The Regulatory Mechanism Of Mitochondrial Deacetylase Sirt3 In The Pathogenesis Of Vitiligo

Background:Vitiligo is a pigment degenerative disease characterized by the destruction of epidermal melanocyte.Vitiligo,as one of the most common skin diseases,has an incidence of 0.5-2% in different RACES and regions in the world,and is increasing year by year.However,the pathogenesis of epidermal melanocyte destruction in vitiligo is not fully elucidated.There are different theories about the pathogenesis of vitiligo,most people think that vitiligo is a multifactor disease,including genetics,environment,autoimmune and oxidative stress factors such as joint action leads to the occurrence of vitiligo.Oxidative stress is usually considered to be the cause of vitiligo.There is a lot of evidence that there are anti-oxidative defects in the whole or local epidermis of vitiligo patients,and local ROS accumulation,causing the destruction of melanocyte.Mitochondria are the main source of ROS in cells.However,the specific molecular mechanism for the mitochondrial damage of melanocytes in vitiligo has not been fully elucidated.Therefore,further study of the key molecules and the specific mechanism for mitochondrial oxidative damage resistance,is great important to protect melanocytes against oxidative stress damage and look for effective treatment of vitiligo targets.Sirtuin3 is one of the NAD+-dependent protein deacetylases,mainly located in the mitochondria.It can regulate the acetylation of a variety of mitochondrial proteins and play a variety of important biological functions.Sirt3 can regulate cell metabolism and oxidative stress,repair mitochondrial oxidative damage and resist mitochondrial apoptosis.However,the specific molecular mechanism has not been elucidated.Previous studies have shown that Sirt3 can increase its activity and decrease ROS level in cells by deacetylation to important antioxidant SOD2.At the same time,the mitochondrial fusion protein OPA1 can be modified by deacetylation to maintain the mitochondrial dynamics,thus protecting the cells.Therefore,we propose a new research hypothesis: Sirt3 may play an important role in protecting mitochondrial function against melanocyte oxidative damage by deacetylation of SOD2 and OPA1.When the expression and activity of Sirt3 were abnormal,the oxidative damage of the mitochondria was irreversible,leading to the increase of mitochondrial dependent apoptosis and the occurrence of vitiligo.Aims:1.To investigate the role of Sirt3 in antagonising the oxidative damage of melanocytes.2.To investigate the specific mechanism of aberrant Sirt3 expression and activity in melanocytes of vitiligo.3.To elucidate the molecular mechanism of Sirt3 in protecting mitochondria against oxidative stress of melanocyte.Methods:1.We detected the expression of 8-OHd G,Sirt3 and Ace-SOD2 in both vitiligo tissue and normal healthy skin tissue by the immunofluorescence,and then carried out quantitative analysis.2.We used RT-PCR and Western blot to detect the expression of Sirt3 m RNA level and protein level in NHEM and PIG1 cells treated by H₂O₂ respectively.Then we detected the deacetylated activity of Sirt3 by comparing the Ace-SOD2/SOD2 ratio between before and after treatment of H₂O₂.3.We used the Sirt3-si RNA to knock out the expression of Sirt3 in NHEM cells,and treated the cells with H₂O₂.Then we detected the cell apoptosis,the level of endometrial ROS and mitochondrial membrane potential by flow cytometry.At the same time,Western blot was used to detect the expressions of PARP,Bax,bcl-2,caspase-9.4.We treated PIG1 cells and vitiligo melanocyte cell lines PIG3 V with H₂O₂ to detect the expression and deacetylated activity of Sirt3;In addition,mitochondria were isolated from PIG1 and PIG3 V cells,and mitochondrial proteins were extracted to detect changes in the level of mitochondrial acetylation before and after treatment of H₂O₂.5.We detected the changes of MDA and 4-HNE of lipid peroxidation products before and after treatment of H₂O₂ in PIG1 and PIG3 V respectively.RT-PCR and Western blot were used to detect the expression of PGC1? before and after H₂O₂ treatment,and then the expression of PGC1? in normal skin tissues and vitiligo tissues was detected by immunofluorescence.The expression of Sirt3 m RNA level and protein level was detected after knock out the expression of PGC1? by si RNA interference in PIG1 cells.The total carbonylation level of the cells before and after treatment of H₂O₂ was detected in PIG1 and PIG3 V by oxidative modification of carbonylation test kit.6.We knock out the expression of Sirt3 with si RNA interference in PIG1 cells.The cytoplasm and mitochondrial protein were separated in PIG1 cells treated with H₂O₂,and detected the release to cytoplasm of cytochrome C by Western blot.Then we observed the mitochondrial morphological changes by electron microscopy,and detected the mitochondrial dynamics by immunofluorescence.We used Sirt3 overexpression plasmid to transfect PIG3 V,and detected the release to cytoplasm of cytochrome C,mitochondrial morphological changes,and mitochondrial dynamics.Then the cell apoptosis level,mitochondrial membrane ROS level and mitochondrial membrane potential were measured by flow cytometry.7.We knock out the expression of Sirt3 with si RNA interference in PIG1 cells.After H₂O₂ treatment,Western blot was used to detect the expression of mitochondria fusion proteins MFN1,MFN2 and OPA1 as well as the expression of mitochondrial fission protein DRP1,then Co-IP was used to detect OPA1 acetylation level.We knocked out the expression ofSirt3 and over-expressed OPA1.After H₂O₂ treatment,the cell apoptosis level and mitochondrial membrane potential were measured by flow cytometry;the mitochondrial dynamics was detected by immunofluorescence;the expressions of PARP,Bax,bcl-2,caspase-9 and the cytoplasm of cytochrome C were detected by Western blot.8.We treated PIG3 V cells with Sirt3 specific agonist honokiol.After H₂O₂ treatment,CCK-8 was used to detect the cell vitality;the expression and deacetylated activity of Sirt3 was detected by Western blot;the mitochondrial dynamics was detected by immunofluorescence after interference of OPA1 and HKL treatment;the expressions of PARP,Bax,bcl-2,caspase-9 and the cytoplasm of cytochrome C were detected by Western blot after interference of OPA1 and HKL treatment.Results:1.Compared with normal healthy skin tissue,advanced oxidative stress in vitiligo patients was found by detection of DNA damage product 8-OHd G.Then we speculated Sirt3 abnormal expression and activity in vitiligo by detection of Sirt3 and Ace-SOD2 expression,and which is closely related under oxidative stress.2.The expression and deacetylated activity of the Sirt3 were significantly increased in NHEM and PIG1 cells treated with H₂O₂,which indicated that normal melanocyte expression and acetylation enzyme activity were significantly enhanced under oxidative stress.3.We found that knocked out of Sirt3 in NHEM cells treated with H₂O₂ could significantly increase the cell apoptosis rate and the level of mitochondrial membrane ROS,and decreased the level of mitochondrial membrane potential.At the same time,the expression of cleaved-PARP,Bax,cleaved-caspase9 expression was up-regulated,and the expression of anti-apoptotic protein bcl-2 was down-regulated,suggesting that sirt3 could protect the mitochondrial function of melanocytes and inhibit cell apoptosis under oxidative stress.4.We found that the expression of Sirt3 had no obvious change in PIG3 V cells after H₂O₂ treatment,but the Ace-SOD2/SOD2 ratio significantly increased than PIG1 cells.In addition,compared with PIG,the level of mitochondrial acetylation of PIG3 V cells increased significantly after H₂O₂ treatment,indicating that there was the activation barrier of Sirt3 in vitiligo melanocytes under oxidative stress.5.After H₂O₂ treatment,the MDA and 4-HNE levels in PIG3 V cells were significantly increased.H₂O₂ could induced the expression of Sirt3 transcription factor PGC1? in PIG1 cells,while PIG3 V does not change significantly,and the expression of PGC1?in vitiligo tissues is significantly down-regulated.In addition,PGC1? can regulate the expression of Sirt3 in PIG1 cells.Then the total carbonylation level in PIG3 V cells was significantly increased compared with PIG1 cells treated with H₂O₂.It is suggested that the expression of Sirt3 in vitiligo melanocytes under oxidative stress is regulated by its transcription factor PGC1?,which is caused by the carbonyl modification of 4-hne mediated by lipid peroxidation product.6.We found that knock out the expression of Sirt3 in PIG1 cells under oxidative stress can aggravate the mitochondria damage and increase cell apoptosis.On the contrary,over-expression of Sirt3 in PIG3 V cells can significantly inhibit cell mitochondria damage and inhibit cell apoptosis.It is indicated that Sirt3 can maintain the mitochondria structure and mitochondrial dynamics and protect mitochondrial function,so as to resist the cell apoptosis induced by mitochondrial oxidative damage.7.We found that knock out the expression of Sirt3 in PIG1 cells under oxidative stress had no effect on the expression of mitochondrial fusion protein MFN1,MFN2 and OPA1 and mitochondria fission protein DRP1.The results of Co-IP assay showed that the expression of knock out the expression of Sirt3 can significantly increase the acetylation level of OPA1.The Sirt3 can maintain dynamic balance,protect mitochondrial function and reduce cell apoptosis by OPA1-Cyto.c pathway.It is indicated that Sirt3 can protect mitochondrial function of melanocytes to inhibit cell apoptosis by acetylating OPA1 under oxidative stress.8.Sirt3 specific agonist honokiol have an obvious protective effect on vitiligo melanocytes under oxidative stress by activating Sirt3-OPA1 pathway.Conclusion:In summary,we first provide evidence that the Sirt3 has a protect role of vitiligo melanocytes against the oxidative damage.Sirt3 activation barrier exists in vitiligo melanocytes under oxidative stress.We elucidate the specific mechanism of Sirt3 abnormal expression and activity in vitiligo melanocytes under oxidative stress.Then we demonstrate that Sirt3 can protect the mitochondrial function of melanocyte by deacetylation OPA1.It is established a foundation to further understand the mitochondrial oxidative damage mechanism of melanocytes in vitiligo.This study not only enriched and improved the pathogenesis of the oxidative stress in vitiligo,but also provided a theoretical basis for finding new targets for clinical treatment.