The Fuction Of Autophagy In Melanocyte At The Oxidative Stress Environment

objective:The oxidative damage of melanocytes are involved in a variety of the pigment related diseases' pathogenesis,such as vitiligo,melanoma and so on.Melanocytes are particularly vulnerable to the overproduction reactive oxygen species that induced by the oxidative stress that induced by exogenous factors and indigenous factors,owing to their specialized function and especial location.Reactive oxygen species can attack melanocytes and interfere with normal metabolism,proliferation,and differentiation of melanocytes,causing cell apoptosis or malignant transformation.Reactive oxygen species also can cause mitochondria dysfuction,DNA damage and protein modification changed.Autophagy is a highly conservative degradition way that depends on lysosomes,widesly existing in eukaryotic cells.It can digest macromolecule proteins,injured organelles,extraneous microbes.Recently plenty of studies indicate the closed relationship between autophagy and oxidative stress.The disorder of autophagy has a closed relationship with various diseases mediated by oxidative stress,such as,Parkinson's disease,Alzheimer's disease and cardiac disease.But the role of autophagy in melanocyte oxidative damage is not studied among world.Our study detect the change of autophagy level and the autophagy correlation indexes in melanocytes under oxidative damage.We aim to discuss the role of autophagy in melanocyte under oxidative stresssuperficially.Methods :1?Firstly,we separated the normal human primary epidermal melanocytes from children who are younger than ten years old.And we separately cultured the human primary epidermal melanocytes and the human melanocyte cell line PIG1 in 254 medium containing 5% serum.Putting both of them in incubator box containing 37 degree centigrade,5% CO2,changing the medium once three days or four days depends on the condition of the floated cells and medium.And digesting cells with 0.25% trypsase when they were adhered.We accepted logarithmic phase cells to further experiment.2?Constructing melanocyte oxidative stress models.We utilized 1.0nM H₂O₂ to treat the the normal human primary epidermal melanocytes and the cell line PIG1 for 24 hours.Then,we tested the cell viability by MTS method.3?The protein expressions of light chain 3?LC3?and p62 were detected by Western blot.The autophagosome of oxidative stress melanocytes were observed under laser confocal microscope.4?The melanocytes were pretreated with autophagy promoter and inhibitor.The level of apoptosis,ROS,mitochondria membrane potential were detected by flow cytometry.Results:1?The melanocytes' cell viability were descended after treated by1.0mM H₂O₂ for 24 hours,the difference is statistic significantly?P<0.05?.2?The autophagy related protein LC3 II was up-regulated expression,LC3 I was down-regulated expression,p62 was also down-regulation after treated with 1.0mM H₂O₂.The number of autophagosome was significantly increased.When autophagy was inhibited,the level of ROS and apoptosis was higher than the control group,the mitochondria membrane potential was decreased.While promoting the autophagy,the level of ROS and apoptosis was lower than the control group,the mitochondria membrane potential is increased?P<0.05?.Conclusions:1?1.0mM H₂O₂ can make melanocytes oxidative stress and injure the cells.2?The level of autophagy is increasing when the melanocytes under oxidative stress.3?Autophagy of the melanocytes can protect them from injuring resulting from oxidative stress.