IRS-2通过PI3K/AKT通路调控肝细胞焦亡的实验研究

摘要/Abstract

摘要： 目的探讨胰岛素受体底物(IRS-2)在过氧化氢(H₂O₂)诱导肝细胞焦亡中的作用及分子机制。方法 H₂O₂刺激HepG2与L02细胞系。构建IRS-2 siRNA,在HepG2与L02细胞系中抑制IRS-2基因表达,Western印迹法检测细胞IRS-2与焦亡相关蛋白表达水平。CCK-8法检测细胞活性,流式细胞仪检测线粒体膜电位变化,电镜下观测细胞形态、线粒体与焦亡小体,Mito-Track Green观测线粒体形态与数量。IRS-2 siRNA单独或联合PI3K/AKT通路激动剂刺激HepG2与L02细胞系,检测PI3K/AKT通路蛋白与焦亡相关蛋白表达水平。结果与对照组相比,H₂O₂可降低肝细胞活率,降低IRS-2蛋白表达,提高细胞焦亡相关蛋白表达。下调细胞内IRS-2表达可导致肝细胞线粒体功能障碍,肝细胞形态发生破坏,焦亡小体数量增多,上调细胞焦亡相关蛋白表达水平,P-PI3K/PI3K与P-AKT/AKT比值下调。激活PI3K/AKT通路可逆转IRS-2下调导致的肝细胞焦亡相关蛋白表达。结论 H₂O₂刺激肝细胞能降低IRS-2蛋白表达,诱导细胞焦亡。抑制IRS-2表达可能通过减少PI3K/AKT通路激活导致线粒体功能障碍,诱导肝细胞焦亡。

关键词: IRS-2, PI3K/AKT通路, 焦亡, 肝细胞, 线粒体功能障碍

Abstract: Objective To explore the role and molecular mechanism of insulin receptor substrate (IRS) -2 in hydrogen peroxide (H₂O₂)-induced hepatocyte pyrotosis. Methods HepG2 and L02 cells were stimulated with H₂O₂, and the expressions of IRS-2 and pyroptosis-related proteins were assessed by Western blot analysis. IRS-2 siRNA was synthesized and employed to suppress the expression of IRS-2 gene in both HepG2 and L02 cell lines. The expression levels of IRS-2 and pyroptosis-related proteins were subsequently evaluated using Western blot analysis. Cell viability was determined using the CCK-8 assay, while changes in mitochondrial membrane potential were analyzed via flow cytometry. Cell morphology, mitochondria structure, and pyroptosomes were visualized under an electron microscope, with mitochondria morphology and quantity observed using Mito-Track Green staining. HepG2 and L02 cells were treated with IRS-2 siRNA alone or in combination with a PI3K/AKT pathway agonist to assess the expression levels of PI3K/AKT pathway proteins and pyroptosis-related proteins, Data analysis was conducted using independent sample t tests or one-way analysis of variance(ANOVA) where appropriate. Results In comparison to the control group, exposure to H₂O₂ led to decreased viability of hepatocytes, downregulated expression of IRS-2 protein, and increased expression of pyroptosis-related proteins. Reduced expression of IRS-2 resulted in mitochondrial dysfunction, disrupted hepatocyte morphology, increased pyroptosome numbers, and up-regulate expression of pyroptosis-related proteins. Additionally, the ratio of P-PI3K/PI3K and P-AKT/AKT was decreased. Activation of the PI3K/AKT pathway reversed the expression of pyroptosis-related proteins induced by IRS-2 downregulation. Conclusion Stimulation with H₂O₂ can decrease the expression of IRS-2 protein and induce pyroptosis in hepatocytes. Inhibiting IRS-2 expression may induce mitochondrial dysfunction by reducing PI3K/AKT pathway activation, ultimately leading to hepatocyte pyroptosis.

Key words: IRS-2, PI3K/AKT pathway, Pyroptosis, Hepatocyte, Mitochondrial dysfunction

郭庆鑫, 姚婷, 沈乐而, 陈金梅, 胡微微, 张毅, 陈小华. IRS-2通过PI3K/AKT通路调控肝细胞焦亡的实验研究[J]. 肝脏, 2024, 29(5): 561-566.

GUO Qing-xin, YAO Ting, SHEN Le-er, CHEN Jin-mei, HU Wei-wei, ZHANG Yi, CHEN Xiao-hua. Impact of IRS-2 on the regulation of hepatocyte pyroptosis via the PI3K/AKT pathway[J]. Chinese Hepatolgy, 2024, 29(5): 561-566.

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