化瘀通络中药对糖尿病肾病大鼠肾脏和AMPK/Nox4信号通路的作用研究

Effect of Huayu Tongluo Chinese medicines on kidney and AMPK/Nox4 signaling pathway of rats with diabetic nephropathy

目的探讨化瘀通络中药对糖尿病肾病(diabetic nephropathy,DN)大鼠肾脏的保护作用以及对腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)/烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(nicotinamide adenine dinucleotide phosphatase oxidase 4,Nox4)信号通路的干预作用。方法随机选取7只SD大鼠作为对照组,其余大鼠采用高糖高脂饲料喂养联合ip链脲佐菌素(35 mg/kg)建立DN模型,DN大鼠随机分为模型组、化瘀通络中药(4.41 g/kg)组和厄贝沙坦(13.5 mg/kg)组。给予化瘀通络中药16周后,取尿液检测大鼠24h尿蛋白(urinetotalprotein,UTP)和8-羟基脱氧鸟苷(8-hydroxydeoxyguanosine,8-OHdG)水平;考察各组大鼠肾组织病理变化;考察各组大鼠肾组织超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛水平;采用免疫组化法检测各组大鼠肾组织8-OHdG和纤维连接蛋白(fibronectin,FN)蛋白表达;采用qRT-PCR法检测肾组织转化生长因子-β1(transforming growth factor-β1,TGF-β1)m RNA表达;采用Western blotting法检测各组大鼠肾组织TGF-β1、AMPKα、磷酸化AMPKα(p-AMPKα)、Nox4蛋白表达。结果与模型组比较,化瘀通络中药组大鼠尿液24 h UTP和8-OHdG水平明显降低(P<0.01);肾脏病理损伤缓解,肾组织细胞外基质(extracellular matrix,ECM)堆积减少,肾组织胶原面积明显降低(P<0.01);肾组织SOD活性明显升高(P<0.05),肾组织丙二醛水平显著下降(P<0.01);肾组织8-OHd G和FN表达减少;肾组织TGF-β1 mRNA和蛋白表达水平显著降低(P<0.05、0.01);肾组织p-AMPK/AMPK蛋白表达水平明显升高(P<0.05),Nox4蛋白表达水平明显降低(P<0.05)。结论化瘀通络中药能够通过作用于AMPK/Nox4信号通路,从而抑制DN大鼠氧化应激、减少ECM大量堆积、降低UTP排泄、保护肾脏。

Objective To explore the protective effect of Huayu Tongluo Chinese medicines on kidneys of diabetic nephropathy(DN)rats and intervention on AMP-activated protein kinase(AMPK)/nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4)signaling pathway.Methods Seven SD rats were randomly selected as control group,rest rats were fed with high-sugar and high-fat diet combined with ip streptozotocin(35 mg/kg)to establish DN model.DN rats were randomly divided into model group,Huayu Tongluo Chinese medicines(4.41 g/kg)group and irbesartan(13.5 mg/kg)group.After ig Huayu Tongluo Chinese medicines for 16 weeks,urine was collected to test levels of 24 h urine total protein(UTP)and 8-hydroxydeoxyguanosine(8-OHdG)in rats;Pathological changes in kidney tissue of rats in each group was investigated.Superoxide dismutase(SOD)activity and malondialdehyde level in kidney of rats in each group were investigated;Immunohistochemistry was used to detect the expression of 8-OHdG and fibronectin(FN)in kidney of rats in each group.TGF-β1 m RNA level in kidney was detected by q RT-PCR.Expressions of TGF-β1,AMPKα,p-AMPKα,and Nox4 in kidney of rats in each group were detected by Western blotting.Results Compared with model group,24 h UTP and 8-OHdG levels in urine of rats in Huayu Tongluo Chinese medicines group were significantly reduced(P<0.01);Kidney pathological damage was relieved,extracellular matrix(ECM)accumulation in kidney was reduced,kidney collagen area was significantly reduced(P<0.01);SOD activity in kidney was significantly increased(P<0.05),malondialdehyde level was significantly decreased(P<0.01);8-OHdG and FN expression were decreased in kidney;TGF-β1 mRNA and protein expression in kidney were significantly reduced(P<0.05,0.01);p-AMPKα/AMPKαexpression was significantly increased(P<0.05),Nox4 expression was significantly reduced(P<0.05).Conclusion Huayu Tongluo Chinese medicines can inhibit oxidative stress of DN rats,reduce the accumulation of ECM,reduce UTP excretion,and protect kidneys by acting on AMPK/Nox4 signaling pathway.