siRNA干扰HIF-1α表达对高糖高胰岛素诱导的肥大心肌细胞的影响

Effect of HIF-1α-siRNA on cardiomyocyte hypertrophy induced by high glucose and insulin in neonatal rats

目的研究胰岛素受体底物1(insulin receptor substrate 1,IRS1)和缺氧诱导因子1a(hypoxia inducible factor 1a,HIF-1a)在高糖高胰岛素诱导的肥大心肌细胞中的表达及其之间的关系;观察siRNA沉默HIF-1a基因对高糖高胰岛素诱导的心肌细胞肥大的影响。方法新生大鼠心肌细胞培养48h后,换用无血清DMEM培养液并分别加入高糖高胰岛素、高糖高胰岛素+HIF-1a-siRNA培养48h,未加入任何药物的心肌细胞在无血清DMEM培养液中继续培养48h作为对照。通过心肌细胞表面积、总蛋白含量指标检测心肌细胞肥大,并利用Real time PCR检测转染前后HIF-1a mRNA表达变化及免疫细胞化学方法检测HIF-1a及IRS1蛋白水平的表达。结果高糖高胰岛素可增加心肌细胞表面积、总蛋白含量、HIF-1a mRNA以及HIF-1a表达,并降低IRS1表达。转染siRNA后使HIF-1a基因的表达下降,能部分抑制心肌细胞的肥大,降低心肌细胞表面积和总蛋白含量,但对IRS1表达的影响不明显。在对正常对照组和高糖高胰岛素组中IRS1表达量与HIF-1a表达量进行相关分析表明,两者的表达量成负相关。结论通过siRNA技术对HIF-1a的有效沉默可明显地抑制高糖高胰岛素诱导大鼠乳鼠心肌细胞肥大,并且这种作用可能是通过作用于IRS1/PI3K/Akt/MTOR途径来实现的。

Objective The expression of insulin receptor substrate 1（IRS1）and hypoxia inducible factor 1a（HIF-1a） in myocardial hypertrophy induced by high glucose and insulin as well as their relationship were studied;The effect of siRNA silencing HIF-1a gene on such cardiomyocyte hypertrophy was observed.Methods Cardiomyocytes of neonatal rats were cultured for 48 hours,and then treated with high glucose plus high insulin or high glucose plus high insulin plus HIF-1a-siRNA in serum free DMEM for another 48 hours,while control cardiomyocytes were continuously cultured in serum free DMEM for another 48 hours.Cardiomyocyte hypertrophy was determined by myocardial cell surface area and total protein content.HIF-1a mRNA was examined by real time PCR,and the expressions of HIF-1a and IRS1 proteins were detected by immunocytochemistry.Results The cellular surface area,total protein content,HIF-1a and HIF-1a mRNA were significantly increased after treatment with high glucose and insulin compared with those of the controls,while IRS1 was significantly decreased.After HIF-1a-siRNA was transfected to cardiac cells of neonatal rats,the cellular surface area and total protein content were decreased,but IRS1 was not significantly affected.The expressions of HIF-1a and IRS1 showed a negative correlation in the control group and the group of high glucose and insulin.Conclusion The application of siRNA targeting HIF-1a can effectively lessen cardiac hypertrophy of neonatal rats induced by high glucose and high insulin,and this effect may be achieved by acting on the IRS1/PI3K/Akt/MTOR pathway.