Role Of Autophagy In Regulating Melanin Metabolism By Shufeng-Huoxue Formulation In Vitro

ObjectiveTo elucidate the effect of Shufeng Huoxue Formulation on melanin metabolism in vitro;To explore the role of autophagy in the metabolism of melanin;To explore the role of autophagy in modulating melanin metabolism by Shufeng Huoxue Formulation in vitro.MethodThe melanocytes were identified by L-Dopa staining,and the morphology of the cells was observed daily.The cell proliferation was measured by CCK-8.The synthesis of melanin was measured by NaOH method.The activity of TYR was measured by cell lysis.The expression of autophagy related protein Beclinl and LC-3? were measured by Western blot.Result1.Compared with the C group,Shufeng Huoxue Formulation has inhibitory effect on the proliferation of B16 in vitro(P<0.05),with the same concentration of RS group compared with R group,B16 cell proliferation rate is lower,but the difference was not significant(P>0.05);Shufeng Huoxue Formulation promoted the tyrosinase activity and melanin synthesis(P<0.05),with the same concentration of RS group compared with R group,the activity of tyrosinase and melanin content was higher in B16,but the difference was not significant(P>0.05);rapamycin inhibitde the proliferation of B16 in vitro(P<0.05);prometed tyrosinase activity and melanin synthesis(P<0.05);Shufeng Huoxue Formulation and rapamycin group increased content of autophagy related protein LC3B and Beclinl,the order that up-regulated protein LC3B and Beclin1 from lowest to highest were 0.98mg/ml S group 0.98mg/ml RS group and Rap 50 group.2.Compared with the C group,Shufeng Huoxue Formulation inhibited the proliferation of normal human epidermal melanocytesin vitro(P<0.05),with the same concentration of RS group compared with R group,the melanocyte cell proliferation rate was higher,but the difference was not significant(P>0.05);group S promoted on tyrosinase activity at low concentration(P<0.05);and inhibited in high concentration,but without statistical significance(P>0.05);low concentration of S group promoted the melanin synthesis(P<0.05),0.49mg/ml of S group had no significant effect on melanin synthesis(P>0.05),high concentration(0.98mg/ml)of S group inhibited melanin synthesis(P<0.05).With same concentration of RS group compared with R group,the melanin content were higher than that in S group,but the difference was not significant(P>0.05).Different concentrations of rapamycin promoted proliferation on cultured melanocytes cell in vitro(P>0.05),and increased tyrosinase activity and melanin synthesis(P<0.05).Shufeng Huoxue Formulation and rapamycinroup increased content of autophagy related protein LC3B and Beclinl,from lowest to highest followed by 0.25mg/mlS group,0.25mg/ml group RS group Rap 50.Conclusion1.Shu Feng Huoxue Formulation regulates melanogenesis of B16 melanoma cells through autophagy,inhibits cell proliferation,promotes tyrosinase activity and melanogenesis,and up-regulates autophagy related proteins LC3B and Beclinl.2.Shufeng Huoxue Formulation regulates melanogenesis of melanocytes through autophagy pathway,inhibits the melanocytes proliferation;primarily promotes tyrosinase activity and melanin synthesis;upregulates autophagy related protein LC3B and Beclin1.3.Autophagy inducer rapamycin plays an important role in the regulation of melanin metabolism,inhibits the proliferation of B16 cells,promotes the proliferation of human epidermal melanocytes;increases tyrosinase activity and melanin synthesis of B16 and melanocytes,and upregulates autophagy related protein LC3B and Beclin1.