Pigmentation Effects of Kaliziri Standard Extracts on Melanocytes, C57BL/6 mice of Hydroquinone-Induced and Guinea Pigs of Hydrogen Peroxide-Induced Hyperpigmentation Model

Background: Vitiligo is a depigmentation disorder characterized by losing functional melanocytes, leading to skin and/or hair depigmentation. Kaliziri (Vernonia anthelmintica (L.) Willd) seeds have been traditionally used to treat pigmentation disorders in Central Asia. The extracts of these seeds increased melanin content and tyrosinase activity and upregulated melanogenesis-related proteins in B16 melanoma cells. We isolated the main active compound Kaliziri standard Extracts (encoded as CAM-Y7) from the Kaliziri seeds in a previous study. Our goal was to reveal whether CAM-Y7 promotes melanogenesis in melanocytes and restored pigmentation in animal models of vitiligo. Methods: Herein, we analyzed the melanogenic effects of CAM-Y7 on melanocytes and the skin pigmentation of hydroquinone (H.Q.)-induced mouse model and hydrogen peroxide (H 2 O 2 )-induced guinea pig model of vitiligo. Results: CAM-Y7 promotes Melanogenesis up-Regulation MITF-induced Tyrosinase Expression via p38MAPK signal pathway in melanocytes. Oral administration of CAM-Y7 progressively darkened the dorsal skin and hair of the C57BL/6 mice and guinea pigs. Lillie staining and hematoxylin-eosin staining further showed that CAM-Y7 induced melanogenesis in the epidermis and hair follicles of the animals. Conclusion: Taken together, these results suggest that CAM-Y7 regulation of melanogenesis may be mediated by the activation of p38 MAPK and restored pigmentation in an HQ-Induced C57BL/6 mouse and H 2 O 2 -induced guinea pig models of vitiligo. CAM-Y7 might be valuable and promising therapeutic as an agent for vitiligo. Subsequent clinical experiments using are needed to verify our results.


Introduction
Melanogenesis, or the production of melanin, occurs in the melanocytes of the epidermal basal layer. The melanin granules formed in the basal layer are transferred and stored in the overlying keratinocytes [1,2] .
Dysfunctional melanogenesis or the absence of melanocytes can lead to skin hypopigmentation [3] .
Vitiligo is a depigmentation disorder involving selective dysfunction and destruction of melanocytes, leading to loss of melanin in the skin and hair [4] .
The expression and activity of MITF were regulated by MAPKs, which were key signaling molecules involved in regulating melanogenesis, including the c-Jun N terminal kinase (JNK)1/2, extracellular signal-regulating kinase (ERK), and the p38 MAPK signaling cascades [10] . Previous studies have shown that the JNK and ERK stress-activated protein kinase pathways to downregulated melanin synthesis [11] .
By contrast, the phosphorylation of p38 MAPK activates MITF to stimulate melanogenesis ultimately.
Vernonia anthelmintica (L.) Wild was a famous medicine for vitiligo and known as Kaliziri in Traditional Uyghur Medicine (TUM); the seeds of Kaliziri have been used to treat skin depigmentation diseases in TUM for over a century. The seed extracts promoted melanin production and tyrosinase activity and induced melanogenesis-related proteins in B16 melanoma cells [12] . In a previous study, we isolated the main active compound Kaliziri standard Extracts (encoded as CAM-Y7), including sesquiterpenoids, caffeoylquinic acids, and active avonoid compounds V. Kaliziri seeds. Herein, we found that CAM-Y7 successfully accelerates melanogenesis in B16 cells, subsequently the mechanism of promoting effect on melanogenesis via p38 MAPK signal pathway up-Regulation MITF-induced Tyrosinase Expression and restored pigmentation in an HQ-Induced C57BL/6 mouse and H 2 O 2 -induced guinea pig models of vitiligo.

Drug analysis
Fifty milligrams of the sample powder were dissolved in 10 ml methanol and ltered through 0.22μm nylon membrane micro lters (Shimadzu-GL, Japan) and chromatographically analyzed using a Shimadzu LC-2030c HPLC tted with a photodiode detector (PDA) (Shimadzu-GL, Japan) (Fig. 1A). Reversed-phase separation was performed on a Shimadzu Shim-Pack GIST C18 column (4.6 × 250 mm, 5 μm, Shimadzu-GL, Japan) at 35°C. The mobile phases consisted of (A) 0.1% phosphoric acid in water and (B) acetonitrile. The sample was injected (5μl injection volume) onto the column and eluted at 1 ml/min ow. The fractions were detected using U.V. light at 210 nm, 254 nm, and 330 nm. Quantitative analysis was performed using HPLC.
Cell viability assay Cells seeded at a density of 5×10 3 cells/well in 96-well were incubated with CAM-Y7 for 24h. Then the culture medium was replaced with the CCK-8 solution (10 µl), and the cells were further cultured for 2 h at 37˚C. Plates were read at 450 nm using a microplate reader Spectra Max M5 (Molecular Devices company, San Diego, CA, USA). An equal volume of cells without the treatment was used as a blank control. All the experiments were repeated three times.

Tyrosinase activity
The cellular tyrosinase activity was estimated by measuring the rate of L-3, 4dihydroxyphenylalanine (L-DOPA) oxidase activity [13] . brie y, the process involved culturing the B16 cells plated on six-well plates (3.5 × 10 5 cells per well) and treated with CAM-Y7 for 24 h at 37°C. The cells were then washed with cold PBS and lysed in a PBS buffer containing 1% TritonX-100 + 1% sodium deoxycholate. The cell lysates were centrifuged at 12,000 × g for 20 min, and 90 µl of this supernatant and 10 µl of 10 mM L-DOPA solution were mixed and plated in 96-well plates incubated at 37 ∘ C for 30 min. The optical densities were measured at 490 nm using a microplate reader.

Melanin contents measurement
The melanin contents were measured using a previously described method with a slight modi cation [14] .
Brie y, the B16 cells plated on six-well plates (2× 10 5 cells per well) were incubated with various (1-10 µg/ml) concentrations of CAM-Y7 for 48 h at 37°C. Following this, the cells were lysed in RIPA Lysis buffer and centrifuged at 12,000×g for 20 min. 3µl supernatants of the cell extracts were used to measure the total protein content by the BCA kit assay. Then discarding the remaining part, 190 µl 1N NaOH was added at 80°C for 1h. The optical density of the supernatant was measured at 405nm. For the tissue melanin assays, as with previous reports [15,16] , tissue was weighed before boiling in 1N NaOH for 1 h and centrifuged at 12 000 rpm for 20 min discarding the insoluble materials. Following this, the optical density of the supernatants was measured at 405 nm and normalized to the weight of tissue.

Western blot analysis
The protein of the B16 cells was prepared as mentioned in the previous section above. All protein per lane were separated by 10% SDS-PAGE, transferred to PVDF membrane, blocked with 5% skim milk or 5% BSA, and exposed overnight at 4°C with appropriate antibodies. Following incubation with the second antibody, the protein bands were detected using an ECL (enhanced chemiluminescence) western blotting detection kit and quanti ed with a Chemi Doc MP Imaging system (Bio-Rad Laboratories, Inc, Hercules, CA, USA). All experiments were performed three times. Establishment of vitiligo model and treatment regimen C57BL/6 mice and Black dorsal skin guinea pigs were supplied by the Vital River Laboratory Animal Technology Co. Ltd., Beijing (Approval ID: SCXK-(jing) 2014-0013). All animals were housed at 22 ± 4°C and 50±10% humidity with ad libitum access to food and water. The animals were acclimatized for 1 week and randomly divided into six groups (n =12 per group). The dorsal side of the animals was shaved a day before the experiment using electric clippers. As previously described, dorsal hypopigmentation in the mice was induced by H.Q 17 . Brie y, each mouse's 2 cm × 2 cm depilated region was smeared with 0.5 ml 5% H.Q. (CAS:23-31-9) once a day for 50 days. 2 ml 5% H 2 O 2 was topically applied once a day for 50 days to induce hypopigmentation in the guinea pigs. A control group was also included wherein the animals were smeared with distilled water for 20 days. In C57BL/6 mice, the vitiligo models were treated with 720mg/kg Vitiligo Capsule (Chinese Drug Approval Number: Z12020225) as a positive group, distilled water as a model group, or 61.5mg/kg, 123mg/kg, or 246mg/kg CAM-Y7 per day by gastric administration for 30 days. The control group was given distilled water during this period. In the guinea pig, the vitiligo models were treated with 270mg/kg Vitiligo Capsule as a positive control, distilled water as model control, or 22mg/kg, 44mg/kg, or 88mg/kg CAM-Y7 per day by gastric administration for 30 days. The control group was given distilled water during this period. The dorsal skin of the animals was photographed every 10 days for macroscopic evaluation. At the end of the 30-day treatment, blood was withdrawn retro-orbitally from the mice and the abdominal aorta in guinea pigs under pentobarbital sodium. In addition, dorsal skin samples were also cut from the animals.
Enzyme-linked immunosorbent assay (ELISA) The levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), Tyrosinase (TYR), and monoamine oxidase (MAO) and malondialdehyde (MDA) in the mice or guinea pig sera were measured using speci c ELISA kits according to the manufacturers' instructions. The absorbance at 450nm was measured with the Spectra Max M5 microplate reader (Molecular Devices company, San Diego, CA, USA).

Immunohistochemistry (IHC)
Due to the lack of guinea pig-speci c antibodies, IHC was performed only on murine skin. The skin samples were xed, embedded in para n, and cut into 4μm thick sections. After quenching the endogenous peroxidases with hydrogen peroxide (KIT-9730-A), the sections were incubated overnight with an anti-tyrosinase antibody (1:50, sc-20035) at 4°C, followed by the peroxidase-labeled goat anti-rabbit IgG polymer PV-6001. The sections were washed, treated with DAB solution, and viewed under a microscope (Leica Microsystems, CMS GmbH DM6000B, Germany); the protein expression was determined using Image J software (National Institutes of Health, Bethesda, MD, USA).

Histological examination
H&E staining was performed on skin sections as per standard protocols. The thickness of the epidermis was measured as the distance from the basal layer to the surface of the epidermis. The melanincontaining hair follicles (H.F.) in murine skin and basal cells in Guinea pig skin were evaluated by Lillie stanning. One hundred cells were observed under high magni cation (×200) (Olympus Optical Co., Ltd., Tokyo, Japan). The number of melanin-containing Hair follicles was counted.

Statistics
All results are expressed as mean ± S.D., and statistical analysis was performed with one-way ANOVA, followed by Tukey's multiple comparisons test. Statistical analysis was performed using GraphPad Prism 6 (La Jolla, CA, USA). P values < 0.05 were considered to be statistically signi cant.

Characterization of drug components
According to a previous study on V. anthelmintica, 20 compounds were identi ed in the seed extracts, including caffeoylquinic acids, sesquiterpenoids, and avonoids (Fig. 1A). The structures are shown in Fig. 1B. The contents of these components were calculated by the one-point external standard method (Table 1). We used the CCK-8 cell viability assay to evaluate the cytotoxic effects of CAM-Y7 on B16 cells. Although B16 cells were treated with various concentrations of CAM-Y7 (0-10µg/ml), they did not show apparent cytotoxic effects or an increase in cell number ( Figure 2B and 2A). Next, we evaluated the melanin contents and tyrosinase activity of CAM-Y7 to determine the melanogenic effect of CAM-Y7. As shown in Figure2C, 2D, the CAM-Y7 increased the cellular melanin contents (P<0.001) in a dose-dependent manner and showed a concentration-dependent increase in cellular TYR activity.

CAM-Y7 promotes the expression of MITF and TRPs in B16
We evaluated the expressions of TYR, TRP-1, TRP-2, and MITF, which act as transcription regulators during the melanogenesis process. CAM-Y7 treatment showed a dose-dependent increase in TYR, TRP-1, but not TRP-2, which indicated that the CAM-Y7 could stimulate melanogenesis by enhancing the expression of TYR, TRP-1. Additionally, CAM-Y7 could also increase the expression of MITF in B16 cells. These results indicated that CAM-Y7 could induce melanin biosynthesis in B16 cells via up-Regulation MITF-induced Tyrosinase Expression ( Figure 2E, 2F).

CAM-Y7 promotes melanin synthesis by activation of p38 MAPK
The phosphorylation of p38 MAPK was reported to be one of the signaling pathways involved in hyperpigmentation. Therefore, western blot analysis was performed to determine ERK, p38, and JNK in B16 cells in the presence or absence of CAM-Y7. As shown in Fig. 3A and B, phosphorylation of JNK and ERK in B16 cells revealed no change by CAM-Y7 treatments; the p38 MAPK levels were signi cantly increased after treatment with CAM-Y7 in B16 cells compared with the control group. These data indicated that CAM-Y7 treatment accelerated melanogenesis in B16 cells by regulating the p38 MAPK signaling pathway. To further con rm the p38 MAPK pathways involved in CAM-Y7-mediated melanogenesis promotion, p38 inhibitor SB203580 was used to investigate. After treatment with CAM-Y7 for 24 h, co-treatment with 10 µM p38 MAPK inhibitor SB203580 at 37˚C for 2 h signi cantly inhibited CAM-Y7 triggered melanin content and Tyrosinase activity, respectively (Fig. 3C). According to these results, p38 MAPK signaling might be involved in the melanogenesis pathway mediated by CAM-Y7.
CAM-Y7 restores pigmentation of murine epidermis hair exposed to H.Q.
H.Q. induces hypopigmentation by inhibiting Tyrosinase [19,20] , which prevents the conversion of DOPA to melanin and causes melanosome degradation and loss of functional melanocytes [21,22] . The epidermal melanocytes in C57BL/6 mice are primarily located within the hair follicles, which lends the black color to the fur [23] . Thus, topical application of 5% H.Q. on the dorsal skin of C57BL/6 mice resulted in signi cant depigmentation of mice skin and hair. However, treatment with CAM-Y7 visibly darkened the affected region over 30 days. Compared to the H.Q. group, the CAM-Y7-treated mice had signi cantly lower vitiligo scores and higher melanin content in the affected area ( Figure 4A,4B,4C). Continuous exposure to H.Q. also triggers DNA damage in the keratinocytes and melanocytes, resulting in considerable tissue damage. Consistent with this, topical administration of 5% H.Q. signi cantly thickened the epidermal stratum corneum, which was alleviated upon CAM-Y7 treatment (Fig. 4D,4G).
Furthermore, Lillie staining showed an increase in the number of melanic hair follicles in the CAM-Y7treated skin (Fig. 4E, 4H). Together, H.Q. successfully induced vitiligo in a mouse model, and CAM-Y7 reversed the depigmentation. Tyrosinase is the core enzyme of the melanogenic pathway [24] , and the depigmentation effects of H.Q. depend on the inhibition of this enzyme. Consistent with this, the HQtreated skin showed signi cantly lower tyrosinase expression and level in situ than the control group ( Fig  4F, 4I, 4J), which matched with the lower overall skin levels of Tyrosinase in the former. CAM-Y7 treatment restored tyrosinase levels, with the hair follicles showing the highest tyrosinase expression concentration. The protective effects of CAM-Y7 eventually manifest as increased melanin production and pigmentation.

CAM-Y7 restores H 2 O 2 -induced depigmentation of guinea pig skin
The HQ-induced hypopigmentation model in C57BL/6 mice does not truly re ect the clinic-pathological state of vitiligo since it mainly affects melanin production in the hair follicles rather than skin [25] . Guinea pig skin has an epidermis similar to that of human skin and harbors numerous melanocytes. Therefore, H 2 O 2 -induced depigmentation in the guinea pig can simulate vitiligo more accurately [26] . Topical 5% H 2 O 2 resulted in signi cant whitening of the dorsal skin and hair similar to that seen with vitiligo and was alleviated by CAM-Y7 treatment (Fig. 5A). In addition, H 2 O 2 exposure signi cantly increased the vitiligo score compared to the control group, which was reduced by CAM-Y7 ( Fig 5B). Consistent with this, the melanin content of the H 2 O 2 -treated regions also increased signi cantly following CAM-Y7 treatment (Fig 5C).
Furthermore, H 2 O 2 caused signi cant epidermal damage characterized by loose and thick stratum corneum and detachment of the upper epidermal layer, in addition to signi cant decrease in melanin particles in the basal layer. CAM-Y7 treatment signi cantly alleviated this cellular damage (Fig. 5D, 5F). Lillie staining further showed a signi cant decrease in granular melanin in the H 2 O 2 -exposed skin (Fig.   5E,5G), while IHC revealed lower in situ tyrosinase levels (Fig. 5H), both of which were restored by CAM-Y7. Taken together, CAM-Y7 alleviated H 2 O 2 -induced depigmentation of guinea pig skin, indicating its potential as an anti-vitiligo drug.

CAM-Y7 protects guinea pig melanocytes from H 2 O 2 -induced damage
The induction and maintenance of vitiligo are associated with local in ammation 27,28 . The proin ammatory cytokines TNF-α and IL-6 inhibit pigmentation, and their mRNA levels are increased in biopsies of the vitiligo epidermis. TNF-α induces apoptosis in keratinocytes, resulting in the loss of melanocytes [29] . The serum levels of TNF-α and IL-6 were signi cantly higher in the H 2 O 2-treated animals and decreased following CAM-Y7 treatment (Fig. 5K,5J). Based on these ndings, we surmised that H 2 O 2 penetrates the stratum corneum and damages the underlying epidermal layers, triggering an in ammatory reaction that further exacerbates tissue injury. Furthermore, oxidative stress is another key mechanism of H 2 O 2 -induced melanocyte destruction and depigmentation [30,31] . H 2 O 2 also induces apoptosis in melanocytes [32] by increasing the cellular ROS levels [33,34] . Consistent with this, the epidermal and serum levels of both MAO and MDA increased in the H 2 O 2 -induced vitiligo model and were reduced signi cantly by CAM-Y7 (Fig. 5L, 5I, 5M). Therefore, CAM-Y7 not only restores tyrosinase activity but also protects melanocytes against H 2 O 2 -induced toxicity and restores skin pigmentation.

Discussion
The pathophysiology of vitiligo is not completely clear, although there is evidence of melanocyte loss from the cutaneous epidermis or dysfunction that loses the ability to secrete melanin. Therefore, the primary focus of treating vitiligo is to reverse the loss or hypoactivity of melanocytes, which is the basis of depigmentation. Kaliziri seeds have been used to treat skin pigmentation diseases in Central Asia for over a century. The seed extracts promoted tyrosinase activity and melanin production and induced the expression of melanogenesis-related proteins in the B16 melanoma cells. We found that CAM-Y7, a bioactive compound isolated from Kaliziri seed extracts, not only successfully accelerate melanogenesis in B16 cells, subsequently the mechanism of promoting effect on melanogenesis via p38 MAPK signal pathway to up-Regulation of MITF-induced Tyrosinase Expression, but also reversed HQ-induced depigmentation in C57BL/6 mice skin and hair by restoring tyrosinase activity and glutathione levels, and alleviating melanocyte apoptosis and melanosome degradation. In addition, CAM-Y7 also reversed the thickening of the epidermal stratum corneum induced by H.Q. and increased the number of melanic hair follicles. However, the C57BL/6 mice harbor numerous proliferative melanocytes in their hair follicles, which are the primary targets of H.Q. Therefore, HQ-induced depigmentation in the C57BL/6 mouse model mainly involves hair follicles and does not accurately simulate the epidermal changes seen in vitiligo [35] .   (F). The Quantity One program measured the band densities of proteins, and the expression levels of each protein were normalized against β-actin expression. The data were shown as mean ± S.D. and analyzed by one-way analysis of variance (ANOVA) followed by Turkey's test. **<0.01, ***p<0.001, ***p<0.0001 compared to control group. All experiments were performed three times.