基于Nrf2/ARE信号通路探讨桂枝茯苓丸对糖尿病小鼠氧化应激因子及肾纤维化的机制

Mechanism of Guizhi Fulingwan on Oxidative Stress Factors and Renal Fibrosis in Diabetic Mice Based on Nrf2/ARE Signaling Pathway

目的:基于核因子E2相关因子2/抗氧化反应元件(Nrf2/ARE)信号通路,研究桂枝茯苓丸对糖尿病小鼠氧化应激和肾纤维化的影响,探讨其作用机制。方法:采用高脂饲料喂养联合腹腔注射链脲佐菌素建立糖尿病小鼠模型,造模成功小鼠分成模型组、二甲双胍组(250 mg·kg^(-1))、桂枝茯苓丸低、中、高剂量组(400.4、800.8、1601.6)mg·kg^(-1),每组10只,另设10只正常组小鼠,连续给药8周,记录小鼠体质量、血糖,计算肾脏指数;检测甘油三酯(TG)、总胆固醇(TC)、尿素氮(BUN)、血肌酐(SCr)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平;苏木素-伊红、高碘酸-席夫、马松染色观察肾脏组织病理变化;免疫组化观察α-平滑肌肌动蛋白(α-SMA)、纤连蛋白(FN)、转化生长因子-β_(1)(TGF-β_(1))蛋白表达,蛋白免疫印迹法(Western blot)检测肾组织Nrf2、Kelch样ECH关联蛋白1(Keap1)、SOD1、NAD(P)H单核苷酸氧化酶1(NQO1)、血红素加氧酶-1(HO-1)蛋白表达。结果:与正常组比较,模型组小鼠血糖、肾脏指数、TG、TC、BUN、SCr水平升高,肾小球基底膜增厚,肾组织胶原纤维增多,SOD活性降低,MDA含量升高,FN、TGF-β_(1)、α-SMA、Keap1蛋白表达显著增加,Nrf2、SOD1、NQO1、HO-1表达显著减少(P<0.01);与模型组比较,桂枝茯苓丸及二甲双胍组血糖、肾脏指数、TG、TC、BUN、SCr水平明显降低,肾组织病理损伤有不同程度的改善,SOD活性显著增加,MDA含量明显降低,FN、TGF-β_(1)、α-SMA、Keap1表达明显减少,Nrf2、SOD1、NQO1、HO-1表达明显增加(P<0.05,P<0.01)。结论:桂枝茯苓丸能改善氧化应激、抑制肾纤维化,其机制可能与Nrf2/ARE信号通路的激活有关。

Objective To investigate the effects of Guizhi Fulingwan on oxidative stress and renal fibrosis in diabetic mice based on the nuclear factor E_(2)-associated factor 2/antioxidant response element(Nrf2/ARE)pathway,and to explore its mechanism of action.Method A diabetic mouse model was established by feeding high-fat diet combined with intraperitoneal injection of streptozotocin.The diabetic mice were divided into a model group,a metformin group(250 mg·kg^(-1)),and Guizhi Fulingwan low,medium,and high dose groups(400.4,800.8,1601.6 mg·kg^(-1)),with 10 mice in each group.Another 10 mice were included in a normal group.The treatment was conducted continuously for 8 weeks.Body mass and blood glucose were recorded,and the renal index was calculated.Triglyceride(TG),total cholesterol(TC),urea nitrogen(BUN),serum creatinine(SCr),superoxide dismutase(SOD),and malondialdehyde(MDA)levels were measured.Pathological changes in renal tissues were observed using Hematoxylin-eosin,periodate-Schiff,and Masson staining.The protein levels ofα-smooth muscle actin(α-SMA),fibronectin(FN),and transforming growth factor-β_(1)(TGF-β_(1))were detected by immunohistochemistry.The protein levels of Nrf2,Kelch-like ECH-associated protein 1(Keap1),SOD1,NAD(P)H single-nucleotide oxidase 1(NQO1),and heme oxygenase-1(HO-1)were detected by Western blot.Result Compared with the normal group,the model group showed elevated glucose,renal index,TG,TC,BUN,and SCr levels,thickened glomerular basement membrane,increased collagen fibers in renal tissues,decreased SOD activity,elevated MDA content,increased protein levels of FN,TGF-β_(1),α-SMA,and Keap1,and decreased protein levels of Nrf2,SOD1,NQO1,and HO-1(P<0.01).Compared with the model group,the Guizhi Fulingwan and metformin groups showed reduced blood glucose,renal index,TG,TC,BUN,and SCr levels,improved renal histopathological injury,significantly increased SOD activity,significantly reduced MDA content,reduced protein levels of FN,TGF-β_(1),α-SMA,and Keap1,and increased protein levels of Nrf2,SOD1,NQO1,and HO-1(P<0.05,P<0.01).Conclusion Guizhi Fulingwan can improve oxidative stress and inhibit renal fibrosis,and its mechanism may be related to the activation of the Nrf2/ARE signaling pathway.