关键词: 白癜风, 氧化性应激, 线粒体, 活性氧, 膜电位, 线粒体, 腺苷三磷酸, 异鼠李素, HaCaT细胞

Abstract: 【Abstract】 Objective To evaluate protective effects of isorhamnetin on mitochondrial structure and function in HaCaT cells under oxidative stress. Methods HaCaT cells served as the research object, and were divided into 4 groups: control group receiving conventional culture, isorhamnetin group treated with 60 μmol/L isorhamnetin, H2O2 group treated with 600 μmol/L H2O2 , and isorhamnetin + H2O2 group pretreated with 60 μmol/L isorhamnetin for 12 hours, followed by medium replacement and 12-hour treatment with 600 μmol/L H2O2 . Flow cytometry was performed to detect cellular reactive oxygen species （ROS） levels, transmission electron microscopy to observe mitochondrial ultrastructure, confocal fluorescence microscopy to evaluate mitochondrial membrane potential, real-time fluorescence-based quantitative PCR （qRT-PCR） to determine the mitochondrial DNA copy number, and adenosine triphosphate （ATP） assay kit was used to determine the mitochondrial ATP content. One-way analysis of variance was used for comparisons among multiple groups, and least significant difference-t test for multiple comparisons. Results Oxidative stress was provoked in HaCaT cells after the treatment with H2O2 . Compared with the control group, the H2O2 group showed significantly increased ROS levels （10 725.0 ± 845.8 vs. 1 708.0 ± 69.4, t = 18.4, P ＜ 0.001）, but significantly decreased fluorescence intensity of mitochondrial membrane potential, mitochondrial ATP content, and expression of ND-1 （a characteristic gene of mitochondrial DNA） （t = 4.58, 4.48, 6.11, P = 0.01, 0.01, 0.003, respectively）. After the pretreatment with isorhamnetin followed by H2O2 treatment, the isorhamnetin+ H2O2 group showed significantly decreased ROS levels （7 640.0 ± 922.7） compared with the H2O2 group （t = 4.27, P = 0.013）, but significantly increased fluorescence intensity of mitochondrial membrane potential, mitochondrial ATP content, and expression of ND-1 compared with the H2O2 group （t = 4.59, 4.58, 5.61, P = 0.01, 0.01, 0.005, respectively）. Under the electron microscope, the mitochondrial structure was clearer and more complete in the isorhamnetin+ H2O2 group than in the H2O2 group; there was no swelling or slight swelling of mitochondrial cristae, as well as no vacuolization of mitochondria in the isorhamnetin+ H2O2 group; in addition, autophagosomes engulfing damaged mitochondria were observed in the isorhamnetin+ H2O2 group. Conclusion Isorhamnetin may reduce ROS levels by inducing autophagy, and has a protective effect against the H2O2-induced mitochondrial structural and functional damage in HaCaT cells.

Key words: Vitiligo, Oxidative stress, Mitochondria, Reactive oxygen species, Membrane potential, mitochondrial, Adenosine triphosphate, Isorhamnetin, HaCaT cells