三焦祛湿方对膜性肾病小鼠肾组织ZO-1和synaptopodin蛋白表达的影响

Effects of Sanjiao Qushi Formula on ZO-1 and synaptopodin protein expression in mice with membranous nephropathy

关键词:  三焦祛湿方  膜性肾病  实验研究  紧密连接蛋白ZO-1  骨架蛋白synaptopodin

Key words:  Sanjiao Qushi Formula  membranous nephropathy  experimental research  ZO-1  synaptopodin

摘要:

[目的] 探讨三焦祛湿方对膜性肾病小鼠肾组织的保护作用以及对紧密连接蛋白ZO-1和骨架蛋白synaptopodin表达的影响。[方法] 将60只雌性Balb/c小鼠随机分为对照组10只和造模组50只。造模组预免疫2周后，开始正式免疫。给予尾静脉注射阳离子化牛血清白蛋白（C-BSA）（6.5 mg/kg），隔日1次，持续6周，复制膜性肾病小鼠模型。6周后，测造模组所有小鼠24 h尿蛋白定量（24 h-UTP）显示均为阳性，提示造模成功；并随机选取4只作肾脏病理，观察到基底膜增厚并伴有免疫复合物的沉积，证实膜性肾病小鼠模型建模成功。将造模组随机分为模型组10只、中药低剂量组10只、中药高剂量组10只和盐酸贝那普利组10只，开始灌胃。空白组和模型组给予0.2 mL生理盐水灌胃，三焦祛湿方高低剂量组以及盐酸贝那普利组分别给予相应药物治疗（中药低剂量组3.71 g/kg、中药高剂量组7.42 g/kg、盐酸贝那普利组1.3 mg/kg），每日1次，持续4周。实验结束后，麻醉小鼠，腹主动脉取血，收集血液标本检测总胆固醇（TC）、血清白蛋白（Alb）、血肌酐（Scr）、尿素氮（BUN）；光镜下观察肾组织病理形态；免疫荧光检测肾组织免疫球蛋白G（IgG）沉积情况；蛋白免疫印迹法（Western blot）检测肾组织中ZO-1和synaptopodin的表达水平。[结果] 与空白组相比，模型组小鼠24 h-UTP、TC显著升高（P<0.05），Alb显著降低（P<0.05）；与模型组相比，各治疗组小鼠24 h-UTP、TC显著降低（P<0.05），Alb显著升高（P<0.05）；各治疗组组间比较，24 h-UTP、TC、Alb无显著性差异（P>0.05）。各组小鼠Scr、BUN相比无显著性差异（P>0.05）。与空白组相比，模型组小鼠肾组织ZO-1、synaptopodin的表达显著减少（P<0.05）；与模型组相比，各治疗组小鼠肾组织ZO-1、synaptopodin的表达显著升高（P<0.05），各治疗组间ZO-1、synaptopodin的表达无显著性差异（P>0.05）。[结论] 三焦祛湿方对膜性肾病小鼠的肾脏具有保护作用，其机制可能与上调ZO-1和synaptopodin蛋白的表达量有关。

Abstract:

[Objective] To investigate the protective effect of Sanjiao Qushi Formula on renal tissueand the expression oftight junction protein ZO-1 andsynaptopodin in membranous nephropathy mice.[Methods] Sixty female Balb/c mice were randomly divided into control group (N group) (n=10) and modeling group (n=50). After 2 weeks of pre-immunization,formal immunization began.Cationized bovine serum albumin(C-BSA)(6.5 mg/kg) was injected by tail vein every other day for 6 weeks to replicate the mouse model of membranous nephropathy.After 6 weeks,the 24-hour urine protein quantification (24h-UTP) of all mice in the modeling group was positive,indicating that the modeling was successful.Four mice were randomly selected for renal pathology.Thickening of basement membrane accompanied by deposition of immune complex was observed,confirming the successful modeling of membranous nephropathy mouse model.The model group was randomly divided into model group (M group) (n=10),Chinese medicine low-dose group (L group) (n=10),Chinese medicine high-dose group (H group) (n=10) and Benazepril hydrochloride group (X group) (n=10). N group and M group were given 0.2 mL normal saline intragaedally.The high and low dose group of Sanjiao Qushi Formula and Benazepril hydrochloride group were treated with corresponding drugs (3.71 g/kg in group L,7.42 g/kg in group H and 1.3 mg/kg in group X),once a day,for 4 weeks. After the experiment,the mice were anesthetized,abdominal aorta blood was collected,and blood samples were collected to detect total cholesterol(TC),serum albumin (Alb),serum creatinine (Scr) and urea nitrogen (BUN).The pathological morphology of renal tissue was observed under light microscope.Immunofluorescence was used to detect the deposition of immunoglobulin G(IgG) in renal tissues. The expression levels of ZO-1 and synaptopodin in renal tissues were detected by Western blot.[Results] Compared with N group,24 h-UTP and TCin M group were significantly increased (P<0.05),Alb was significantly decreased (P<0.05);Compared with M group,24 h-UTP and TCin treatment groups were significantly decreased (P<0.05),and Alb was significantly increased (P<0.05);There were no significant differences in 24 h-UTP,TC and Albamong all treatment groups (P>0.05).There were nosignificant differences in Scr and BUN among all groups (P>0.05).Compared with N group,the expressions of ZO-1 and synaptopodin in renal tissues of M group were significantly decreased (P<0.05);Compared with M group,the expressions of ZO-1 and synaptopodin in kidney tissues of all treatment groups were significantly increased (P<0.05),but there were no significant differences in ZO-1 and synaptopodin expressions among all treatment groups (P>0.05).[Conclusion] Sanjiao Qushi Formula has protective effect on the kidney of membranous nephropathy mice,the mechanism may be related to the up-regulation of ZO-1 and synaptopodin protein expression.