MitoQ Alleviates H₂O₂-induced Mitochondrial Dysfunction In Keratinocytes Through The NRF2/PINK1 Pathway

Objective Oxidative stress is one of the pathogenic factors of vitiligo,and oxidative damage of keratinocytes plays an important role in the pathogenesis.The aim of this study is to clarify the role of NRF2,a central antioxidant regulator,in the regulation of PINK 1/Parkin which is the classical mitophagy signaling pathway,and to explore its effect on keratinocyte abnormalities and the intervention role of the mitochondrial antioxidant MitoQ.It is expected to enrich the theoretical basis of the pathogenesis of vitiligo and provide a new strategy.Methods 1 Detection of clinical samples:epidermal tissues were collected from healthy subjects and vitiligo patients,and immunofluorescence was used to observe the expression of NRF2,.PINK1,Parkin and LC3.2 cell experiments:(1)CCK8 was used to screen the optimal concentration of H2O2 for treating human immortalized keratinocyte cell lines(HaCaT cells);(2)The level of ROS was detected by DCFH-DA fluorescent probe.(3)Western blot was used to detect the expression of NRF2,PINK1,Parkin and LC3.(4)DAPI fluorescence staining was used to observe cell apoptosis;(5)GreenNucTM live cell Caspase-3 activity assay kit was used to detect cell apoptosis;(6)Mitochondrial membrane potential and cell apoptosis were detected by Mito-Tracker Red CMXRos staining and Annexin V-FITC staining.(7)Mitochondrial morphology was observed by transmission electron microscope.(8)Cell apoptosis was detected by flow cytometry.Results 1.Immunofluorescence results showed that the expression of NRF2,PINK1,Parkin and LC3 in the skin lesions of patients with vitiligo was lower than that of healthy controls.2.The optimal concentration of H2O2 for inducing damage to HaCaT cells was 900μM.3.In HaCaT cells treated with H2O2,the expressions of NRF2,PINK1,Parkin and LC3 were down-regulated,cell apoptosis was increased,and ROS levels were elevated,MitoQ upregulated the expression of NRF2,PINK1,Parkin and LC3 in H2O2-treated HaCaT cells,protected against H2O2-induced apoptosis and reduced H2O2-induced ROS production.4.MitoQ alleviated H2O2-induced abnormal mitochondrial morphology,down-regulated autophagy and increased apoptosis through NRF2 pathway.5.MitoQ relieved H2O2-induced mitochondrial membrane potential decline and cell apoptosis through PINK1.Conclusion This study confirmed that NRF2 and PINK1/Parkin axis were down-regulated in vitiligo lesions.By regulating the NRF2/PINK1 pathway,MitoQ,attenuated H2O2-induced mitochondrial abnormalities,promoted the upregulation of mitophagy related proteins,enhanced cell viability,reduced cell apoptosis,and protected cells from oxidative damage.This study provides an important experimental basis for further understanding the involvement of keratinocyte oxidative damage in the pathogenesis of vitiligo,and provides a new strategy for clinical treatment of vitiligo.