308 2011

[9] D Park G Yang D K Bae et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve cognitive functionand physical activity in ageing micerdquo Journal of NeuroscienceResearch vol 91 no 5 pp 660ndash670 2013

[10] S Muller-Rover B Handjiski C van der Veen et al ldquoA com-prehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stagesrdquoThe Journal of InvestigativeDermatology vol 117 no 1 pp 3ndash15 2001

[11] T Andl S T Reddy T Gaddapara and S E Millar ldquoWNT sig-nals are required for the initiation of hair follicle developmentrdquoDevelopmental Cell vol 2 no 5 pp 643ndash653 2002

[12] DM Danilenko B D Ring D Yanagihara et al ldquoKeratinocytegrowth factor is an important endogenousmediator of hair folli-cle growth development and differentiation normalization ofthe nunu follicular differentiation defect and amelioration ofchemotherapy-induced alopeciardquo American Journal of Pathol-ogy vol 147 no 1 pp 145ndash154 1995

[13] G S KimDHKim J J Lim et al ldquoBiological and antibacterialactivities of the natural herb Houttuynia cordata water extractagainst the intracellular bacterial pathogen Salmonella withinthe RAW 2647 macrophagerdquo Biological amp PharmaceuticalBulletin vol 31 no 11 pp 2012ndash2017 2008

[14] X-Y Yang G-S Park M H Lee et al ldquoToll-like receptor 4-mediated immunoregulation by the aqueous extract of MoriFructusrdquo Phytotherapy Research vol 23 no 12 pp 1713ndash17202009

[15] H-S Lee J-H Choi Y-E Kim I-H Kim B-M Kim and C-H Lee ldquoEffects of the cynanchum wilfordii ethanol extract onthe serum lipid profile in hypercholesterolemic ratsrdquo PreventiveNutrition and Food Science vol 18 no 3 pp 157ndash162 2013

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

Ctrl FIN

FDE-high FDE-low

DE-high DE-low

ML

HF

DP IRS

ORS

(a)

Ctrl FIN High Low High LowFDE DE

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast lowastlowastlowast

0

10

20

30

40

Hai

r fol

licle

coun

ts

(b)

Figure 2 Comparison of hair follicle growth in C57BL6N mice (a) Hematoxylin-eosin staining of dorsal skin from mice administered thefermented herbal extract after decoction (FDE) the nonfermented herbal extract after decoction (DE) or finasteride for 20 days was analyzedArrows are muscle layer (ML) dermal papilla (DP) outer root sheath (ORS) and inner root sheath (IRS) (b) The number of hair follicles indeep subcutis Values are mean plusmn standard deviations lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrl control group

(low 140mgkg high 280mgkg) were administered andpatterns of dorsal hair growth were examined on days0 3 6 9 12 15 18 and 20 (Figure 1(a)) As shown inFigure 1(a) the FDE and DE caused a gray hair color on day12 after induction and the hair shafts were visible on day15 whereas hair in the control group remained unpigmenteduntil day 15 Interestingly regrowth of hair in FDE-treatedmice was as much as that seen in the FIN positive controlwhich is a representative oral hair loss drug used worldwide(Figure 1(b))

32 Effects of FDEDE on Hair Follicle Structure Hair folliclegrowth (anagen) between groups was compared in accor-dance with accepted morphological guidelines [10] Ourresults revealed that FDE and DE increased the number andsize of hair follicles which are markers for transition of folli-cles from the telogen to anagen phase of hair growth whereashair in the control group was in the early anagen phasein which enlarged dermal papilla and the bulb located inthe dermis are distinct characteristics (Figure 2(a)) NotablyHampE staining of FDE and DE mice well demonstrated that

6 Evidence-Based Complementary and Alternative Medicine

Ctrl FIN Low High Low High00

05

10

15

20

25

FDE DE

Rela

tive c

once

ntra

tion

(Wnt

3 m

RNA

)no

rmal

ized

to120573

-act

in

lowastlowast

lowastlowastlowast

(a)

Rela

tive c

once

ntra

tion

(Wnt

10a m

RNA

)no

rmal

ized

to120573

-act

in

0

1

2

3

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(b)

Rela

tive c

once

ntra

tion

(Wnt

10b

mRN

A)

norm

aliz

ed to

120573-a

ctin

0

2

4

6

8

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(c)

Rela

tive c

once

ntra

tion

(Lef

1 m

RNA

)no

rmal

ized

to120573

-act

in

0

2

4

6

8

10

Ctrl FIN Low High Low HighFDE DE

lowastlowast

lowastlowast

lowastlowastlowast

(d)

Relat

ive c

once

ntra

tion

(FG

F7 m

RNA

)no

rmal

ized

to120573

-act

in

00

05

10

15

20

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)

Figure 3 Comparative analysis of the expression of hair regrowth-related genes at the mRNA level in dorsal skin tissue The tissues werecollected on day 21 andmRNAswere harvested using TRIZOLWnt310a10b Lef1 and fibroblast growth factor (FGF) 7 geneswere comparedby real-time quantitative polymerase chain reaction Results are expressed as means plusmn standard deviations lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

the hair follicles were at least in the anagen IIIc-IV phaserepresenting thinner dermal papilla maximal hair bulb sizeand volume and newly formed hair shafts The number ofhair follicles in the longitudinal sections of the FDE-treatedmice increased similar to that observed in the positive FINcontrol (Figure 2(b))

33 Expression of Wnt Fibroblast Growth Factor (FGF) 7 andLef1 Genes in C57BL6N Dorsal Skin Primary Wnt (Wnt3)

and secondary Wnts (Wnt10a and Wnt10b) are essential forhair follicle initiation morphogenesis and development [11]and were examined to determine whether FDE and DEincreased expression of dermal Wnt genes implicated inthe first signal essential for inducing hair follicles Figures3(a)ndash3(c) indicate that oral administration of FDE and DEcontributed largely to differentiation of the hair medullaand regulation of matrixprecortex cells by stimulating Wntgenes (3 10a and 10b) at the higher concentration Moreover

Evidence-Based Complementary and Alternative Medicine 7

Ctrl FINFDE DE

120573-cateninPhospho-Akt

AktActin

Low High Low High

(a)

0

50

100

150

120573-c

aten

ina

ctin

()

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

0

100

200

300

p-A

ktA

kt (

)Ctrl FIN Low High Low High

FDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(c)

Figure 4 Western blot analysis of 120573-catenin and Akt phosphorylation (a) Immunoblotting analysis of 120573-catenin and phospho-Akt proteinlevels was conducted in C57BL6N dorsal skin tissue sampled on day 21 after treatment (b) A bar graph shows the quantification of 120573-cateninactin and (c) phospho-AktAkt ratio Finasteride (FIN) was used as the positive control lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrlcontrol group

FDE and DE upregulated expression of Lef1 which is anessential regulatory gene in the Wnt signaling pathway thatcontrols cell growth and differentiation through a signalingcascade from Wnts to Lef1 (Figure 3(d)) The FGF7 genewas also overexpressed in the FDE- and DE-treated groups(Figure 3(e)) suggesting a prolonged anagen phase anddelayed progression into the catagen phase in dermal papillacells [12]

34 Activation of 120573-Catenin and Akt Signaling by FDE andDE Cytosolic 120573-catenin an essential molecule in the Wntsignaling pathway translocates into the nucleus where itinduces transcription of target genes Thus we investigatedwhether FDE or DE upregulates 120573-catenin levels in dorsalskin tissues 120573-catenin level increases during the initial stagesof hair regeneration We also compared the protein levelof phosphorylated Akt a downstream target of phospho-inositide 3-kinase with that in the FIN group 120573-cateninexpression increased in response to the high doses of FDEand DE to levels comparable to those in the FIN groupMoreover Akt activation which promotes hair regrowth byregulating dermal papilla cell proliferation in the hair folliclewas upregulated in the presence of FDE and DE with moreupregulation in the FDE than that in the DE (Figures 4(a)ndash4(c))

35 Cytotoxicity and Profiles of Wnts mRNAs in hMSCs Thecytotoxicity induced by the FDE and DE was quantified bymeasuring LDH release at varying ranges of concentration

(1ndash1000120583gmL) Incubating hMSCs with each of the studysamples did not result in cell cytotoxicity except at the highestdose (1000 120583gmL) Figures 5(a) and 5(b) show that the FDEand DE did not significantly increase LDH release for 1ndash100 120583gmL exposure for up to 24 h but a higher concen-tration (1000 120583gmL) induced a significant increase in LDHrelease Figures 5(c)ndash5(e) clearly show that FDE significantlyincreased Wnt3 Wnt10a and Wnt10b mRNA in hMSCs Asexpected results of this in vitro study coincidewith the in vivodorsal skin results

4 Discussion

The hair follicle is a mammalian skin organ that produceshair which is repeatedly renewed through canonical sig-naling for proper growth and patterning Otherwise thehair cycle is disrupted and hair is lost from the head orbody which is called alopecia To date two representativetypes of drugs have been prescribed for pattern baldnessFIN which is a 5 alpha-reductase inhibitor and minoxi-dil which is an antihypertensive vasodilator are the mostfrequently prescribed medications for alopecia The selectedherbal medicines used in this study have been reportedto have diverse pharmacological activities such as antihy-percholesterolemia immunoregulation antiallergy antibac-teria antioxidation anticancer hepatoprotection and anti-insomnia [13ndash18]

As C57BL6N mice have been shown to be in a syn-chronized telogen stage of the hair cycle at 8 weeks of age

8 Evidence-Based Complementary and Alternative Medicine

1 10 100 1000

Cyto

toxi

city

()

hMSCs

0

20

40

60

80

100ve

rsus

Ctr

l

lowastlowastlowast

lowastlowastlowast

H2O2FDE (120583gmL)

(a)

Cyto

toxi

city

()

hMSCs

20

40

60

80

100

vers

us C

trl

lowastlowastlowast

lowastlowastlowast

1 10 100 10000

H2O2DE (120583gmL)

(b)

Ctrl PMA FDE DE

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

00

05

10

15

20

(Wnt

3 m

RNA

)

lowastlowastlowast

lowastlowast

(c)

hMSCs

Rela

tive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

(Wnt

10a m

RNA

)

Ctrl PMA FDE DE00

lowastlowast

(d)

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

25

(Wnt

10b

mRN

A)

Ctrl PMA FDE DE00

lowast

lowastlowast

(e)

Figure 5 Effect on Wnts (Wnt3 10a and 10b) gene expression in hMSCs (a-b) Cytotoxicity assays were performed in a 96-well plate for24 h and the results were expressed as the percent cytotoxicity for identical treatments of HDE and DE (1ndash1000 120583gmL) For gene expressionanalysis cells were seeded on a 6-well plate and treated with the HDE (100120583gmL) or DE (100120583gmL) in the presence or absence of 50120583Mphorbolmyristic acetate (PMA) for 24 h (cndashe)Wnt3Wnt10a andWnt10bmRNAwere quantified by fold units using the real-time polymerasechain reaction Results are expressed as means plusmn standard deviations from three separate experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

their hair follicle regeneration and regrowth have been welldescribed The high dose of FDE promoted hair regrowthduring the 20 consecutive days of administration that wasequal to that seen in the positive FIN controlWnt signaling isrequired to establish the hair follicle [19] and plays a key roleactivating bulge stem cells to progress toward hair formationand this signal is relayed by 120573-catenin and Lef1 [5]

In recent studies a hierarchy of Wnts that control hairfollicle development has been introduced According tothe prior reports among 19 Wnts Wnts 3 4 and 6 wereconsidered to mediate hair follicle initiation and Wnts 2 7b10a and 10b were considered to depend on the epidermalactivation [6 10 20] In line with thisWnt secretionmediateshair follicle development by acting as signalingmolecules and

Evidence-Based Complementary and Alternative Medicine 9

they can be classified as primary Wnts (Wnts 3 4 and 6)and secondary Wnts (Wnts 2 7b 10a and 10b) which areessential for initiating hair follicle growth and are involvedin development of the hair follicle respectively [10 21]In particular matrix and precortex cells express Wnt3 andWnt10ab which send a signaling cascade to Lef1 [22 23]Importantly120573-catenin which is a key regulator of hair folliclegrowth is involved in inducing the transition from telogento anagen [23] Thus it is important that a hair regrowthcandidate should be associated with Wnt signaling HampEstaining demonstrated that FDE and DE actively promotedhair follicle regrowth by increasing the number and size ofhair follicles which is an indicator for the transition of hairgrowth from the telogen to anagen phases at particular timesThe hair of FDE-treated mice was more prone to transitioninto the late anagen phase (ie IIIc) in which the dermalpapilla reaches the deepest position and rests close to themuscle layer along with the distinguishable inner and outerroot sheaths [10]

The increase in Wnt310a10b and Lef1 gene expressionin the dorsal skin strongly indicates that hair follicles wereactively developing because these genes are predominantlyexpressed in the hair placode epithelium during induction ofthe hair follicle [24ndash26] Coincident data were obtained fromqPCR analysis in hMSCs which are known to express all ofthe common stem cell markers and can be differentiated intovarious types of specialized cells under appropriate growthconditions [27] Taken together FDE could play an essentialrole in the initiation and development of hair follicles asshown by theHampE staining results Furthermore the increasein FGF7 mRNA helps understand how FDE prolongs theanagen phase and delays progression into the catagen phase

5 Conclusion

Our data clearly demonstrate that FDE effectively promoteshair regrowth by enhancing Wnt signaling and activatingAkt together with overexpression of hair regrowth-relatedprimary and secondaryWnts (Wnt310a10b) Lef1 and FGF7Although more in-depth chemical screening studies arerequired it is certain that the extract from eight selectedherbal medicines could be a promising hair regrowth candi-date when fermented after decoction

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Su Kil Jang and Seung Tae Kim equally contributed to thispaper

Acknowledgment

This study was supported by a grant from the Korea Health-care Technology RampD Project Ministry for Health Welfareand Family Affairs Republic of Korea (A091121)

References

[1] R Paus andGCotsarelis ldquoThebiology of hair folliclesrdquoTheNewEngland Journal of Medicine vol 341 no 7 pp 491ndash497 1999

[2] E Fuchs B J Merrill C Jamora and R Dasgupta ldquoAt the rootsof a never-ending cyclerdquo Developmental Cell vol 1 no 1 pp13ndash25 2001

[3] S Claudinot M Nicolas H Oshima A Rochat and Y Bar-randon ldquoLong-term renewal of hair follicles from clonogenicmultipotent stem cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 41 pp14677ndash14682 2005

[4] M Ohyama A Terunuma C L Tock et al ldquoCharacterizationand isolation of stem cell-enriched human hair follicle bulgecellsrdquoTheJournal of Clinical Investigation vol 116 no 1 pp 249ndash260 2006

[5] W E Lowry C Blanpain J A Nowak G Guasch L Lewis andE Fuchs ldquoDefining the impact of 120573-cateninTcf transactivationon epithelial stem cellsrdquo Genes amp Development vol 19 no 13pp 1596ndash1611 2005

[6] S Reddy T Andl A Bagasra et al ldquoCharacterization of Wntgene expression in developing and postnatal hair follicles andidentification of Wnt5a as a target of Sonic hedgehog in hairfollicle morphogenesisrdquo Mechanisms of Development vol 107no 1-2 pp 69ndash82 2001

[7] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[8] J C Ra I S Shin S H Kim et al ldquoSafety of intravenousinfusion of human adipose tissue-derived mesenchymal stemcells in animals and humansrdquo Stem Cells and Development vol20 no 8 pp 1297ndash1308 2011

[9] D Park G Yang D K Bae et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve cognitive functionand physical activity in ageing micerdquo Journal of NeuroscienceResearch vol 91 no 5 pp 660ndash670 2013

[10] S Muller-Rover B Handjiski C van der Veen et al ldquoA com-prehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stagesrdquoThe Journal of InvestigativeDermatology vol 117 no 1 pp 3ndash15 2001

[11] T Andl S T Reddy T Gaddapara and S E Millar ldquoWNT sig-nals are required for the initiation of hair follicle developmentrdquoDevelopmental Cell vol 2 no 5 pp 643ndash653 2002

[12] DM Danilenko B D Ring D Yanagihara et al ldquoKeratinocytegrowth factor is an important endogenousmediator of hair folli-cle growth development and differentiation normalization ofthe nunu follicular differentiation defect and amelioration ofchemotherapy-induced alopeciardquo American Journal of Pathol-ogy vol 147 no 1 pp 145ndash154 1995

[13] G S KimDHKim J J Lim et al ldquoBiological and antibacterialactivities of the natural herb Houttuynia cordata water extractagainst the intracellular bacterial pathogen Salmonella withinthe RAW 2647 macrophagerdquo Biological amp PharmaceuticalBulletin vol 31 no 11 pp 2012ndash2017 2008

[14] X-Y Yang G-S Park M H Lee et al ldquoToll-like receptor 4-mediated immunoregulation by the aqueous extract of MoriFructusrdquo Phytotherapy Research vol 23 no 12 pp 1713ndash17202009

[15] H-S Lee J-H Choi Y-E Kim I-H Kim B-M Kim and C-H Lee ldquoEffects of the cynanchum wilfordii ethanol extract onthe serum lipid profile in hypercholesterolemic ratsrdquo PreventiveNutrition and Food Science vol 18 no 3 pp 157ndash162 2013

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Ctrl FIN Low High Low High00

05

10

15

20

25

FDE DE

Rela

tive c

once

ntra

tion

(Wnt

3 m

RNA

)no

rmal

ized

to120573

-act

in

lowastlowast

lowastlowastlowast

(a)

Rela

tive c

once

ntra

tion

(Wnt

10a m

RNA

)no

rmal

ized

to120573

-act

in

0

1

2

3

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(b)

Rela

tive c

once

ntra

tion

(Wnt

10b

mRN

A)

norm

aliz

ed to

120573-a

ctin

0

2

4

6

8

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

(c)

Rela

tive c

once

ntra

tion

(Lef

1 m

RNA

)no

rmal

ized

to120573

-act

in

0

2

4

6

8

10

Ctrl FIN Low High Low HighFDE DE

lowastlowast

lowastlowast

lowastlowastlowast

(d)

Relat

ive c

once

ntra

tion

(FG

F7 m

RNA

)no

rmal

ized

to120573

-act

in

00

05

10

15

20

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)

Figure 3 Comparative analysis of the expression of hair regrowth-related genes at the mRNA level in dorsal skin tissue The tissues werecollected on day 21 andmRNAswere harvested using TRIZOLWnt310a10b Lef1 and fibroblast growth factor (FGF) 7 geneswere comparedby real-time quantitative polymerase chain reaction Results are expressed as means plusmn standard deviations lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

the hair follicles were at least in the anagen IIIc-IV phaserepresenting thinner dermal papilla maximal hair bulb sizeand volume and newly formed hair shafts The number ofhair follicles in the longitudinal sections of the FDE-treatedmice increased similar to that observed in the positive FINcontrol (Figure 2(b))

33 Expression of Wnt Fibroblast Growth Factor (FGF) 7 andLef1 Genes in C57BL6N Dorsal Skin Primary Wnt (Wnt3)

and secondary Wnts (Wnt10a and Wnt10b) are essential forhair follicle initiation morphogenesis and development [11]and were examined to determine whether FDE and DEincreased expression of dermal Wnt genes implicated inthe first signal essential for inducing hair follicles Figures3(a)ndash3(c) indicate that oral administration of FDE and DEcontributed largely to differentiation of the hair medullaand regulation of matrixprecortex cells by stimulating Wntgenes (3 10a and 10b) at the higher concentration Moreover

Evidence-Based Complementary and Alternative Medicine 7

Ctrl FINFDE DE

120573-cateninPhospho-Akt

AktActin

Low High Low High

(a)

0

50

100

150

120573-c

aten

ina

ctin

()

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

0

100

200

300

p-A

ktA

kt (

)Ctrl FIN Low High Low High

FDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(c)

Figure 4 Western blot analysis of 120573-catenin and Akt phosphorylation (a) Immunoblotting analysis of 120573-catenin and phospho-Akt proteinlevels was conducted in C57BL6N dorsal skin tissue sampled on day 21 after treatment (b) A bar graph shows the quantification of 120573-cateninactin and (c) phospho-AktAkt ratio Finasteride (FIN) was used as the positive control lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrlcontrol group

FDE and DE upregulated expression of Lef1 which is anessential regulatory gene in the Wnt signaling pathway thatcontrols cell growth and differentiation through a signalingcascade from Wnts to Lef1 (Figure 3(d)) The FGF7 genewas also overexpressed in the FDE- and DE-treated groups(Figure 3(e)) suggesting a prolonged anagen phase anddelayed progression into the catagen phase in dermal papillacells [12]

34 Activation of 120573-Catenin and Akt Signaling by FDE andDE Cytosolic 120573-catenin an essential molecule in the Wntsignaling pathway translocates into the nucleus where itinduces transcription of target genes Thus we investigatedwhether FDE or DE upregulates 120573-catenin levels in dorsalskin tissues 120573-catenin level increases during the initial stagesof hair regeneration We also compared the protein levelof phosphorylated Akt a downstream target of phospho-inositide 3-kinase with that in the FIN group 120573-cateninexpression increased in response to the high doses of FDEand DE to levels comparable to those in the FIN groupMoreover Akt activation which promotes hair regrowth byregulating dermal papilla cell proliferation in the hair folliclewas upregulated in the presence of FDE and DE with moreupregulation in the FDE than that in the DE (Figures 4(a)ndash4(c))

35 Cytotoxicity and Profiles of Wnts mRNAs in hMSCs Thecytotoxicity induced by the FDE and DE was quantified bymeasuring LDH release at varying ranges of concentration

(1ndash1000120583gmL) Incubating hMSCs with each of the studysamples did not result in cell cytotoxicity except at the highestdose (1000 120583gmL) Figures 5(a) and 5(b) show that the FDEand DE did not significantly increase LDH release for 1ndash100 120583gmL exposure for up to 24 h but a higher concen-tration (1000 120583gmL) induced a significant increase in LDHrelease Figures 5(c)ndash5(e) clearly show that FDE significantlyincreased Wnt3 Wnt10a and Wnt10b mRNA in hMSCs Asexpected results of this in vitro study coincidewith the in vivodorsal skin results

4 Discussion

The hair follicle is a mammalian skin organ that produceshair which is repeatedly renewed through canonical sig-naling for proper growth and patterning Otherwise thehair cycle is disrupted and hair is lost from the head orbody which is called alopecia To date two representativetypes of drugs have been prescribed for pattern baldnessFIN which is a 5 alpha-reductase inhibitor and minoxi-dil which is an antihypertensive vasodilator are the mostfrequently prescribed medications for alopecia The selectedherbal medicines used in this study have been reportedto have diverse pharmacological activities such as antihy-percholesterolemia immunoregulation antiallergy antibac-teria antioxidation anticancer hepatoprotection and anti-insomnia [13ndash18]

As C57BL6N mice have been shown to be in a syn-chronized telogen stage of the hair cycle at 8 weeks of age

8 Evidence-Based Complementary and Alternative Medicine

1 10 100 1000

Cyto

toxi

city

()

hMSCs

0

20

40

60

80

100ve

rsus

Ctr

l

lowastlowastlowast

lowastlowastlowast

H2O2FDE (120583gmL)

(a)

Cyto

toxi

city

()

hMSCs

20

40

60

80

100

vers

us C

trl

lowastlowastlowast

lowastlowastlowast

1 10 100 10000

H2O2DE (120583gmL)

(b)

Ctrl PMA FDE DE

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

00

05

10

15

20

(Wnt

3 m

RNA

)

lowastlowastlowast

lowastlowast

(c)

hMSCs

Rela

tive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

(Wnt

10a m

RNA

)

Ctrl PMA FDE DE00

lowastlowast

(d)

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

25

(Wnt

10b

mRN

A)

Ctrl PMA FDE DE00

lowast

lowastlowast

(e)

Figure 5 Effect on Wnts (Wnt3 10a and 10b) gene expression in hMSCs (a-b) Cytotoxicity assays were performed in a 96-well plate for24 h and the results were expressed as the percent cytotoxicity for identical treatments of HDE and DE (1ndash1000 120583gmL) For gene expressionanalysis cells were seeded on a 6-well plate and treated with the HDE (100120583gmL) or DE (100120583gmL) in the presence or absence of 50120583Mphorbolmyristic acetate (PMA) for 24 h (cndashe)Wnt3Wnt10a andWnt10bmRNAwere quantified by fold units using the real-time polymerasechain reaction Results are expressed as means plusmn standard deviations from three separate experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

their hair follicle regeneration and regrowth have been welldescribed The high dose of FDE promoted hair regrowthduring the 20 consecutive days of administration that wasequal to that seen in the positive FIN controlWnt signaling isrequired to establish the hair follicle [19] and plays a key roleactivating bulge stem cells to progress toward hair formationand this signal is relayed by 120573-catenin and Lef1 [5]

In recent studies a hierarchy of Wnts that control hairfollicle development has been introduced According tothe prior reports among 19 Wnts Wnts 3 4 and 6 wereconsidered to mediate hair follicle initiation and Wnts 2 7b10a and 10b were considered to depend on the epidermalactivation [6 10 20] In line with thisWnt secretionmediateshair follicle development by acting as signalingmolecules and

Evidence-Based Complementary and Alternative Medicine 9

they can be classified as primary Wnts (Wnts 3 4 and 6)and secondary Wnts (Wnts 2 7b 10a and 10b) which areessential for initiating hair follicle growth and are involvedin development of the hair follicle respectively [10 21]In particular matrix and precortex cells express Wnt3 andWnt10ab which send a signaling cascade to Lef1 [22 23]Importantly120573-catenin which is a key regulator of hair folliclegrowth is involved in inducing the transition from telogento anagen [23] Thus it is important that a hair regrowthcandidate should be associated with Wnt signaling HampEstaining demonstrated that FDE and DE actively promotedhair follicle regrowth by increasing the number and size ofhair follicles which is an indicator for the transition of hairgrowth from the telogen to anagen phases at particular timesThe hair of FDE-treated mice was more prone to transitioninto the late anagen phase (ie IIIc) in which the dermalpapilla reaches the deepest position and rests close to themuscle layer along with the distinguishable inner and outerroot sheaths [10]

The increase in Wnt310a10b and Lef1 gene expressionin the dorsal skin strongly indicates that hair follicles wereactively developing because these genes are predominantlyexpressed in the hair placode epithelium during induction ofthe hair follicle [24ndash26] Coincident data were obtained fromqPCR analysis in hMSCs which are known to express all ofthe common stem cell markers and can be differentiated intovarious types of specialized cells under appropriate growthconditions [27] Taken together FDE could play an essentialrole in the initiation and development of hair follicles asshown by theHampE staining results Furthermore the increasein FGF7 mRNA helps understand how FDE prolongs theanagen phase and delays progression into the catagen phase

5 Conclusion

Our data clearly demonstrate that FDE effectively promoteshair regrowth by enhancing Wnt signaling and activatingAkt together with overexpression of hair regrowth-relatedprimary and secondaryWnts (Wnt310a10b) Lef1 and FGF7Although more in-depth chemical screening studies arerequired it is certain that the extract from eight selectedherbal medicines could be a promising hair regrowth candi-date when fermented after decoction

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Su Kil Jang and Seung Tae Kim equally contributed to thispaper

Acknowledgment

This study was supported by a grant from the Korea Health-care Technology RampD Project Ministry for Health Welfareand Family Affairs Republic of Korea (A091121)

References

[1] R Paus andGCotsarelis ldquoThebiology of hair folliclesrdquoTheNewEngland Journal of Medicine vol 341 no 7 pp 491ndash497 1999

[2] E Fuchs B J Merrill C Jamora and R Dasgupta ldquoAt the rootsof a never-ending cyclerdquo Developmental Cell vol 1 no 1 pp13ndash25 2001

[3] S Claudinot M Nicolas H Oshima A Rochat and Y Bar-randon ldquoLong-term renewal of hair follicles from clonogenicmultipotent stem cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 41 pp14677ndash14682 2005

[4] M Ohyama A Terunuma C L Tock et al ldquoCharacterizationand isolation of stem cell-enriched human hair follicle bulgecellsrdquoTheJournal of Clinical Investigation vol 116 no 1 pp 249ndash260 2006

[5] W E Lowry C Blanpain J A Nowak G Guasch L Lewis andE Fuchs ldquoDefining the impact of 120573-cateninTcf transactivationon epithelial stem cellsrdquo Genes amp Development vol 19 no 13pp 1596ndash1611 2005

[6] S Reddy T Andl A Bagasra et al ldquoCharacterization of Wntgene expression in developing and postnatal hair follicles andidentification of Wnt5a as a target of Sonic hedgehog in hairfollicle morphogenesisrdquo Mechanisms of Development vol 107no 1-2 pp 69ndash82 2001

[7] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[8] J C Ra I S Shin S H Kim et al ldquoSafety of intravenousinfusion of human adipose tissue-derived mesenchymal stemcells in animals and humansrdquo Stem Cells and Development vol20 no 8 pp 1297ndash1308 2011

[9] D Park G Yang D K Bae et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve cognitive functionand physical activity in ageing micerdquo Journal of NeuroscienceResearch vol 91 no 5 pp 660ndash670 2013

[10] S Muller-Rover B Handjiski C van der Veen et al ldquoA com-prehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stagesrdquoThe Journal of InvestigativeDermatology vol 117 no 1 pp 3ndash15 2001

[11] T Andl S T Reddy T Gaddapara and S E Millar ldquoWNT sig-nals are required for the initiation of hair follicle developmentrdquoDevelopmental Cell vol 2 no 5 pp 643ndash653 2002

[12] DM Danilenko B D Ring D Yanagihara et al ldquoKeratinocytegrowth factor is an important endogenousmediator of hair folli-cle growth development and differentiation normalization ofthe nunu follicular differentiation defect and amelioration ofchemotherapy-induced alopeciardquo American Journal of Pathol-ogy vol 147 no 1 pp 145ndash154 1995

[13] G S KimDHKim J J Lim et al ldquoBiological and antibacterialactivities of the natural herb Houttuynia cordata water extractagainst the intracellular bacterial pathogen Salmonella withinthe RAW 2647 macrophagerdquo Biological amp PharmaceuticalBulletin vol 31 no 11 pp 2012ndash2017 2008

[14] X-Y Yang G-S Park M H Lee et al ldquoToll-like receptor 4-mediated immunoregulation by the aqueous extract of MoriFructusrdquo Phytotherapy Research vol 23 no 12 pp 1713ndash17202009

[15] H-S Lee J-H Choi Y-E Kim I-H Kim B-M Kim and C-H Lee ldquoEffects of the cynanchum wilfordii ethanol extract onthe serum lipid profile in hypercholesterolemic ratsrdquo PreventiveNutrition and Food Science vol 18 no 3 pp 157ndash162 2013

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Ctrl FINFDE DE

120573-cateninPhospho-Akt

AktActin

Low High Low High

(a)

0

50

100

150

120573-c

aten

ina

ctin

()

Ctrl FIN Low High Low HighFDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

0

100

200

300

p-A

ktA

kt (

)Ctrl FIN Low High Low High

FDE DE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(c)

Figure 4 Western blot analysis of 120573-catenin and Akt phosphorylation (a) Immunoblotting analysis of 120573-catenin and phospho-Akt proteinlevels was conducted in C57BL6N dorsal skin tissue sampled on day 21 after treatment (b) A bar graph shows the quantification of 120573-cateninactin and (c) phospho-AktAkt ratio Finasteride (FIN) was used as the positive control lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus Ctrl Ctrlcontrol group

FDE and DE upregulated expression of Lef1 which is anessential regulatory gene in the Wnt signaling pathway thatcontrols cell growth and differentiation through a signalingcascade from Wnts to Lef1 (Figure 3(d)) The FGF7 genewas also overexpressed in the FDE- and DE-treated groups(Figure 3(e)) suggesting a prolonged anagen phase anddelayed progression into the catagen phase in dermal papillacells [12]

34 Activation of 120573-Catenin and Akt Signaling by FDE andDE Cytosolic 120573-catenin an essential molecule in the Wntsignaling pathway translocates into the nucleus where itinduces transcription of target genes Thus we investigatedwhether FDE or DE upregulates 120573-catenin levels in dorsalskin tissues 120573-catenin level increases during the initial stagesof hair regeneration We also compared the protein levelof phosphorylated Akt a downstream target of phospho-inositide 3-kinase with that in the FIN group 120573-cateninexpression increased in response to the high doses of FDEand DE to levels comparable to those in the FIN groupMoreover Akt activation which promotes hair regrowth byregulating dermal papilla cell proliferation in the hair folliclewas upregulated in the presence of FDE and DE with moreupregulation in the FDE than that in the DE (Figures 4(a)ndash4(c))

35 Cytotoxicity and Profiles of Wnts mRNAs in hMSCs Thecytotoxicity induced by the FDE and DE was quantified bymeasuring LDH release at varying ranges of concentration

(1ndash1000120583gmL) Incubating hMSCs with each of the studysamples did not result in cell cytotoxicity except at the highestdose (1000 120583gmL) Figures 5(a) and 5(b) show that the FDEand DE did not significantly increase LDH release for 1ndash100 120583gmL exposure for up to 24 h but a higher concen-tration (1000 120583gmL) induced a significant increase in LDHrelease Figures 5(c)ndash5(e) clearly show that FDE significantlyincreased Wnt3 Wnt10a and Wnt10b mRNA in hMSCs Asexpected results of this in vitro study coincidewith the in vivodorsal skin results

4 Discussion

The hair follicle is a mammalian skin organ that produceshair which is repeatedly renewed through canonical sig-naling for proper growth and patterning Otherwise thehair cycle is disrupted and hair is lost from the head orbody which is called alopecia To date two representativetypes of drugs have been prescribed for pattern baldnessFIN which is a 5 alpha-reductase inhibitor and minoxi-dil which is an antihypertensive vasodilator are the mostfrequently prescribed medications for alopecia The selectedherbal medicines used in this study have been reportedto have diverse pharmacological activities such as antihy-percholesterolemia immunoregulation antiallergy antibac-teria antioxidation anticancer hepatoprotection and anti-insomnia [13ndash18]

As C57BL6N mice have been shown to be in a syn-chronized telogen stage of the hair cycle at 8 weeks of age

8 Evidence-Based Complementary and Alternative Medicine

1 10 100 1000

Cyto

toxi

city

()

hMSCs

0

20

40

60

80

100ve

rsus

Ctr

l

lowastlowastlowast

lowastlowastlowast

H2O2FDE (120583gmL)

(a)

Cyto

toxi

city

()

hMSCs

20

40

60

80

100

vers

us C

trl

lowastlowastlowast

lowastlowastlowast

1 10 100 10000

H2O2DE (120583gmL)

(b)

Ctrl PMA FDE DE

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

00

05

10

15

20

(Wnt

3 m

RNA

)

lowastlowastlowast

lowastlowast

(c)

hMSCs

Rela

tive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

(Wnt

10a m

RNA

)

Ctrl PMA FDE DE00

lowastlowast

(d)

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

25

(Wnt

10b

mRN

A)

Ctrl PMA FDE DE00

lowast

lowastlowast

(e)

Figure 5 Effect on Wnts (Wnt3 10a and 10b) gene expression in hMSCs (a-b) Cytotoxicity assays were performed in a 96-well plate for24 h and the results were expressed as the percent cytotoxicity for identical treatments of HDE and DE (1ndash1000 120583gmL) For gene expressionanalysis cells were seeded on a 6-well plate and treated with the HDE (100120583gmL) or DE (100120583gmL) in the presence or absence of 50120583Mphorbolmyristic acetate (PMA) for 24 h (cndashe)Wnt3Wnt10a andWnt10bmRNAwere quantified by fold units using the real-time polymerasechain reaction Results are expressed as means plusmn standard deviations from three separate experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

their hair follicle regeneration and regrowth have been welldescribed The high dose of FDE promoted hair regrowthduring the 20 consecutive days of administration that wasequal to that seen in the positive FIN controlWnt signaling isrequired to establish the hair follicle [19] and plays a key roleactivating bulge stem cells to progress toward hair formationand this signal is relayed by 120573-catenin and Lef1 [5]

In recent studies a hierarchy of Wnts that control hairfollicle development has been introduced According tothe prior reports among 19 Wnts Wnts 3 4 and 6 wereconsidered to mediate hair follicle initiation and Wnts 2 7b10a and 10b were considered to depend on the epidermalactivation [6 10 20] In line with thisWnt secretionmediateshair follicle development by acting as signalingmolecules and

Evidence-Based Complementary and Alternative Medicine 9

they can be classified as primary Wnts (Wnts 3 4 and 6)and secondary Wnts (Wnts 2 7b 10a and 10b) which areessential for initiating hair follicle growth and are involvedin development of the hair follicle respectively [10 21]In particular matrix and precortex cells express Wnt3 andWnt10ab which send a signaling cascade to Lef1 [22 23]Importantly120573-catenin which is a key regulator of hair folliclegrowth is involved in inducing the transition from telogento anagen [23] Thus it is important that a hair regrowthcandidate should be associated with Wnt signaling HampEstaining demonstrated that FDE and DE actively promotedhair follicle regrowth by increasing the number and size ofhair follicles which is an indicator for the transition of hairgrowth from the telogen to anagen phases at particular timesThe hair of FDE-treated mice was more prone to transitioninto the late anagen phase (ie IIIc) in which the dermalpapilla reaches the deepest position and rests close to themuscle layer along with the distinguishable inner and outerroot sheaths [10]

The increase in Wnt310a10b and Lef1 gene expressionin the dorsal skin strongly indicates that hair follicles wereactively developing because these genes are predominantlyexpressed in the hair placode epithelium during induction ofthe hair follicle [24ndash26] Coincident data were obtained fromqPCR analysis in hMSCs which are known to express all ofthe common stem cell markers and can be differentiated intovarious types of specialized cells under appropriate growthconditions [27] Taken together FDE could play an essentialrole in the initiation and development of hair follicles asshown by theHampE staining results Furthermore the increasein FGF7 mRNA helps understand how FDE prolongs theanagen phase and delays progression into the catagen phase

5 Conclusion

Our data clearly demonstrate that FDE effectively promoteshair regrowth by enhancing Wnt signaling and activatingAkt together with overexpression of hair regrowth-relatedprimary and secondaryWnts (Wnt310a10b) Lef1 and FGF7Although more in-depth chemical screening studies arerequired it is certain that the extract from eight selectedherbal medicines could be a promising hair regrowth candi-date when fermented after decoction

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Su Kil Jang and Seung Tae Kim equally contributed to thispaper

Acknowledgment

This study was supported by a grant from the Korea Health-care Technology RampD Project Ministry for Health Welfareand Family Affairs Republic of Korea (A091121)

References

[1] R Paus andGCotsarelis ldquoThebiology of hair folliclesrdquoTheNewEngland Journal of Medicine vol 341 no 7 pp 491ndash497 1999

[2] E Fuchs B J Merrill C Jamora and R Dasgupta ldquoAt the rootsof a never-ending cyclerdquo Developmental Cell vol 1 no 1 pp13ndash25 2001

[3] S Claudinot M Nicolas H Oshima A Rochat and Y Bar-randon ldquoLong-term renewal of hair follicles from clonogenicmultipotent stem cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 41 pp14677ndash14682 2005

[4] M Ohyama A Terunuma C L Tock et al ldquoCharacterizationand isolation of stem cell-enriched human hair follicle bulgecellsrdquoTheJournal of Clinical Investigation vol 116 no 1 pp 249ndash260 2006

[5] W E Lowry C Blanpain J A Nowak G Guasch L Lewis andE Fuchs ldquoDefining the impact of 120573-cateninTcf transactivationon epithelial stem cellsrdquo Genes amp Development vol 19 no 13pp 1596ndash1611 2005

[6] S Reddy T Andl A Bagasra et al ldquoCharacterization of Wntgene expression in developing and postnatal hair follicles andidentification of Wnt5a as a target of Sonic hedgehog in hairfollicle morphogenesisrdquo Mechanisms of Development vol 107no 1-2 pp 69ndash82 2001

[7] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[8] J C Ra I S Shin S H Kim et al ldquoSafety of intravenousinfusion of human adipose tissue-derived mesenchymal stemcells in animals and humansrdquo Stem Cells and Development vol20 no 8 pp 1297ndash1308 2011

[9] D Park G Yang D K Bae et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve cognitive functionand physical activity in ageing micerdquo Journal of NeuroscienceResearch vol 91 no 5 pp 660ndash670 2013

[10] S Muller-Rover B Handjiski C van der Veen et al ldquoA com-prehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stagesrdquoThe Journal of InvestigativeDermatology vol 117 no 1 pp 3ndash15 2001

[11] T Andl S T Reddy T Gaddapara and S E Millar ldquoWNT sig-nals are required for the initiation of hair follicle developmentrdquoDevelopmental Cell vol 2 no 5 pp 643ndash653 2002

[12] DM Danilenko B D Ring D Yanagihara et al ldquoKeratinocytegrowth factor is an important endogenousmediator of hair folli-cle growth development and differentiation normalization ofthe nunu follicular differentiation defect and amelioration ofchemotherapy-induced alopeciardquo American Journal of Pathol-ogy vol 147 no 1 pp 145ndash154 1995

[13] G S KimDHKim J J Lim et al ldquoBiological and antibacterialactivities of the natural herb Houttuynia cordata water extractagainst the intracellular bacterial pathogen Salmonella withinthe RAW 2647 macrophagerdquo Biological amp PharmaceuticalBulletin vol 31 no 11 pp 2012ndash2017 2008

[14] X-Y Yang G-S Park M H Lee et al ldquoToll-like receptor 4-mediated immunoregulation by the aqueous extract of MoriFructusrdquo Phytotherapy Research vol 23 no 12 pp 1713ndash17202009

[15] H-S Lee J-H Choi Y-E Kim I-H Kim B-M Kim and C-H Lee ldquoEffects of the cynanchum wilfordii ethanol extract onthe serum lipid profile in hypercholesterolemic ratsrdquo PreventiveNutrition and Food Science vol 18 no 3 pp 157ndash162 2013

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

1 10 100 1000

Cyto

toxi

city

()

hMSCs

0

20

40

60

80

100ve

rsus

Ctr

l

lowastlowastlowast

lowastlowastlowast

H2O2FDE (120583gmL)

(a)

Cyto

toxi

city

()

hMSCs

20

40

60

80

100

vers

us C

trl

lowastlowastlowast

lowastlowastlowast

1 10 100 10000

H2O2DE (120583gmL)

(b)

Ctrl PMA FDE DE

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

00

05

10

15

20

(Wnt

3 m

RNA

)

lowastlowastlowast

lowastlowast

(c)

hMSCs

Rela

tive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

(Wnt

10a m

RNA

)

Ctrl PMA FDE DE00

lowastlowast

(d)

hMSCs

Relat

ive c

once

ntra

tion

norm

aliz

ed to

GA

PDH

05

10

15

20

25

(Wnt

10b

mRN

A)

Ctrl PMA FDE DE00

lowast

lowastlowast

(e)

Figure 5 Effect on Wnts (Wnt3 10a and 10b) gene expression in hMSCs (a-b) Cytotoxicity assays were performed in a 96-well plate for24 h and the results were expressed as the percent cytotoxicity for identical treatments of HDE and DE (1ndash1000 120583gmL) For gene expressionanalysis cells were seeded on a 6-well plate and treated with the HDE (100120583gmL) or DE (100120583gmL) in the presence or absence of 50120583Mphorbolmyristic acetate (PMA) for 24 h (cndashe)Wnt3Wnt10a andWnt10bmRNAwere quantified by fold units using the real-time polymerasechain reaction Results are expressed as means plusmn standard deviations from three separate experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt0001 versus Ctrl Ctrl control group

their hair follicle regeneration and regrowth have been welldescribed The high dose of FDE promoted hair regrowthduring the 20 consecutive days of administration that wasequal to that seen in the positive FIN controlWnt signaling isrequired to establish the hair follicle [19] and plays a key roleactivating bulge stem cells to progress toward hair formationand this signal is relayed by 120573-catenin and Lef1 [5]

In recent studies a hierarchy of Wnts that control hairfollicle development has been introduced According tothe prior reports among 19 Wnts Wnts 3 4 and 6 wereconsidered to mediate hair follicle initiation and Wnts 2 7b10a and 10b were considered to depend on the epidermalactivation [6 10 20] In line with thisWnt secretionmediateshair follicle development by acting as signalingmolecules and

Evidence-Based Complementary and Alternative Medicine 9

they can be classified as primary Wnts (Wnts 3 4 and 6)and secondary Wnts (Wnts 2 7b 10a and 10b) which areessential for initiating hair follicle growth and are involvedin development of the hair follicle respectively [10 21]In particular matrix and precortex cells express Wnt3 andWnt10ab which send a signaling cascade to Lef1 [22 23]Importantly120573-catenin which is a key regulator of hair folliclegrowth is involved in inducing the transition from telogento anagen [23] Thus it is important that a hair regrowthcandidate should be associated with Wnt signaling HampEstaining demonstrated that FDE and DE actively promotedhair follicle regrowth by increasing the number and size ofhair follicles which is an indicator for the transition of hairgrowth from the telogen to anagen phases at particular timesThe hair of FDE-treated mice was more prone to transitioninto the late anagen phase (ie IIIc) in which the dermalpapilla reaches the deepest position and rests close to themuscle layer along with the distinguishable inner and outerroot sheaths [10]

The increase in Wnt310a10b and Lef1 gene expressionin the dorsal skin strongly indicates that hair follicles wereactively developing because these genes are predominantlyexpressed in the hair placode epithelium during induction ofthe hair follicle [24ndash26] Coincident data were obtained fromqPCR analysis in hMSCs which are known to express all ofthe common stem cell markers and can be differentiated intovarious types of specialized cells under appropriate growthconditions [27] Taken together FDE could play an essentialrole in the initiation and development of hair follicles asshown by theHampE staining results Furthermore the increasein FGF7 mRNA helps understand how FDE prolongs theanagen phase and delays progression into the catagen phase

5 Conclusion

Our data clearly demonstrate that FDE effectively promoteshair regrowth by enhancing Wnt signaling and activatingAkt together with overexpression of hair regrowth-relatedprimary and secondaryWnts (Wnt310a10b) Lef1 and FGF7Although more in-depth chemical screening studies arerequired it is certain that the extract from eight selectedherbal medicines could be a promising hair regrowth candi-date when fermented after decoction

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Su Kil Jang and Seung Tae Kim equally contributed to thispaper

Acknowledgment

This study was supported by a grant from the Korea Health-care Technology RampD Project Ministry for Health Welfareand Family Affairs Republic of Korea (A091121)

References

[1] R Paus andGCotsarelis ldquoThebiology of hair folliclesrdquoTheNewEngland Journal of Medicine vol 341 no 7 pp 491ndash497 1999

[2] E Fuchs B J Merrill C Jamora and R Dasgupta ldquoAt the rootsof a never-ending cyclerdquo Developmental Cell vol 1 no 1 pp13ndash25 2001

[3] S Claudinot M Nicolas H Oshima A Rochat and Y Bar-randon ldquoLong-term renewal of hair follicles from clonogenicmultipotent stem cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 41 pp14677ndash14682 2005

[4] M Ohyama A Terunuma C L Tock et al ldquoCharacterizationand isolation of stem cell-enriched human hair follicle bulgecellsrdquoTheJournal of Clinical Investigation vol 116 no 1 pp 249ndash260 2006

[5] W E Lowry C Blanpain J A Nowak G Guasch L Lewis andE Fuchs ldquoDefining the impact of 120573-cateninTcf transactivationon epithelial stem cellsrdquo Genes amp Development vol 19 no 13pp 1596ndash1611 2005

[6] S Reddy T Andl A Bagasra et al ldquoCharacterization of Wntgene expression in developing and postnatal hair follicles andidentification of Wnt5a as a target of Sonic hedgehog in hairfollicle morphogenesisrdquo Mechanisms of Development vol 107no 1-2 pp 69ndash82 2001

[7] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[8] J C Ra I S Shin S H Kim et al ldquoSafety of intravenousinfusion of human adipose tissue-derived mesenchymal stemcells in animals and humansrdquo Stem Cells and Development vol20 no 8 pp 1297ndash1308 2011

[9] D Park G Yang D K Bae et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve cognitive functionand physical activity in ageing micerdquo Journal of NeuroscienceResearch vol 91 no 5 pp 660ndash670 2013

[10] S Muller-Rover B Handjiski C van der Veen et al ldquoA com-prehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stagesrdquoThe Journal of InvestigativeDermatology vol 117 no 1 pp 3ndash15 2001

[11] T Andl S T Reddy T Gaddapara and S E Millar ldquoWNT sig-nals are required for the initiation of hair follicle developmentrdquoDevelopmental Cell vol 2 no 5 pp 643ndash653 2002

[12] DM Danilenko B D Ring D Yanagihara et al ldquoKeratinocytegrowth factor is an important endogenousmediator of hair folli-cle growth development and differentiation normalization ofthe nunu follicular differentiation defect and amelioration ofchemotherapy-induced alopeciardquo American Journal of Pathol-ogy vol 147 no 1 pp 145ndash154 1995

[13] G S KimDHKim J J Lim et al ldquoBiological and antibacterialactivities of the natural herb Houttuynia cordata water extractagainst the intracellular bacterial pathogen Salmonella withinthe RAW 2647 macrophagerdquo Biological amp PharmaceuticalBulletin vol 31 no 11 pp 2012ndash2017 2008

[14] X-Y Yang G-S Park M H Lee et al ldquoToll-like receptor 4-mediated immunoregulation by the aqueous extract of MoriFructusrdquo Phytotherapy Research vol 23 no 12 pp 1713ndash17202009

[15] H-S Lee J-H Choi Y-E Kim I-H Kim B-M Kim and C-H Lee ldquoEffects of the cynanchum wilfordii ethanol extract onthe serum lipid profile in hypercholesterolemic ratsrdquo PreventiveNutrition and Food Science vol 18 no 3 pp 157ndash162 2013

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

they can be classified as primary Wnts (Wnts 3 4 and 6)and secondary Wnts (Wnts 2 7b 10a and 10b) which areessential for initiating hair follicle growth and are involvedin development of the hair follicle respectively [10 21]In particular matrix and precortex cells express Wnt3 andWnt10ab which send a signaling cascade to Lef1 [22 23]Importantly120573-catenin which is a key regulator of hair folliclegrowth is involved in inducing the transition from telogento anagen [23] Thus it is important that a hair regrowthcandidate should be associated with Wnt signaling HampEstaining demonstrated that FDE and DE actively promotedhair follicle regrowth by increasing the number and size ofhair follicles which is an indicator for the transition of hairgrowth from the telogen to anagen phases at particular timesThe hair of FDE-treated mice was more prone to transitioninto the late anagen phase (ie IIIc) in which the dermalpapilla reaches the deepest position and rests close to themuscle layer along with the distinguishable inner and outerroot sheaths [10]

The increase in Wnt310a10b and Lef1 gene expressionin the dorsal skin strongly indicates that hair follicles wereactively developing because these genes are predominantlyexpressed in the hair placode epithelium during induction ofthe hair follicle [24ndash26] Coincident data were obtained fromqPCR analysis in hMSCs which are known to express all ofthe common stem cell markers and can be differentiated intovarious types of specialized cells under appropriate growthconditions [27] Taken together FDE could play an essentialrole in the initiation and development of hair follicles asshown by theHampE staining results Furthermore the increasein FGF7 mRNA helps understand how FDE prolongs theanagen phase and delays progression into the catagen phase

5 Conclusion

Our data clearly demonstrate that FDE effectively promoteshair regrowth by enhancing Wnt signaling and activatingAkt together with overexpression of hair regrowth-relatedprimary and secondaryWnts (Wnt310a10b) Lef1 and FGF7Although more in-depth chemical screening studies arerequired it is certain that the extract from eight selectedherbal medicines could be a promising hair regrowth candi-date when fermented after decoction

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Su Kil Jang and Seung Tae Kim equally contributed to thispaper

Acknowledgment

This study was supported by a grant from the Korea Health-care Technology RampD Project Ministry for Health Welfareand Family Affairs Republic of Korea (A091121)

References

[1] R Paus andGCotsarelis ldquoThebiology of hair folliclesrdquoTheNewEngland Journal of Medicine vol 341 no 7 pp 491ndash497 1999

[2] E Fuchs B J Merrill C Jamora and R Dasgupta ldquoAt the rootsof a never-ending cyclerdquo Developmental Cell vol 1 no 1 pp13ndash25 2001

[3] S Claudinot M Nicolas H Oshima A Rochat and Y Bar-randon ldquoLong-term renewal of hair follicles from clonogenicmultipotent stem cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 41 pp14677ndash14682 2005

[4] M Ohyama A Terunuma C L Tock et al ldquoCharacterizationand isolation of stem cell-enriched human hair follicle bulgecellsrdquoTheJournal of Clinical Investigation vol 116 no 1 pp 249ndash260 2006

[5] W E Lowry C Blanpain J A Nowak G Guasch L Lewis andE Fuchs ldquoDefining the impact of 120573-cateninTcf transactivationon epithelial stem cellsrdquo Genes amp Development vol 19 no 13pp 1596ndash1611 2005

[6] S Reddy T Andl A Bagasra et al ldquoCharacterization of Wntgene expression in developing and postnatal hair follicles andidentification of Wnt5a as a target of Sonic hedgehog in hairfollicle morphogenesisrdquo Mechanisms of Development vol 107no 1-2 pp 69ndash82 2001

[7] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[8] J C Ra I S Shin S H Kim et al ldquoSafety of intravenousinfusion of human adipose tissue-derived mesenchymal stemcells in animals and humansrdquo Stem Cells and Development vol20 no 8 pp 1297ndash1308 2011

[9] D Park G Yang D K Bae et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve cognitive functionand physical activity in ageing micerdquo Journal of NeuroscienceResearch vol 91 no 5 pp 660ndash670 2013

[10] S Muller-Rover B Handjiski C van der Veen et al ldquoA com-prehensive guide for the accurate classification of murine hairfollicles in distinct hair cycle stagesrdquoThe Journal of InvestigativeDermatology vol 117 no 1 pp 3ndash15 2001

[11] T Andl S T Reddy T Gaddapara and S E Millar ldquoWNT sig-nals are required for the initiation of hair follicle developmentrdquoDevelopmental Cell vol 2 no 5 pp 643ndash653 2002

[12] DM Danilenko B D Ring D Yanagihara et al ldquoKeratinocytegrowth factor is an important endogenousmediator of hair folli-cle growth development and differentiation normalization ofthe nunu follicular differentiation defect and amelioration ofchemotherapy-induced alopeciardquo American Journal of Pathol-ogy vol 147 no 1 pp 145ndash154 1995

[13] G S KimDHKim J J Lim et al ldquoBiological and antibacterialactivities of the natural herb Houttuynia cordata water extractagainst the intracellular bacterial pathogen Salmonella withinthe RAW 2647 macrophagerdquo Biological amp PharmaceuticalBulletin vol 31 no 11 pp 2012ndash2017 2008

[14] X-Y Yang G-S Park M H Lee et al ldquoToll-like receptor 4-mediated immunoregulation by the aqueous extract of MoriFructusrdquo Phytotherapy Research vol 23 no 12 pp 1713ndash17202009

[15] H-S Lee J-H Choi Y-E Kim I-H Kim B-M Kim and C-H Lee ldquoEffects of the cynanchum wilfordii ethanol extract onthe serum lipid profile in hypercholesterolemic ratsrdquo PreventiveNutrition and Food Science vol 18 no 3 pp 157ndash162 2013

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

[16] X-YWang Z-L Yu S-Y Pan et al ldquoSupplementation with theextract of schisandrae fructus pulp seed or their combinationinfluences the metabolism of lipids and glucose in mice fedwith normal and hypercholesterolemic dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID472638 11 pages 2014

[17] Y Shi J Dong J Zhao L Tang and J Zhang ldquoHerbal insomniamedications that target GABAergic systems a review of thepsychopharmacological evidencerdquo Current Neuropharmacol-ogy vol 12 no 3 pp 289ndash302 2014

[18] Z Pang Z Zhi-Yan W Wang et al ldquoThe advances in researchon the pharmacological effects of Fructus Ligustri LucidirdquoBioMed Research International vol 2015 Article ID 281873 5pages 2015

[19] L Alonso and E Fuchs ldquoStem cells in the skin waste not Wntnotrdquo Genes amp Development vol 17 no 10 pp 1189ndash1200 2003

[20] J Fu and W Hsu ldquoEpidermal Wnt controls hair follicle induc-tion by orchestrating dynamic signaling crosstalk between theepidermis and dermisrdquo Journal of Investigative Dermatologyvol 133 no 4 pp 890ndash898 2013

[21] Y Zhang P Tomann T Andl et al ldquoReciprocal requirementsfor EDAEDARNF-120581B and Wnt120573-catenin signaling pathwaysin hair follicle inductionrdquo Developmental Cell vol 17 no 1 pp49ndash61 2009

[22] S EMillar KWillert P C Salinas et al ldquoWNT signaling in thecontrol of hair growth and structurerdquo Developmental Biologyvol 207 no 1 pp 133ndash149 1999

[23] A E Oro and K Higgins ldquoHair cycle regulation of Hedgehogsignal receptionrdquo Developmental Biology vol 255 no 2 pp238ndash248 2003

[24] C van Genderen R M Okamura I Farinas et al ldquoDevel-opment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient micerdquoGenes amp Development vol 8 no 22 pp 2691ndash2703 1994

[25] P Zhou C Byrne J Jacobs and E Fuchs ldquoLymphoid enhancerfactor 1 directs hair follicle patterning and epithelial cell faterdquoGenes amp Development vol 9 no 6 pp 700ndash713 1995

[26] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[27] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom