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目的:探讨砷暴露诱导细胞氧化应激的分子机制。方法:采用人正常肝细胞进行亚砷酸钠和砷酸钠的暴露处理,并设相应对照组,采用SOD模拟物MnTMPyP和还原型谷胱甘肽(reduced glutathione,GSH)预处理,检测细胞超氧阴离子(O2·-)和细胞整体ROS的水平。Western Blot方法检测细胞氧化/抗氧化重要酶微粒体谷胱甘肽硫转移酶(microsomal glutathione S-transferase-1,Mgst-1)、半胱氨酸双加氧酶1(cysteine dioxygenase 1,Cdo1)和NADPH氧化酶的催化亚基NOX4的表达。针对NADPH氧化酶,采用特异性抑制剂(diphenyleneiodonium chloride,DPI)进行预处理,观察对砷暴露引起的细胞ROS水平及细胞凋亡的影响。结果:砷暴露能够显著诱导细胞超氧阴离子的产生,提高细胞整体ROS水平,其中三价砷(亚砷酸钠,As3+)诱导氧化应激作用显著强于五价砷(砷酸钠,As5+)。亚砷酸钠能够显著提高NOX4的表达。针对NADPH氧化酶的抑制剂DPI能够显著抑制砷暴露引起的细胞ROS水平升高以及细胞凋亡的增加。结论:NADPH氧化酶是砷暴露诱导人肝细胞的作用靶点,砷能够通过NADPH氧化酶产生大量超氧阴离子,提高ROS水平,造成氧化应激,诱导人正常肝细胞凋亡。
Objective: To investigate the molecular mechanism of arsenic exposure in inducing cellular oxidative stress. Methods: Human normal hepatocytes were exposed to sodium arsenite and sodium arsenate. Corresponding control group was treated with MnTMPyP and reduced glutathione (GSH) Oxygen Anions (O2 · -) and overall cellular ROS levels. Western Blot was used to detect the expression of microsomal glutathione S-transferase-1 (Mgst-1), cysteine dioxygenase 1 (Cdo1) And the catalytic subunit NOX4 expression of NADPH oxidase. In this study, NADPH oxidase was pretreated with diphenyleneiodonium chloride (DPI) to observe the effect of arsenic exposure on ROS level and cell apoptosis. Results: Arsenic exposure significantly induced the production of superoxide anion and increased the overall ROS level. Among them, the effects of arsenic trioxide (As3 +) on oxidative stress were significantly stronger than that of pentavalent arsenite (As5 +), . Sodium arsenite can significantly increase NOX4 expression. DPI, an inhibitor of NADPH oxidase, significantly inhibited the increase of cellular ROS levels and the increase of apoptosis induced by arsenic exposure. CONCLUSION: NADPH oxidase is a target of arsenic exposure to induce human hepatocytes. Arsenic can produce a large amount of superoxide anion through NADPH oxidase, increase ROS level, cause oxidative stress and induce apoptosis in normal human hepatocytes.