补肾泄浊化瘀通络方调控膜性肾病足细胞转分化保护肾功能机制研究

Mechanism of Bushen Xiezhuo Huayu Tongluo Formula(补肾泄浊化瘀通络方)in Regulating the Podocyte EMT and Protecting Renal Function in Membranous Nephropathy

目的:研究补肾泄浊化瘀通络方对阳离子化牛血清白蛋白(Cationized Bovine Serum Albumin,C-BSA)诱导的膜性肾病(Membranous Nephropathy,MN)大鼠肾保护作用及对MN足细胞转分化(Epithelial-Mesenchymal Transition,EMT)的调节作用。方法:SD大鼠采用C-BSA尾静脉注射法制备MN大鼠模型,造模成功后随机分为模型对照组、盐酸贝那普利10 mg/kg组、补肾泄浊化瘀通络方10.2、21.3、42.6 g/kg组,另设正常对照组,各组灌胃给药相应药物或蒸馏水,1次/d,给药4 w后取材检测。采用光镜(MASSON染色、PAS染色)、电镜、IgG免疫荧光进行肾组织病理观察;记录大鼠24 h尿蛋白定量(24-UTP)、血清总蛋白(TP)、白蛋白(ALB)、三酰甘油(TG)、总胆固醇(TC)、尿素氮(BUN)、肌酐(Scr)含量;应用免疫组化及Real-time PCR法检测肾组织突触足蛋白(Synaptopodin)、转化生长因子-β1(TGF-β1)蛋白及mRNA的表达情况。结果:与正常对照组比较,模型对照组大鼠出现较为明显的肾组织病理损伤,肾小球基底膜不均匀增厚,上皮下免疫复合物沉积伴特有的“钉突”样改变,IgG弥漫性、条带状沉积于基底膜区;24-UTP、TG、TC含量明显升高(P<0.05),血清TP、ALB含量明显降低(P<0.05),肾组织足细胞标志物Synaptopodin蛋白及mRNA表达下调,EMT相关标志物TGF-β1蛋白及mRNA表达上调(P<0.05);与模型对照组比较,补肾泄浊化瘀通络方10.2、21.3、42.6 g/kg组明显减轻MN模型大鼠肾组织损伤程度,明显降低24-UTP、TG、TC含量(P<0.05),增加血清TP、ALB含量(P<0.05),上调肾组织Synaptopodin蛋白及mRNA表达,下调TGF-β1蛋白及mRNA表达。结论:补肾泄浊化瘀通络方对C-BSA致MN大鼠具有肾保护作用,可能与其能够上调肾组织中Synaptopodin表达,下调TGF-β1表达,抑制足细胞EMT、修复足细胞损伤有关。

Objective:To investigate the protective effect of Bushen Xiezhuo Huayu Tongluo Formula(补肾泄浊化瘀通络方)on membrane nephropathy(MN)induced by cationic bovine serum albumin(C-BSA)and the regulation of podocyte epithelial-mesenchymal transition(EMT)in MN.Methods:The MN model was induced by tail vein injection of C-BSA in SD rats,and the model rats were randomly divided into a model control group,a benazepril hydrochloride(10 mg/kg)group,and low-,medium-,and high-dose Bushen Xiezhuo Huayu Tongluo Formula(BXHT)groups(10.2 g/kg,21.3 g/kg,and 42.6 g/kg).A normal control group was also set up.Rats were administered with corresponding drugs or distilled water,once a day.After 4 weeks of administration,samples were collected for detection.The pathological changes in renal tissues were observed by MASSON staining,PAS staining,electron microscopy,and immunofluorescence.The 24-hour urine total protein(24-UTP),serum total protein(TP),albumin(ALB),triglyceride(TG),total cholesterol(TC),blood urea nitrogen(BUN),and serum creatinine(Scr)were recorded.Then immunohistochemistry and Real-time PCR were used to detect the protein and mRNA expression of synaptopodin and transforming growth factor-β1(TGF-β1)in renal tissues.Results:Compared with the normal control group,the model control group showed obvious pathological damage in renal tissues(such as uneven thickening of the glomerular basement membrane,deposition of subepithelial immune complex with spikes projecting,and diffuse and band-like deposition of IgG in basement membrane),increased content of 24-UTP,TG,and TC(P<0.05),decreased content of TP and ALB(P<0.05),down-regulated protein and mRNA expression of synaptopodin,and up-regulated protein and mRNA expression of EMT-related protein TGF-β1(P<0.05).Compared with the model control group,the BXHT groups showed improved renal injury in MN model rats,decreased content of 24-UTP,TG,and TC(P<0.05),increased content of TP and ALB(P<0.05),up regulated protein and mRNA expression of synaptopodin,and down regulated protein and mRNA expression of TGF-β1.Conclusion:BXHT has a protective effect against MN induced by C-BSA in rats,and this effect is presumably achieved by up-regulating synaptopodin expression,down-regulating TGF-β1 expression,inhibiting the podocyte EMT,and repairing the podocyte injury.