The Regulatory Mechanism Of AHR Pathway In The Oxidative Stress Pathogenesis Of Vitiligo

Background: Vitiligo is a chronic depigmentation disorder resulting from melanocyte destruction, which is difficult to cure as the pathogenesis iscomplicated and remains unclear.Accumulating dataemphasize the crucial role of oxidative stress in the initiation ofvitiligo. Multiple factors might disturb oxidation-antioxidation balance and then product excessive reactive oxygen species(ROS), which could further induce oxidative damage of DNA, protein, lipid and organelles, espiacally mitochondria. Previous studies have demonstrated that mitochondrial damage could aggravate oxidative stress and induce mitochondrial apoptosis pathway to mediate melanocyte loss in vitiligo. However, the mechanism underlying mitochondrial damage in vitiligo remains unclear, rigirous research is warranted to explore the key signal pathway in mitochondrial regeneration.The aryl hydrocarbon receptor(AHR) is a ligand-activated transcription factor. Upon binding ligand, AHR translocatesinto the nucleus to govern target genes. AHR plays vital roles in immuneregulation, melanocyte homeostasis and antioxidation.Additionally, AHR could protect mitochondrial functions to aviod cellular apoptosis. However, thebiologicalcharacterization underlying such effects is not fully certain. Recent study has indicated that impaired AHR pathway might be involved in vitiligo, by showing significantly declined expressions of AHR and its target genesin the epidermis of vitiligo patients. Taken together, we hypothesized that AHR signal pathway cloud regulate mitochondrial homeostasis and therefore protect melanocyte against oxidative damage, abnormal of which might be involve in melanocyte destruction in vitiligo.Aims: 1. To investigate the association of aberrant AHR signal with vitiligo.2. To evaluate the protectiverole of AHR againsit oxidative stress in melanocyte.Methods: 1. We conducted molecular epidemiology research and functional studies to determine the association of AHR polymorphisms with vitiligo risk.2. We treated PIG1 cells with AHR ligands to assess whether AHR is functional in human normal melanocyte.3. We treated PIG1 cells with H2O2 to investigate whether oxidative stress could activate AHR signal pathway.4. To characterize the protective role of AHR pathway against oxidative damage in PIG1 cells, ROS level, cell cycle and apoptosis were detected after co-treatment with AHR ligand or antagonist and H2O2.5. To test whether AHR pathway regulates mitochondrial regeneration in PIG1 cells, we assessed mitochondrial ROS level, mitochondrial membrane potential, ATP production, mitochondrial DNA and mitochondrial modulators in PIG1 cells co-treated with AHR ligand or antagonist and H2O2.6. We detected the expression or activation patterns of AHR in vitiligo melanocyte cell lines PIG3 V and in peripheral blood monouclear cells(PBMCs) isolated from vitiligo patients.Results: 1. AHR functionalvariation(rs10249788;-129C>T) was closely associated with vitiligo risk. The promoter variantaffected AHR promoter activity through influencing the binding activityof transcriptional regulator SP1. We then found decreased peripheral expressions of AHRand SP1 invitiligo. Further genetic analysis showed that the-129C>T variant had an impact on the AHRtranscript expression.2. PIG1 cells express a functional AHR signal pathway. 3. H2O2 promoted nuclear translocation of AHR and induced the expression of AHR target genes CYP1A1 and CYP1B1 in PIG1 cells, which was abolished by AHR antagonist.4. H2O2 induced significantly increased ROS level and enhanced cell death in PIG1 cells, which were exacerbated unber co-treatment with AHR antagonist.5. H2O2 led to increased mitochondrial ROS level and declined levels of mitochondrial membrane potential, ATP production and mitochondrial DNA copy number, which were more obviously after co-treatment with AHR antagonist. Conversely, after treatment with AHR ligand FICZ, ATP production and mitochondrial DNA level were profoundly upregulated in PIG1 cells under oxidative stress. The expression of Nfr1 and its downstream effectors Tfam or CYCS was also regulated by AHR in PIG1 cells.6. Abnormal expression and activation of AHR were observed in vilitigo melanocyte cell line. Moreover, apparently decreased AHR m RNA level with negative correlation of disease duration was observed in PBMCs ofvitiligo patients.Conclusion: In summary, we provide evidence that AHR signal pathway could defend against oxidative stress in melanocyte through improving mitochondrion biosynthesis, and genetic mutation or abnormal expression of AHRmight be involved in vitiligo. Therefore, our findings have not only advancedour understanding on AHR physiological function but also provided significantinsight into the pathogenesis of vitiligo.