IRS-2通过PI3K/AKT通路调控肝细胞焦亡的实验研究  

Impact of IRS-2 on the regulation of hepatocyte pyroptosis via the PI3K/AKT pathway

作　　者：郭庆鑫 姚婷 沈乐而 陈金梅 胡微微 张毅[1] 陈小华[1] GUO Qing-xin;YAO Ting;SHEN Le-er;CHEN Jin-mei;HU Wei-wei;ZHANG Yi;CHEN Xiao-hua(Department of Infection Disease,Shanghai Sixth People′s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200233,China)

机构地区：[1]上海交通大学医学院附属第六人民医院感染科,200233

出　　处：《肝脏》2024年第5期561-566,共6页Chinese Hepatology

基　　金：国家科学自然基金(82070615);上海市科学技术委员会科研计划项目(21140901100)。

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。Objective To explore the role and molecular mechanism of insulin receptor substrate(IRS)-2 in hydrogen peroxide(H 2O 2)-induced hepatocyte pyrotosis.Methods HepG2 and L02 cells were stimulated with H 2O 2,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 H 2O 2 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 H 2O 2 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.

关 键 词：IRS-2 PI3K/AKT通路 焦亡 肝细胞 线粒体功能障碍 

分 类 号：R575[医药卫生—消化系统]