参芪颗粒及其拆方对足细胞损伤的保护作用  

Protective effects of Shenqi Granules and its component formulas on injured podocytes

作　　者：吴燕升 郭小平 魏丽凤 徐睿 王琳[1] WU Yansheng;GUO Xiaoping;WEI Lifeng;XU Rui;WANG Lin(Department of Nephrology,Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院肾病科,上海200032

出　　处：《上海中医药杂志》2024年第12期153-162,共10页Shanghai Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81273730);上海申康医院发展中心资助项目(SHDC2020CR2048B);上海中医药大学科技发展基金项目(23KFL078)。

摘　　要：目的观察参芪颗粒及其拆方[益气方、活血方、清热化湿方(以下简称“化湿方”)]对足细胞损伤的保护作用,探寻各拆方的作用靶点,为参芪颗粒各成分的有效性提供现代药理学依据。方法采用细胞计数试剂盒(CCK-8)检测不同浓度嘌呤霉素氨基核苷(PAN)对小鼠肾足细胞MPC-5存活率的影响。选择合适浓度PAN刺激MPC-5的同时,用不同浓度参芪颗粒及益气方、活血方、化湿方,干预细胞24 h后,根据CCK-8检测得出各中药处理组药物浓度,将细胞分为10组,分别为:正常对照组、模型组、环孢素组、参芪颗粒组、益气方组、活血加化湿方组、活血方组、益气加化湿方组、化湿方组、益气加活血方组。干预24 h后,通过细胞划痕试验检测CCK-8检测获得的最佳浓度拆方对MPC-5细胞划痕愈合的作用。进一步以Western blot法检测足细胞黏附蛋白(nephrin)、p-nephrin(Tyr1217)的表达水平;免疫荧光检测足细胞nephrin及F-肌动蛋白(F-actin)的表达水平;透射电镜观察足细胞形态、细胞器、细胞骨架等超微结构变化,研究参芪颗粒对PAN诱导的损伤足细胞MPC-5的保护作用,以环孢素作为阳性对照,将不同治则进行组合,明确益气、活血、清热化湿与足细胞增殖、骨架蛋白表达、细胞骨架结构的关系。结果①CCK-8检测发现,PAN以时间和浓度依赖的方式降低足细胞的存活率,在所试验的浓度范围内,益气方和活血方对足细胞存活率没有影响,参芪颗粒的细胞毒性主要是来自化湿方。25 mg/L PAN为造模最佳浓度,0.5 g/L参芪颗粒、0.21 g/L益气方、0.09 g/L活血方、0.2 g/L化湿方为各药物干预最佳浓度。②划痕试验显示,与模型组比较,参芪颗粒及其拆方促进了足细胞的增殖和迁移(P<0.05)。治疗组之间,益气方与活血加化湿方比较,化湿方组与益气加活血方组比较,伤口愈合面积无明显差异,但益气加化湿方在促进伤口愈合方面优于�Objective To observe the protective effects of Shenqi Granules and its decomposed formulas[Yiqi Formula,Huoxue Formula,and Qingre Huashi Formula(Huashi Formula)]on injured podocytes,explore their targets,and provide modern pharmacological evidence for the efficacy of each component of Shenqi Granules.Methods The Cell Counting Kit-8(CCK-8)assay was used to evaluate the effects of different concentrations of puromycin aminonucleoside(PAN)on the viability of mouse podocyte clone 5(MPC-5).Following the determination of the optimal PAN concentration for stimulating MPC-5 cells,the cells were treated with varying concentrations of Shenqi Granules,Yiqi Formula,Huoxue Formula,and Huashi Formula for 24 h.Based on the CCK-8 assay results,the appropriate drug concentrations for each treatment group were determined,and the cells were divided into 10 groups:normal control group,model group,cyclosporine group,Shenqi Granules group,Yiqi Formula group,Huoxue plus Huashi Formula group,Huoxue Formula group,Yiqi plus Huashi Formula group,Huashi Formula group,and Yiqi plus Huoxue Formula group.After 24 hours of incubation,a scratch assay was performed to assess the effects of the optimal concentrations of the component formulas,as determined by the CCK8 assay,on the scratch wound healing of MPC-5.Further analysis was conducted using Western blot to detect the expression levels of podocyte adhesion protein(nephrin)and phosphorylated nephrin(p-nephrin Tyr1217).Immunofluorescence was used to assess the expression of nephrin and F-actin in podocytes.Transmission electron microscopy was used to observe ultrastructural changes in podocytes,including morphology,organelles,and cytoskeleton.These methods were used to investigate the protective effects of Shenqi Granules on PAN-induced podocyte MPC-5 injury,with cyclosporine serving as a positive control.Combinations of different therapeutic principles were tested to clarify the relationships between benefiting qi,activating blood circulation,clearing heat,resolving dampness,and their effects

关 键 词：特发性膜性肾病 足细胞损伤 细胞骨架 参芪颗粒 中药研究 

分 类 号：R285.5[医药卫生—中药学]