Alpha-lipoic Acid Protect Human Melanocytes Against Oxidative Stress By Inhibiting Autophagy

Background and objective:Vitiligo is a common acquired depigmentation disease characterized by progressive loss of functional melanocytes in the epidermis,and its mechanism has not been fully elucidated.At present,more and more studies have shown that oxidative stress plays a vital role in the pathogenesis of vitiligo.The accumulation of reactive oxygen species in melanocytes disturbing its normal physiological functions,causing the structural destruction,and eventually leading to depigment.a-lipoic acid is currently the only known water and fat-soluble antioxidant,which is known as a universal antioxidant together with its reduced form dihydro-lipoic acid.In addition to scavenging reactive oxygen and radicals directly,it can also improve the antioxidant capacity by increasing the production of endogenous antioxidants.In recent years,it has been widely used in the prevention and treatment of diabetes and its complications,ischemia-reperfusion,cardiovascular disease and cognitive disorders.Clinical trials have confirmed thata-lipoic acid can significantly improve the treatment effect of vitiligo patients.This study was designed to investigate the protective effects ofa-lipoic acid on melanocytes under oxidative stress and to explore its possible mechanism of action.Methods:Normal human melanocytes were treated with 500mM H₂O₂ for 6h,or400mMa-lipoic acid pretreated for 12h,then followed by 500mM H₂O₂ for 6h.Determination of cell viability was performed by CCK-8.Flow cytometry was used to detect intracellular reactive oxygen species,calcium level,mitochondrial membrane potential,cell cycle.The retraction velocity of melanocyte dendrites was measured by image analysis software.Transmission electron microscopy was used to observe cell ultrastructure.The P62,LC3-II/LC3-I ratio,an indicator of autophagy level,as well as the expression of mTOR signaling pathways was analyzed by western blot.Results:The cell viability of melanocytes was decreased after treated with H₂O₂,whilea-lipoic acid pretreatment could increase the cell viability of melanocytes under oxidative stress,significantly compared with the H₂O₂ group?0.74±0.08,0.51±0.03,p<0.01?.The intracellular ROS content?159.43±16.53?and calcium ion level?32.87±2.68?induced by H₂O₂ were higher compared with control group?110.43±2.23?and?13.75±1.58?.However,a-lipoic acid pretreatment rescued the increased level of ROS and calcium ion level under oxidative stress significantly?P<0.01,P<0.01?.After exposure to H₂O₂,the dendrites of melanocytes retracted obviously compared with the control group?0.39±0.02,0.02±0.01,P<0.01?.Over the same time course,a-lipoic acid pretreatment induced only slight dendritic retractions compared with the H₂O₂ group?0.18±0.03,0.39±0.02,P<0.01?.Meanwhile,a-lipoic acid pretreatment could prevent the decrease of mitochondrial membrane potential induced by oxidative stress,which was significantly different from that of H₂O₂ group?P<0.05?.After expose to H₂O₂,the integrity of cell membrane was destroyed,at the same time,the mitochondria number was reduced accompanied by the intima swelling and even vacuolation and rupture.Moreover,the damaged structure of melanocyte could be rescued bya-lipoic acid.Compared with the control group,oxidative stress can arrest the cell cycle in G2 phase?P<0.01?and reduced the number of cells in the S phase?P<0.01?,while,a-lipoic acid could reduce the number of cells blocked in G2 phase,and promote cells to enter S phase.The results of western blot showed that the ratio of LC3II/LC3I in H₂O₂-treated melanocytes were up-regulated significantly compared with control group?1.19±0.19,0.45±0.10,P<0.01?.However,the pretreatment ofa-lipoic acid could reduce the level of autophagy under oxidative stress compared with the H₂O₂ group?0.62±0.10,1.19±0.19,P<0.05?.a-lipoic acid could protect melanocytes by promoting the phosphorylation of mTOR and reducing the levels of hyperactive autophagy under oxidative stress conditions.Conclusions:These results demonstrate that the protective effective ofa-lipoic acid in melanocytes under oxidative stress may be achieved by eliminating intracellular ROS and reduce the level of overactivated autophagy.