温阳活血利水方含药血清对嘌呤霉素损伤小鼠永生系足细胞组织蛋白酶L表达的影响  

Effect of Wenyang Huoxue Lishui Recipe Containing Serum on Expression of Cathepsin L in Puromycin Aminonucleoside-induced Injury of Mouse Glomerular Podocytes

作　　者：邱雯雯[1,2] 袁军[3,4] 杨柳[1,2] 

机构地区：[1]湖北中医药大学中医临床学院,武汉430061 [2]湖北中医药大学研究生院,武汉430061 [3]湖北中医药大学临床医学院,武汉430061 [4]湖北省中医院肾内科,武汉430061

出　　处：《中国中西医结合杂志》2016年第5期602-607,共6页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：2012年教育部高等学校博士学科点专项科研基金(新教师类)资助项目(No.20124230120003)

摘　　要：目的观察温阳活血利水方含药血清对嘌呤霉素损伤的小鼠永生系足细胞组织蛋白酶L(Cathepsin L,CatL)表达的影响。方法体外培养小鼠永生系足细胞,将其分为正常对照组、模型组、地塞米松组、10%温阳活血利水方血清组(简称10%中药血清组)、20%中药血清组及中药血清空白对照组。正常对照组以培养液孵育足细胞24 h,模型组应用嘌呤霉素45 mg/L作用于足细胞24 h;在模型组干预基础上,地塞米松组加用地塞米松1μmol/L共孵育24 h;10%及20%中药血清组分别加用10%及20%的中药血清共孵育24h,并设中药血清空白对照组(单用中药血清孵育24 h)。采用细胞免疫荧光染色观察各组足细胞CatL及其底物Synaptopodin荧光表达改变;异硫氰酸荧光素(FITC)-鬼笔毒环肽(phalloidin)染色标记足细胞纤维肌动蛋白(F-actin),并采用F-actin外周环评分(cortical F-actin score,CFS)半定量分析足细胞F-actin分布。结果与正常对照组比较,模型组足细胞Synaptopodin表达水平明显降低,CatL表达水平升高,F-actin排列紊乱,逐渐向细胞外周分布形成F-actin环,CFS评分明显升高(P<0.01)。与模型组比较,地塞米松组、10%、20%中药血清组足细胞Synaptopodin蛋白表达升高,CatL表达水平降低,CFS评分亦降低(P<0.05,P<0.01)。与地塞米松组比较,10%中药血清组Synaptopodin表达降低(P<0.05),20%中药血清组CFS评分明显降低(P<0.05)。结论温阳活血利水方能上调嘌呤霉素损伤的足细胞Synaptopodin表达,下调CatL表达水平,减少骨架蛋白F-actin的重构,有助于稳定足细胞actin骨架,改善足细胞融合。Objective To observe the effect of Wenyang Huoxue Lishui Recipe（WHLR） containing serum on the expression of cathepsin L（Cat L） in puromycin aminonucleoside-induced injury of mouse glomerular podocytes. Methods Mouse podocyte cells（MPCs） in vitro cultured were divided into the normal control group, the model group, the dexamethasone（DEX） group, 10% WHLR containing serum group, 20% WHLR containing serum group, the vehicle serum control group. MPCs in the normal control group were cultured at 37 ℃ culture solution for 24 h. 45 mg/L puromycin was acted on MPCs in the model group for 24 h. On the basis of puromycin intervention, 1 μmol/L DEX was co-incubated in MPCs of the DEX group for 24 h;10% or 20% WHLR containing serum was co-incubated in MPCs of the 10% WHLR containing serum group and 20% WHLR containing serum group for 24 h. The vehicle serum control group was also set up by incubating with WHLR containing serum alone for 24 h. The expression of Cat L and its substrate Synaptopodin in podocytes were detected by cell immunofluo-rescence staining. FITC-conjugated phalloidin was used to stain F-actin. A cortical F-actin score index（CFS index）was designed to quantify the degree of cytoskeletal reorganization in cultured podocytes. Results Compared with the normal control group, the expression of synaptopodin significantly decreased and the expression of Cat L sig-nificantly increased in the model group. F-actin arranged in disorder, gradually forming pericellular F-actin ring. CFS index was obviously elevated（P〈0. 01）. Compared with the model group, the expression of synaptopodin increased, the expression of Cat L decreased, and CFS index also decreased in the DEX group, 10% WHLR containing serum group, and 20% WHLR containing serum group（P〈0. 05, P〈0. 01）. Compared with the DEX group,the expression of synaptopodin decreased in 10% WHLR containing serum group, CFS index also decreased in20% WHLR containing serum group（P〈0. 05）. Conclusions WHLR could up-regulate the expres

关 键 词：温阳活血利水方 嘌呤霉素 足细胞 组织蛋白酶L 

分 类 号：R285.5[医药卫生—中药学]