丹红注射液对膜性肾病模型大鼠足细胞跨膜蛋白表达的影响

Effect of Danhong injection on expression of nephrin and podocin in podocyte of renal tissue in rats with membranous nephropathy

目的观察丹红注射液对膜性肾病模型大鼠肾组织足细胞跨膜蛋白肾病蛋白(nephrin)与足突蛋白质(podocin)表达的影响。方法将34只SD大鼠按随机数字表法分为正常组8只,造模组26只。采用尾静脉注射阳离子化牛血清白蛋白制备膜性肾病大鼠模型。造模成功后,将造模组大鼠随机分为模型组、氯沙坦组及丹红组,每组8只。氯沙坦组大鼠灌胃氯沙坦40 mg/kg,丹红组大鼠尾静脉注射丹红注射液4 ml/kg,连续给药4周。末次给药后收集大鼠尿液,采用分光光度计测定24 h尿蛋白定量;采用全自动生化分析仪测定血清白蛋白、TC、TG水平;采用HE染色观察肾脏病理学改变;采用免疫组化染色法检测肾组织nephrin、podocin及WT1标记足细胞的表达;采用实时定量荧光PCR及Western blot法检测肾组织nephrin、podocin表达,并对结果进行相关性分析。结果与模型组比较,丹红组大鼠血清白蛋白升高(P<0.01),24 h尿蛋白定量、TC、TG降低(P<0.01);丹红组大鼠肾组织nephrin蛋白[(1.12±0.03)比(0.57±0.09)]、podocin蛋白[(0.99±0.04)比(0.53±0.08)]、nephrin mRNA [(0.49±0.08)比(0.14±0.02)]、podocin mRNA[(0.50±0.11)比(0.18±0.05)]表达升高(P<0.01),WT1标记的足细胞数[(14.88±1.73)%比(9.63±1.69)%]增多(P<0.05)。相关性分析结果显示,大鼠24 h尿蛋白定量与nephrin蛋白(r=-0.908)、podocin蛋白(r=-0.916)、nephrin mRNA(r=-0.930)、podocin mRNA(r=-0.923)表达、血清白蛋白(r=-0.934)及足细胞数(r=-0.778)呈负相关(P<0.01),与TC(r=0.814)、TG(r=0.870)呈正相关(P<0.01)。结论丹红注射液可能通过上调膜性肾病模型大鼠肾组织足细胞跨膜蛋白nephrin、podocin蛋白和mRNA表达,降低膜性肾病大鼠的尿蛋白水平。

Objective To observe the effect of Danhong injection on the expression of nephrin podocin protein and gene in the kidney of membranous nephropathy rats. Methods A total of 34 male clean level SD rats were divided at random into the normal control group (8 rats), model group (26 rates). The model group were injected the C-BSA into caudal vein to establish the model of the membranous nephropathy. After four week of succession of the models, two model rats were taken to confirm the success of the model. Another 24 models divided at random into model group, Losartan potassium treatment group and Danhong injection treatment group, 8 in each group. In the losartan group, the losartan 40 mg/kg was administered, and the Danhong group was injected with 4 ml/kg of Danhong injection in the tail vein for 4 weeks. The rat urine was collected after the last administration, and 24 h urine protein was determined by spectrophotometer. The serum total protein, albumin, TC and TG levels were determined by automatic biochemical analyzer. The renal pathological changes were observed by HE staining. The expressions of nephrin, podocin and WT1 labeled podocytes were detected by immunohistochemical staining. The expressions of nephrin and podocin in renal tissue were detected by real-time quantitative PCR and Western blot, and the results were correlated. Results Compared with the model group, the content of serum albumin increased in the Danhong group (P<0.01), 24 h urine protein quantitation, TC, TG significantly decreased (P<0.01). The expression of nephrin protein (1.12 ± 0.03 vs. 0.57 ± 0.09), podocin protein (0.99 ± 0.04 vs. 0.53 ± 0.08), nephrin mRNA (0.49 ± 0.08 vs. 0.14 ± 0.02), podocin mRNA (0.50 ± 0.11 vs. 1.18 ± 0.05) in Danhong group significantly increased (P<0.01), and the number of podocytes labeled with WT1 (14.88%± 1.73% vs. 9.63%± 1.69%) significantly increased (P<0.05). Correlation analysis showed that 24 h urine protein quantitation and expression of nephrin protein (r=-0.908), podocin protein (r=-0.916), nephrin mRNA (r=-0.930), podocin mRNA (r=-0.923). There was a negative correlation between serum albumin (r=-0.934) and podocyte count (r=-0.778)(P<0.01), and positive correlation with TC (r=0.814) and TG (r=0.870)(P<0.01). Conclusions The Danhong injection may reduce the urine protein of membranous nephropathy rats by up-regulating the expression of nephrin, podocin mRNA and protein in renal cells of membranous nephropathy.