【摘要】 目的探讨金属蛋白酶组织抑制因子3(TIMP3)在高糖诱导的人脐静脉内皮细胞(HUVECs)凋亡过程中的作用机制。方法细胞分为对照组(Con)、高糖组(HG)、22.0 mmol/L葡萄糖+活性氧簇(ROS)清除剂组(NAC)、22.0 mmol/L葡萄糖+caspase 8特异性抑制剂组(IETD)、22.0 mmol/L葡萄糖+caspase 9特异性抑制剂组(LEHD)和22.0 mmol/L葡萄糖+si-TIMP3组(si-TIMP3),各组培养48 h,噻唑蓝实验检测细胞存活率;Annexin V/PI双染法检测细胞凋亡;DCHF-DA染色法检测细胞内ROS的生成水平;Caspase特异性抑制剂预处理以预估细胞凋亡途径,检测线粒体凋亡通路蛋白细胞色素c(Cyt c)和凋亡诱导因子(AIF)的蛋白表达;JC-1染色分析细胞膜电位的丢失情况。高糖刺激前予TIMP3敲低以观察其对上述事件的影响。结果高糖浓度依赖性促进TIMP3表达和细胞活力降低(P<0.05或P<0.01)。与Con组比较,HG组细胞质Cyt c和AIF表达升高,线粒体膜电位降低(P<0.01)。与HG组比较,LETD组细胞活性升高(P<0.01),IETD组差异无统计学意义(P>0.05),NAC、si-TIMP3组细胞活性升高,细胞凋亡降低,细胞质中Cyt c和AIF表达降低,线粒体膜电位升高(P<0.01)。结论 TIMP3敲低通过抑制ROS生成,进而抑制高糖诱导的线粒体凋亡通路,以对抗高糖诱导的HUVECs细胞损伤。更多还原

【Abstract】 Objective To explore the effect and the underlying mechanisms of TIMP3 in high glucose-induced apoptosis in HUVECs.Methods Cells were divided into control group(Con),high glucose group(HG),22.0 mmol/L glucose +ROS scavenger group(NAC),22.0 mmol/L glucose +caspase 8 specific inhibitor group(IETD),22.0 mmol/L glucose +caspase 9 specific inhibitor group(LEHD)and 22.0 mmol/L glucose +si-TIMP3 group(si-TIMP3).All the cells were cultured for 48 h.The cell viability was evaluated by MTT assay.Cell apoptosis was confirmed by Annexin V/PI double staining.The amount of intracellular ROS was evaluated by DCHF-DA staining.Caspase specific inhibitor pretreatment was aimed to explore the apoptotic pathway.The Cyt c and AIF was detected by western blotting.The mitochondrial transmembrane potential was examined by JC-1 staining.The effect of TIMP3 in the process above was confirmed by TIMP3 knockdown followed by high glucose-stimulation.Results High glucose stimulation decreased cell viability and promoted TIMP3 expression in HUVECs in a dose-dependent manner(P<0.05 or P<0.01).Cytoplasmic Cyt c and AIF expression increased,while the mitochondrial membrane potential decreased in HG group than in Con group(P<0.01).Compared with HG group,LETD group showed an increase in cell viability(P<0 01);IETD group had a similar cell activity(P>0.05);NAC and si-TIMP3 group both had an increase in cell activity,decrease in cell apoptosis,lower expression of Cyt c and AIF in cytoplasm,and higher mitochondrial membrane potentia(lP<0.01).Conclusion By inhibiting ROS production,TIMP3 knockdown can inhibit the mitochondrial apoptosis pathway induced by high glucose to fight against the cell injury and plays a protective role in HUVECs.更多还原