黄芪多糖通过AMPK/SIRT1/PGC-1α途径对糖尿病肾病肾小管上皮细胞线粒体的影响

Effect of astragalus polysaccharide on mitochondrial function of renal tubular epithelial cells with diabetic nephropathy through AMPK/SIRT1/PGC-1α pathway

目的探讨黄芪多糖(APS)对糖尿病肾病肾小管上皮细胞线粒体功能的影响以及机制。方法人近端肾小管上皮细胞分为对照组、高糖组(30 mmol/L葡萄糖)、APS组(200 mg/L APS+30 mmol/L葡萄糖)、Ex 527组(10μmol/L Ex 527+200 mg/L APS+30 mmol/L葡萄糖)。采用CCK8法检测细胞生存率,比色法检测LDH漏出率,化学自发光法检测ATP含量,荧光分析法检测Caspase-9活性,realtime PCR法检测沉默调节因子1(SIRT1)mRNA含量,Western blotting法检测腺苷酸活化蛋白激酶(AMPK)、p-AMPK、SIRT1以及过氧化酶体增殖物激活受体γ共激活因子-1α(PGC-1α)含量。结果与对照组相比,高糖组细胞活性降低,LDH漏出率增加,ATP含量下降,p-AMPK、SIRT1以及PGC-1α蛋白表达下降,SIRT1 mRNA水平下降,Caspase-9活性增加(P均<0.05)。而与高糖组相比,APS组细胞活性提高,LDH漏出率降低,ATP含量提高,p-AMPK、SIRT1以及PGC-1α蛋白表达上调,SIRT1 mRNA水平下调,Caspase-9活性降低(P均<0.05)。结论APS可以通过AMPK/SIRT1/PGC-1α途径维持线粒体功能,对糖尿病肾病肾小管上皮细胞发挥保护作用。

Objective To investigate the effect and mechanism of astragalus polysaccharide(APS)on mitochondrial function of renal tubular epithelial cells with diabetic nephropathy.Methods Human proximal renal tubular epithelial cells were divided into the control group,high glucose group(30 mmol/L glucose),APS group(200 mg/L APS+30 mmol/L glucose),and Ex 527 group(10μmol/L Ex 527+200 mg/L APS+30 mmol/L glucose).CCK8 was used to detect the cell survival rate,colorimetric method was used to detect LDH leakage rate,chemiluminescence method was used to detect ATP content,fluorescence method was used to detect the Caspase-9 activity,real-time PCR was used to detect sirtuin1(SIRT1)mRNA content,Western blotting was used to detect AMP-activated protein kinase(AMPK),p-AMPK,SIRT1 and eroxisome proliferator-activated receptor-γcoactivator 1α(PGC-1α)content.Results Compared with the control group,the cell survival rate decreased,LDH leakage rate increased,ATP content decreased,p-AMPK,SIRT1 and PGC-1αprotein expression decreased,SIRT1 mRNA level decreased and Caspase-9 activity increased in the high glucose group(all P<0.05).APS could improve cell survival rate,reduce LDH leakage rate,increase ATP content,up-regulate the expression of p-AMPK,SIRT1 and PGC-1αprotein,down-regulate SIRT1 mRNA level,and reduce Caspase-9 activity(all P<0.05).Conclusion APS can maintain the mitochondrial function through AMPK/SIRT1/PGC-1αpathway and protect renal tubular epithelial cells with diabetic nephropathy.