升阳益胃汤合补阳还五汤对特发性膜性肾病大鼠肾脏的保护作用及对Nephrin、CD2AP表达的影响

Decoction for upraising Yang and replenishing stomach with Decoction for Invigorating Yang for Recuperation protects kidney in rats with idiopathic membranous nephropathy via up-regulating protein expressions of Nephrin and CD2AP

目的观察升阳益胃汤合补阳还五汤对特发性膜性肾病大鼠肾脏的保护作用及对肾组织中肾病蛋白(Nephrin)、CD2相关蛋白(CD2AP)表达的影响。方法取40只雄性SD大鼠,随机选择10只大鼠作为正常组,余30只大鼠尾静脉一次性注射羊抗大鼠F×1A血清0.4 mL/100 g制备特发性膜性肾病模型。将造模成功后大鼠随机分为模型组、升阳合补阳组、贝那普利组,每组10只。升阳合补阳组给予升阳益胃汤合补阳还五汤全方颗粒剂溶液9.2 g/(kg·d)灌胃,贝那普利组给予盐酸贝那普利溶液0.00088 g/(kg·d)灌胃,正常组和模型组给予等量蒸馏水灌胃,均1次/d,连续灌胃6周。于灌胃6周末收集各组大鼠24 h尿液,用以检测24 h尿蛋白水平;腹主动脉取血,检测血白蛋白(ALB)、总胆固醇(TC)、肌酐(Cr)、尿素氮(BUN)水平;取肾组织,HE染色和Masson染色观察肾脏病理形态,免疫组化法和Western blot法检测肾脏组织中Nephrin、CD2AP表达情况。结果与正常组比较,模型组大鼠24 h尿蛋白及血清TC水平均明显升高(P均<0.05),血清ALB水平明显降低(P<0.05);与模型组比较,升阳合补阳组、贝那普利组大鼠24 h尿蛋白及血清TC水平均明显降低(P均<0.05),血清ALB水平均明显升高(P均<0.05);各组间血清Cr、BUN水平比较差异均无统计学意义(P均>0.05)。HE染色和Masson染色显示,模型组大鼠肾小球体积增大,肾小球基底膜明显增厚,大量嗜复红蛋白沉积;与模型组相比,升阳合补阳组和贝那普利组大鼠肾小球体积明显减小,肾小球内嗜复红蛋白沉积较模型组明显减少。模型组大鼠肾脏组织中Nephrin、CD2AP阳性表达平均光密度值和蛋白相对表达量均明显低于正常组(P均<0.05),升阳合补阳组和贝那普利组大鼠肾脏组织中Nephrin、CD2AP阳性表达平均光密度值和蛋白相对表达量均明显高于模型组(P均<0.05)。结论升阳益胃汤合补阳还五汤具有保护特发性膜性肾病大鼠肾脏的作用,作用机制可能与上调Nephrin、CD2AP蛋白表达有关。

Objective It is to observe the protective effect of Decoction for upraising Yang and replenishing stomach with Decoction for Invigorating Yang for Recuperation on the kidney of rats with idiopathic membranous nephropathy(IMN)rats and its effect on the expressions of Nephrin and CD2AP proteins in renal tissue.Methods Forty male SD rats were taken,in which 10 rats were randomly selected as the normal group,and the remaining 30 rats were given sheep anti-rat Fx1A serum 0.4 mL/100 g by one-time tail vein injection to establish models of IMN.The successfully modeled rats were randomly divided into model group,Chinese medicine group and benazepril group,with 10 rats in each group.The Chinese medicine group was given whole formula granule solution of Decoction for upraising Yang and replenishing stomach with Decoction for Invigorating Yang for Recuperation 9.2 g/(kg·d)by gavage,the benazepril group was given benazepril hydrochloride solution 0.00088 g/(kg·d)by gavage,and the normal group and model group were given equal amount of distilled water by gavage,all once daily,continuously treated for 6 weeks.At the end of 6 weeks of gavage,24 h urine of rats in each group was retained to determine the levels of 24 h urine protein(24-UTP),blood was taken from abdominal aorta to determine the serum levels of albumin(ALB),total cholesterol(TC),creatinine(Cr),and urea nitrogen(BUN),kidney tissues were taken to observe the pathological morphology of kidneys by HE staining and Masson staining,and the expressions of Nephrin and CD2AP in renal tissue were detected by immunohistochemistry and Western blot.Results Compared with the normal group,the levels of 24-UTP and serum TC of rats in the model group were significantly higher(all P<0.05),and the level of serum ALB was significantly lower(P<0.05);compared with the model group,the levels of 24-UTP and serum TC of rats in the Chinese medicine group and benazepril group were significantly lower(all P<0.05),and the level of serum ALB was significantly higher(all P<0.05);the differences in the levels of serum Cr and BUN were not statistically significant among every groups(all P>0.05).HE staining and Masson staining showed that the glomerular volume of rats in the model group was increased,with significantly thickened basement membrane and a large amount of deposited rephosphingosine;compared with the model group,the glomerular volume of rats in the the Chinese medicine group and benazepril group were significantly decreased,and the rephosphingosine deposition in the glomerulus was significantly reduced.The average optical density and relative protein expression of Nephrin and CD2AP in the renal tissues of rats in the model group were significantly lower than those in the normal group(all P<0.05),while the average optical density and relative protein expression of Nephrin and CD2AP in the renal tissues of rats in the Chinese medicine group and benazepril group were significantly higher than those in the model group(all P<0.05).Conclusion Decoction for upraising Yang and replenishing stomach with Decoction for Invigorating Yang for Recuperation can protect the kidney of rats with idiopathic membranous nephropathy,the mechanism maybe related to up-regulating the expressions of Nephrin and CD2AP proteins.