益肾通络方对膜性肾病大鼠的肾保护作用及足细胞骨架相关蛋白的影响

Effect of Yishen Tongluo Formula on kidney protection and podocyte skeleton related proteins in rats with membranous nephropathy

目的探讨益肾通络方对膜性肾病大鼠的肾脏保护作用及可能的机制。方法健康雄性SD大鼠60只随机分为对照组10只和造模组50只,造模组采用尾iv阳离子化牛血清白蛋白(C-BSA)的方法复制膜性肾病大鼠模型。造模成功后再随机分为模型组、盐酸贝那普利组和益肾通络方低、中、高剂量(6.61、13.22、26.44 g/kg)组,各组按照相应剂量ig给药,每天1次,连续给药4周。给药结束后检测大鼠24 h尿蛋白定量(UTP)、血清总胆固醇(TC)、总蛋白(TP)、白蛋白(ALB)、尿素氮(BUN)、肌酐(Scr)水平;免疫荧光检测各组大鼠肾组织Ig G免疫复合物沉积情况,电镜下观察肾小球基底膜及足细胞形态结构;免疫组化及实时荧光定量PCR(q RT-PCR)法检测各组大鼠肾足细胞骨架相关蛋白ezrin、synaptopodin的表达。结果与模型组比较,各治疗组大鼠UTP、TC水平均显著下降(P<0.01),TP、ALB水平均显著上升(P<0.01);益肾通络方中、高剂量组与盐酸贝那普利组疗效相当,效果优于益肾通络方低剂量组;各组大鼠BUN、Scr相比无明显差异。与对照组比较,模型组大鼠肾足细胞骨架相关蛋白ezrin、synaptopodin m RNA表达显著减少(P<0.01);与模型组比较,各治疗组大鼠肾足细胞相关蛋白ezrin、synaptopodin m RNA表达均有不同程度增加(P<0.01);益肾通络方中、高剂量组大鼠肾组织ezrin、synaptopodin m RNA表达量与盐酸贝那普利组相当,均高于益肾通络方低剂量组。结论益肾通络方对膜性肾病大鼠具有治疗作用,其机制可能与防止足细胞骨架相关蛋白ezrin、synaptopodin降解、维持足细胞骨架及足突结构完整性相关。

Objective To explore the kidney protection and possible mechanism of Yishen Tongluo Formula（YTF） in rats with membranous nephropathy（MN）.Methods A total of 60 SD healthy male rats were randomly divided into 10 for normal group and 50 for MN rat model group.The MN rat model was established by tail iv cationic bovine serum albumin（C-BSA）.After successful modeling,they were randomly divided into model group,benazepril group,and YTF groups at low,medium,and high doses（6.61,13.22,and 26.44 g/kg）.Rats in each group were ig administrated once daily for continuous four weeks according to the corresponding dose.At the end of administration,the 24 h urine total protein（UTP）,total cholesterol（TC）,total protein（TP）,albumin（ALB）,blood urea nitrogen（BUN）,and serum creatinine（Scr） levels were measured.Immunofluorescence was used to detect the deposition of Ig G immune complexes in renal tissue.The glomerular basement membrane and podocyte morphology were observed under electron microscope.Immunohistochemistry and q RT-PCR were used to detect the expression of cytoskeleton-related proteins ezrin and synaptopodin in rat kidneys.Results Compared with the model group,the UTP and TC levels in the rats in each treatment group decreased significantly（P 0.01）,and the TP and ALB levels increased significantly（P 0.01）.The middle and high dose groups of YTF were similar to the benazepril group,and the effect was better than the low dose group of YTF.There was no significant difference in the BUN and Scr among the groups.Compared with the control group,the expression of ezrin and synaptopodin m RNA in the kidney of the model group was significantly decreased（P 0.01）.Compared with the model group,the expression of ezrin and synaptopodin m RNA in the podocytes of different treatment groups was increased in different degrees（P 0.01）.The expression levels of ezrin and synaptopodin m RNA in the kidney of rats in the middle and high dose groups of YTF were similar to those in the benazepril group,which were higher than that in the low dose group of YTF.Conclusion YTF has a therapeutic effect on membranous nephropathy in rats.The mechanism may be related to the inhibition of the degradation of podocyte skeleton related proteins ezrin and synaptopodin and the maintenance of the integrity of the podocyte skeleton and foot process.