The Role Of Oxidative Stress-induced Autophagy On The Pathogenesis Of Vitiligo And Its Underlying Mechanism

Background: Vitiligo is acommon pigmentary disorder involving skin and mucous membrane.Lesions of vitiligo often affect exposure sites and can be psychologically devastating and stigmatizing especially for the severe cases.To date,the mechanism of vitiligo remains unclear,but it is widely accepted that the ultimate pathological changes are caused by the local melanocytes damage.Recently,compelling evidences have shown that oxidative stress is a pivotal etiological aspect for the destruction of epidermal melanocytes in patients with vitiligo.Under the internal or/and external environmental stimuli-induced oxidative stress,the generation of excessive reactive oxygen species?ROS?in melanocytes can disturb cell metabolism,proliferation and differentiation,which could further induce autoimmune response towards melanocytes,leading to their reversible cell damage.Previous studies have found that melanocytes in patients with vitiligo are more susceptible to oxidative damage than that in healthy individuals,but the mechanism has not been elucidated.Therefore,to clarify the mechanism underlying the susceptibility of vitiligo melanocytes to oxidative stress would improve the understanding of the pathogenesis of vitiligo,and be of great importance for the development of effective clinical treatment for vitiligo.Autophagy is a conservative biological phenomenon in eukaryotic cells that degrades aggregated proteins,damaged organelles and exogenous microorganisms.Previous studies have shown that in cells with the status of elevated intracellular ROS under oxidative stress,multiple signaling pathways can protect cells from oxidative damage by activating autophagy.Within those pathways,Nrf2-ARE?N uclear factor E2-related factor 2-Antioxidant response element?signaling axis has been demonstrated to be an essential antioxidant pathway in melanocytes that are activated under oxidative stress.Increasing intracellular ROS during oxidative stress promotes the translocation of Nrf2 into the cell nucleus.By binding to the promoter region of ARE on multiple phase II detoxification enzymes and antioxidant molecule genes,Nrf2 promotes the expression of ARE to exert its effect on protecting cells from oxidative damage.Recent studies have shown that there is an ARE binding site in the promoter region of p62,which is a critical participant in autophagy.Nrf2 binding to ARE in the promoter region of p62 can upregulates the expression of p62 and thus participates in autophagy.In the previous study,we have determined the functional impairment of Nrf2-ARE pathway in melanocytes from patients with vitiligo.Therefore,in present study,we propose a hypothesis that ROS can promote autophagy by activating Nrf2-P62 pathway in normal melanocytes to protect melanocytes from oxidative damage.In melanocytes from patients with vitiligo,the functional defect of Nrf2-P62 pathway following ROS activation leading to the disorder of autophagy,which causes vitiligo melanocytes susceptible to oxidative damage.Objective:1.To clarify the role of autophagy in oxidative stress in normal human melanocytes under oxidative stress;2.To analyze the difference of autophagy and compare its effect on melanocytes under oxidative stress from patients with vitiligo and healthy individuals.3.To investigate the regulation of autophagy in melanocytes under oxidative stress from patients with vitiligo and healthy individuals via Nrf2-p62 pathway.Methods:1.Normal human melanocyte cell line PIG1 were treated with different concentrations of H₂O₂ and then subjected to CCK8 assays to determine the optimal level of stimuli for the induction of oxidative stress in melanocytes.2.The levels of autophagy were detected using immunoblotting,as well as under the transmission electron microscopy and laser confocal microscopy in normal melanocytes,normal human melanocyte cell line PIG1 and melanoma cell line PIG3 V treated with H₂O₂ at different time points.3.Normal melanocytes,normal human melanocyte cell line PIG1 and melanoma cell line PIG3 V were treated with H₂O₂,autophagy inhibitor and autophagy accelerator respectively,and then subjected to flow cytometry to detect cell apoptosis,mitochondrial membrane potential and the generation of intracellular ROS.4.Normal human melanocyte cell line PIG1 and vitiligo melanocyte cell line PIG3 V were firstly transfected with short interfering RN A targeting Nrf2 or p62,or with Nrf2 or p62 overexpression plasmid,and then treated with H₂O₂.Cells were subsequently subjected to immunoblotting,Real-time RT-PCR or under the laser focused microscopy to detect the status of autophagy.5.Vitiligo melanocyte line PIG3 V were firstly transfected with short interfering RNA targeting Nrf2 or p62,or with Nrf2 or p62 overexpression plasmid,and then treated with H₂O₂.Cells were subsequently subjected to flow cytometry,immunoblotting and laser confocal microscopy to detect cell apoptosis,mitochondrial membrane potential,intracellular ROS generation and the status of autophagy.Result:1.In vitro observation revealed that the activity of normal human melanocytes cell line PIG1 and primary normal human melanocytes were both decreased in a dose-dependent manner under H₂O₂ stimulation,and 0.5 mM H₂O₂ for 24 h was optimal for the induction of oxidation stress model in melanocytes;2.H₂O₂ significantly upregulated the ratio of autophagy-related protein LC3II/I expression and increased the number of autophagosomes in both normal human melanocyte cell line PIG1 and primary normal human melanocytes in a time-dependent manner;3.Pretreatment with autophagy accelerator rapamycin in normal human melanocyte cell line PIG1 and primary normal human melanocytes under H₂O₂-induced oxidation stress significantly reduced the percentage of cell apoptosis and intracellular ROS levels,but increased the level of mitochondrial membrane potential.In contast,pretreatment with autophagy inhibitor chloroquine in normal human melanocyte cell line PIG1 and primary normal human melanocytes significantly increased the percentage of cell apoptosis and intracellular ROS levels,but downregulated the level of mitochondrial membrane potential under H₂O₂-induced oxidation stress;4.Under the H₂O₂-induced oxidative stress,innormal human melanocyte line PIG1,the ratio of autophagy-related protein LC3II/I expression,the number of autophagosomes and the level of mitochondrial membrane potential were significantly higher than that in vitiligo melanocyte cell line PIG3 V.In the meanwhile,the percentage of cell apoptosis and the level of intracellular ROS in normal human melanocyte cell line PIG1 were significantly lower compared with that in vitiligo melanocyte cell line PIG3V;5.Knockdown of Nrf2 in normal human melanocytic cell line PIG1 siginificantly reduced the the H₂O₂-induced expression of p62,the ratio of autophagy-related protein LC3II/I expression and the number of autophagosomes compared to the control group.Moreover,knockdown of p62 in normal human melanocytic cell line PIG1 siginificantlydownregulatedthe H₂O₂-inducedthe ratio of autophagy-related protein LC3II/I expression and the number of autophagosomes compared to the control group.Overexpression of p62 in normal human melanocyte cell line PIG1 with knockdown of Nrf2 significantly increased the expression of H₂O₂-induced ratio of autophagy-related protein LC3II/I expression and the number of autophagosomes compared to cells with knockdown of Nrf2 alone;6.Different cell groups were pretreatment with chloroquine to inhibitor autop hagic protein degradation and then stimulated with H₂O₂.The cell expression of Nrf2 and P62,especially Nrf2 expression within cell nuclear in the normal human melanocyte cell line PIG1 cells was significantly enhanced compared with that in vitiligo melanocyte cell line PIG3 V.7.Overexpression of p62 in vitiligo melanocyte line PIG3 V significantly upregulated the ratio of intracellular autophagy-related protein LC3II/I and the number of autophagy-lysosomes induced by H₂O₂.Moreover,compared to the control group,the percentage of cell apoptosis and intracellular ROS level were significantly reduced,while the level of mitochondrial membrane potential was significantly upregulated in vitiligo melanocyte line PIG3 V with overexpression of p62 under H₂O₂-induced oxidation stress.Conclusion:In this study,we firstly found that H₂O₂-induced oxidative stress enhances autophagic flux in primary human melanocytes against oxidative damage,and then established that Nrf2-p62 is a key pathway in protecting melanocytes against H₂O₂-induced oxidative damage.Moreover,we confirmed that vitiligo melanocytes under H₂O₂-induced oxidative stress conditions showed lower autophagic flux owing to the dysfunction of Nrf2-p62 pathway,which resulted in increased susceptibility to excessive ROS.Giving the first evidence,our data provide new insight into the pathogenesis of vitiligo as well as novel therapeutic target for vitiligo management.