Research ArticleERK1/2 Pathway Is Involved in the Enhancement of FattyAcids from Phaeodactylum tricornutum Extract (PTE) on HairFollicle Cell Proliferation

Lei Xiao ,1,2 Xia Zhang,1 Zhiyi Chen,3 Yuting Li,4 Bing Li ,1 and Lin Li 1,4

1School of Food Science and Engineering, Guangdong Province Key Laboratory for Green Processing of Natural Products andProduct Safety, South China University of Technology, Guangzhou 510640, China2Infinitus (China) Co., China3Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Functional Foods,Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory of Agricultural Products Processing,Guangzhou 510610, China4School of Chemical Engineering and Energy Technology, Dongguan University of Technology, College Road 1,Dongguan 523808, China

Correspondence should be addressed to Bing Li; bli@scut.edu.cn and Lin Li; lilin@dgut.edu.cn

Received 2 July 2020; Revised 21 September 2020; Accepted 27 September 2020; Published 31 October 2020

Academic Editor: Junyan Liu

Copyright © 2020 Lei Xiao et al. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Extractions from Phaeodactylum tricornutum have been widely studied and evaluated to various biological effects. The aim of thisstudy was to investigate the promotional effect of P. tricornutum extract (PTE) on the ERK1/2 signaling pathway involved in hairfollicle cell proliferation. In order to illuminate the enhancement of PTE on hair growth by promoting proliferation of hair folliclecells, the activities of human hair follicle outer root sheath cell (HFORSC), human hair follicle germinal matrix cells (HFGMC), andhair epithelial melanocytes (HEM) were observed under PET treatment. Levels of keratins, PKCζ, ERK1/2, and p38 MAPK in hairfollicle cells were determined by Western blotting to illustrate the mechanisms of PTE effects on hair growth. Analyzed by GC-MS,the main polyunsaturated fatty acids which were 9.43% of total fatty acids in PTE were linolenic acid, linoleic acid, eicosapentaenoicacid, and docosahexaenoic acid. Melanin content and tyrosinase activity in HEM were measured. The results showed that PTEexhibited remarkable enhancement on cell proliferation. Melanin production was inhibited by PTE treatment, while keratin-14,keratin-15, and keratin-17 levels on hair follicle cells were elevated at different concentrations. The promotions of ERK1/2 andp38 MAPK levels indicated that the ERK1/2 signaling pathway is involved in the proliferation of hair follicle cells. These resultsare the evidence that PTE potentially deserves further study as a new natural candidate for hair care applications.

1. Introduction

Phaeodactylum tricornutum (P. tricornutum), an interestingnatural material, is a common type of marine diatoms withthree different morphotypes, of which the genomes havebeen fully sequenced [1]. P. tricornutum constituents withmultiple components such as proteins, carotenoids like fuco-xanthin, polyphenols like (epi-)catechin, vitamins, minerals,and several kinds of lipophilic materials of polyunsaturatedfatty acids (PUFAs), such as eicosapentaenoic acid (EPA)

and docosahexaenoic acid (DHA), which might mediatephysiological values [2–6].

The antioxidant capacity, cytotoxicity, immunomodula-tory activity, and antimicrobial ability, as well as antiobesityand antidiabetic properties of the extractions from P.tricornutum (PTE) have been widely studied and evaluated.PTE showed abilities to stimulate proteasome activity onhuman keratinocytes under UVA and UVB radiations, asevidence indicated that carbonyl content of total proteinand oxidized protein level was reduced in PTE-treated cells

HindawiBioMed Research InternationalVolume 2020, Article ID 2916104, 11 pageshttps://doi.org/10.1155/2020/2916104

both before and after UV irradiation to further preserve pro-teasome function in lowering the irradiation-induced proteinoxidative damage [7]. PET also showed the capability to haveprotective effects on protein oxidation level in human stra-tum corneum post UVA and UVB exposures [8]. There arepublications and patents that archived PTE antioxidationand proteasome peptidase activities from the detrimentaleffects of UV exposure [9]. Beneficial effects such as anti-inflammation were evaluated [10, 11]. New compounds, suchas carotenoid, from PTE exhibited health benefit effects ofinflammatory reduction, alleviation of oxidative stress, andinterference with cancer cells [12]. PTE, rich with fucoxan-thin, comprises several lipophilic constituents and showedantiobesity and antidiabetic properties that have beenreported in cell and in vivo studies [13–15]. The antiobesityeffects of ω-3 long-chain PUFAs comprise decreased lipogen-esis and the enhancement of fatty acid oxidation in liver andadipose tissues, as well as the influence of intestinal lipidabsorption and lipid metabolism [16, 17]. As the result, theinteresting properties of PTE were described above andfurther studies on PTE-related biological effects may lead toimportant clinical as well as economic significances.

Hair, as the skin appendage growing on the surface of thescalp with its appearance and protective functions, has beenconsidered to play important roles in human daily mainte-nance. The human hair follicle (HF) is highly dynamic thatrepeatedly and perpetually undergoes tightly programmedcyclical transformations through periods of active hairgrowth (anagen), apoptosis-driven involution (catagen), hairshedding (exogen), and relative resting (telogen) [18, 19].During HF growth and hair production, the activity of factorspromoting the proliferation, differentiation, and survival pre-dominates, whereas HF regression is characterized by theactivation of a variety of signaling pathways that induce apo-ptosis in HF cells [20]. Continuation and transition of eachphase of hair growth result from elaborate interactionsbetween mesenchymal dermal papilla, located at the base ofthe hair follicle, and adjacent follicular epithelium.

Hair problems, such as hair loss and grey hair, may resultfrom hair cell imbalances of metabolisms or outer injuries ofphysical and chemical interferences. The regeneration andself-healing of HF are vital in maintaining normal hairgrowth. Among the follicular epithelium are HF outer rootsheath cells (HFORSC) that form the HF and are the repair-men of epidermis cells [18]. HF germinal matrix cell(HFGMC), located at the base of the bulb and the mainsource for hair growth, goes through cornification to form alayer of keratin to suppress hair loss. And HF inner rootsheath cells (HFIRSC) keep the hair steady in HF. In partic-ular, mesenchyme-derived dermal papilla cells (DPC) havepotential to induce hair follicle formation [21]. A numberof signals from DPC may directly influence HFORSC in thebulge area to regulate hair growth cycle [22]. Besides, keratinsincluding keratin-14, keratin-15, keratin-17, and keratin-19related to HFORSC have properties in the formation of HF[23]. Hair pigmentation is coupled to the HF growth cyclethat occurs only during anagen and stops very early in theanagen-catagen transition [24]. Tyrosinase is detected in hairbulb melanocytes, pointing to a melanin-producing subpop-

ulation of the follicular melanocytes, while the mechanism ofthe pigment incorporating into the hair shaft or beingremoved from the hair bulb during early catagen is not clear[25, 26]. Epidermal hairs are usually nursed by hair care rou-tines. However, some of the routines could contribute tosome degrees of hair problems.

It would be meaningful to find better materials or rou-tines with less damage on hair growth. Some natural extractshave been believed to regulate hair cell growth with less dam-age on HF cells. In order to obtain better insight into thepossible benefits of PTE influences on human hair growth,we focused on its biological effect on HF cell growth, levelof key protein including keratin, and HF cell-related melaninproduction. This work will be helpful in broadening ideas forfuture investigations of PTE on hair care applications.

2. Results

2.1. Influences of PTE on Hair Follicle Cell Viability andGrowth. The influences of PTE on HFORSC, HFGMC, andHEM proliferations were evaluated with MTT assays. Cellswere treated with serial dilutions of PTE at the range from0.03μg/mL to 16μg/mL in correspondent culture media for24 hours at 37°C in a CO2 incubator. The results showed sig-nificant reduction effect on both HFORSC and HFGMC pro-liferations at higher concentrations between 4μg/mL and16μg/mL, while there was statistical significance of cellgrowth-promoting effects on both HFORSC and HFGMCproliferations at lower concentrations ranged from 1μg/mLto 2μg/mL in comparison with their control (Figures 1(a)and 1(b)). The proliferation of HEM was not affected byPTE treatment at concentrations ranged from 1μg/mL to8μg/mL but was significantly induced at concentrationsranged from 0.125μg/mL to 0.5μg/mL (Figure 1(c)). Thisresult may indicate that PTE exhibited some kind of promot-ing effects on proliferation of hair follicle cells.

Cell colony formation is a primmorph counting of theproliferation from single cell in vitro. The adaption abilityof a colony is stronger than the single cell under signals orstimulations. A colony forming experiment is a kind of testto challenge individual cells under low cell density, whichshows higher sensibility in analyzing the cell viability. Statis-tical data of stained colonies are presented in Figure 1(d). Itshowed a significant rise of the HFGMC colony formationat 0.125μg/mL of PTE, while a significant increase of theHFORSC colony formation at 0.0625μg/mL. PTE at otherconcentrations could also raise the colony numbers butshowed no statistical significances. The results indicated thatPTE could enhance the colony multiplications on HFORSCand HFGMC, especially at concentration ranges from0.0625μg/mL to 0.125μg/mL.

In order to evaluate long-term influences of PTE effectson hair follicle cells, the growth profiles of HFORSC andHFGMC were evaluated under PTE treatment with the con-centration of 0.0625μg/mL obtained from cell colony forma-tion assay. The results showed that there was no significantdifference in cell growth levels between the PTE-treatedgroup and the control group on both cells (Figures 1(e) and

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1(f)), which indicates that PTE has no long-term effect on thegrowth of hair follicle cells at low concentrations.

2.2. Influences of PTE on Keratin Level. Keratins, expressed inHFORSC and dermal papilla, are the main components ofthe cortex and medulla, which have great influences on hairstructural strength and thickness, where keratin-15 andkeratin-19 are epidermal stem signs that reflect the cellabilities of differentiation and proliferation. Influences ofPTE on HFORSC keratin-14, keratin-15, keratin-17, andkeratin-19 were examined (Figure 2). The results indicatedthat expression levels of keratin-14, keratin-15, and keratin-17 in HFORSC were enhanced with PTE at various concen-trations. There were significant increases of 77%, 49%, and131% in keratin-14, keratin-15, and keratin-17, respectively,with PTE at 0.25μg/mL.

2.3. Influences of PTE on Melanin Production and TyrosinaseActivity of HEM. In this experiment, 8-methoxypsoralen (8-MOP) was chosen as a positive control since it is photosensi-tive with melanin-promoting and tyrosinase-activatingabilities. PTE treatment groups had no melanin inductionsin the treatment concentrations but exhibited melanin reduc-tion significantly at concentrations of 0.25μg/mL and0.50μg/mL, as well as the 8-MOP treatment had melanin-promoting abilities on HEM (Figure 3(a)). On the otherhand, tyrosinase activities were significantly induced withthe PTE treatment at concentrations of 0.125μg/mL, ofwhich the tyrosinase activity was higher than the 8-MOPtreatment group (Figure 3(b)), although the melanin levelshowed no significant differences with PTE treatment at thesame concentration.

2.4. Influences of PTE on ERK1/2, p38, and PKCζ Expressionin HEM. It has been reported that the activation of extracel-lular signal-regulated kinase (ERK) reduces melanin synthe-

sis, as well as the inhibition of p38 MAPK (mitogen-activatedprotein kinase) blocks melanogenesis [27]. Protein kinase Czeta (PKCζ) activation plays a predominant role in the for-mation of melanocyte dendricity [28]. Influences of PTE onHEM protein expressions of ERK1/2, p38 MAPK, and PKCζare presented in Figure 4. The expressions of ERK1/2 wereinduced by 56% with PTE treatment at a concentration of0.5μg/mL, while the expression level of p38 MAPK exhibitedan insignificant increase at about 17%. PTE treatmentshowed no significant influence on PKCζ expression levelsin HEM at all tested concentrations.

3. Discussion

3.1. Enhancement of PTE on Hair Follicle Cell Growth. Hairfollicle is composed of the epithelium and leather parts. Theepithelial part includes the inner root sheath and outer rootsheath. The leather part includes the dermal papillae and der-mal sheath, of which the morphology of hair follicle genera-tion is induced by interactions of the epidermis and dermis.The cyclicality stages of hair growth includes anagen, cata-gen, telogen, and exogen, and the cyclic growth cycles repeatthe hair follicle’s whole life [29]. The outer root sheath cellswhich are composed by a large number of undifferentiatedcutin with long proliferation cycle and stem cell propertiesare involved in the formation of the hair follicle and repair-men of epidermis cells [27]. The inner root sheath cells arethe one layer of cells that cling to hair, which play an impor-tant role in keeping the hair steady in the hair follicle.HFGMC, located at the base of the hair bulb around the nip-ples, is the main support for hair growth and hair melanintransfer. In the active stages of hair growth, quick cell growthand differentiation contribute to hair growth. HFGMC goesthrough cornification to form a layer of keratin to suppresshair loss. During the cyclicality, all of the above mentionedcells interact with each other to support the growth of hair.

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Figure 1: Influences of PTE on cell viability and growth of HFORSC, HFGMC, and HEM. (a–c) Proliferation activities of HFORSC, HFGMC,and HEM determined by MTT assay; (d) colony formations of HFORSC and HFGMC; (e, f) long-term influences of PTE at 0.0625μg/mL onHFORSC and HFGMC growth. PTE: extractions from Phaeodactylum tricornutum; HFORSC: outer root sheath cells of human hair follicle;HFGMC: germinal matrix cell of human hair follicle; HEM: hair epithelial melanocytes. ∗P < 0:05 versus non-PTE-treated controls.

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In our current studies, the HFORSC and HFGMC were cho-sen to evaluate the PTE abilities to influence on hair follicleregeneration and hair growth.

HFORSC is connected with epidermal basal layer cellsanatomically that is widely accepted to have the characteris-tics of stem cells, while HFGMC is considered as the mainresource of hair growth [30]. Cell viability study results indi-cated that PTE treatments could effectively increase theproliferation abilities on HFORSC and HFGMC at concen-trations from 0.0625μg/mL to 0.125μg/mL. Therefore, PTEeffects on HFORSC and HFGMC proliferation inductionscould possibly relate to influence on hair follicle proliferationand hair growth.

3.2. Influences of PTE on Keratin Level in Hair Follicle Cell.Keratins of the cortex are essential factors to support thetenacity and elasticity of hair. Thus, the general hair quality

is believed to have some correlations to the keratin levels ofHFORSC and HFIRSC. Keratin-14, a type I keratin in normalepidermis which is mainly expressed in the basal layer cellwith fission and proliferation abilities, is of great importancein maintaining the proliferation and differentiation of skinbasal layer cells and the integrity of cell morphology [31,32]. Keratin-15, as a sign of hair follicle stem cells, is specifi-cally expressed in HFORSC [33]. Keratin-19 is generallyconsidered as one of the symbols of epidermal stem cells.The self-renewal, proliferation, and differentiation abilitiesof cells could be implied by its expression level. Keratin-15and keratin-19 are remarkably expressed in the gatheringarea of epidermal stem cells and hair follicle outer root sheathuplift area (bulge) [34]. Previous research showed thatkeratin-15 and keratin-19 have no expression in vitro gener-ation of cultured HFORSC, which implies loose of differenti-ation ability [35]. PTE enhanced various degrees of protein

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Figure 2: Influences of PTE on protein levels of keratin-14, keratin-15, keratin-17, and keratin-19 in HFORSC. (a)Western blot of keratin-14,keratin-15, keratin-17, and keratin-19 with PET treatment at concentrations of 0.0625, 0.125, and 0.25μg/mL; (b) normalized densities ofkeratin-14, keratin-15, keratin-17, and keratin-19 by ImageJ. PTE: extractions from Phaeodactylum tricornutum; HFORSC: outer rootsheath cells of human hair follicle. ∗P < 0:05 versus non-PTE-treated controls.

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expressions of keratin-14, keratin-15, and keratin-17 onHFORSC in this study (Figure 2) which could be a possibleindication that PTE is involved in regulating hair tenacityand elasticity. Further, the increased protein expression levelsof keratin-14, keratin-15, and keratin-17 on HFORSC underthe treatments of PTE might indicate regulatory effects atmolecular levels to improve the renewals of hair follicle andhair growth based on the mechanisms of proliferation anddifferentiation.

So far, there were studies that indicated a total of 54 kindsof keratins in human beings, 37 of which exist in the epithe-lial cell and 17 kinds in the hair. Furthermore, 9 keratins arespecifically expressed in hair follicle epithelial cells. As a maincomponent of the cytoskeleton, hair follicle keratins are ofgreat importance in keeping the mechanical stability andintegrity of the epithelial cell as well as the structure of hairfollicles [33]. There are 5 kinds of hair follicle keratins relatedto hereditary hair disorders [36]. Among them, there werelots of attention on keratin-17, a kind of keratin which sup-ports the structure of cells and could promote the hair folliclegrowth by reducing the TNF-α-induced apoptosis [37]. Micelacking keratin-17 had severe moulting, brittle hair, apopto-sis of hair follicle stromal cells, and hair follicle histologychange [38]. The expression of keratin-17 could be easilyinduced in stratified epithelial cells to regulate the growth

of the cells and renovation of the skin [39]. In this study, a131% expression level increase of keratin-17 on HFORSCby PTE indicated that PTE could act as an enhancer to influ-ence hair follicle activities effectively and act as a protector tomaintain hair normal structure.

3.3. Influences of PTE on Melanogenesis. The synthesis ofmelanin initiates in mature melanocytes in the hair follicleactive growth phase, and then the melanin were transferredinto the hair cortex. The cellular tyrosine is catalyzed bytyrosinase into the molecules of Dopa and then Dopaqui-none. Thus, the activity of tyrosinase could be an indicatorto predict melanin formation following melanosome transferof melanin into horn cells by synapse regulated by signalPKCζ. On the other hand, melanin level could be inhibitedby activation of the ERK pathway [27]. The current studyshowed that the activity of tyrosinase, the expression levelof ERK1/2, and the melanin levels in HEM cells were allinfluenced by PTE. A study showed that PTE concentrationsat 0.125μg/mL to 0.5μg/mL could significantly increase theproliferation of HEM (Figure 1(c)), but the development ofmelanin is inhibited (Figure 3(a)). The promotions of mela-nophore and tyrosinase are supposed to bring increases ofmelanin level, but our current study showed melanindecrease and tyrosinase increase induced by PTE at

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Figure 3: Influence of PTE onmelanin levels and tyrosinase activity of HEM. (a) Melanin levels; (b) tyrosinase activity. PTE: extractions fromPhaeodactylum tricornutum; HEM: hair epithelial melanocytes; 8-MOP: 8-methoxypsoralen. ∗P < 0:05 versus non-PTE-treated controls.

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0.125μg/mL. Established cocultures of melanocytes andkeratinocytes could provide a closer model to the bodyphysiological state to understand the interactions betweenmelanophore and keratinocyte [40]. Therefore, PTE specula-tions on melanin metabolisms could be further evaluatedwith the coculture method.

PKCζ is a vital regulator of synapse formation of the 5kinds of PKC subtypes expressed by melanophore, especiallyprostaglandin E2 [28]. The mitoses, cell polarity, and differ-entiation are all managed by PKCζ. A study indicated thatthe activation of PKCζ could also increase the developmentof synapses and transfer the amount of melanosome intoadjoining cells [41]. Our current experiment showed less

significance of the PTE effect on the PKCζ level (Figure 4).This result might be a biological benefit with the mainte-nance of normal expression of PKCζ, which is of greatimportance to normal metabolism without overexpressionof PKCζ that could trigger tumors [42].

ERK1/2, which is related to cell proliferation and apopto-sis, is a main pathway of MAPK pathways. Research hadproven that the activation of ERK and Akt could protectthe Mel-Ab cells from the apoptosis induced by UVB [43].Other studies found that the activation of ERK could inhibitthe activity of melanin enzyme and generation of melanin[44, 45]. Kim et al. chose the human melanophore as a mate-rial and concluded that the activation of ERK and inhibition

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Figure 4: Influences of PTE on protein levels of ERK1/2, p38 MAPK, and PKCζ in HEM. (a) Western blot of ERK1/2, p38, and PKCζ withPET treatment at concentrations of 0.125, 0.25, and 0.5 μg/mL; (b) normalized densities of ERK1/2, p38, and PKCζ by ImageJ. PTE:extractions from Phaeodactylum tricornutum; HEM: hair epithelial melanocytes. ∗P < 0:05 versus non-PTE-treated controls.

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of p38 MAPK could both reduce the generation of melanin[27]. Our current study showed that PTE promoted 56% ofthe ERK1/2 level (Figure 4) which was was consistent withthe reduction of the melanin level. The level of p38 MAPKwas slightly increased but had no significant influence onthe generation of melanin. Considering the PTE influenceson promotions of the level of ERK1/2 and p38 MAPK, andthe enhancement of HEM growth, we believed that melano-genesis could be regulated by PTE at proper concentrations.

4. Conclusions

The liposoluble extract from Phaeodactylum tricornutumexhibited activities on hair growth by promoting the growthof hair follicle cells and showed influence on the melanogen-esis in melanophore. Thus, this extract could provide a newnatural compound for hair care applications by regulatinghair cell growth.

5. Methods

5.1. Materials. P. tricornutum was collected from a salted lakeand isolated by the South Central University for Nationalitiesin China. The cultivation was conducted in a modified f/2medium and lasted for 10 days. Then, the microalgal cellswere harvested by centrifuge and freeze-dried for extraction.To prepare the PTE extract, disperse 20 g of frozen dry massin 500mL of 95% ethanol rendered alkaline to pH 13 by add-ing 30% aqueous sodium hydroxide solution. Reflux at 79°Cfor 30min under a nitrogen atmosphere and cool to 18°C.Remove the insoluble materials by filtration. Add 150mL dis-tilled water and agitate to form an aqueous-alcoholic phase.Wash the aqueous-alcoholic phase with heptane by aliquid-liquid process and get the lower phase. Repeat twotimes and acidify the lower phase to a pH value of 2.5 bythe addition of sulfuric acid. Wash with heptane three times.Remove heptane with a rotary evaporator to get 0.197 g ofPTE extract with dark yellow oily appearance.

Dulbecco’s modified Eagle’s medium (DMEM) with highglucose, fetal bovine serum (FBS) and cell culture supple-ments of penicillin, streptomycin, and L-glutamine were pur-chased from Gibco® Life Technologies. Cell culturedisposable sterile materials with the name brand of Corning®were purchased from Thermo Fisher. Mouse anti-cytokeratin14 monoclone antibody (Ab49806), rabbit anti-cytokeratin15 monoclone antibody (Ab139840), and rabbit anti-KRT71 polyclone antibody (Ab133817) were purchased fromAbcam; mouse anti-cytokeratin 19 monoclone antibody (Sc-374192), mouse anti-cytokeratin 17 monoclone antibody(Sc-393091), and rabbit anti-PKCζ polyclonal serum (Sc-216) were purchased from Santa Cruz Biotechnology; andrabbit anti-P44/42 MAPK (ERK1/2) monoclonal antibody(CST#4695), rabbit anti-tubulin monoclonal antibody(CST#2125), and rabbit anti-p38 MAPK (D13E1) monoclo-nal antibody (CST#8690) were purchased from Cell Signal-ing Technology. The PTE extract was prepared by themethods of Nizard et al. with some modification as indicatedabove [9]. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO),

Nonidet P-40, L-3,4-dihydroxyphenylalanine (L-Dopa), 8-methoxypsoralen (8-MOP), and other chemical reagents, ifnot specified, were purchased from Sigma-Aldrich Co.

5.2. Determination of Fatty Acid Composition of PTE. Deriv-atization of fatty acids in PTE was prepared by the transester-ification method [46]. Then, the fatty acid methyl esters wereanalyzed by GC-MS. Decanoic acid methyl ester was used asthe internal standard. Samples were analyzed by usingAgilent Technology 7820A GC and 5977E MSD systemsequipped with an autosampler. Chromatographic separa-tions were carried out by using a TG-Wax MS column(Thermo Scientific™) with 30m length, 0.25mm internaldiameter, and 0.25μm column phase thickness. Nitrogenwas used as carrier gas, and 1μg volume of the sample wasinjected to the inlet heated to 280°C with a splitless injectionmode. The oven temperature was programmed at 120°Cinitially and held for 3min and raised up to 240°C at a speedof 5°C/min, until it reaches 240°C with an 8min hold time.Conditions used for the mass spectrometer were a sourcetemperature of 230°C, scan range 40-650m/z, and operationin positive electron impact mode with ionization energy of70 eV. Chromatographic and mass spectral data were ana-lyzed by the instrument built in MSD ChemStation (AgilentTechnologies, USA). Quantification of fatty acids was calcu-lated by the internal standard with the relationship of relativeresponse factors. The result showed that PTE contained aunique fatty acid profile (Table 1), especially some polyunsat-urated fatty acids such as linolenic acid (C18:3n3), linoleicacid (C18:2n6c), eicosapentaenoic acid (EPA, C20:5), anddocosahexaenoic acid (DHA, C22:6).

5.3. Cell Cultures. HFORSC, HFGMC, and HEM werebought from Beijing Yuhengfeng Biotech Co., Ltd. Beforeincubating, epidermal growth factor and mesenchymal stemcell growth factor are added to stabilize cell amplificationsin the culture media. The cells were maintained and culti-vated in DMEM containing 10% FBS, with 5% CO2 at 37

°C.Cells from the third to sixth passages of subcultures wereused for experiments.

5.4. MTT Assay for Cell Viability and Proliferation. Influenceof PTE on cell proliferations was measured by the MTTmethod as previously described [47]. PTE extract was steril-ized by filtration with 0.22μm microporous membrane anddiluted with culture media containing 10% FBS to have aninitial concentration of 16μg/mL. The media of cell culturesat 80% confluence in 96-well cell culture plates were replacedby the PTE serial dilutions, respectively, with 5 parallel wellsat each concentration. MTT assays were conducted on thecells after 24-hour treatments with PTE dilutions. The resultswere expressed as a percentage of the untreated controls, andthe values were represented as the mean ± SD.

5.5. Colony Formation Test and Cell Growth Curve. InoculateHFORSC and HFGMC into a 6-well plate at concentrationsof 2:00 × 102 cells per well and 5:00 × 102 cells per well,respectively. The cell culture media were replaced withPTE dilutions at 0.0625μg/mL, 0.125μg/mL, and0.25μg/mL after the cell adhering onto the bottom of the

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plates with 3 parallel wells of each concentration. After 6-day incubation at 37°C with 5% CO2, cells were stainedwith 5% crystal violet for 4 hours, the excessive dye waswashed away with PBS, and the cell colonies were countedunder a light microscope.

2mL of HFORSC and HFGMC cell culture suspension(about 2:25 × 104 cells/mL) was inoculated into a 3.5 cmculture dish. The culture media was replaced with PTE at aconcentration of 0.0625μg/mL after the cultured cell adheredonto the bottom of the culture dish. Cell numbers werecounted every day for six days in order to establish the cellgrowth curves.

5.6. Western Blotting. HF cells were incubated in culturemedia with 10% FBS until the cell culture was at 30% in con-

fluence in the culture dish. The culture media were replacedwith the PTE dilutions at the concentrations of 0.125μg/mL,0.25μg/mL, and 0.5μg/mL with proper controls and incu-bated at 37°C with 5% CO2 until the cell culture reached at80% confluence. The level of keratin and other proteins incells was assessed by Western blot. Proteins were extractedby cracking cells in a lysis buffer with protease inhibitors onice. The protein concentration was adjusted to the same levelby Bradford assay before being loaded in the SDS-PAGE. α-Tubulin was used as an internal control to normalize otherprotein expression levels in this experiment. The densitiesof the protein images from Western blots were quantifiedby the software ImageJ and normalized in comparison tothe internal control protein of α-tubulin to determine theprotein levels.

Table 1: Fatty acids profile of Phaeodactylum tricornutum extract determined by GC-MS.

Fatty acids Structure Relative content (%)

1 C12:0O

OH10.99

2 C14:0O

OH7.874

3 C16:0O

OH26.43

4 C16:1O

OH34.45

5 C18:0O

OH1.555

6 C18:1n9cO

OH5.034

7 C18:2n6cO

OH3.491

8 C18:3n3O

OH0.507

9 C20:5O

OH5.127

10 C22:1n9O

OH0.695

11 C22:6O

OH0.303

12 C24:0O

OH2.72

13 C24:1O

OH0.825

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5.7. Measurements of Melanin Levels and TyrosinaseActivities. When the HEM cell cultures were at 20% in con-fluence, the culture media were replaced with media contain-ing PTE at different concentrations and 8-MOP at10μmol/L, respectively. The HEM were incubated until thecultured cells reached 90% in confluence at 37°C with 5%CO2. The cultured cells were trypsinized and washed twicewith PBS at 6,000 g centrifugations. For the melanin leveldetermination, the cell numbers were adjusted and pelletedat 1:00 × 106 cells per sample for melanin level measurementswith 0.5mL NaOH (1N) resuspension following 30-minuteincubations at 60°C in a water bath. The NaOH cell suspen-sions were measured with a chroma meter at 405nm formelanin levels. For the tyrosinase activity measurement, thecell numbers were adjusted and pelleted at 2:00 × 105 cellsper sample. The extraction buffer containing 1% NonidetP-40, 0.01% SDS, and proteinase inhibitors (3μg/mL aproti-nin, 2μg/mL pepstatin, 1μg/nL leupeptin, 0.1μMPMSF, and1mM DTT) was added into each sample and treated byultrasound at 4°C for 5 minutes following a 30-minute centri-fugation at 12,800 g to obtain supernatants as the tyrosinaseis extracted. The tyrosinase activity was measured with theabsorbance at 492nm after mixed 70μg tyrosinase extractsand 100μg L-Dopa at 37°C for 30min.

5.8. Statistical Analysis. All values are presented as means ±standard deviations (SD) of triplicate samples. Differencesbetween two groups were assessed using unpaired two-tailed Student’s t-test. In all statistical comparisons, P <0:05 was defined as significant. GraphPad Prism softwarewas used for all calculations.

Data Availability

All data included in this study are available upon request bycontact with the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the research start-up funds ofDongguan University of Technology (GC300502-36) andInfinitus (China) Company Ltd.

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