益气活血化湿方对被动型Heymann肾炎模型大鼠足细胞裂孔膜蛋白α-actinin-4和CD2AP的调节作用

Regulation of Yiqi Huoxue Huashi formula on podocyte slit membrane protein α-actinin-4 and CD2AP in passive Heymann nephritis model rats

目的探讨益气活血化湿方及其拆方对被动型Heymann肾炎(PHN)模型大鼠足细胞裂孔膜蛋白α-actinin-4和CD2AP的调节作用。方法取雄性Wistar大鼠,制备经典的PHN模型。将造模成功的大鼠随机分为模型组、环孢素组、益气活血化湿方全方组、益气方组、活血方组和化湿方组,另取正常大鼠为对照组。各药物组分别灌胃给予相应药液,对照组和模型组大鼠灌胃给予等量0.9%NaCl溶液,连续6周。分别于第0、2、4、6周对各只大鼠眼眶静脉丛采血,代谢笼收集24 h尿液。全自动生化分析仪检测血清样本白蛋白(Alb)、肌酐(SCr)、尿素氮(BUN)水平以及尿液样本24 h尿蛋白定量(UPRO)。干预结束后,取双侧肾脏。HE染色后光镜下观察肾组织形态学变化,透射电镜下观察肾小球基底膜、足细胞及足突的形态学变化。PCR和Western blot检测肾组织中α-actinin-4和CD2AP的mRNA及蛋白表达水平。结果①与对照组相比,模型组大鼠在各时间点UPRO和SCr水平均明显升高(P<0.05),Alb水平明显下降(P<0.05)。与模型组相比,环孢素组第4、6周UPRO水平明显下降(P<0.05),第2、4、6周Alb水平明显下降(P<0.05),但第2、4、6周SCr和BUN水平明显升高(P<0.05)。与模型组相比,全方组仅第6周UPRO水平明显下降(P<0.05)。与环孢素组相比,全方组第2、4、6周SCr和BUN水平明显降低(P<0.05)。②与对照组相比,模型组光镜下观察可见肾小球明显肿大,肾小球基底膜弥漫性增厚,毛细血管襻受压,部分肾小管上皮细胞肿胀;电镜下显示上皮细胞下大量电子致密物沉积,足突广泛融合或消失。与模型组相比,各药物干预组的肾小球病变程度均有减轻。③与对照组相比,模型组α-actinin-4 mRNA和蛋白表达水平显著升高(P<0.05),CD2AP mRNA和蛋白表达水平显著降低(P<0.05)。与模型组相比,环孢素组α-actinin-4 mRNA和蛋白表达水平显著下调(P<0.05),CD2AP mRNA和蛋白表达水平显著上调(P<0.05)。与模型组相比,全方组α-actinin-4蛋白表达水平显著下调(P<0.05)。④与模型组比较,各拆方组在各时间点的UPRO、Alb、SCr和BUN水平均无明显变化(P>0.05)。病理学观察显示各拆方组肾小球损伤程度较模型组明显减轻。与模型组相比,益气方组α-actinin-4 mRNA和蛋白表达水平显著下调(P<0.05),活血方组CD2AP mRNA和蛋白表达显著升高(P<0.05)。结论益气活血化湿方可明显降低PHN模型大鼠尿蛋白水平,减轻肾小球损伤,其机制可能与下调裂孔膜蛋白α-actinin-4表达、上调CD2AP表达有关。其拆方益气方具有抑制α-actinin-4表达的作用,活血方具有促进CD2AP表达的作用。

Objective To investigate the regulatory effects of Yiqi Huoxue Huashi formula and its decomposed recipes on the podocyte slit membrane proteinα-actinin-4 and CD2AP in rats with passive Heymann nephritis(PHN).Methods Male Wistar rats were used to establish the classical PHN model.The successful modeling rats were randomly assigned into model group,cyclosporine group,Yiqi Huoxue Huashi formula group,Yiqi recipe group,Huoxue recipe group and Huashi recipe group.The normal rats were used as controls.The drug treatment groups were treated with the corresponding drug by intragastric administration,and the control and model groups were treated with equal volume of 0.9%NaCl solution.The treatment course was 6 weeks.Blood was drawn from the orbital venous plexus of rats at weeks 0,2,4 and 6,and 24-hour urine was collected using metabolic cages.The serum levels of albumin(Alb),creatinine(SCr)and urea nitrogen(BUN),and the 24-hour urinary protein quantification(UPRO)were measured by automatic biochemical analyzer.After intervention,the kidneys were collected.The morphological changes of kidney tissues were observed under the light microscope after HE staining,and the morphological changes of glomerular basement membrane,podocytes and podocytic process were observed under the transmission electron microscope.The mRNA and protein expression levels ofα-actinin-4 and CD2AP in kidney tissues were detected by PCR and Western blot.Results①Compared with the control group,the levels of UPRO and SCR increased significantly(P<0.05)and the level of Alb decreased significantly(P<0.05)in the model group at each time point.Compared with the model group,the level of UPRO decreased significantly at weeks 4 and 6(P<0.05),and the level of Alb decreased significantly at weeks 2,4 and 6(P<0.05),while the levels of SCr and BUN increased significantly at weeks 2,4 and 6(P<0.05)in the cyclosporine group.Compared with the model group,the level of UPRO decreased significantly in the Yiqi Huoxue Huashi formula group only at week 6(P<0.05).Compared with the cyclosporine group,the levels of SCr and BUN decreased significantly in the Yiqi Huoxue Huashi formula group at weeks 2,4 and 6.②Compared with the control group,results observed under the light microscope showed in the model group,the glomerulus was obviously enlarged,the basement membrane was diffusely thickened,the capillary loops were compressed and some renal tubular epithelial cells were swollen;under the electron microscope,it was revealed in the model group that there were a large number of electron dense deposits under the epithelium and the podocytic process were widely fused or disappeared.Compared with the model group,the glomerular lesions in all drug treatment groups were alleviated.③Compared with the control group,the mRNA and protein expression levels ofα-actinin-4 increased significantly(P<0.05),while those of CD2AP decreased significantly(P<0.05)in the model group.Compared with the model group,the mRNA and protein expression levels ofα-actinin-4 were down-regulated significantly(P<0.05),while those of CD2AP were up-regulated significantly(P<0.05)in the cyclosporine group.Compared with the model group,the protein expression level ofα-actinin-4 was down-regulated significantly in the Yiqi Huoxue Huashi formula group(P<0.05).④Compared with the model group,the levels of UPRO,Alb,SCr and BUN showed no obvious changes in all of the decomposed recipe groups at each time point(P>0.05).Pathological observation showed that glomerular injury was obviously relieved in all of the decomposed recipe groups compared with the model group.Compared with the model group,the mRNA and protein expression levels ofα-actinin-4 were down-regulated significantly in the Yiqi recipe group(P<0.05),while those of CD2AP were up-regulated significantly in the Huoxue recipe group(P<0.05).Conclusion Yiqi Huoxue Huashi formula significantly reduces the urinary protein level and alleviates glomerular injury in PHN model rats,and the mechanism may be related to down-regulation ofα-actinin-4 expression and up-regulation of CD2AP expression.Moreover,among its decomposed recipes,Yiqi recipe can inhibitα-actinin-4 expression and Huoxue recipe can promote CD2AP expression.