| Background:Vitiligo is a chronic acquired skin disease characterized by depigmented macules and disappearance of functional melanocytes, which is a refractory disease, and the incidence rate is increasing. The pathogenesis of vitiligo has not been clearly elucidated so far and the results of studies are involved in multiple theories. In recent years, a growing number of studies have suggested that the injury of melanocytes in vitiligo is a result of genetic and environmental interaction. On the basis of individual specific and genetic context, oxidative stress in vitiligo may be the earliest event, which promotes apoptosis of melanocytes, and then induces specific immunologic response against auto-antigen. Many studies have shown that the excess accumulation of H2O2 in the epidermis of vitiligo can lead to the oxidative injury of melanocytes. In this research, we established an oxidative model induced by H2O2 in melanocytes to further study the complex pathogenesis of vitiligo.The risk of vitiligo is partly determined by genetic variation, for genetic studies have showed that SNPs and mutations located in the coding regions may affect the expression of genes and then have impact on the functional roles. In several candidate genes detected by the method of molecular epidemiology, mutations found in PDGFRA in familial vitiligo vulgaris are significantly higher than that in the general Chinese population, supporting the hypothesis that this gene may play a role in some patients with vitiligo. Meanwhile, researches have showed that oxidative stress can induce tyrosine phosphorylation of PDGFRα, and PDGFRα is correlated with a protective role in cell death induced by H2O2. Therefore, we mainly focused on whether PDGFRα functioned in survival of melanocyte under oxidative stress induced by H2O2 in this study. Aims:It has been reported that PDGFRα plays important roles in cell growth, differentiation and survival. Therefore, observation of the effects of PDGFRα on apoptosis and autophagy, and detection of the expression of some related proteins, can enhance the understanding of biological function of PDGFRα in melanocytes and further elucidate the interacted roles that genetic and environmental factors played in vitiligo. Methods:1. We detected the m RNA and proteinic levels of PDGFRA in primary cultured human melanocytes, immortalized epidermal melanocytes PIG1 from normal human and immortalized epidermal melanocytes PIG3 V from vitiligo patient, by the methods of reverse transcription PCR and Western-blot.2. We established a cellular model of high degree of cytotoxicity induced by H2O2 in melanocytes PIG1 and melanocytes PIG3 V respectively. By the method of CCK-8 to detect the cell vitality, we ensured the optimal concentration of H2O2. After treatment with H2O2 in different final concentrations and different time points, we used Western-blot to determine the proteinic levels of PDGFRA in melanocytes PIG1.3. We transfected the melanocytes PIG1 and melanocytes PIG3 V with si RNAs and over-expression plasmids of PDGFRA respectively, and used quantitative real-time PCR and Western-blot to detected the m RNA and proteinic levels of PDGFRA.4. After down- and up-regulated the expression of PDGFRA in melanocytes PIG1 and melanocytes PIG3 V, we used H2O2 to induce the oxidative stress for 24 h. Then detected the apoptosis of both the melanocytes by the method of Flow cytomtry, and detected expression levels of main proteins related to survival by the method of Western-blot.5. We treated the melanocytes PIG1 with optimal PDGF-AA, H2O2 or both of them, and then detected the expression levels of PDGFRα, main proteins related to autophagy, melanin-regulating, and PI3K/AKT/m TOR pathway by Western-blot.6. We transfected the melanocytes PIG1 and melanocytes PIG3 V with plasmids expressing autophagosomal marker LC3, treated the cells with H2O2 and PDGF-AA, and then observed the formation of autophagosomes in the cytoplasm of both melanocytes using fluorescence microscope.7. Furthermore, we used PI3 K, AKT and m TOR inhibitors with optimal concentrations to treat melanocytes PIG1 and to detect the expression levels of the main proteins related to autophagy and PI3K/AKT/m TOR pathway. Results:1. We found that PDGFRA m RNA and protein were both expressed in primary cultured melanocytes, normal melanocytes PIG1 and vitiligo melanocytes PIG3 V.2. In cellular models under H2O2-mediated oxidative stress in vitro, we observed that the optimal concentrations of H2O2 in melanocytes PIG1 and melanocytes PIG3 V. After treatment of melanocytes PIG1 with different concentrations of H2O2, the results of Western-blot showed that the proteinic levels of PDGFRA was positive correlated with the concentration of H2O2. After treatment of melanocytes PIG1 with H2O2 in different times, the results of Western-blot showed that the proteinic level of PDGFRA reached to the peak at the time point of 30 min.3. In melanocytes PIG1 and melanocytes PIG3 V, we selected the most efficient si RNA of PDGFRA, which could significantly down-regulated PDGFRA m RNA and proteinic expression; meanwhile, we transfected the p CMV6-Entry-PDGFRΑ plasmids into both of the melanocytes and observed the obvious increased levels of m RNA and proteinic expression of PDGFRA.4. After down- and up-regulated PDGFRA levels in both cell lines, we used H2O2 to induce oxidative stress. The results of Flow cytomtry showed that, the apoptosis of melanocytes in PDGFRΑ-si RNA2 transfected group was significantly increased than that in the control group(P<0.01)in both cell lines, while the apoptosis of melanocytes in p CMV6-Entry-PDGFRΑ plasmids transfected group was significantly reduced compared to the control group(P<0.05). The results of Western-blot also showed that, compared to the control group, transfection of PDGFRΑ-si RNA2 increased the expression of autophagosomal marker LC3II/I and autophagy-forming regulated gene Beclin-1, and decreased anti-apoptosis gene Bcl-2 in both melanocytes; By contrast, transfection of p CMV6-Entry-PDGFRΑ plasmids decreased the expression of LC3II/I and Beclin-1, and increased Bcl-2 in both melanocytes.5. We used Western-blot to detect the main functional protein in melanocytes and the result showed that, compared to the control group, PDGFRΑ-si RNA2 significantly down-regulated the MITF, gp100, TYR and TRP-1 in both melanocytes; while over-expression of PDGFRΑ significantly up-regulated MITF, gp100, TYR and TRP-1 in melanocytes PIG1, and up-regulated MITF and gp100 in melanocytes PIG3 V, whereas the TYR and TRP-1 expression were not significantly changed in melanocytes PIG3 V.6. After treatment of different concentrations of PDGF-AA, the results of Western-blot showed that the proteinic level of p-PDGFRα, p-AKT, p-m TOR were increased as the elevation of the concentration, and treatment of PDGF-AA resulted in significant differences between the treated group and control group. Compared to the control group, melanocytes treated by H2O2 and PDGF-AA displayed higher proteinic levels of p-PDGFRα, p-AKT, p-m TOR and MITF, and lower proteinic levels of LC3II/I, Beclin-1.7. After transfected m Cherry-LC3B-p CDNA3.1 plasmids and followed by H2O2 induction, we observed that the autophagosomes in PDGF-AA treated group was less than that in control group using fluorescence microscope.8. The proteinic levels of p-AKT, p-m TOR were significantly reduced after treatments of PI3 K inhibitor and AKT inhibitor, while the proteinic levels of p-m TOR was significantly reduced after treatment of m TOR inhibitor. Moreover, the proteinic levels of LC3II/I and Beclin-1 were significantly increased in all the inhibitor-treated groups in melanocytes PIG1. Conclusions:1. PDGFRA is expressed in primary cultured melanocytes, normal melanocytes PIG1 and vitiligo melanocytes PIG3 V. Its expression can be induced by H2O2 in a dose-dependent manner.2. In melanocytes PIG1 and melanocytes PIG3 V,down-regulation of PDGFRA increased the expression levels of LC3II/I and Beclin-1, and decreased the expression levels of MITF, gp100, TYR and TRP-1; promoted activations of apoptosis and autophagy were observed, which worsened survival of melanocytes under oxidative stress. On the contrary, up-regulation of PDGFRA decreased the expressions of LC3II/I and Beclin-1, and increased the expression levels of MITF, gp100, TYR and TRP-1 in melanocytes PIG1; and up-regulation of PDGFRA increased the expressions of MITF, gp100 in melanocytes PIG3V; inhibited activations of apoptosis and autophagy were observed, which benefited survival of melanocytes under oxidative stress.3. Under the circumstances of oxidative stress induced by H2O2, PDGF-AA/PDGFRα can improve the survival of melanocytes via PI3K/AKT/m TOR pathway and inhibit cellular autophagy. In conclusion, PDGF-AA/PDGFRα can protect human melanocytes from oxidative stress. |
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