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24 h尿蛋白（urine total protein，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） mRNA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-OHdG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 mRNA level in kidney was detected by qRT-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/AMPKα expression 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.

R285.5

国家自然科学基金资助项目（81373804）