温阳活血利水方调控Klotho/TRPC6/CatL通路稳定补体损伤足细胞骨架  

Impact of Wenyang Huoxue Lishui Formula on the Klotho/TRPC6/CatL Pathway and Cytoskeletal Stability after Podocyte Injury Induced by the Terminal Complement Factor C5b-9 in Vitro

作　　者：薛雪 吴健[4] 付彤飞 谭瑶 袁军 XUE Xue;WU Jian;FU Tong-fei;TAN Yao;YUAN Jun(First Clinical College,Hubei University of Traditional Chinese Medicine,Wuhan,430061;Department of Nephrology,Hubei Provincial Hospital of Traditional Chinese Medicine,Wuhan,430061;Department of Nephrology,Hubei Provincial Academy of Traditional Chinese Medicine,Wuhan,430074;Clinical College of Chinese Medicine,Hubei University of Traditional Chinese Medicine,Wuhan,430061)

机构地区：[1]湖北中医药大学第一临床学院,武汉430061 [2]湖北省中医院肾病科,武汉430061 [3]湖北省中医药研究院肾病科,武汉430074 [4]湖北中医药大学中医临床学院,武汉430061

出　　处：《中国中西医结合杂志》2022年第2期202-209,共8页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金面上项目(No.82074364)。

摘　　要：目的体外观察温阳活血利水方含药血清对膜攻击复合物C5b-9介导的非致死性足细胞损伤的影响,探讨该方药治疗特发性膜性肾病(IMN)的疗效机制。方法体外组装C5b-9建立足细胞亚溶破模型,将足细胞分为正常对照组、模型组、他克莫司组、10%及20%含药血清组,并设他克莫司及中药血清空白对照组。激光共聚焦显微镜观测足细胞Klotho、瞬时受体电位通道(TRPC6)、组织蛋白酶L(CatL)及其底物Synaptopodin表达;采用荧光探针检测细胞内Ca^(2+)水平;活性定量检测试剂盒测定RhoA及Rho关联含卷曲螺旋蛋白激酶(ROCK)活性;Western Blot检测LIM结构域激酶1(LIMK1)、cofilin蛋白及其磷酸化蛋白表达。结果与正常对照组比较,模型组足细胞Klotho及Synaptopodin表达下调(P<0.01);TRPC6、CatL、Ca^(2+)、RhoA、ROCK、p-LIMK1/LIMK1及p-cofilin/cofilin表达上调(P<0.01);骨架微丝明显减少,排列紊乱。与模型组比较,各干预组足细胞Klotho与Synaptopodin的表达上调(P<0.01);而TRPC6、CatL、Ca^(2+)、RhoA、ROCK、p-LIMK1/LIMK1及p-cofilin/cofilin表达下调(P<0.01);中药血清和他克莫司均改善了足细胞骨架的重构。结论温阳活血利水方含药血清可能通过调控Klotho/TRPC6/CatL通路,从而稳定非致死性C5b-9介导的细胞骨架紊乱。Objective The effect of serum containing drug of Wenyang Huoxue Lishui(WHL)Formula on non-fatal podocyte injury mediated by membrane attack complex C5 b-9 was observed in vitro,and the therapeutic mechanism of this recipe in idiopathic membranous nephropathy(IMN)was explored.Methods Terminal complement complex C5 b-9 was formed on pedocytes using purified C5 b-6 and C7-C9.Podocytes were divided into normal control group,model group,tacrolimus group and 10%and 20%concentrations of WHL-containing serum,as well as tacrolimus blank group and WHL blank group.The expression of Klotho,transient receptor potential channel 6(TRPC6),cathepsin L(CatL)and its substrate synaptopodin were observed by laser confocal microscope.Fluorescent probe was applied to detect intracellular Ca^(2+)level.The activities of RhoA and Rho-associated coiled-coil containing kinases(ROCK)of podocytes were measured by the activity quantitative detection kit.The expression of LIM domain kinase1(LIMK1),phosphorylatedLIMK1(p-LIMK1),cofilin protein and p-cofilin protein of podocyte were detected by Western Blot.Results As compared with normal control in podocytes,expression of Klotho and Synaptopodin were down-regulated(P<0.01)as well as expression of TRPC6,CatL,Ca^(2+)level,RhoA,ROCK,p-LIMK1/LIMK1 and p-cofilin/cofilin in podocytes were up-regulated in model group(P<0.01).Moreover,Treatment of podocytes with C5 b-9 caused F-actin disorderliness and rarefaction of filaments.As compared with model group,expression of Klotho and Synaptopodin were up-regulated(P<0.01)as well as expression of TRPC6,CatL,Ca^(2+),RhoA,ROCK,p-LIMK1/LIMK1 and p-cofilin/cofilin in podocytes were down-regulated by treatment of tacrolimus or 10%and 20%WHL containing serum(P<0.01).Additionally,disrupted actin stress fiber was partially reversed by treatment of tacrolimus or different concentration of WHL containing serum.Conclusion The WHLR containing serum may regulate the Klotho/TRPC6/CatL pathway,thus stabilizing non-fatal C5 b-9 induced actin cytoskeleton disruption.

关 键 词：温阳活血利水方 膜性肾病 膜攻击复合物C5b-9 足细胞 KLOTHO 瞬时受体电位通道 组织蛋白酶L 中药复方 

分 类 号：R285[医药卫生—中药学]