Journal of Ethnopharmacology 143 (2012) 639–647

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Alcohol extract from Vernonia anthelmintica (L.) willd seed enhances melaninsynthesis through activation of the p38 MAPK signaling pathway in B16F10cells and primary melanocytes

Jia Zhou a,b,1, Jing Shang a,b,n,1, Fengfeng Ping a,b, Guorui Zhao a,b

a Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, Chinab State Key Laboratory of Natural Medicines, China Pharmaceutical University, China

a r t i c l e i n f o

Article history:

Received 20 April 2012

Received in revised form

20 June 2012

Accepted 13 July 2012Available online 31 July 2012

Keywords:

Vernonia anthelmintica (L.) willd extract

(AVE)

Microphthalmia-associated transcription

factor (MITF)

Tyrosinase

P38 mitogen-activated protein kinase (p38

MAPK)

Protein kinase A (PKA)

41/$ - see front matter & 2012 Elsevier Irelan

x.doi.org/10.1016/j.jep.2012.07.030

esponding author at: Center for Drug Scree

ity, No.24, Tongjiaxiang, Nanjing 210009,

086 25 83271500; fax: þ0086 25 85303260.

ail address: shangjing2006@yahoo.com.cn (J.

ese two authors contributed equally to this

red co-first authors.

a b s t r a c t

Ethnopharmacological relevance: Vernonia anthelmintica (L.) willd has been used in folk medicine to treat

leukoderma in China for centuries. The promoting activities of its extract (AVE) in melanogenesis and

possible signaling pathways were investigated in this article.

Materials and methods: The improving activities of AVE were examined by melanin synthesis,

tyrosinase activity assay and Western blot in B16F10 mouse melanoma cells and normal human

primary melanocytes (NHMC).

Results: AVE increased the tyrosinase activity and melanin content in a dosage-dependent manner at

concentrations of 1–40 mg/ml and treatment with 20 mg/ml AVE enhanced the expression of tyrosinase

time-dependently in both B16F10 cells and NHMC. Whether AVE affects the expression of micro-

phthalmia-associated transcription factor (MITF), which is required for tyrosinase expression, was

investigated. Our results showed that AVE induced MITF protein expression up-regulation. Besides,

Western blot analysis revealed that AVE promoted the level of phosphorylation of p38 mitogen-

activated protein kinase (p38 MAPK) markedly at 0–6 h, while the level of phosphorylation of CREB at

0–2 h. The special p38 MAPK inhibitor, SB203580, and protein kinase A (PKA) inhibitor, H89, both

attenuated the AVE-induced up-regulation of MITF and tyrosinase expression in B16F10 cells and

NHMC. However, SB203580 could significantly decrease the melanin biosynthesis induced by AVE, but

not H89.

Conclusion: AVE exerts its improving effect on melanogenesis mainly by p38 MAPK activation and MITF

induction of tyrosinase.

& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Melanin serves a number of valuable functions, the mostimportant being photoprotection of the human skin from ultra-violet (UV) radiation (Eller et al., 1994; Schallreuter et al., 2008).Taking it in this sense, the presence and distribution of melanin,resulting in skin, hair and eyes pigmentation of mammals is themajor physiological defense against solar irradiation. Melanin isderived from dopaquinone and synthesized in the melanosomesof melanocytes (Hearing and Tsukamoto, 1991; Schallreuter et al.,2008; Schiaffino, 2010). There are more than 100 distinct genesinvolved in the regulation of melanogenesis (Yamaguchi et al.,

d Ltd. All rights reserved.

ning, China Pharmaceutical

JiangSu Province, PR China.

Shang).

work and should be

2007). Melanin synthesis is regulated by enzymatic cascade, suchas tyrosinase, tyrosinase-related protein 1 (TRP-1) and TRP-2(Kameyama et al., 1995; Fang et al., 2001; Park et al., 2004; Yeet al., 2010). Tyrosinase is regarded as the rate-limiting enzyme ofmelanogenesis (Hearing, 1987; Park et al., 2004; Ye et al., 2010),which modulates this process by catalyzing the hydroxylation oftyrosine into 3,4-dihydroxyphenylalanine (DOPA) and the furtheroxidation of DOPA into dopaquinone (Hearing and Jimenez,1999; Park et al., 2004). The tyrosinase family genes TYR, TRP-1,TRP-2 responsible for pigmentation are transcriptionally regu-lated by microphthalmia-associated transcription factor (MITF)(Tachibana, 2001; Levy et al., 2006; Ye et al., 2010; Tsang et al.,2012)

Many approaches have been used to clarify the mechanisms ofmelanogenesis over the past decades. Mitogen-activated proteinkinases (MAPKs) belong to a super family of serine/threoninekinases, including the extracellularly responsive kinase (ERK), thec-Jun N-terminal kinase (JNK), which is also known as stress-activated protein kinase (SAPK), and the p38 MAPK (Seger and

J. Zhou et al. / Journal of Ethnopharmacology 143 (2012) 639–647640

Krebs, 1995; Cohen, 1997; Jiang et al., 2009). Recent investiga-tions suggest that p38 MAPK positively contributes to pigmenta-tion. The activation of p38 MAPK pathway induces MITFexpression (Singh et al., 2005; Saha et al., 2006; Hirata et al.,2007) and promotes the transcription of tyrosinase (Corre andGalibert, 2005). Some Chinese medicines such as San-bai-tangand Qian-wang-hong-bai-san, the Chinese herbal formulae, havebeen reported to have anti-melanogenic activity via inhibiting thep38 MAPK signaling pathway (Tsang et al., 2012; Ye et al., 2010),and 2,3,5,40-tetrahydroxystilbene-2-O-b-D-glucoside (THSG), thewater-soluble active component extracted from dried tuber rootof Polygonum multiflorum, has been shown to affect tyrosinaseexpression by activation of p38 MAPK (Jiang et al., 2009).

The same effect of protein kinase A (PKA) signaling has beendemonstrated in the melanin biosynthesis (Busc �A and Ballotti,2000). PKA activation can lead to the activation of MITF activityvia the phosphorylation of CREB.

Leukoderma, also named vitiligo, is an acquired, progressive,depigmenting disorder characterized by the development of whitemacules in the skin due to the chronic, selective loss of functionalmelanocytes in the epidermis (Alikhan et al., 2010; Sandoval-Cruzet al., 2011). Depigmentation may be the source of severe psycho-logical distress, diminished quality of life, and increased risk ofpsychiatric morbidity. However, the clinical treatment of this dis-ease is largely absent from the effective medicines. Vernonia

anthelmintica (L.) willd is a kind of plant growing only in the highaltitude localities of southern Xinjiang and minority regions ofPakistan and India. Its fruits extract (AVE) is one of the most popularuygur medicines used for leukoderma and initially recorded in ‘Yao

Yong Zong Ku’ around 300 years ago, nonetheless, its effect andunderlying mechanisms on melanogenesis are not very clear.

Hence, the aim of this study was to clarify the impact of AVEand its molecular mechanism on melanins biosynthesis in B16F10cells and normal human primary melanocytes (NHMC). Sincethere exist some functional discrepancies between cell lines andprimary cells, our study validates whether the effectiveness ofAVE on melanogenesis was stable via contrast of underlyingmechanisms in these two kinds of cells.

2. Materials and methods

2.1. Reagents

Dimethylsulfoxide (DMSO), L-3, 4-dihydroxyphenylalanine(L-DOPA), 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazo-lium bromide (MTT), 3-isobutyl-1-methylxanthine (IBMX), phor-bol esters (TPA), cholera toxin (CT) L-DOPA, melanin, phospho-p38mapk inhibitor SB203580, b-actin antibodies, and horseradishperoxidase-conjugated secondary antibodies were purchasedfrom Sigma–Aldrich (MO, USA). Tyrosinase (C-19) antibody wasfrom Santa Cruz Biotechnology (CA, USA). A polyclonal antibodyto MITF was purchased from Bioworld Technology (USA). Anti-bodies against phospho-specific p38 MAPK, p38, phospho-specificERK1/2, ERK1/2, phospho-JNK (Thr183/Tyr185), JNK, P-CREB(Ser 133) and CREB were from Cell Signaling Technology (USA),enhanced BCA protein assay kit, phenylmethylsulfonyl fluoride(PMSF) and cell lysis buffer for Western and IP were fromBeyotime Institute of Biotechnology (China), and total proteinextraction kit was from Applygen Technologies Inc. (China). Otherreagents were of the highest quality available.

2.2. Preparation of AVE

AVE was prepared by solvent extraction and extracted by 60%ethanol. After being degreased and dried, the dark brown powder

was obtained. In the HPLC profile of AVE, as we reported, morethan 13 characteristic peaks were identified (Jia et al., 2011).Stock solutions of AVE (2.56 mg/ml) were prepared in PBSfor cells.

2.3. Cell culture

Murine B16 melanoma cell lines (B16F10) were obtained fromCAS (Chinese Academy of Sciences, China). The B16F10 cells weregrown in DMEM medium (GIBICO, USA) supplemented with 10%heat-inactivated fetal bovine serum (GIBICO, USA), 100 U/mlpenicillin and 100 mg/ml streptomycin (GIBCO, USA) in a humi-dified atmosphere with 5% CO2 at 37 1C. Normal human melano-cytes (NHMC) were derived from young male adult foreskinsobtained at circumcision following standard protocols. Briefly,foreskins were cut into strips and digested with 0.25% trypsin at4 1C for 20 h. Epidermis was separated from dermis. The NHMCsuspension was filtered and cells were washed twice at 1,500 rpmfor 5 min prior to resuspension in MCDB153 medium (Sigma),supplemented with IBMX, TPA, CT and 100 U/ml penicillin and100 mg/ml streptomycin (GIBCO, USA). In experiments usingSB203580 p38 MAPK inhibitor and H89 PKA inhibitor, B16F10cells were serum-starved for 24 h, and then either pretreated with10 mM SB203580 and 10 mM H89 for 1 h or not before AVE wasapplied for the indicated time at 20 mg/ml.

2.4. Cell viability assay

Cell viability was determined using the MTT assay. B16F10cells were plated in 96-well dishes at a density of 2�103 cells perwell, while NHMC 104 cells per well. After different concentra-tions of AVE added, the cells were incubated for 48 h. Then, 10 mlof MTT solution (5 mg/ml in PBS) was added into each well andcells were incubated at 37 1C for another 4 h. Following mediumremoval, 100 ml of DMSO was added to each well and plates weregently shaken for 5 min. Optical absorbance was determined at570 nm with a microplate spectrophotometer (BD Bioscience,USA). Absorbance of cells without treatment was regarded as100% of cell survival. Each treatment was performed in quintu-plicate and each experiment was repeated three times.

2.5. Tyrosinase activity and melanin contents assay

Tyrosinase activity was estimated by measuring the rate ofL-DOPA oxidation as reported (Tomita and Tagami, 1992). B16F10cells and NHMC were treated with AVE for 48 h, washed with ice-cold PBS, lysed by incubation in cell lysis buffer [1 mM PMSF] at4 1C for 20 min, and then lysates were centrifuged at 14,000 rpmfor 15 min to obtain the supernatant for activity assay andcentrifugation for melanin contents assay. Protein concentrationswere determined by BCA kit with bovine serum albumin (BSA) asa standard. 100 ml of supernatant containing the same 30 mg totalproteins was added to each well in 96-well plate, and then mixedwith 100 ml of 0.1% L-DOPA in PBS (pH 6.8). After incubation at37 1C for 1 h, the dopachrome was monitored by measuring theabsorbance at 475 nm.

Total melanin in the cell pellet was dissolved in 100 ml of 1 NNaOH/10% DMSO for 1 h at 80 1C, and solubilized melanin wasmeasured at 405 nm. Melanin content was calculated from astandard curve using synthetic melanin.

2.6. Western blot analysis

The protein suspension was obtained as the method men-tioned above. An equal amount of protein of each sample wasadded to sodium dodecyl sulfate sample buffer and proteins were

J. Zhou et al. / Journal of Ethnopharmacology 143 (2012) 639–647 641

separated by polyacrylamide gel (10%) electrophoresis. Followingelectrotransfer to nitrocellulose membranes, the membraneswere blocked with 2% BSA and 0.1% Tween 20 in 0.01 M tris-buffered saline (TBS) for 2 h at room temperature. After threewashes with TBS containing 0.1% Tween 20 (TBST), membraneswere incubated with tyrosinase (C-19), MITF, phospho-p38 MAPK,p38 MAPK, phospho-ERK, ERK, phospho-JNK, JNK, P-CREB, CREBand b-actin antibodies (diluted 1:1000) with TBST containing 5%BSA, for 3 h at room temperature. After five washes, the mem-branes were then incubated with horseradish peroxidase-conjugated secondary anti-bodies at a dilution of 1:4000 for1.5 h at room temperature. After washing with TBST, proteins

Fig. 1. Effect of AVE on cell viability, tyrosinase activity and melanin synthesis in B16

(0–40 mg/ml) of AVE for 48 h, cell viability was determined using a MTT assay, B16F10

oxization as described in ‘Materials and methods’. Stimulation of tyrosinase activity

performed as described in ‘Materials and methods’, B16F10 cells (E) and NHMC (F), the s

of three independent experiments. Data were analyzed by One-Way Analysis of Varian

control.

were visualized using an enhanced chemiluminescence detectionsystem. Densitometric analysis was again performed using theQuantity One (Bio-Rad) to scan the signals. Western blot assayresults reported here are representative of at least three inde-pendent experiments.

2.7. Statistical analysis

All data were expressed as means7SEM. Statistical analysiswas performed with one-way ANOVA followed by Tukey’s post

hoc test for multiple comparisons tests. Significant differenceswere accepted when Po0.05.

F10 melanoma cells and NHMC. After incubation of with various concentrations

melanoma cells (A) and NHMC (B). Tyrosinase activity was determined by L-DOPA

of B16F10 cells (C) and NHMC (D) by AVE at 0–40 mg/ml. Melanin content were

ame by AVE at 0–40 mg/ml. Results shown are means7SEM and are representative

ce (ANOVA) followed by post hoc Turkey test. *Po0.05, **Po0.01, compared with

J. Zhou et al. / Journal of Ethnopharmacology 143 (2012) 639–647642

3. Results

3.1. Effect of AVE on tyrosinase activity and melanin amount

in B16F10 cells and NHMC

To investigate the effect of AVE on melanogenesis, MTT assaywas performed as the first step to examine whether AVE wascytotoxic to B16F10 cells and NHMC. As shown in Fig. 1A and B,AVE was not cytotoxic to these two kinds of cells at concentra-tions of 1–40 mg/ml, rather the opposite, B16F10 cells prolifera-tion was observely increased at this concentration range, andat a concentration of 10 mg/ml, AVE played a promoting role inNHMC proliferation. Therefore, to investigate the effect of AVE on

Fig. 2. Effect of AVE on the protein levels of MITF and tyrosinase in B16F10 cells and NH

blot assays were performed to examine MITF and tyrosinase expression levels, B16F10 c

shown are means7SEM and are representative of three independent experiments. Da

Turkey test. **Po0.01, compared with control.

tyrosinase activity and melanin synthesis, B16F10 cells andNHMC were then exposed to AVE ranging from 0 to 40 mg/ml.The effects of AVE on tyrosinase was measured by L-DOPAoxidation, shown in Fig. 1C and D. As compared to treatmentwith medium only (untreated condition), treatment with AVE atdosages 1–20 mg/ml resulted in a dose-dependent increase oftyrosinase activity in B16F10 cells (Fig. 1C) and NHMC (Fig. 1D). Inmelanin content assay, to exclude the possibility that raise inmelanin content may be induced by the cell proliferating effect ofAVE, the absorbance of the same number of cells across AVEconcentrations (1–40 mg/ml) was measured and results showedthat melanin levels were increased in a dose-dependent mannerby AVE treatment in B16F10 cells (Fig. 1E) and NHMC (Fig. 1F).

MC. The cells were treated with 20 mg/ml of AVE for the indicated times. Western

ells (A) and NHMC (B). Results were normalized against b-actin expression. Results

ta were analyzed by One-Way Analysis of Variance (ANOVA) followed by post hoc

J. Zhou et al. / Journal of Ethnopharmacology 143 (2012) 639–647 643

At 40 mg/ml of AVE, melanin content only increased slightly, so20 mg/ml was chosen as an effective concentration of AVE in laterexperiments.

3.2. Effect of AVE on MITF and tyrosinase protein expression

in B16F10 cells and NHMC

Since AVE increased tyrosinase activity and melanin synthesis,we further explored whether AVE affected the expression of MITF,which plays a critical role in tyrosinase gene expression andmelanogenesis. We studied MITF levels after AVE (20 mg/ml)treatment. Our data showed that MITF protein expression wasenhanced significantly 24 h after AVE addition both in B16F10(Fig. 2A) and NHMC (Fig. 2B). The effect of AVE on tyrosinaseexpression in B16F10 cells and NHMC was also examined byWestern blot analysis. As shown in Fig. 2A and B, levels of tyrosinaseprotein expression were up-regulated by AVE in a time-dependentmanner in B16F10 cells and NHMC, respectively. These resultssuggested that AVE increased melanin synthesis through MITF andtyrosinase expression up-regulation in both B16F10 cells and NHMC.

3.3. Effect of AVE on phosphorylation of p38 MAPK, ERK1/2 and

JNK in B16F10 cells and NHMC

To further clarify the mechanism underlying the effect of AVEon melanogenesis, the mitogen-activated protein kinase (MAPK)intracellular signal transduction cascade was examined. Theactivation of p38 MAPK, ERK1/2, and JNK was detected byWestern blot analysis. As shown in Fig. 3A, the phosphorylationof p38 MAPK was significantly enhanced at 1–6 h in B16F10 cells,whereas no effect was observed in the phosphorylation of ERK1/2or JNK, and the same effect of AVE on NHMC was shown in Fig. 3B.

Fig. 3. Effect of AVE on the activity of MAPK signaling pathways in B16F10 cells

and NHMC. Cells were treated with AVE (20 mg/ml) for the indicated time period

(0–360 min), and the phosphorylation of the p38 MAPK, ERK, and JNK were measured

by Western blot, B16F10 cells (A) and NHMC (B). The band densities of phospho-p38

MAPK were measured by the Quantity One 1D analysis software program. Expression

of b-actin was used as an internal control. Results shown are means7SEM and are

representative of three independent experiments. Data were analyzed by One-Way

Analysis of Variance (ANOVA) followed by post hoc Turkey test. *Po0.05, **Po0.01,

compared with control.

3.4. Effects of the inhibitor of p38 MAPK on AVE-induced

pigmentation in B16F10 cells and NHMC

Given that the p38 MAPK was activated in both B16F10 cellsand NHMC, whether it was involved in the up-regulation of MITFand tyrosinase expression or not was then assessed. As shown inFig. 4, SB203580, the specific inhibitor of p38 MAPK, markedlyattenuated AVE-induced improvement of MITF and tyrosinaseexpression in B16F10 cells (Fig. 4A) and NHMC (Fig. 4B). Thisindicated that AVE-induced MITF and tyrosinase up-regulationwas correlated with p38 MAPK activation. The increase ofmelanin amount by AVE was also abolished by the presence of10 mM SB203580, revealing the involvement of p38 MAPK signal-ing in AVE-induced melanogenesis in B16F10 cells (Fig. 4C) andNHMC (Fig. 4D).

3.5. Effect of AVE on phosphorylation of CREB in B16F10 cells and

NHMC

To further clarify the mechanism underlying the effect of AVEon melanogenesis, CREB signaling pathway was also examined.The activation of CREB was detected by Western blot analysis. Asshown in Fig. 5A, the phosphorylation of CREB was significantlyenhanced at 60 min, and the same effect of AVE on NHMC was at60–120 min, shown in Fig. 5B.

3.6. Effects of the inhibitor of PKA on AVE-induced pigmentation

in B16F10 cells and NHMC

Given that the CREB was activated in both B16F10 cells andNHMC, whether it was involved in the up-regulation of MITF andtyrosinase expression or not was then assessed. As shown in Fig. 6,

H89, an inhibitor of PKA, attenuated AVE-induced improvement ofMITF and tyrosinase expression in B16F10 cells (Fig. 6A) and NHMC(Fig. 6B). But the increase of melanin amount by AVE was not

Fig. 4. Effect of the inhibitor of p38 MAPK on AVE-induced MITF and tyrosinase expression and melanin contents in B16F10 cells and NHMC. B16F10 cells and NHMC were

serum-starved for 24 h, and then either pretreated or not with 10 mM SB203580 for 1 h before AVE was applied for 24 h at 20 mg/ml. Western blotting for the anti-MITF

antibody was performed, B16F10 cells (A) and NHMC (B). Melanin contents were measured, as described in ‘Materials and methods’, B16F10 cells (C) and NHMC (D).

Results shown are means7SEM and are representative of three independent experiments. Data were analyzed by One-Way Analysis of Variance (ANOVA) followed by post

hoc Turkey test. **Po0.01, compared with control.

Fig. 5. Effect of AVE on the activity of PKA signaling pathways in B16F10 cells and NHMC. Cells were treated with AVE (20 mg/ml) for the indicated time period

(0–120 min), and the phosphorylation of the CREB were measured by Western blot, B16F10 cells (A) and NHMC (B). The band densities of phospho-CREB were measured by

the Quantity One 1D analysis software program. Expression of b-actin was used as an internal control. Results shown are means7SEM and are representative of three

independent experiments. Data were analyzed by One-Way Analysis of Variance (ANOVA) followed by post hoc Turkey test. *Po0.05, **Po0.01, compared with control.

Fig. 6. Effect of the inhibitor of PKA on AVE-induced MITF and tyrosinase expression and melanin contents in B16F10 cells and NHMC. B16F10 cells and NHMC were

serum-starved for 24 h, and then either pretreated with 10 mM H89 for 1 h or not before AVE was applied for 24 h at 20 mg/ml. Western blotting for the anti-MITF and anti-

tyrosinase antibody was performed, B16F10 cells (A) and NHMC (B). Melanin contents were measured, as described in ‘Materials and methods’, B16F10 cells (C) and NHMC

(D). Results shown are means7SEM and are representative of three independent experiments. Data were analyzed by One-Way Analysis of Variance (ANOVA) followed by

post hoc Turkey test.

J. Zhou et al. / Journal of Ethnopharmacology 143 (2012) 639–647 645

influenced significantly by the presence of 10 mM H89, revealingthe secondarily involvement of PKA signaling in AVE-inducedmelanogenesis in B16F10 cells (Fig. 6C) and NHMC (Fig. 6D).

4. Discussion

Vernonia anthelmintica (L.) willd is a well-known herb tradi-tionally used as a pigmentation improving medicine for leuko-derma in China. Its alcohol extract (AVE) is rich in flavonoidscompounds. Though it has been used for centuries as a folkmedicine, its mechanisms of action in melanogenesis still remainunkown.

In the current study, the improving effect of AVE on melano-genesis in B16F10 cell lines and normal human primary melano-cytes (NHMC) was investigated to clarify its underlying molecularmechanism. To evaluate the promoting activity on melaninsynthesis, AVE was prepared using 60% EtOH from its fruits,then degreased and dried. The dark brown powder was got for

experiments. Firstly, we evaluated whether there existed poten-tial cytotoxicity of AVE on B16F10 cells and NHMC or not. Asshown in Fig. 1, AVE had no cytotoxic effect at concentrations of1–40 mg/ml, inversely, it observably increased B16F10 cells pro-liferation, and at a concentration of 10 mg/ml it played a promot-ing role in NHMC proliferation. The differences of AVE effect oncell proliferation may be due to the discrepancy of characteristicsbetween cell lines and primary cells.

Since it has been reported that melanin content correlateddirectly with the activity of tyrosinase and the protein levels ofthis enzyme (Shibahara et al., 2000), the effect of AVE on tyro-sinase activity and expression was next explored. As expected,AVE significantly increased both tyrosinase activity and melaninsynthesis in a concentration-dependent manner (Fig. 1). Theseresults suggested that AVE up-regulated tyrosinase activity andenhanced cellular melanin synthesis in B16F10 cells and NHMC.AVE was effective on tyrosinase activity at coincident concentra-tions in these two kinds of cells, both 10 mg/ml and 20 mg/ml(Fig. 1C and D). And yet, when referring to the effect on melanin

J. Zhou et al. / Journal of Ethnopharmacology 143 (2012) 639–647646

synthesis, B16F10 cells seemed less susceptible to AVE induce-ment. The improvement could be achieved even at 1 mg/ml inNHMC (Po0.05), and more significantly at 10 mg/ml and 20 mg/ml (Po0.01) while onset concentration at 10 mg/ml (Po0.05) andmore valid at 20 mg/ml (Po0.01) in B16F10 cells. So 20 mg/ml waschosen for further study.

To clearly elucidate the molecular mechanisms of AVE-induced promoting actions, the effect of AVE on melanogenicprotein expression was examined. As MITF plays an importantrole in melanogenesis as the major transcriptional regulator oftyrosinase (Costin and Hearing, 2007; Zhu et al., 2009; Sensi et al.,2011), the expression of MITF and tyrosinase treated by 20 mg/mlAVE at 0–48 h was examined. As shown in Fig. 2, AVE significantlyincreased MITF and tryosinase levels at 24 h in the two cells.

According to reports in the literature, the expression andfunction of MITF were regulated by several signaling pathwaysand MAPK and PKA signaling pathway was embraced. As men-tioned in the introduction, the MAPK family, including p38 MAPK,ERK, and JNK, played an important role in the regulation ofmelanogenesis (Hirata et al., 2007; Bellei et al. 2010). Activationsof the ERK and the JNK/SAPK pathways were related to the down-regulation of melanogenesis (Costin and Hearing, 2007; Zhu et al.,2009; Sensi et al., 2011). The involvement of p38 MAPK inmelanogenic differentiation has been reported in a number ofresearches (Hirata et al., 2007; Jiang et al., 2009; Ye et al., 2010;Wu et al., 2011; Tsang et al., 2012). We therefore examined theinfluence of AVE treatment on the activations of p38 MAPK, ERKJNK and PKA attempting to further understand the molecularmechanisms involved in the pigmentation property of AVE byWestern blot assay. As our data shown, treatment with AVE at20 mg/ml for 0–6 h, phosphorylation of p38 MAPK was signifi-cantly affected, while not in JNK or ERK, both in B16F10 cells andNHMC (Fig. 3). Given the phosphorylation of the p38 was earlierin B16F10 cells at 30 min (Fig. 3A) and 180 min (Fig. 3B) in NHMC,the same sensitivity to AVE of NHMC was revealed. We demon-strated that p38 MAPK pathway, but not the ERK or the JNK,participated in AVE-mediated enhancement of melanogenesis inboth two cells. And treatment with AVE at 20 mg/ml for 0–2 h,phosphorylation of CREB was significantly affected, both inB16F10 cells and NHMC (Fig. 5)

Additionally, to confirm our results, we examined the effect ofSB203580, the specific inhibitor of p38 MAPK, and H89, theinhibitor of PKA on expression of MITF and tyrosinase andmelanin synthesis in presence of 20 mg/ml AVE for 24 h inB16F10 cells and NHMC. As shown in Fig. 4A and B, co-treatmentwith SB203580 and AVE, decreased AVE-mediated MITF andtyrosinase expression in both B16F10 cells and NHMC. And thesimilar effect was present when co-treatment with H89 and AVEin both B16F10 cells and NHMC, shown in Fig. 6A and B. However,AVE-induced melanogenesis was attenuated significantly bySB203580, but not H89. All these results suggested that AVEenhanced melanin synthesis mainly by up-regulating p38 MAPK,secondarily by activating PKA signaling, and subsequently increasingthe expression of MITF in B16F10 cells and NHMC. Since themolecular mechanisms of melanin biosynthesis in these two kindsof cells are pretty similar, we think that the impact of AVE onmelanogenesis is jarless.

Besides, the compounds in AVE have been measured by highperformance liquid chromatography and more than 13 character-istic peaks were identified (Jia et al., 2011). Subsequently, fivemonomeric compounds were isolated and identified from AVE,which all belong to flavonoids. Serum pharmacochemistry of AVEalso has been studied and nine compounds were found with highcontent (Jia et al., 2011). In the next phase of the study, thesources and structures of these compounds need to be confirmedto find out which are the active materials.

5. Conclusion

In conclusion, present study showed that improving thephosphorylation of p38 MAPK, then sequentially inducing theexpression of MITF contributed to the melanogenic effect of AVEon B16F10 cells and NHMC. These findings shed light on themolecular mechanisms underlying the promoting activity of AVEon melanin biosynthesis. Consequently, more wide-ranging testsare successful, Vernonia anthelmintica (L.) willd may be an effec-tive alternative treatment for vitiligo in the future.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (30873156; 30672524), New Century ExcellentTalents in University (NCET-07–0851) and Mega-project of ScienceResearch for the 11th Five-Year Plan of China (2009ZX09302-002),2011 Program for Excellent Scientific and Technological InnovationTeam of Jiangsu Higher Education.

References

Alikhan, A., Felsten, L.M., Daly, M., Petronic-Rosic, V., 2010. Vitiligo: A compre-hensive overview: part I. introduction, epidemiology, quality of life, diagnosis,differential diagnosis, associations, histopathology, etiology, and work-up.Journal of the American Academy of Dermatology 65, 473–491.

Bellei, B., Maresca, V., Flori, E., Pitisci, A., Larue, L., Picardo, M., 2010. p38 regulatespigmentation via proteasomal degradation of tyrosinase. Journal of BiologicalChemistry 285, 7288–7299.

Busc �A, R., Ballotti, R., 2000. Cyclic AMP a key messenger in the regulation of skinpigmentation. Pigment Cell Research 13, 60–69.

Cohen, P., 1997. The search for physiological substrates of MAP and SAP kinases inmammalian cells. Trends in Cell Biology 7, 353–361.

Corre, S., Galibert, M.D., 2005. Upstream stimulating factors: highly versatilestress-responsive transcription factors. Pigment Cell & Melanoma Research 18,337–348.

Costin, G.E., Hearing, V.J., 2007. Human skin pigmentation: melanocytes modulateskin color in response to stress. FASEB Journal 21, 976–994.

Eller, M.S., Yaar, M., Gilchrest, B.A., 1994. DNA damage and melanogenesis. Nature372, 413–414.

Fang, D., Kute, T., Setaluri, V., 2001. Regulation of tyrosinase-related protein-2(TYRP2) in human melanocytes: relationship to growth and morphology.Pigment Cell & Melanoma Research 14, 132–139.

Hearing, V., Tsukamoto, K., 1991. Enzymatic control of pigmentation in mammals.The Federation of American Societies for Experimental Biology Journal 5,2902–2909.

Hearing, V.J., Jimenez, M., 1999. Biochemical control of melanogenesis andmelanosomal organization. The Journal of Investigative Dermatology Sympo-sium Proceedings 4, 24–28.

Hirata, N., Naruto, S., Ohguchi, K., Akao, Y., Nozawa, Y., Iinuma, M., Matsuda, H.,2007. Mechanism of the melanogenesis stimulation activity of (-)-cubebinin murine B16 melanoma cells. Bioorganic & Medicinal Chemistry 15,4897–4902.

Jia, Zhou, Sha, Liao, Fengfeng, Ping, Jing, Shang, 2011. Serum pharmacochemistryof Vernonia anthelmintica. Central South Pharmacy 9, 561–563.

Jiang, Z., Xu, J., Long, M., Tu, Z., Yang, G., He, G., 2009. 2, 3, 5, 40-tetrahydroxy-stilbene-2-O-b-D-glucoside (THSG) induces melanogenesis in B16 cells by MAPkinase activation and tyrosinase upregulation. Life Sciences 85, 345–350.

Kameyama, K., Sakai, C., Kuge, S., Nishiyama, S., Tomita, Y., Ito, S., Wakamatsu, K.,Hearing, V.J., 1995. The expression of tyrosinase, tyrosinase-related proteins1 and 2 (TRP1 and TRP2), the silver protein, and a melanogenic inhibitor inhuman melanoma cells of differing melanogenic activities. Pigment Cell &Melanoma Research 8, 97–104.

Levy, C., Khaled, M., Fisher, D.E., 2006. MITF: master regulator of melanocytedevelopment and melanoma oncogene. Trends in Molecular Medicine, 12.

Park, S.H., Kim, D.S., Kim, W.G., Ryoo, I.J., Lee, D.H., Huh, C.H., Youn, S.W., Yoo, I.D.,Park, K.C., 2004. Terrein: a new melanogenesis inhibitor and its mechanism.Cellular and Molecular Life Sciences 61, 2878–2885.

Saha, B., Singh, S.K., Sarkar, C., Bera, R., Ratha, J., Tobin, D.J., Bhadra, R., 2006.Activation of the Mitf promoter by lipid-stimulated activation of p38-stresssignalling to CREB. Pigment Cell & Melanoma Research 19, 595–605.

Sandoval-Cruz, M., Garcıa-Carrasco, M., Sanchez-Porras, R., Mendoza-Pinto, C.,Jimenez-Hernandez, M., Munguıa-Realpozo, P., Ruiz-Arguelles, A., 2011.Immunopathogenesis of vitiligo. Autoimmunity Reviews 10, 762–765.

Schallreuter, K.U., Kothari, S., Chavan, B., Spencer, J.D., 2008. Regulation ofMelanogenesis—Controversies and New Concepts. Blackwell Publishing Ltd.,pp. 395–404.

J. Zhou et al. / Journal of Ethnopharmacology 143 (2012) 639–647 647

Schiaffino, M.V., 2010. Signaling pathways in melanosome biogenesis and patho-logy. The International Journal of Biochemistry & Cell Biology 42, 1094–1104.

Seger, R., Krebs, E.G., 1995. MAPK signalling cascade. FASEB Journal 9, 726–735.Sensi, M., Catani, M., Castellano, G., Nicolini, G., Alciato, F., Tragni, G., De Santis, G.,

Bersani, I., Avanzi, G., Tomassetti, A., Canevari, S., Anichini, A., 2011. Humancutaneous melanomas lacking MITF and melanocyte differentiation antigensexpress a functional Axl receptor kinase. Journal of Investigative Dermatology131, 2248–2257.

Shibahara, S., Yasumoto, K.-I., Amae, S., Udono, T., Watanabe, K.-I., Saito, H.,Takeda, K., 2000. Regulation of pigment cell-specific gene expression by MITF.Pigment Cell Research 13, 98–102.

Singh, S.K., Sarkar, C., Mallick, S., Saha, B., Bera, R., Bhadra, R., 2005. Humanplacental lipid induces melanogenesis through p38 MAPK in B16F10 mousemelanoma. Pigment Cell & Melanoma Research 18, 113–121.

Tachibana, M., 2001. Cochlear melanocytes and MITF signaling. Journal ofInvestigative Dermatology Symposium Proceedings 6, 95–98.

Tomita, Y.M.K., Tagami, H., 1992. Melanocyte-stimulating properties of arachido-nic acid metabolites: possible role in postinflammatory pigmentation. PigmentCell Research 5, 357–361.

Tsang, T.-F., Ye, Y., Tai, W.C.-S., Chou, G.-X., Leung, A.K.-M., Yu, Z.-L., Hsiao, W.-L.W.,2012. Inhibition of the p38 and PKA signaling pathways is associated with theanti-melanogenic activity of Qian-wang-hong-bai-san, a Chinese herbal for-

mula, in B16 cells. Journal of Ethnopharmacology 141, 622–628.Wu, L.C., Yang, S.Y., Weng, Y.T., Tsai, Y.T., 2011. Antimelanogenic effect of c-

phycocyanin through modulation of tyrosinase expression by upregulation ofERK and downreguraltion of p38 MAPK signaling pathways. Journal ofBiomedical Science 18, 74.

Yamaguchi, Y., Brenner, M., Hearing, V.J., 2007. The regulation of skin pigmenta-tion. Journal of Biological Chemistry 282, 27557–27561.

Ye, Y., Chu, J.-H., Wang, H., Xu, H., Chou, G.-X., Leung, A.K.-M., Fong, W.-F., Yu, Z.-L.,2010. Involvement of p38 MAPK signaling pathway in the anti-melanogenic

effect of San-bai-tang, a Chinese herbal formula, in B16 cells. Journal ofEthnopharmacology 132, 533–535.

Zhu, S., Wurdak, H., Wang, Y., Galkin, A., Tao, H., Li, J., Lyssiotis, C.A., Yan, F., Tu, B.P.,Miraglia, L., Walker, J., Sun, F., Orth, A., Schultz, P.G., Wu, X., 2009. A genomicscreen indentifies TYRO3 as MITF regulator in melanoma. Proceedings of the

National Academy of Sciences 106, 17025–17030.