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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 study the mechanism of APBBR regulating mi R-126-3p to improve the insulin resistance of INS-1 cells (IR-INS-1). Methods: Insulin resistance was induced by high glucose in INS-1 cells. The levels of insulin in IR-INS-1 cells were detected by radioimmunoassay (AP), berberine (BBR) and APBBR. The bioavailability of mi R-126-3p was detected by RT-PCR. The levels of IRS1 mRNA of mi R-126-3p target gene in each group were determined by RT-PCR. Mi R-126-3p mimic and mi R -126-3p mi After IR-INS-1 cells were transfected with R-inhibitor, the target gene IRS m RNA and its target protein expression levels were detected by RT-PCR and Western blot. RESULTS: There was no significant difference in basic insulin secretion (BIS) between AP and BBR groups compared with model group. GSIS of AP and BBR were significantly different from that of model group (P <0.05). The BIS value and GSIS value in APBBR group were significantly different from those in model group (P <0.05, P <0.01). Bioinformatics analysis showed that IRS1 is a potential target of mi R-126-3p. The results of RT-PCR showed that the expression of IRS1 in model group was significantly lower than that in control group (P <0.01). The expression of IRS1 in AP group, BBR group and APBBR group was significantly higher than that in model group (P <0.05, P <0.01). The level of IRS1 was significantly decreased after transfected with mi R-126-3p mimics (P <0.01); IRS1 level was not significantly decreased after transfection with mi R-126-3p inhibitor; IR-INS-1 cells reduce the level of IRS1 significant inhibitory effect. Western blot results showed that IRS1 protein expression in control group was significantly higher than that in model group and IR negative control group (IR-NC). IRS1 protein expression in 126M + APBBR and 126I + APBBR group was significantly higher than that in model group and IR-NC group . Conclusion: APBBR can significantly promote the insulin secretion of IR-INS-1 cells; mi R-126-3p overexpression can promote insulin resistance in INS-1 cells. APBBR may down-regulate the expression of mi R-126-3p in IR-INS-1 cells So as to increase the level of IRS1 m RNA and its protein expression and improve insulin resistance.