补肾消渴方对糖尿病肾病模型大鼠TGF-β_(1)/Smad信号通路的影响

Effect of Bushen Xiaoke Decoction on TGF-β_(1)/Smad signaling pathway in diabetic nephropathy model rats

目的:观察中药补肾消渴方煎剂对高脂高糖联合链脲菌素(STZ)诱导的糖尿病肾病模型大鼠肾脏TGF-β1/Smad信号通路的影响,探讨其作用的机制。方法:健康雄性SD大鼠随机分为正常对照组(6只)给予常规饲料和生理盐水灌胃,模型对照组、补肾消渴方组和肾炎康复片组采用高脂高糖联合小剂量STZ(30 mg/kg)腹腔注射的方法建立糖尿病肾病模型大鼠。模型对照组(6只)给予生理盐水灌胃;补肾消渴方组(7只)给予补肾消渴方水煎剂浓缩液灌胃4.2 g/(kg·d);肾炎康复片组(7只)将肾炎康复片研磨溶于生理盐水中灌胃2.4 g/(kg·d)。灌胃8周末收集大鼠尿液,测定24 h尿微量白蛋白排泄率(UAER);处死大鼠,腹主动脉采血测定血清中的肌酐(Scr)、尿素氮(BUN)含量;光镜观察HE染色肾脏病理改变;应用免疫组化和Western blot检测肾组织转化生长因子(TGF-β1)、Smad2、Smad 3、P-Smad2、P-Smad3、α平滑肌肌动蛋白(α-SMA)蛋白的表达。结果:补肾消渴方组大鼠血Scr、BUN和24 h UAER较模型对照组明显下降(P<0.01)。肾脏病理结果显示补肾消渴方组肾组织病理改变较模型对照组明显减轻。肾组织免疫组化显示补肾消渴方肾组织TGF-β1、α-SMA蛋白的表达较模型对照组均有降低(P<0.01)。肾组织Western blot实验结果显示补肾消渴方组较模型对照组肾组织TGF-β1、P-Smad2、P-Smad3、α-SMA蛋白表达明显降低(P<0.01)。结论:补肾消渴方可能是通过抑制糖尿病肾病大鼠肾组织TGF-β1/Smad信号通路、下调TGF-β1、P-Smad2、P-Smad3、α-SMA蛋白的表达而起到降低蛋白尿、改善肾脏肾功能的作用。

Objective:To observe the effect of the the Bushen Xiaoke Decoction on the expression of TGF-β1/Smad signaling pathway in diabetic nephropathy model rats induced by fat-diet combined with STZ and investigate its effect and mechanism.Methods:Healthy male SD rats were randomly divided into normal control group(n=6)and given normal diet and normal saline by gavage.The rats in model control group,Bushen Xiaoke Decoction group and Shenyan Kangfu tablet group were injected intraperitoneally low-dose STZ(30 mg/kg)combined with high-fat and high-glucose diet to establish the DN rat model.The model control group(n=6)was given normal saline by gavage;the Bushen Xiaoke Decoction group(n=7)was given Bushen Xiaoke Decoction concentrate by gavage 4.2 g/(kg·d);the Shenyan Kangfu tablet group(n=7)was given Shenyan Kangfu tablet by gavage in normal saline 2.4 g/(kg·d).At the end of 8 weeks,the rats urine were collected to evaluate 24-hour urinary albumin excretion rate.Arterial blood samples from abdominal aortic were used for the evaluation of serum creatinine and blood urea nitrogen.Renal pathological lesion was observed by light microscopy and electron microscopy.TGF-β1,Smad2,Smad3,P-Smad2,P-Smad3,α-smooth muscle protein in renal tissue were detected by immunohistochemistry and western blot.Results:Compared with the model control group,the 24-hour urinary albumin excretion rate,blood Scr and BUN were significantly decreased in the Bushen Xiaoke Decoction group(P<0.01).Renal pathological results show that renal tissue structure was nearly lightened in the Bushen Xiaoke Decoction group compared with the model control group.The results of renal immunohistochemical staining show that the expression of TGF-β1,α-SMA were significantly decreased in Bushen Xiaoke Decoction groups compared with the model control group(P<0.01).The results of Western blot show that the protein expression of TGF-β1,P-Smad2,P-Smad3,α-SMA were significantly decreased in Bushen Xiaoke Decoction groups compared with the model control group(P<0.01).Conclusions:The Bushen Xiaoke Decoction could plays the role of reducing proteinuria and improving renal pathological changes by depressing TGF-β1/Smad singal pathway and reducing the expression of TGF-β1,P-Smad2,P-Smad3,α-SMA in the kidney from rats.It can improve renal function.