pingmu decoction induces orbital preadipocytes apoptosis in vitro · 2019. 7. 30. · ent effect of...

Research ArticlePingmu Decoction Induces Orbital PreadipocytesApoptosis In Vitro

Yali Zhang1 Hong Li2 Long Gao2 Xia Zhang2 and RuiFang Xie3

1Department of Gastroenterology Long Hua Hospital Affiliated to Shanghai University of Traditional Chinese MedicineThe Institute of Digestive Disease Affiliated to Shanghai University of Traditional Chinese Medicine South Wan Ping Road No 725Shanghai 200032 China2Department of Endocrinology Long Hua Hospital Affiliated to Shanghai University of Traditional Chinese MedicineSouth Wan Ping Road No 725 Shanghai 200032 China3Department of Pharmacy Long Hua Hospital Affiliated to Shanghai University of Traditional Chinese MedicineSouth Wan Ping Road No 725 Shanghai 200032 China

Correspondence should be addressed to Hong Li shanhongli126com

Received 1 November 2016 Revised 23 February 2017 Accepted 20 March 2017 Published 18 April 2017

Academic Editor Pradeep Kumar

Copyright copy 2017 Yali Zhang 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

Pingmu Decoction is the Traditional Chinese Medicine which has treated Gravesrsquo Ophthalmopathy (GO) in the inactive stage formore than ten yearsThis studywas to explore themechanismof PingmuDecoction of inhibiting preadipocytes inGOpatients fromdifferentiating into mature adipocytes Human orbital preadipocytes were isolated and cultured through tissue explant methodOrbital preadipocytes were induced into mature adipocytes The medicinal serum was prepared from rats The cells were treatedwith medicinal serumwhich were divided into three groups low dose group (5) medium dose group (10) and high dose group(20) The cells viabilities were observed by Oil Red O staining MTT method and Annexin Vpropidium iodide (PI) doublestaining Effect of PingmuDecoction on cell apoptosis rate of orbitalmatured adipocyteswasmeasured by flow cytometryThe genesFas and Fas L from cell groups were tested by RT-PCR and Western blotting The expression of master adipogenic transcriptionfactors including peroxisomeproliferation-activity receptor (PPAR) 120574 andCCAATenhancer binding protein (CEBP)120572 was testedby Western blotting Pingmu Decoction could reduce orbital preadipocytes viability and induce apoptosis of mature adipocyte viaFasFas L signaling pathway Pingmu Decoction reduced lipid accumulation and downregulated the expression of PPAR 120574 andCEBP 120572 Pingmu Decoction may play a therapeutic effect by reducing the accumulation of orbital adipocytes

1 Introduction

Gravesrsquo Ophthalmopathy (GO) also known as thyroid-associated ophthalmopathy is an autoimmune disease char-acterized by invasion of retrobulbar and orbital tissues [12] About 20ndash50 of GO patients also present clinicallywith Gravesrsquo Disease (GD) which is characterized by dif-fuse goiter with hyperthyroidism [3ndash5] In recent years theincidence of GD has undergone an annual increase due tothe influence of hereditary and environmental factors thatcontribute to autoimmune disorders [6] GO affects adultsworldwide and has become one of the major causes ofblindness However the etiology and pathogenesis of GOhave not been completely defined An increase in orbital

adipose tissue content may increase the orbital pressure andlead to proptosis Thus abnormal proliferation of orbitaladipose tissue and the occurrence of fat in GO play animportant role in disease development [7 8] Furthermorean increase in orbital preadipocyte volume and quantity maylead to the characteristic increase in adipose tissues [9]Recent studies have confirmed that preadipocytes that residein orbital tissues of patients can transform into fatty cellsunder certain conditions and contribute to the developmentof GO [10 11] Therefore an imbalance in the proliferationdifferentiation and apoptosis of orbital preadipocytes maybe the main factor regulating orbital exenteration increaseorbital pressure increase and the occurrence of proptosis inpatients with GO

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 2109249 10 pageshttpsdoiorg10115520172109249

2 Evidence-Based Complementary and Alternative Medicine

GO can be divided into active and inactive stages accord-ing to its development In the inactive stage the clinicalcurative effect is not very good with no available specific andeffectivemethods of treatment byWesternmedicine PingmuDecoction has been used in Chinese medicine to treat GO inthe inactive stage for more than ten years with good clinicalcurative effects A preliminary experimental study demon-strated that serum from rats treated with Pingmu Decoctionand its components can act on orbital preadipocytes frompatients with GO in the inactive period Pingmu Decoctionserum promotes the apoptosis of adipocytes and reduces theaccumulation of orbital adipocytes by increasing the expres-sion of Caspase-3 Caspase-8 and Caspase-9 protein [12]However its apoptotic effect on differentiated adipocytesfrom GO patients remains uncharacterized Intracellularpathways mediated by FasFas L in mammals can induceapoptosis [13 14] We cultured orbital preadipocytes frompatients by the tissue explant method and differentiated theminto orbital adipocytes The effect and molecular mechanismof Pingmu Decoction on the apoptosis of orbital adipocyteswere explored by a variety of methods

2 Materials and Methods

21 Experimental Animals and Drugs Six-week-old specificpathogen-free male Sprague-Dawley rats weighing 200plusmn10 gwere purchased from Shanghai Silaike Experimental Ani-mal Co Ltd (certification number SCXK (Hu) 2012-0002)Animal experiment ethical approval was obtained from theAffiliated Long Hua Hospital of Shanghai University ofTraditional Chinese Medicine

Pingmu Decoction was composed of 30 g of Huangqi(Astragalus membranaceus) 15 g of Xianlingpi (Herba Epime-dii) 15 g of Chuanxiong (Ligusticum wallichii) 15 g of Baijiezi(Brassica alba Boiss) 15 g of stir-baked Biejia (CarapaxTrionycis) and 30 g of Cheqianzi (Semen plantaginis) Theseherbs were provided by the Chinese Medicine Pharmacyof the Affiliated Long Hua Hospital of Shanghai Universityof Traditional Chinese Medicine (TCM) The Decoctionof the above herbs was prepared twice according to theoriginal formula and then filtered and concentrated in acombined liquor The dose of rat administration is based onthe human mouse equivalent dosage conversion accordingto the textbook Pharmacology and Pharmacology Experimentof TCM (edited by Zhang Dafang Shanghai Science andTechnology Press 2002)The crude drug (4 gmL) was storedat 4∘C

22 GO Patients Four cases (4 eyes) of postbulbar adiposetissues from GO inpatients were collected from ShanghaiEENT Hospital Of the four GO cases in this study twofemale and two male patients (age ranging from 25 to 48years old) had a GOhistory of three to eight years Diagnosticcriteria of GOwere in accordance with the clinical score stan-dards described in the Chinese Guideline for Diagnosis andTreatment of Thyroid Disease published in 2008 The clinicalactivity score (CAS) of GO in clinical practice was based onthe clinical score standards ofChinese Guideline for Diagnosisand Treatment of Thyroid Disease including spontaneous

retrobulbar pain pain with eye movement eyelid erythemaconjunctival hyperemia chemosis swollen lacrimal caruncleand swollen eyelid Each symptom accounted for one pointand the total score obtained was up to seven in each caseTheinactive phase of GO referred to active exophthalmos withthe clinical score of lt3 Classification of exophthalmos inthis study was in accordance with the classification crite-ria of GO proposed by the American Thyroid Association(ATA) The patients were confirmed to have GO paralleledorbital decompression by the Ophthalmology Department atShanghai EENTHospital All patientswere in theGO inactivestage which is in line with the diagnostic criteria Otherdiseases such as autoimmune diseases and cancer wereruled out Approval was obtained from the Medical EthicalCommittee and patient consent was obtained in writing priorto collection of tissue samples

23 Medicinal Serum Preparation The Sprague-Dawley ratswere randomly divided into 2 groups with 5 rats per groupThe rats in the PingmuDecoction group were given 15mLkgPingmu Decoction and the rats in the control group weregiven an equal volume of distilled water by intragastricadministration twice a day for 3 daysThefinal concentrationsof medicinal serum (final concentration = volume added toserumtotal volume times 100) were 5 (low dose) 10(medium dose) and 20 (high dose)

Bloodwas collected from the abdominal aortae of the ratsunder ether anesthesia 15 h after the last administrationTheblood was incubated for 2 h at 4∘C and then centrifuged at4∘C at 3000 rpmmin for 15min The sera from each groupof mice were combined and inactivated for 30min at 56∘C ina water bath filtered with 022120583m aperture micropore filmpacked with frozen pipes and stored at minus20∘C

24 Culture of Primary Cells from Patient Tissue Sterilizedorbital adipose tissues from the operating table were washed3 timeswith sterile phosphate buffer solution (PBS) to removethe blood and then were placed into culture bottles con-taining complete medium As soon as possible after transferto the laboratory (within 4 h) the tissues were processedfor experimentation at room temperature Human orbitalpreadipocytes were cultured by the tissue explant methodThe adipose tissue with visible connective tissue or bloodclots was removed and the remaining tissue was harvestedinto Petri dishes with D-Hankrsquos solutionThe tissues were cutinto pieces of approximately 1mm times 1mm times 1mm in sizewith ophthalmic scissors and were seeded evenly at thebottom of culture bottles Then the bottles were gentlyturned upside down and 2mL complete culture solutionwas added The bottles were placed askew into an incubatorwith 5 CO

2at 37∘C for culturing After the tissue explants

adhered tightly to the culture bottle 5mL complete culturesolution was added and the solution was replaced withfresh solution every 2-3 d At 80 confluence the cells werepassaged 1 3 (every 3-4 d) Cells were used in experimentsafter 3ndash6 generations The morphology and growth status ofthe cultured primary cells were observed every day Oil RedO staining and microscopic photography were performed toassess the formation of lipid droplets Healthy preadipocytes

Evidence-Based Complementary and Alternative Medicine 3

were seeded at a density of 3ndash5 times 107L on coverslips in a 6-well plate When they approached confluence the cells werefixed in 4 paraformaldehyde for 10 minutes at normal tem-perature and stained by indirect immunofluorescent assayThe prepared cell slides were washed 3 times with PBS for 3minutes incubated for 1 h at 37∘C with PBS containing 2bovine serum albumin and Pref-1 antibody (diluted 1 100)and then incubated at 4∘C overnight After 4 washes withPBS for 5 minutes FITC-secondary antibody (diluted 1 500)was added for 30min at room temperature in darkness andthe slides were washed 4 times with PBS for 5 minutes Theslides were then stained with 5 120583gmL DAPI for 2min andantiquenching mounting medium was added before exami-nation under a fluorescent inverted microscope

25 Identification of Mature Adipocytes Healthy preadipo-cytes were seeded at a density of 1 times 105 in 6-well platesAfter the cells adhered themediumwas replaced with Differ-entiation Medium I containing Dexamethasone (1 120583molL)IBMX (02mmolL) and insulin (10 120583gmL) to induce dif-ferentiation After 4 days the medium was replaced withDifferentiationMedium II which has noDexamethasone andIBMX and the cells were cultured for 4 additional daysThenmedium was replaced with complete medium and the cellswere cultured for 8 d with the medium replaced every 2 dThe entire differentiation period was 16 d

After differentiation the mature adipocytes were washed3 times with PBS and fixed with 4 paraformaldehyde for 10minutesThe excess liquidwas removed by aspiration andOilRed O solution was added The cells were rinsed with 75ethanol to facilitate observation The resulting orange lipiddroplets were observable for 10min under the fluorescentinverted microscope

26 Cell Viability Assessment by the MTT Method Cells inthe logarithmic phase were digested with trypsin to produceamonocell suspension in complete culturemediumThe cellswere seeded in 96-well plates at about 10000 cells200 120583L perwell Then the 96-well plates were cultured in 5 CO

2in an

incubator at 37∘C The cells in each group (group 1 controlgroup group 2 Pingmu Decoction low dose group group3 Pingmu Decoction medium dose group group 4 PingmuDecoction high dose group and group 5 Dexamethasonegroup) were cultured for 24 h 48 h 72 h and 96 h A zerocontrol (culture media MTT and DMSO) and a blankcontrol (cells culture medium without serum MTT andDMSO)were also assayed in triplicate After 4 additional daysof culture 20120583L MTT solution (5mgmL in PBS) was addedto each well The culture supernatant was carefully aspiratedand then 150 120583LDMSOwas added to eachwell with low speedoscillation to allow the crystals to dissolve adequately Theoptical density value of each group was measured at 490 nmand a cell growth curve was drawn using time as the 119909-axisand light absorption as the 119910-axis

27 Apoptosis Assessment by the Annexin VPropidium Iodide(PI) Double Staining Method Cells treated with differentamounts of Pingmu Decoction serum were cultured for2 d The cell culture medium in each well of a 6-well plate

was aspirated and 400 120583L trypsin was added After 3min1mL complete culture medium was added to terminate thedigestion and the nonadherent cells were collectedThe wellswere washed with 500 120583L culture media to recover all ofthe cells and cells from both washes were combined andcentrifuged at 2000 rpmmin for 5min The supernatant wasdiscarded and the cells were washed with chilled sterile PBSresuspended in PBS counted and adjusted to 1 times 106mLThe cell suspensions were collected mixed gently with 5 120583LAnnexin V-FITC and incubated at room temperature for15min in darkness Then 5 120583L PI staining solution was addedgently and the cells were incubated in an ice bath in darknessfor 5min Flow cytometry was performed immediately afteradding 300 120583L binding buffer

28 Real-Time PCR Assessment of Fas and Fas L mRNAExpression TotalmRNAwas extracted by the TrizolmethodThe samples (2 120583L per well) were plated with DEPC water asthe backgroundThen the sample concentration was assessedusing a micro reader RNA of sufficient purity (A260A280value 18ndash2) was used for subsequent experimentation Theconcentrationswere adjusted to 1120583g120583L and thenmRNAwasheat-denatured for 5min at 65∘C cooled immediately andreverse transcribed to cDNA using the Sensiscript RT KitReverse transcription was performed in a 20120583L volume (9 120583Lnuclease-free water 4 120583L 5x RT Buffer 1120583L RT Enzyme 1 120583LPrimer Mix and 5120583L cDNA) Reaction conditions were asfollows 37∘C for 15min 98∘C for 5min and 4∘C for 5min

For PCR the primer sequences selected were designedbased on the information in GenBank The upstreamand downstream sequences of the primers and predictedproduct size were as follows Fas 51015840-TTCTGCCATAAG-CCCTGTCC-31015840 and 51015840-CTAAGCCATGTCCTTCATCAC-AC-31015840 (169 bp) Fas L 51015840-GGATGTTTCAGCTCTTCC-ACCTAC-31015840 and 51015840-TGTTAAATGGGCCACTTTCCTC-3 (152 bp) GAPDH 51015840-GCACCGTCAAGGCTGAGAAC-31015840 and 51015840-TGGTGAAGACGCCAGTGGA-31015840 (138 bp) Real-time PCR reactions (50 120583L total) included 5120583L cDNA sample2 120583L upstream primers (10120583M) 2 120583L downstream primers(10 120583M) 16 120583L distilled water and 25120583L SYBR Green Theconditions of PCR amplification were 95∘C for 15 s 60∘Cfor 15 s and 72∘C for 45 s (40 cycles) The relative quantityof the target genes was determined by the comparative Ctvalue2minusΔΔCt method with GAPDH as control

29 Assessment of Fas Fas L PPAR 120574 and CEBP 120572 ProteinExpression by Western Blotting Cells were cultured for 2 dunder different conditions (control group PingmuDecoctionlow moderate and high dose group and Dexamethasonegroup) 2 times 106 adipocytes were washed twice with PBS and100 120583L precooling protein lysis buffer (50mM Tris-HCl150mM NaCl 002 NaN

3 1 Triton X-100 1mM PMSF

1 120583gmL aprotinin and 1120583gmL leupeptin) was added to theplates on ice for 10ndash20min The cells were then scraped andcollected into EP tubes at 12000 rpm for 5min at 4∘C Thenthe supernatants were transferred into another centrifugetube and a small amount of supernatant was used forprotein quantitation by the Bradford Assay Fifty microlitersof protein from each sample was separated by SDS-PAGE


(a) (b)

Figure 1 Verification of Pref-1 expression by preadipocytes Positive Pref-1 expression by preadipocytes was confirmed by indirectimmunofluorescent assay (a-b) Merged image of Pref-1 stain (green) and nuclear stain (blue) (a) Nucleus was stained by DAPI (blue) (b)

and after transfer PVDF membranes were incubated at 4∘Covernight with primary antibody against Fas Fas L PPAR 120574or CEBP 120572 (1 1000 diluted in TBST) Fluorescence-labeledsecondary antibody (1 2000) was added after washing andthe membranes were incubated for 2 h at room temperatureThe membranes were rinsed with TBST ECL luminescencereagents (reagent Amix with equal volume of reagent B) wereadded and the membranes were dynamically integrated on agel imaging instrument for 5ndash15min with analysis by Gel-Proanalysis software

210 Statistical Analysis Statistical analysis was performedusing SPSS 180 software Data measurement was expressedas mean values plusmn standard deviation Normal distributionand homogeneity of variance assessments were carried outfirst For experimental data thatmet the criteria comparisonsbetween multiple groups were performed using single-factoranalysis of variance (analysis of variance ANOVA) andcomparisons among groups were performed using LSD-t(least significant difference-t) For experimental data thatdid not meet the normal distribution and homogeneity ofvariance criteria nonparametric tests were adopted 119875 lt 005was considered statistically significant

3 Results

31 Primary Culture of Preadipocytes Growth Characteris-tics after Passage and Morphological Observation To deter-mine the molecular effects of Pingmu Decoction on orbitaladipocytes from GO patients we collected tissue frompatients undergoing surgery Cells cultured from the tissuesamples emerged from the tissue block on the fourth day andwere spindle shaped with oval or near-circular nuclei Thecells morphology was close to that of fibrocytes with gradualproliferation of cells After passaging the preadipocytes grad-ually arranged themselves and grew 3-4 d before requiringsubsequent passage In this study orbital adipose tissue from4 cases of GO was cultured and cells emerged from theadipose tissue block in all cases within 4-5 d The cells werepassagedwhen they reached confluence (within 9-10 d) under

the sterile conditions Subsequently the cells were passagedonce every 3-4 d

32 Identification of Preadipocytes To verify that the culturedcells represented preadipocytes we performed immunofluo-rescent staining assays with Pref-1 antibody The membranesof the cultured preadipocytes positively expressed Pref-1(green fluorescence) as identified by indirect immunofluores-cent assay (Figures 1(a) and 1(b)) Based on this finding it canbe verified that the cultured primary cells were preadipocytes

33 Induction of Differentiation of Preadipocytes and theEffects of PingmuDecoction on Differentiation To induce dif-ferentiation of the preadipocytes into mature adipocytes wecultured them in differentiation liquidThe cells retracted thecell space became larger and proliferation stopped graduallyFurthermore the morphology of the cells gradually changedto oval or nearly circular and the volume of the cells graduallyincreased An obvious change in morphology was generallyobserved after a week or so Lipid granules presented as fatdroplets in the cytoplasm as observed under the light micro-scope On the 8th day of the differentiation the cells swelledprominently and their shape became short or polygonal Fatdroplets became enlarged and gradually increased in numberas observed by Oil Red O staining On the 16th day of thedifferentiation the cell differentiation was close to 80ndash90complete and the nuclei shifted to one side Fat dropletsin the cytoplasm stained red with Oil Red O and could beseen under the inverted microscope (Figure 2(a)) Based onthis finding it could be verified that the preadipocytes weresuccessfully differentiated as mature adipocytes

Next we treated the mature adipocytes with three dif-ferent doses of Pingmu Decoction serum (low 5 medium10 and high 20) As a control cells were untreated(blank) or treated with 30 120583gmL Dexamethasone (Dexa)The results demonstrate that Pingmu Decoction serum caninhibit differentiation (Figures 2(b)ndash2(f)) These findingssuggest that the explanted preadipocytes can be differenti-ated but that exposure with PingmuDecoction serumduringthe differentiation blocks maturation


(a) (b)

(c) (d)

(e) (f)

Figure 2 Fat accumulation in mature adipocytes Lipid droplets in adipocytes were stained with Oil Red O Staining was performed fornormal mature adipocytes (a) and for preadipocytes treated with or without Pingmu Decoction serum during differentiation as indicated(bndashf) Blank blank control group Dexa Dexamethasone group Pingmu low PingmuDecoction low dose group Pingmumoderate PingmuDecoction medium dose group Pingmu high Pingmu Decoction high dose group

34 Effect of Pingmu Decoction Serum on Cell Viability Tofurther explore the effects of Pingmu Decoction serum wetreated differentiated adipocytes for up to 96 h The growthcurves of the Pingmu Decoction low moderate and highgroups compared with the Blank and Dexa groups were sig-nificantly reduced which indicates that the cell viability wasdecreased (Figure 3) There were no significant differencesbetween the Blank and Dexa groups The OD values of eachPingmu Decoction group compared with the control groupwere decreased at 48 h (119875 lt 005) The OD values werefurther decreased in the Pingmu Decoction groups at 96 hwith the most dramatic decrease observed for the medium

dose group (119875 lt 005) These results verify the time-depend-ent effect of Pingmu Decoction serum in suppressing thegrowth of adipocytes from GO patients

35 Effect of Pingmu Decoction Serum on Apoptosis of MatureAdipocytes To determine whether the decrease in cell via-bility by Pingmu Decoction serum occurs at the level ofapoptosis we performed flow cytometry of cells assessed byAnnexin VPI double stainingThe results show that the earlyand total apoptosis rates ofmature adipocyteswere 065 and153 in the blank control group However the cell apoptosisof each Pingmu Decoction group was increased (119875 lt 005)


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24 48 72 96(h)

OD

val

ue

BlankDexaPingmu low

Pingmu modPingmu high

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast lowast

lowast

Figure 3 MTT cell viability assay of control adipocytes andadipocytes treated with Pingmu Decoction serum Blank blankcontrol group Dexa Dexamethasone group Pingmu low PingmuDecoction low dose group Pingmu moderate Pingmu Decoctionmedium dose group Pingmu high Pingmu Decoction high dosegroup lowast119875 lt 005 versus the blank control group (119899 = 3 per group)

with the early and total apoptosis rates in the PingmuDecoction medium dose group being the highest (Figure 4)There were no significant differences between Dexa andblank groups There were statistical differences between themedium dose group and low dose group (119875 lt 005) but notthe high dose group in the early apoptosis rate These resultssuggest that Pingmu Decoction serum induces apoptosis ofmature GO adipocytes with the most dramatic effect occur-ring at the medium dose

36 Effect of Pingmu Decoction Serum on the Expression ofFas and Fas L mRNA To further verify the apoptotic effectof Pingmu Decoction serum and to determine whether theeffect might be explained by alterations in the expression ofthe apoptosis modulators Fas and Fas L we performed RT-PCRThe relative expression of FasmRNA in adipocytes fromeach PingmuDecoction group was higher than blank controlgroup (low 137-fold increase medium 150-fold increasehigh 142-fold increase) and the difference between the lowdose group and the blank group was statistical (119875 lt 005)Furthermore the relative expression quantity of Fas LmRNAin adipocytes from each Pingmu Decoction was higher thanblank control (119875 lt 005) (low 132-fold increase medium146-fold increase high 142-fold increase) (Figure 5) Therewere no significant differences in the expression of Fas or FasL mRNA between the Blank and Dexa groups

37 Effect of Pingmu Decoction Serum on the Expression ofFas and Fas L Protein The protein expression of Fas andFas L was further assessed by Western blotting Comparedwith the blank control group the Fas and Fas L expressionof each Pingmu Decoction group was increased statistically(119875 lt 005) (Figure 6) Among the Pingmu Decoction groups

the low dose group had the highest expression of Fas butthe lowest expression of Fas L and the high dose group hasthe lowest expression of Fas but the highest expression ofFas L There was no significant difference between the Blankand Dexa groups These results verify the RT-PCR resultssuggesting that Pingmu Decoction may induce apoptosis ofGO adipocytes by increasing FasFas L signaling but suggestthat the relationship may not be strictly dose-dependent

38 Pingmu Decoction Decreases Protein Expression of Adi-pogenic Transcription Factors To identify the effect ofPingmu Decoction on adipogenic transcription factors theprotein expression of PPAR 120574 and CEBP 120572 was furtherassessed by Western blotting Compared with the blankcontrol group the PPAR 120574 and CEBP 120572 expression of eachPingmu Decoction group was decreased statistically (119875 lt005) (Figure 7) Among the PingmuDecoction groups therewas no significant difference And also there was no signifi-cant difference between the Blank and Dexa groups

4 Discussion

The clinical symptoms of patients with GO are caused bya shift in the balance between limited orbital volume andincreasing amounts of the orbital adipose tissue [15] Theincrease in orbital adipose tissue can directly increase orbitalpressureThe abnormal proliferation of orbital adipose tissueand fat accumulation play a major role in the occurrence anddevelopment of GO Accordingly the role of orbital adiposetissue in GO has received increasing attention [16ndash18] CTexamination has indicated that the increase in orbital fatin GO is consistent with the degree of exophthalmos andinconsistent with orbital muscle enlargement [19] Potgieserand others [20] have demonstrated that the increase inextraocular muscle volume in GO is obviously lower than theincrease in orbital adipose tissue volume and the correlationcoefficient between total orbital adipose volume and thedegree of exophthalmos is higher than the correlation coeffi-cient between extraocular muscle volume and exophthalmosKumar and others [21] found evidence of new adipose tissuein GOpatients Furthermore someGOpatients with obviousexophthalmos are observed to only manifest with an increasein the retrobulbar adipose tissue without the enlargement ofextraocular muscle Patients with this variety of GO mainlymanifest with exophthalmos which will affect appearance Inthis case the patientsrsquo total cross-sectional area of extraocularmuscle is not large [22 23]

To examine the mechanisms of growth control inadipocytes by Pingmu Decoction serum we culturedpreadipocytes Preadipocytes were shown to be culturedsuccessfully by the tissue explant method in this study Cellsemerged from the adipose tissue block on the 4th day Theprimary cells were spindle shaped with oval or near-circularnuclei which is similar to the morphology of fibroblastsunder the microscopeThe cells proliferated gradually at firstand then proliferated more rapidly after passage We verifiedthe identification of human preadipocytes from the primaryculture of orbital adipose tissue by staining for Pref-1 a


0

2

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6

Total Early

Apop

tosis

rate

()

lowastlowast

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(e)

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Annexin V-FITC-A

Gate P1

PI-A

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Gate P1Dead11

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1072

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Annexin V-FITC-A1037 105 106 1072

Figure 4 Apoptosis rates of the matured adipocytes treated with Pingmu Decoction serum Apoptosis rates were determined by AnnexinVPI staining and flow cytometry Blank control group (a) Dexamethasone group (b) Pingmu Decoction low dose group (c) PingmuDecoction medium dose group (d) Pingmu Decoction high dose group (e) lowast119875 lt 005 versus the blank control group (119899 = 3 per group)


00

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Gen

e exp

ress

ion

relat

ive t

o G

APD

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Fas Fas L

DexaPingmu low


lowast

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Figure 5 Effect of Pingmu Decoction serum on the expressionof Fas and Fas L mRNA Expression was quantified by RT-PCRBlank blank control group Dexa Dexamethasone group Pingmulow Pingmu Decoction low dose group Pingmumoderate PingmuDecoction medium dose group Pingmu high Pingmu Decoctionhigh dose group lowast119875 lt 005 versus the blank control group (119899 = 3per group)

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lowast lowast

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Figure 6 Effect of Pingmu Decoction serum on the expressionof Fas and Fas L protein Protein expression was quantified byWestern blotting Blank blank control groupDexaDexamethasonegroup Pingmu low Pingmu Decoction low dose group Pingmumoderate Pingmu Decoction medium dose group Pingmu highPingmu Decoction high dose group lowast119875 lt 005 versus the blankcontrol group (119899 = 3 per group)


PPAR 훾

PPAR 훾

CEBP 훼

CEBP 훼

GAPDH

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Prot

ein

relat

ive e

xpre

ssio

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Figure 7 Effect of Pingmu Decoction serum on the expression ofPPAR 120574 and CEBP 120572 protein Protein expression was quantified byWestern blotting Blank blank control groupDexaDexamethasonegroup Pingmu low Pingmu Decoction low dose group Pingmumoderate Pingmu Decoction medium dose group Pingmu highPingmu Decoction high dose group lowast119875 lt 005 versus the blankcontrol group (119899 = 3 per group)

marker expressed preferentially in preadipocytes ratherthan mature adipocytes Pref-1 inhibits the differentiationof the preadipocytes and reduces the generation of fat [24]and glucocorticoid can promote the differentiation of theadipocytes by inhibiting the expression of Pref-1 [25] Ourexamination revealed that Pref-1 was positively expressed incultured cells which verifies that these cells were preadipo-cytes Oil Red O staining a specific method for staininglipid droplets also was used to confirm that preadipocytescould be differentiated into mature adipocytes which furthervalidates our isolation and culture methods

The effects of Pingmu Decoction on cell viability wereassessed byMTT assay Our results show that the cell viabilityof preadipocytes could be reduced by Pingmu Decoctionserum at low medium and high doses at 48 h with the mostsignificant effects observedwith themediumdose and at 96 hThe results of flow cytometry revealed that the early and totalapoptosis rates of matured adipocytes were also increasedobviously for cells exposed to Pingmu Decoction serum (119875 lt005) with the most dramatic effects in the medium and lowdose groups These results suggest that Pingmu Decoctioncan promote the apoptosis of adipocytes from GO patientsIn these experiments the medium dose which was designedto reflect the prescribed human dose was most effective


However additional experiment with a more narrow rangeof doses might verify a dose-effect relationship

We also demonstrated that Pingmu Decoction serummediates an increase in Fas and Fas L which was shownboth at the level of mRNA and proteinThus the mechanismby which Pingmu Decoction serum may act to promote celldeath could involve the activation of death receptor apoptosismediated by Fas and Fas L expression While the currentstudy was limited in the availability of primary tissuesfuture experimentation should be performed to investigatethe responsiveness of the primary adipocytes to Fas L as wellas the Fas L concentration in the orbital locale of patients ascompared to healthy subjects Pingmu Decoction reducedlipid accumulation and downregulated the expression ofPPAR 120574 and CEBP 120572 Icariin is the main constituent ofHerba Epimedii Han et al found that Icariin can inhibitthe adipocyte differentiation through downregulation of theadipogenic transcription factors [26] Zhang et al show thatIcariin could inhibit adipogenic differentiation of BMSCsin the osteoblast-osteoclast coculture [27] And Icariin canalso inhibit the adipogenic transdifferentiation of osteoblasts[28] However despite experimental limitations our studyis consistent with the possibility that Pingmu Decoctionreduces the accumulation of orbital adipocytes in the inactivestage of GO to play a therapeutic role and this effect islikely to be explained by increased FasFas L expression andincreased apoptosis

In this study we used standardized methods to producethe PingmuDecoction serum Components used tomake thePingmu Decoction were produced in factories that adhere toTCM good manufacturing practice Additionally our proto-cols followed methods that are used in clinical application asdescribed in the Pharmacology and Pharmacological Experi-ments with Traditional ChineseMedicine [29] However as forany form of herbal medicine batches of Pingmu Decoctionmay vary according to the source For this reason futurestudies should be done to verify the findings using PingmuDecoction from other sources

5 Conclusion

Our results show that Pingmu Decoction can reduce the cellviability of orbital preadipocytes and inhibit their differen-tiation into adipocytes This effect may be mediated by theactivation of Fas and Fas L by the death signaling pathway topromote the apoptosis of matured adipocytes These resultssuggest a therapeutic mechanism for Pingmu Decoction inreducing the accumulation of orbital adipocytes in GO

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This study is supported by Grants from the National NaturalScience Foundation of China (Grant no 81373617)

References

[1] J Shen Z Li W Li et al ldquoTh1Th2 andTh17 cytokine involve-ment in thyroid associated ophthalmopathyrdquo Disease Markersvol 2015 Article ID 609593 6 pages 2015

[2] K P Cockerham and S S Chan ldquoThyroid eye diseaserdquo Neuro-logic Clinics vol 28 no 3 pp 729ndash755 2010

[3] S S Cury M Oliveira M T Sıbio et al ldquoGene expression ofestrogen receptor-alpha in orbital fibroblasts in Gravesrsquo oph-thalmopathyrdquo Archives of Endocrinology and Metabolism vol59 no 3 pp 273ndash276 2015

[4] F Menconi C Marcocci and M Marino ldquoDiagnosis and clas-sification of Gravesrsquo diseaserdquoAutoimmunity Reviews vol 13 no4-5 pp 398ndash402 2014

[5] L Bartalena ldquoDiagnosis and management of Graves disease aglobal overviewrdquo Nature Reviews Endocrinology vol 9 no 12pp 724ndash734 2013

[6] J Barrio-Barrio A L Sabater E Bonet-Farriol A Velazquez-Villoria and J C Galofre ldquoGravesrsquo ophthalmopathy VISAversus EUGOGO classification assessment and managementrdquoJournal of Ophthalmology vol 2015 Article ID 249125 16 pages2015

[7] R S Bahn ldquoCurrent insights into the pathogenesis of gravesrsquoophthalmopathyrdquoHormone and Metabolic Research vol 47 no10 pp 773ndash778 2015

[8] S Virakul V A SHDalmD Paridaens et al ldquoPlatelet-derivedgrowth factor-BB enhances adipogenesis in orbital fibroblastsrdquoInvestigative Ophthalmology and Visual Science vol 56 no 9pp 5457ndash5464 2015

[9] J A Garrity and R S Bahn ldquoPathogenesis of graves ophthal-mopathy implications for prediction prevention and treat-mentrdquo American Journal of Ophthalmology vol 142 no 1 pp147e2ndash153e2 2006

[10] A Antonelli S M Ferrari S Frascerra et al ldquoPeroxisomeproliferator-activated receptor-120572 agonistsmodulate CXCL9 andCXCL11 chemokines in Gravesrsquo ophthalmopathy fibroblasts andpreadipocytesrdquoMolecular and Cellular Endocrinology vol 349no 2 pp 255ndash261 2012

[11] S Kumar S NadeemMN StanM Coenen andR S Bahn ldquoAstimulatory TSH receptor antibody enhances adipogenesis viaphosphoinositide 3-kinase activation in orbital preadipocytesfrom patients with Gravesrsquo ophthalmopathyrdquo Journal of Molec-ular Endocrinology vol 46 no 3 pp 155ndash163 2011

[12] H Li Y Wang and R Xu ldquoPingmu decoction enhances apop-tosis of orbital adipocytes derived from patients with GravesrsquoophthalmophathyrdquoMolecularMedicine Reports vol 6 no 6 pp1361ndash1366 2012

[13] A Bossowski B Czarnocka A Stasiak-Barmuta K BardadinM Urban and J Dadan ldquoAnalysis of Fas FasL and Caspase-8expression in thyroid gland in young patients with immune andnon-immune thyroid diseasesrdquo Endokrynologia Polska vol 58no 4 pp 303ndash313 2007

[14] C Gajate and F Mollinedo ldquoLipid raft-mediated FasCD95apoptotic signaling in leukemic cells and normal leukocytes andtherapeutic implicationsrdquo Journal of Leukocyte Biology vol 98no 5 pp 739ndash759 2015

[15] P Janusz E Pawlak-Adamska I E Kochanowska and JDaroszewski ldquoThe role of adipose tissue in the pathogenesis andclinical manifestation of gravesrsquo orbitopathyrdquo Postepy Higieny iMedycyny Doswiadczalnej vol 67 pp 1173ndash1181 2013


[16] J S Yoon H J Lee M K Chae and E J Lee ldquoAutophagy isinvolved in the initiation and progression of gravesrsquo orbitopa-thyrdquoThyroid vol 25 no 4 pp 445ndash454 2015

[17] M S Draman F Grennan-Jones L Zhang et al ldquoEffects ofprostaglandin F2120572 on adipocyte biology relevant to gravesrsquoorbitopathyrdquoThyroid vol 23 no 12 pp 1600ndash1608 2013

[18] S E Kim J H Lee M K Chae E J Lee and J S Yoon ldquoTherole of sphingosine-1-phosphate in adipogenesis of gravesrsquo orbit-opathyrdquo Investigative Ophthalmology and Visual Science vol 57no 2 pp 301ndash311 2016

[19] M Comerci A Elefante D Strianese et al ldquosemiautomaticregional segmentation to measure orbital fat volumes in thy-roid-associated ophthalmopathy A validation studyrdquoThe Neu-roradiology Journal vol 26 no 4 pp 373ndash379 2013

[20] P W Potgieser W M Wiersinga N I Regensburg and M PMourits ldquoSome studies on the natural history of Gravesrsquoorbitopathy increase in orbital fat is a rather late phenomenonrdquoEuropean Journal of Endocrinology vol 173 no 2 pp 149ndash1532015

[21] S Kumar M J Coenen P E Scherer and R S Bahn ldquoEvi-dence for enhanced adipogenesis in the orbits of patients withgravesrsquo ophthalmopathyrdquo Journal of Clinical Endocrinology andMetabolism vol 89 no 2 pp 930ndash935 2004

[22] H C Kim S W Yoon and H Lew ldquoUsefulness of the ratio oforbital fat to total orbit area in mild-to-moderate thyroid-associated ophthalmopathyrdquo British Journal of Radiology vol88 no 1053 Article ID 20150164 2015

[23] R I Cho V M Elner C C Nelson and B R Frueh ldquoThe effectof orbital decompression surgery on lid retraction in thyroid eyediseaserdquo Ophthalmic Plastic and Reconstructive Surgery vol 27no 6 pp 436ndash438 2011

[24] K-A Kim J-H Kim Y Wang and H S Sul ldquoPref-1 (pread-ipocyte factor 1) activates the MEKextracellular signal-reg-ulated kinase pathway to inhibit adipocyte differentiationrdquoMolecular and Cellular Biology vol 27 no 6 pp 2294ndash23082007

[25] K Lee J AVillena Y SMoon et al ldquoInhibition of adipogenesisand development of glucose intolerance by soluble preadipocytefactor-1 (Pref-1)rdquo Journal of Clinical Investigation vol 111 no 4pp 453ndash461 2003

[26] Y Y Han M Y Song M S Hwang et al ldquoEpimediumkoreanum Nakai and its main constituent icariin suppresslipid accumulation during adipocytedifferentiation of 3T3-L1preadipocytesrdquoChinese Journal of NaturalMedicines vol 14 no9 pp 671ndash676 2016

[27] S Zhang P Feng G Mo et al ldquoIcariin influences adipogenicdifferentiation of stem cells affected by osteoblast-osteoclastco-culture and clinical research adipogenicrdquo Biomedicine ampPharmacotherapy vol 88 pp 436ndash442 2017

[28] D Zhang C Fong Z Jia L Cui X Yao and M Yang ldquoIcariinstimulates differentiation and suppresses adipocytic transdif-ferentiation of primary osteoblasts through estrogen receptor-mediated pathwayrdquo Calcified Tissue International vol 99no 2 pp 187ndash198 2016

[29] D F Zhang Pharmacology and Pharmacological Experimentswith Traditional Chinese Medicine Shanghai Science and Tech-nology Press Shanghai China 2002

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


GO can be divided into active and inactive stages accord-ing to its development In the inactive stage the clinicalcurative effect is not very good with no available specific andeffectivemethods of treatment byWesternmedicine PingmuDecoction has been used in Chinese medicine to treat GO inthe inactive stage for more than ten years with good clinicalcurative effects A preliminary experimental study demon-strated that serum from rats treated with Pingmu Decoctionand its components can act on orbital preadipocytes frompatients with GO in the inactive period Pingmu Decoctionserum promotes the apoptosis of adipocytes and reduces theaccumulation of orbital adipocytes by increasing the expres-sion of Caspase-3 Caspase-8 and Caspase-9 protein [12]However its apoptotic effect on differentiated adipocytesfrom GO patients remains uncharacterized Intracellularpathways mediated by FasFas L in mammals can induceapoptosis [13 14] We cultured orbital preadipocytes frompatients by the tissue explant method and differentiated theminto orbital adipocytes The effect and molecular mechanismof Pingmu Decoction on the apoptosis of orbital adipocyteswere explored by a variety of methods

2 Materials and Methods

21 Experimental Animals and Drugs Six-week-old specificpathogen-free male Sprague-Dawley rats weighing 200plusmn10 gwere purchased from Shanghai Silaike Experimental Ani-mal Co Ltd (certification number SCXK (Hu) 2012-0002)Animal experiment ethical approval was obtained from theAffiliated Long Hua Hospital of Shanghai University ofTraditional Chinese Medicine

Pingmu Decoction was composed of 30 g of Huangqi(Astragalus membranaceus) 15 g of Xianlingpi (Herba Epime-dii) 15 g of Chuanxiong (Ligusticum wallichii) 15 g of Baijiezi(Brassica alba Boiss) 15 g of stir-baked Biejia (CarapaxTrionycis) and 30 g of Cheqianzi (Semen plantaginis) Theseherbs were provided by the Chinese Medicine Pharmacyof the Affiliated Long Hua Hospital of Shanghai Universityof Traditional Chinese Medicine (TCM) The Decoctionof the above herbs was prepared twice according to theoriginal formula and then filtered and concentrated in acombined liquor The dose of rat administration is based onthe human mouse equivalent dosage conversion accordingto the textbook Pharmacology and Pharmacology Experimentof TCM (edited by Zhang Dafang Shanghai Science andTechnology Press 2002)The crude drug (4 gmL) was storedat 4∘C

22 GO Patients Four cases (4 eyes) of postbulbar adiposetissues from GO inpatients were collected from ShanghaiEENT Hospital Of the four GO cases in this study twofemale and two male patients (age ranging from 25 to 48years old) had a GOhistory of three to eight years Diagnosticcriteria of GOwere in accordance with the clinical score stan-dards described in the Chinese Guideline for Diagnosis andTreatment of Thyroid Disease published in 2008 The clinicalactivity score (CAS) of GO in clinical practice was based onthe clinical score standards ofChinese Guideline for Diagnosisand Treatment of Thyroid Disease including spontaneous

retrobulbar pain pain with eye movement eyelid erythemaconjunctival hyperemia chemosis swollen lacrimal caruncleand swollen eyelid Each symptom accounted for one pointand the total score obtained was up to seven in each caseTheinactive phase of GO referred to active exophthalmos withthe clinical score of lt3 Classification of exophthalmos inthis study was in accordance with the classification crite-ria of GO proposed by the American Thyroid Association(ATA) The patients were confirmed to have GO paralleledorbital decompression by the Ophthalmology Department atShanghai EENTHospital All patientswere in theGO inactivestage which is in line with the diagnostic criteria Otherdiseases such as autoimmune diseases and cancer wereruled out Approval was obtained from the Medical EthicalCommittee and patient consent was obtained in writing priorto collection of tissue samples

23 Medicinal Serum Preparation The Sprague-Dawley ratswere randomly divided into 2 groups with 5 rats per groupThe rats in the PingmuDecoction group were given 15mLkgPingmu Decoction and the rats in the control group weregiven an equal volume of distilled water by intragastricadministration twice a day for 3 daysThefinal concentrationsof medicinal serum (final concentration = volume added toserumtotal volume times 100) were 5 (low dose) 10(medium dose) and 20 (high dose)

Bloodwas collected from the abdominal aortae of the ratsunder ether anesthesia 15 h after the last administrationTheblood was incubated for 2 h at 4∘C and then centrifuged at4∘C at 3000 rpmmin for 15min The sera from each groupof mice were combined and inactivated for 30min at 56∘C ina water bath filtered with 022120583m aperture micropore filmpacked with frozen pipes and stored at minus20∘C

24 Culture of Primary Cells from Patient Tissue Sterilizedorbital adipose tissues from the operating table were washed3 timeswith sterile phosphate buffer solution (PBS) to removethe blood and then were placed into culture bottles con-taining complete medium As soon as possible after transferto the laboratory (within 4 h) the tissues were processedfor experimentation at room temperature Human orbitalpreadipocytes were cultured by the tissue explant methodThe adipose tissue with visible connective tissue or bloodclots was removed and the remaining tissue was harvestedinto Petri dishes with D-Hankrsquos solutionThe tissues were cutinto pieces of approximately 1mm times 1mm times 1mm in sizewith ophthalmic scissors and were seeded evenly at thebottom of culture bottles Then the bottles were gentlyturned upside down and 2mL complete culture solutionwas added The bottles were placed askew into an incubatorwith 5 CO

2at 37∘C for culturing After the tissue explants

adhered tightly to the culture bottle 5mL complete culturesolution was added and the solution was replaced withfresh solution every 2-3 d At 80 confluence the cells werepassaged 1 3 (every 3-4 d) Cells were used in experimentsafter 3ndash6 generations The morphology and growth status ofthe cultured primary cells were observed every day Oil RedO staining and microscopic photography were performed toassess the formation of lipid droplets Healthy preadipocytes






2in an










(a) (b)




3 Results







(a) (b)

(c) (d)

(e) (f)






0010203040506070809

1

24 48 72 96(h)

OD

val

ue

BlankDexaPingmu low


lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast lowast

lowast







4 Discussion




0

2

4

6

Total Early

Apop

tosis

rate

()

lowastlowast

lowast

lowast

lowast

lowast

BlankDexaPingmu low


(e)

(c) (d)

(a) (b)

Apoptosis07

Post-Apo anddead 10

Annexin V-FITC-A

Gate P1

PI-A

Dead04

1072

106

105

104

103

1037 105 106 1072

Apoptosis06

Post-Apo anddead 12

Gate P1Dead04

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis28

Post-Apo anddead 28

Gate P1Dead08

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis16

Post-Apo and dead19

Gate P1Dead03

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

1000Apoptosis

13

Post-Apo anddead 15

Gate P1Dead11

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072



00

05

10

15

20

Blank

Gen

e exp

ress

ion

relat

ive t

o G

APD

H

Fas Fas L

DexaPingmu low


lowast

lowast

lowastlowast


00

02

04

06

Fas

Fas

Fas L

Fas L

GAPDH


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowast lowast

lowast

lowast

lowast lowast

BlankDexaPingmu low




PPAR 훾

PPAR 훾

CEBP 훼

CEBP 훼

GAPDH

00

01

02

03

04

BlankDexaPingmu low


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowastlowast lowast









5 Conclusion




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2in an










(a) (b)




3 Results







(a) (b)

(c) (d)

(e) (f)






0010203040506070809

1

24 48 72 96(h)

OD

val

ue

BlankDexaPingmu low


lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast lowast

lowast







4 Discussion




0

2

4

6

Total Early

Apop

tosis

rate

()

lowastlowast

lowast

lowast

lowast

lowast

BlankDexaPingmu low


(e)

(c) (d)

(a) (b)

Apoptosis07

Post-Apo anddead 10

Annexin V-FITC-A

Gate P1

PI-A

Dead04

1072

106

105

104

103

1037 105 106 1072

Apoptosis06

Post-Apo anddead 12

Gate P1Dead04

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis28

Post-Apo anddead 28

Gate P1Dead08

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis16

Post-Apo and dead19

Gate P1Dead03

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

1000Apoptosis

13

Post-Apo anddead 15

Gate P1Dead11

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072



00

05

10

15

20

Blank

Gen

e exp

ress

ion

relat

ive t

o G

APD

H

Fas Fas L

DexaPingmu low


lowast

lowast

lowastlowast


00

02

04

06

Fas

Fas

Fas L

Fas L

GAPDH


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowast lowast

lowast

lowast

lowast lowast

BlankDexaPingmu low




PPAR 훾

PPAR 훾

CEBP 훼

CEBP 훼

GAPDH

00

01

02

03

04

BlankDexaPingmu low


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowastlowast lowast









5 Conclusion




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)




3 Results







(a) (b)

(c) (d)

(e) (f)






0010203040506070809

1

24 48 72 96(h)

OD

val

ue

BlankDexaPingmu low


lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast lowast

lowast







4 Discussion




0

2

4

6

Total Early

Apop

tosis

rate

()

lowastlowast

lowast

lowast

lowast

lowast

BlankDexaPingmu low


(e)

(c) (d)

(a) (b)

Apoptosis07

Post-Apo anddead 10

Annexin V-FITC-A

Gate P1

PI-A

Dead04

1072

106

105

104

103

1037 105 106 1072

Apoptosis06

Post-Apo anddead 12

Gate P1Dead04

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis28

Post-Apo anddead 28

Gate P1Dead08

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis16

Post-Apo and dead19

Gate P1Dead03

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

1000Apoptosis

13

Post-Apo anddead 15

Gate P1Dead11

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072



00

05

10

15

20

Blank

Gen

e exp

ress

ion

relat

ive t

o G

APD

H

Fas Fas L

DexaPingmu low


lowast

lowast

lowastlowast


00

02

04

06

Fas

Fas

Fas L

Fas L

GAPDH


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowast lowast

lowast

lowast

lowast lowast

BlankDexaPingmu low




PPAR 훾

PPAR 훾

CEBP 훼

CEBP 훼

GAPDH

00

01

02

03

04

BlankDexaPingmu low


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowastlowast lowast









5 Conclusion




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e) (f)






0010203040506070809

1

24 48 72 96(h)

OD

val

ue

BlankDexaPingmu low


lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast lowast

lowast







4 Discussion




0

2

4

6

Total Early

Apop

tosis

rate

()

lowastlowast

lowast

lowast

lowast

lowast

BlankDexaPingmu low


(e)

(c) (d)

(a) (b)

Apoptosis07

Post-Apo anddead 10

Annexin V-FITC-A

Gate P1

PI-A

Dead04

1072

106

105

104

103

1037 105 106 1072

Apoptosis06

Post-Apo anddead 12

Gate P1Dead04

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis28

Post-Apo anddead 28

Gate P1Dead08

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis16

Post-Apo and dead19

Gate P1Dead03

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

1000Apoptosis

13

Post-Apo anddead 15

Gate P1Dead11

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072



00

05

10

15

20

Blank

Gen

e exp

ress

ion

relat

ive t

o G

APD

H

Fas Fas L

DexaPingmu low


lowast

lowast

lowastlowast


00

02

04

06

Fas

Fas

Fas L

Fas L

GAPDH


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowast lowast

lowast

lowast

lowast lowast

BlankDexaPingmu low




PPAR 훾

PPAR 훾

CEBP 훼

CEBP 훼

GAPDH

00

01

02

03

04

BlankDexaPingmu low


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowastlowast lowast









5 Conclusion




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0010203040506070809

1

24 48 72 96(h)

OD

val

ue

BlankDexaPingmu low


lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast lowast

lowast







4 Discussion




0

2

4

6

Total Early

Apop

tosis

rate

()

lowastlowast

lowast

lowast

lowast

lowast

BlankDexaPingmu low


(e)

(c) (d)

(a) (b)

Apoptosis07

Post-Apo anddead 10

Annexin V-FITC-A

Gate P1

PI-A

Dead04

1072

106

105

104

103

1037 105 106 1072

Apoptosis06

Post-Apo anddead 12

Gate P1Dead04

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis28

Post-Apo anddead 28

Gate P1Dead08

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis16

Post-Apo and dead19

Gate P1Dead03

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

1000Apoptosis

13

Post-Apo anddead 15

Gate P1Dead11

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072



00

05

10

15

20

Blank

Gen

e exp

ress

ion

relat

ive t

o G

APD

H

Fas Fas L

DexaPingmu low


lowast

lowast

lowastlowast


00

02

04

06

Fas

Fas

Fas L

Fas L

GAPDH


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowast lowast

lowast

lowast

lowast lowast

BlankDexaPingmu low




PPAR 훾

PPAR 훾

CEBP 훼

CEBP 훼

GAPDH

00

01

02

03

04

BlankDexaPingmu low


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowastlowast lowast









5 Conclusion




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

Total Early

Apop

tosis

rate

()

lowastlowast

lowast

lowast

lowast

lowast

BlankDexaPingmu low


(e)

(c) (d)

(a) (b)

Apoptosis07

Post-Apo anddead 10

Annexin V-FITC-A

Gate P1

PI-A

Dead04

1072

106

105

104

103

1037 105 106 1072

Apoptosis06

Post-Apo anddead 12

Gate P1Dead04

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis28

Post-Apo anddead 28

Gate P1Dead08

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

Apoptosis16

Post-Apo and dead19

Gate P1Dead03

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072

1000Apoptosis

13

Post-Apo anddead 15

Gate P1Dead11

PI-A

1072

106

105

104

103

Annexin V-FITC-A1037 105 106 1072



00

05

10

15

20

Blank

Gen

e exp

ress

ion

relat

ive t

o G

APD

H

Fas Fas L

DexaPingmu low


lowast

lowast

lowastlowast


00

02

04

06

Fas

Fas

Fas L

Fas L

GAPDH


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowast lowast

lowast

lowast

lowast lowast

BlankDexaPingmu low




PPAR 훾

PPAR 훾

CEBP 훼

CEBP 훼

GAPDH

00

01

02

03

04

BlankDexaPingmu low


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowastlowast lowast









5 Conclusion




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

05

10

15

20

Blank

Gen

e exp

ress

ion

relat

ive t

o G

APD

H

Fas Fas L

DexaPingmu low


lowast

lowast

lowastlowast


00

02

04

06

Fas

Fas

Fas L

Fas L

GAPDH


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowast lowast

lowast

lowast

lowast lowast

BlankDexaPingmu low




PPAR 훾

PPAR 훾

CEBP 훼

CEBP 훼

GAPDH

00

01

02

03

04

BlankDexaPingmu low


Prot

ein

relat

ive e

xpre

ssio

n

low mod high

lowastlowast lowast









5 Conclusion




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















pingmu decoction induces orbital preadipocytes apoptosis in vitro · 2019. 7. 30. · ent effect of...

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