NOX4/ROS/STAT3通路对肝星状细胞活化增殖的影响

Effect of NOX4/ROS/STAT3 pathway on the activation and proliferation of hepatic stellate cells

目的 研究肝星状细胞活化及增殖过程中还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)和信号转导与转录激活因子3(STAT3)表达的变化,并探讨NOX4抑制剂GKT137831是否可通过抑制NOX4,减少活性氧(ROS)的产生,抑制STAT3信号通路,抑制肝星状细胞活化与增殖。方法 将大鼠肝星状细胞-T6(HSC-T6)分为4组:对照组、TGF-β1组、TGF-β1+GKT137831组、GKT137831组。DCFH-DA荧光探针法检测HSC-T6细胞内ROS水平,CCK-8法检测细胞增殖,实时荧光定量PCR法检测NOX4、STAT3 mRNA的表达;细胞免疫荧光和蛋白印迹法检测NOX4、STAT3、p-STAT3及α-SMA蛋白的表达。结果 与对照组相比,TGF-β1组HSC-T6细胞中NOX4 mRNA和蛋白表达上调,细胞内ROS水平升高,p-STAT3蛋白表达增加,细胞活化增殖能力升高(P<0.05);给予GKT137831处理后,HSC-T6细胞中NOX4 mRNA和蛋白表达下调,细胞内ROS水平下降,STAT3蛋白磷酸化减少,细胞活化增殖能力下降(P<0.05)。结论 NOX4可能通过产生ROS,促进STAT3磷酸化,导致HSC-T6细胞活化与增殖;GKT137831通过抑制NOX4来减少ROS的产生,抑制STAT3磷酸化,从而抑制HSC-T6细胞活化与增殖。

Objective To investigate changes to the expression of reduced nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)and signal transducer and activator of transcription 3(STAT3)during the activation and proliferation of hepatic stellate cells and to explore whether the NOX4 inhibitor GKT137831 can reduce the production of reactive oxygen species(ROS),as well as inhibit the STAT3 signaling pathway and the activation and proliferation of hepatic stellate cells.Methods Rat hepatic stellate cells-T6(HSCs-T6)were divided into four groups:control group,TGF-β1 group,TGF-β1+GKT137831 group,and GKT137831 group.The ROS levels in HSC-T6 cells were detected by DCFH-DA fluorescent probe method.The CCK-8 method was used to detect cell proliferation,and real-time fluorescent quantitative PCR was used to detect the expression of NOX4 and STAT3 mRNA.The expression of NOX4,STAT3,p-STAT3,andα-SMA proteins was detected by immunofluorescence and Western blot.Results Compared with the control group,HSC-T6 cells in the TGF-β1 group’s expression of NOX4 mRNA and protein were up-regulated,and intracellular ROS levels,expression of p-STAT3 protein,and the cells’activation and proliferation ability increased(P<0.05).After addition of GKT137831,NOX4 mRNA and protein expressions were down-regulated in HSC-T6 cells,and intracellular ROS levels,STAT3 protein phosphorylation,and the cells’activation and proliferation ability were decreased(P<0.05).Conclusions NOX4 may promote the phosphorylation of STAT3 by producing ROS,leading to the activation and proliferation of HSC-T6 cells.By inhibiting NOX4,GKT137831 reduces ROS production and inhibits STAT3 phosphorylation,thereby inhibiting HSC-T6 cell activation and proliferation.