基于玄府理论探索汗法与补法治疗局灶节段硬化大鼠足细胞损伤的TRPC机制

Exploration on TRPC mechanism of diaphoresis and tonifying method on podocyte injury in FSGS rats based on the Xuanfu theory

目的:研究汗法与补法治疗嘌呤霉素氨基核苷(PAN)诱导的局灶节段硬化性肾病(FSGS)大鼠足细胞损伤的疗效及机制。方法:40只雄性Wistar大鼠按体质量随机分为正常组、模型组、越婢汤组、固精方组。首剂静脉注射PAN 9mg/100g,后分别于第13、16、19、22、25天追加PAN 0.9mg/100g建立FSGS大鼠模型,8周后留取尿液、血液及肾组织标本。检测血生化指标,尿蛋白排泄,Real-time PCR、Western blotting、免疫荧光观察TRPC5、TRPC6表达与分布,激光共聚焦显微镜观察足细胞骨架蛋白F-actin,光镜观察肾小球病理改变,透射电镜观察足细胞超微结构。结果:模型组大鼠出现肾小球局灶节段硬化,TRPC5、TRPC6表达显著上调,F-actin表达减少,电镜广泛足突融合。与模型组比较,越婢汤与固精方组血清白蛋白水平升高(P<0.05),固精方组尿蛋白排泄减少(P<0.05),固精方组TRPC5、TRPC6蛋白、mRNA及免疫荧光表达下调(P<0.05,P<0.01),F-actin表达增加(P<0.05);越婢汤组TRPC5蛋白、mRNA及免疫荧光表达下调(P<0.05,P<0.01),TRPC6 mRNA及免疫荧光表达下调(P<0.01),两组足突融合较模型组减轻。结论:越婢汤、固精方可能通过调控TRPC5及TRPC6减轻足细胞损伤,发挥FSGS治疗作用。

Objective： To clarify the efficacy and mechanism of diaphoresis and tonifying method on podocyte injury in rats with focal segmental glomerular sclerosis（FSGS） nephropathy induced by puromycin amino nucleoside（PAN）. Methods： Forty male Wistar rats were randomly divided into control group, model group, Yuebi Decoction group, Gujing Formula group. The FSGS rat model was established by intravenous injection of PAN 9 mg/100 g at the first dose and followed dosage of 0.9 mg/100 g added on the 13 th, 16 th, 19 th, 22 th, 25 th days respectively. The urine, blood and kidney tissue samples were collected after 8 weeks. The blood biochemical indexes and urinary protein excretion were detected, and the expression and distribution of TRPC5 and TRPC6 was detected by Real-time PCR, Western blotting and immunofluorescence staining. The renal glomerulus pathological changes was observed by light microscope and the ultrastructure of podocyte by transmission electron microscope. The skelemin F-actin of podocyte was observed by Laser Scanning Confocal Microscopy. Results： Rats in the model group showed renal glomerular FSGS, and TRPC5/TRPC6 expression significantly up-regulated, F-actin expression decreased, and extensive foot process fusion of podocyte by electron microscope. Compared with the model group, the serum albumin levels of Yuebi Decoction and Gujing Formula group increased, and the urinary protein excretion decreased in Gujing Formula group（P0.05）. The expression of protein, m RNA and immunofluorescence staining of TRPC5 and TRPC6 in Gujing Formula group were down-regulated（P0.05, P0.01）, and F-actin was up-regulated（P0.05）. The expressions of protein, mRNA and immunofluorescence staining of TRPC5 in Yuebi Decoction group were also down-regulated（P0.05, P0.01）, however, only the mRNA and immunofluorescence staining of TRPC6 were down-regulated（P0.01）. The foot processes effacements were alleviated both in Yuebi decoction group and Gujing Formula group. Conclusion： Yuebi Decoction and Gujing Formula may alleviate the podocyte injury and play a therapeutic role of FSGS by regulating TRPC5 and TRPC6.