miR-378a对高糖介导胰岛β细胞损伤的影响

Effects of miR-378a on pancreaticβcell injury induced by high glucose

目的:研究miR-378a对高糖介导胰岛β细胞损伤的影响。方法:体外培养大鼠胰岛β细胞INS-1,采用高糖刺激INS-1细胞,实验分为Con组、HG组、HG+anti-miR-NC组和HG+anti-miR-378a组。实时荧光定量PCR(qPCR)分析各组INS-1细胞中miR-378a的表达情况;噻唑蓝比色法(MTT)检测细胞活力;DCFH-DA法检测细胞内活性氧(ROS)水平;采用试剂盒检测乳酸脱氢酶(LDH)和丙二醛(MDA)含量;酶联免疫吸附实验(ELISA)检测胰岛素分泌情况;流式细胞术分析细胞凋亡情况;蛋白免疫印迹法(Western blotting)分析裂解的半胱氨酸天冬氨酸蛋白酶(Cleaved Caspase 3)的表达水平。结果:与Con组相比,HG组INS-1细胞活力和胰岛素分泌水平明显降低,细胞内ROS水平、细胞凋亡率、miR-378a和Cleaved Caspase 3的表达明显升高,LDH漏出量和MDA含量增多,差异均有统计学意义(均P<0.05);与HG组相比,HG+anti-miR-378a组INS-1细胞活力和胰岛素分泌水平明显升高,细胞内ROS水平、细胞凋亡率、miR-378a和Cleaved Caspase 3的表达明显降低,LDH漏出量和MDA含量减少,差异均有统计学意义(均P<0.05)。结论:高糖刺激能够促进INS-1细胞中miR-378a的表达,抑制miR-378a能够逆转高糖刺激对INS-1细胞的损伤,减轻氧化损伤和凋亡。

Objective:To explore the effect of miR-378a on pancreaticβcell injury induced by high glucose(HG).Methods:Rat pancreatic isletβcells INS-1 were cultured in vitro and stimulated by HG.The cells were divided into control(Con)group,HG group,HG+anti-miR-NC group and HG+anti-miR-378a group.The miR-378a expression was determined by qPCR.The cell viability was detected by MTT assay.The level of reactive oxygen species(ROS)in cells was measured by DCFH-DA method.The contents of lactate dehydrogenase(LDH)and malondialdehyde(MDA)were measured as well.The insulin secretion was detected by enzyme-linked immunosorbent assay(ELISA).The cell apoptosis was evaluated by flow cytometry.The expression level of Cleaved Caspase 3 was determined by Western blotting.Results:Compared with the Con group,the INS-1 cell viability and insulin secretion level in the HG group were significantly reduced,while the intracellular ROS level,the apoptosis rate,the expression of miR-378a and Cleaved Caspase 3,the LDH leakage and the MDA content were significantly increased(P<0.05).Compared with the HG group,the INS-1 cell viability and insulin secretion level in the HG+anti-miR-378a group were significantly increased,while the level of intracellular ROS,the apoptosis rate,the expression of miR-378a and Cleaved Caspase 3,the amount of LDH leakage and MDA content were notably reduced(P<0.05).Conclusion:HG stimulation can promote the expression of miR-378a in INS-1 cells.Inhibition of miR-378a can reverse HG-induced INS-1 cells injury though reducing oxidative damage and cell apoptosis.