Effects Of Proanthocyanidins On Autophagy And Apoptosis Induced By H₂O₂in HaCaT Cells

Objective:Oxidative stress caused by excessive accumulation of reactive Oxygen Species（ROS）is one of the risk factors for the development of a variety of chronic skin diseases.Mitochondrial autophagy is closely related to ROS-mediated oxidative stress injury and is a selective form of autophagy in which the mitochondrial autophagy response mediated by PINK1 and Parkin selectively removes excess or damaged mitochondria;thus maintaining cell homeostasis in the face of oxidative stress conditions,and is one of the survival mechanisms of cells against intrinsic and extrinsic stress.Proanthocyanidins（PAs）are considered powerful scavengers of oxygen free radicals with no significant side effects.It is unclear whether PAs affect the protective effects of the skin through the mitochondrial autophagy pathway.The objective of this study was to explore the preprotective effect of PAs on oxidative stress-induced HaCaT cell damage and the mechanism of action of PAs on HaCaT cells.Methods:In this study,HaCaT cells were taken as the research object,and the cells were divided into blank group,injury group and protection group to construct a cell model.The blank group was not given any treatment,the injury group was given 1000 μmol/L H₂O₂ for 24 hours,and the protection group was given 100 μg/L H₂O₂ for 24 h.ml of PAs was pretreated for 4h and then injured by adding H₂O₂ for 24 h.Cell morphology was observed by inverted microscope;cell viability was detected by CCK-8 method;apoptosis level and cell membrane potential changes were detected by Hoechst staining,PI staining and JC-1 staining;Western Blot was used to detect apoptosis-related proteins Bax,Bcl-2 and the expression of mitochondrial autophagy-related proteins PINK1,Parkin,p62 and LC3.Results:1.H₂O₂ induces apoptosis in HaCaT cells,increased expression of apoptotic protein Bax,and decreased expression of anti-apoptotic protein Bcl2;PAs pretreatment can increase cell survival rate,upregulate Bcl2 protein expression,and inhibit Bax protein expression.2.H₂O₂ induces HaCaT mitochondrial membrane potential reduction,and PAs pretreatment can restore the mitochondrial membrane potential level.3.H₂O₂ induced the increased expression of mitophagy-related proteins PINK1,Parkin,p62,and LC3 II in HaCaT,while PAs pretreatment regulated the expression of mitophagy-related proteins PINK1,Parkin,p62,and LC3 II down-regulated.Conclusion:Proanthocyanidins play a protective role in the H₂O₂-induced HaCaT cellular oxidative stress model,and its mechanism may be to reduce mitochondrial damage and exert anti-apoptotic effects by influencing cellular mitochondrial autophagy.