牛磺酸通过调控miR-126对高糖诱导的大鼠内皮细胞凋亡的影响及其作用机制

Taurine Regulates the Effect of mi R-126 on Rat Endothelial Cell Apoptosis Induced by High Glucose and Its Mechanism

目的 探讨牛磺酸通过调控miR-126对高糖诱导的大鼠内皮细胞凋亡的影响及其作用机制。方法 体外培养的大鼠胸主动脉内皮细胞,分为NC组(葡萄糖浓度5.5 mmol·L^(-1))、HG组(葡萄糖浓度为30.0 mmol·L^(-1))、HG+低浓度牛磺酸组(葡萄糖浓度为30.0 mmol·L^(-1),牛磺酸浓度为5.0μmol·L^(-1))、HG+中浓度牛磺酸组(葡萄糖浓度为30.0 mmol·L^(-1),牛磺酸浓度为10.0μmol·L^(-1))和HG+高浓度牛磺酸组(葡萄糖浓度为30.0 mmol·L^(-1),牛磺酸浓度为20.0μmol·L^(-1))。将模拟物(miR-126 mimics)及其阴性对照(miR-NC)、miR-126抑制物(anti-miR-126)及其阴性对照(anti-miR-NC)分别转染至大鼠胸主动脉内皮细胞。细胞计数试剂盒(CCK-8)检测细胞活力;流式细胞术检测细胞凋亡。实时荧光定量PCR(RT-qPCR)检测miR-126表达水平。蛋白质印迹(Western blot)检测细胞裂解的半胱氨酸天冬氨酸蛋白酶3(Cleaved-caspase3)、半胱氨酸天冬氨酸蛋白酶3前体(Pro-caspase3)蛋白表达。结果 与NC组比较,HG组大鼠胸主动脉内皮细胞活性显著降低(P<0.05);与HG组比较,HG+低浓度牛磺酸组、HG+中浓度牛磺酸组、HG+高浓度牛磺酸组大鼠胸主动脉内皮细胞活性显著升高(P<0.05)。与NC组比较,HG组大鼠胸主动脉内皮细胞凋亡率、Cleavedcaspase3蛋白表达显著上升,Pro-caspase3蛋白表达显著降低(P<0.05);与HG组比较,HG+低浓度牛磺酸组、HG+中浓度牛磺酸组、HG+高浓度牛磺酸组大鼠胸主动脉内皮细胞凋亡率、Cleaved-caspase3蛋白表达显著降低,Pro-caspase3蛋白表达显著升高(P<0.05)。与NC组比较,HG组大鼠胸主动脉内皮细胞中miR-126表达显著降低(P<0.05);与HG组比较,HG+低浓度牛磺酸组、HG+中浓度牛磺酸组、HG+高浓度牛磺酸组大鼠胸主动脉内皮细胞中miR-126表达显著升高(P<0.05)。与HG+miR-NC组比较,HG+miR-126组大鼠胸主动脉内皮细胞中miR-126表达显著升高,细胞活性、Pro-caspase3蛋白表达显著升高(P<0.05);凋亡率、Cleaved-caspase3蛋白表达显著降低(P<0.05)。与HG+牛磺酸组和HG+牛磺酸+anti-miR-NC组比较,HG+牛磺酸+anti-miR-126组大鼠胸主动脉内皮细胞中miR-126表达显著降低,细胞活性、Pro-caspase3蛋白表达显著降低,凋亡率、Cleaved-caspase3蛋白表达显著升高(P<0.05)。结论 牛磺酸通过上调miR-126促进高糖诱导的大鼠内皮细胞凋亡,为治疗糖尿病提供了理论依据。

Objective To investigate the effect of taurine on rat endothelial cell apoptosis induced by high glucose and its mechanism by regulating miR-126.Methods Rat thoracic aortic endothelial cells cultured in vitro were divided into NC group(glucose concentration of 5.5 mmol·L^(-1)), HG group(glucose concentration of 30.0 mmol·L^(-1)), HG + low concentration taurine group(glucose concentration of 30.0 mmol·L^(-1), taurine concentration is 5.0 μmol·L^(-1)), HG +medium concentration taurine group(glucose concentration 30.0 mmol·L^(-1), taurine concentration 10.0 μmol·L^(-1)) and HG + high concentration taurine group(glucose concentration 30.0 mmol·L^(-1), taurine concentration 20.0 μmol·L^(-1)). The mimics(miR-126 mimics) and its negative control(miR-NC), miR-126 inhibitor(anti-miR-126) and its negative control(anti-miR-NC) were transfected into the rat chest arterial endothelial cells. Cell counting kit(CCK-8) detected cell viability;flow cytometry detected cell apoptosis. Real-time fluorescent quantitative PCR(RT-qPCR) was used to detect the expression level of miR-126. Western blot was used to detect the expression of Cleaved-caspase3 and Procaspase3 protein of cell lysis.Results Compared with the NC group, the rat thoracic aortic endothelial cell activity in the HG group was significantly reduced(P<0.05);compared with the HG group, the HG + low concentration taurine group, HG + medium concentration taurine group, HG + high concentration cattle, the endothelial cell activity of the thoracic aorta in the sulfonic acid group increased significantly(P<0.05). Compared with the NC group, the apoptosis rate of thoracic aortic endothelial cells and the expression of Cleaved-caspase3 protein in the HG group increased significantly, and the expression of Pro-caspase3 protein decreased significantly(P<0.05);compared with the HG group,the apoptosis rate and Cleaved-caspase3 protein expression of rat thoracic aortic endothelial cells in the HG + low concentration taurine, HG + medium concentration taurine group and HG + high concentration taurine group were significantly decreased, and the expression of Pro-caspase3 protein was significantly increased(P<0.05). Compared with the NC group, the expression of miR-126 in the endothelial cells of the thoracic aorta of the HG group was significantly reduced(P<0.05);compared with the HG group, the HG + low concentration taurine group, HG + medium concentration taurine group, HG + high concentration taurine group, the expression of miR-126 in the endothelial cells of the thoracic aorta of rats in the taurine group was significantly increased(P<0.05). Compared with the HG + miR-NC group, the expression of miR-126 in the endothelial cells of the thoracic aorta of rats in the HG+miR-126 group was significantly increased, and the cell viability and Pro-caspase3 protein expression were significantly increased(P<0.05);The rate and Cleaved-caspase3 protein expression were significantly reduced(P<0.05). Compared with the HG + taurine group and the HG + taurine + anti-miR-NC group, the expression of miR-126 in rat thoracic aortic endothelial cells in the HG +taurine + anti-miR-126 group was significantly reduced, and the cell activity, Pro-caspase3 protein expression was significantly reduced, and apoptosis rate and Cleaved-caspase3 protein expression were significantly increased(P<0.05).Conclusion Taurine promotes rat endothelial cell apoptosis induced by high glucose by up-regulating miR-126, which provides a theoretical basis for the treatment of diabetes.