金属蛋白酶组织抑制因子3敲低通过抑制活性氧簇启动的线粒体凋亡通路对抗高糖诱导的人脐静脉内皮细胞损伤的研究

TIMP3 knockdown protects against high glucose-induced cell injury through inhibiting ROS-initiated mitochondrial apoptosis in HUVECs

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.

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 mem-brane 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(F>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 potential(P<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.