过表达SOCS3对西格列汀改善胰岛素抵抗作用的影响

Effects of SOCS3 overexpression on the improvement of insulin resistance by sitagliptin

目的:探究过表达SOCS3对西格列汀(sitagliptin, SITA)在改善脂质代谢和氧化应激,以减轻棕榈酸(palrnitic acid, PA)介导的HepG2细胞胰岛素抵抗(insulin resistance, IR)中的作用。方法:MTT法检测PA与SITA对肝癌细胞HepG2增殖活性的影响;将HepG2分为4组:Control组,PA组,PA+SITA+pEX-RB-NC组和PA+SITA+pEX-RB-SOCS3组;油红O染色检测细胞中脂质的积聚水平,RT-PCR检测细胞中SREBP1c和PPARα的mRNA表达,DCFH-DA法检测细胞中ROS的水平,RT-PCR与试剂盒检测细胞中CAT与GPx的mRNA表达与酶活性;Western blot试验检测细胞IR通路中p-IRS-1Ser307/IRS-1、p-AKTSer473/AKT、p-GSK3βSer9/GSK3β的比值。结果:与Control组相比,SITA能削弱PA诱导的HepG2细胞活力损伤(P<0.05),且PA组、PA+SITA+pEX-RB-NC组和PA+SITA+pEX-RB-SOCS3组中富含大量红色脂滴,SREBP1c与PPARα的mRNA水平、ROS、CAT与GPx表达及p-IRS-1Ser307/IRS-1、p-AKTSer473/AKT、p-GSK3βSer9/GSK3β比值均明显升高(P<0.05);但与PA组相比,PA+SITA+pEX-RB-NC组除CAT、GPx及p-IRS-1Ser307/IRS-1、p-AKTSer473/AKT、p-GSK3βSer9/GSK3β比值升高外(P<0.05),其他检测指标均显著降低(P<0.05),PA+SITA+pEX-RB-SOCS3组中上述指标均无明显改变(P>0.05)。结论:SITA可抑制脂质代谢和氧化应激,从而以减轻PA介导的肝细胞IR,而该作用能够被过表达SOCS3所抵消。

OBJECTIVE To investigate the effect of SOCS3 over expression on sitagliptin(SITA) in alleviating lipid metabolism and oxidative stress to alleviate palmitic acid(PA)-mediated insulin resistance(IR) in HepG2 cells.METHODS MTT assay was used to detect the effects of PA and SITA on the proliferation activity of HepG2 cells. The cells were divided into four groups: Control, PA, PA+SITA+pEX-RB-NC and PA+SITA+pEX-RB-SOCS3 group. Oil red O staining was used to detect the accumulation level of lipids in each group. The mRNA expression of SREBP1 c and PPARα was detected by RT-PCR. The level of ROS in HepG2 cells was detected by DCFH-DA method. Meanwhile, the activity of antioxidant enzymes and mRNA expression level of CAT and GPx in HepG2 cells were detected by commercial kit and RT-PCR. The phosphorylation levels of insulin receptor substrate 1Ser307,protein kinase BSer473,and glycogen synthesis kinase 3βSer9(p-IRS-1Ser307/IRS-1,p-AKTSer473/AKT and p-GSK3βSer9/GSK3β) were detected by Western blot.RESULTS Compared with the Control group, SITA could attenuate the PA-induced damage of HepG2 cell viability(P<0.05). The cells in the PA group, PA+SITA+pEX-RB-NC group and PA+SITA+pEX-RB-SOCS3 group were rich in a large number of red lipid droplets. The mRNA expression levels of SREBP1 c and PPARα were significantly increased(P<0.05). The expressions level of ROS,CAT and GPx were significantly increased(P<0.05),and the ratios of p-IRS-1 Ser307/IRS-1,p-AKTSer473/AKT,and p-GSK3βSer9/GSK3β were significantly increased P<0.05). Compared with the PA group, the expression of CAT,GPx and the ratio of p-IRS-1Ser307/IRS-1,p-AKTSer473/AKT,p-GSK3βSer9/GSK3β in the PA+SITA+pEX-RB-NC group increased(P<0.05),other detection indexes were significantly decreased(P<0.05). However, there was no significant changes in the above indexes in PA+SITA+pEX-RB-SOCS3 group(P>0.05).CONCLUSION SITA could inhibit lipid metabolism and oxidative stress to alleviate PA-mediated IR in HepG2 cell, which can be reversed by SOCS3 overexpression.