黄芪多糖小檗碱下调IR-INS-1细胞miR-126-3p改善胰岛素抵抗

Effects of astragalus polysaccharides and berberine on attenuating insulin resistance in IR-INS-1 cells by down-regulating mi R-126-3p

目的:研究黄芪多糖小檗碱(APBBR)调控mi R-126-3p改善胰岛素抵抗INS-1细胞(IR-INS-1)的作用机制。方法:高糖诱导INS-1细胞建立胰岛素抵抗模型,放射免疫法检测黄芪多糖(AP)、小檗碱(BBR)以及APBBR干预后IR-INS-1细胞胰岛素含量;利用生物信息学方法分析mi R-126-3p的潜在靶基因;RT-PCR测定各组细胞mi R-126-3p靶基因IRS1 m RNA水平;mi R-126-3p模拟物mi R-126-3p mimic和抑制剂mi R-126-3p mi R-inhibitor转染IR-INS-1细胞后,用RT-PCR与Western blot方法检测靶基因IRS m RNA及其靶蛋白表达水平。结果:AP组和BBR组的基础胰岛素分泌量(BIS)与模型(model)组比较无显著性差异;AP和BBR的葡萄糖刺激胰岛素分泌量(GSIS)与model组比较均有显著性差异(P<0.05);APBBR组BIS值和GSIS值与model组比较均有显著性差异(P<0.05,P<0.01)。生物信息学方法分析结果显示IRS1为mi R-126-3p潜在靶基因。RT-PCR检测结果表明,model组IRS1表达量显著低于control组(P<0.01);AP组、BBR组及APBBR组IRS1表达量均显著高于model组(P<0.05,P<0.01)。转染mi R-126-3p mimics后,IRS1水平显著降低(P<0.01);转染mi R-126-3p inhibtor后,IRS1水平未明显降低;APBBR对转染mi R-126-3p mimics的IR-INS-1细胞IRS1水平的降低有显著抑制作用。Western blot结果表明,control组的IRS1蛋白表达显著高于model组和IR negative control(IR-NC)组;126M+APBBR和126I+APBBR组的IRS1蛋白表达均显著高于model组和IR-NC组。结论:APBBR能显著促进IR-INS-1细胞的胰岛素分泌;mi R-126-3p过表达能促使INS-1细胞胰岛素抵抗,APBBR可能通过下调IR-INS-1细胞mi R-126-3p的表达从而增加IRS1 m RNA水平及其蛋白的表达,改善胰岛素抵抗。

Objective: To explore the mechanism of astragalus polysaccharides and berberine(APBBR) in improving insulin resistance INS-1(IR-INS-1) cells by regulating mi R-126-3p. Methods: IR-INS-1 model was established by high concentration of glucose. Then detected the insulin content in IR-INS-1 cells after intervening by astragalus polysaccharides(AP), berberine(BBR) and APBBR respectively. Protencial target gene of mi R-126-3p was analyzed using bioinformatics methods, m RNA level of mi R-126-3p target gene IRS1 was detected by RT-PCR. m RNA and protein level of target gene IRS were measured by RT-PCR and Western blot respectively after transfected IR-INS-1 cell with mi R-126-3P and mi R-126-3P inhibitor. Results: No significant difference was found on basal insulin secretion(BIS) in the AP and the BBR group compared with the model group, significant differences were found on glucose stimulates insulin secretion(GSIS) in the AP group and the BBR group as well as on the BIS and GSIS in the APBBR group compared with the model group(P<0.05, P<0.01). Bioinformatics results suggested that IRS1 was the target gene of mi R-126-3p. RT-PCR results showed that IRS1 m RNA level in the model group was significantly lower than that in the control group(P<0.01), the m RNA expression of IRS1 in the AP group and the BBR group were significantly higher than that in the model group(P<0.05), IRS1 expression increased significantly in the APBBR group compared with the model group(P<0.01). IRS1 m RNA level decreased in mi R-126-3p mimics transfected group(P<0.01) while no significant change was found after transfecting with mi R-126-3p inhibtor. APBBR could inhibit the decreased IRS1 m RNA expression in IR-INS-1 cells after mi R-126-3p mimics transfected. Western blot results showed that IRS1 protein expression in the control was significant higher than that in the model and IR negative control(IR-NC) group, IRS1 protein expression in the 126M+APBBR and 126I+APBBR group were significant higher than that in the model and IR-NC group. Conclusion: APBBR could promote insulin secretion in IR-INS-1 cells, mi R-126-3p over expressing could impel insulin resistance in INS-1 cells. APBBR may improve insulin resistance by down-regulating mi R-126-3p expression to raise IRS1 m RNA and protein expression.