水蛭素通过RhoA信号传导保护足细胞骨架改善糖尿病肾病作用机制研究  

Effect and Mechanism of Hirudin in Protecting Podocyte Cytoskeleton through RhoA Signaling to Improve Diabetic Nephropathy

作　　者：陈茂盛[1] 刘琳[1] 徐长亮[2] 邵丽娜[1] 任燕[1] 叶晓兰[1] CHEN Maosheng;LIU Lin;XU Changliang;SHAO Lina;REN Yan;YE Xiaolan(Zhejiang Provincial People's Hospital,Affiliated People's Hospital,Hangzhou Medical College,Hangzhou 310014,Zhejiang,China;Nanjing University of Chinese Medicine,Nanjing 210023,Jiangsu,China)

机构地区：[1]浙江省人民医院,杭州医学院附属人民医院,浙江杭州310014 [2]南京中医药大学,江苏南京210023

出　　处：《辽宁中医药大学学报》2022年第12期30-35,共6页Journal of Liaoning University of Traditional Chinese Medicine

基　　金：浙江省中医药科技计划项目(2015ZZ002);浙江省医药卫生科技计划项目(2019320552,2023KY523);浙江省自然科学基金项目(LY19H050003)。

摘　　要：目的通过构建足细胞损伤模型及糖尿病肾病动物模型,研究水蛭素对高糖所致足细胞骨架蛋白损伤的影响。方法动物模型部分,使用8周龄db/m和db/db雄性小鼠,随机分为3组:(1)db/m组[db/m小鼠每天灌胃0.3 mL磷酸缓冲盐溶液(PBS)];(2)db/db组(db/db小鼠每天灌胃0.3 mL PBS);(3)db/db+水蛭素组(db/db+水蛭素小鼠每日口服0.3 mL PBS中含200 mg/kg水蛭素);最后一次给药后24 h处死小鼠,检测肾功能、尿蛋白定量,以及进行肾脏病理学检查。细胞模型部分,将小鼠足细胞用RPMI-1640培养基培养,用不同剂量的水蛭素或高糖处理细胞,检测足细胞骨架相关的蛋白质nephrin、podocin、synaptopodin和TRPC6等。结果水蛭素治疗的db/db小鼠的24 h尿蛋白水平呈时间依赖性下降,同时血肌酐(Scr)和尿素氮(BUN)水平也下降,肾脏病理改变得到改善;在水蛭素治疗的db/db小鼠中,nephrin、podocin、synaptopodin和TRPC6的水平明显恢复;与db/m组相比,db/db小鼠足细胞中的F-肌动蛋白显著降低,而水蛭素治疗可以维持F-肌动蛋白稳定性;与高糖组相比,水蛭素或RhoA抑制剂(Y-27632)治疗均可一定程度上逆转nephrin、synaptopodin、podocin和TRPC6的表达水平,二者联用效果更加明显。结论水蛭素通过抑制TRPC6依赖的Rho信号通路减轻足细胞损伤,从而改善db/db小鼠的蛋白尿。Objective To study the effects of hirudin on the damage of podocyte protein induced by high glucose in mice by establishing podocyte injury model and animal model of diabetic nephropathy.Methods Healthy 8-week-old db/m and db/db male mice,after a week of acclimatization,weightmatched mice were randomly divided into three groups:(1)db/m[db/m mice,n=6,received 0.3 mL of phosphate-buffered saline(PBS)daily by oral gavage];(2)db/db(db/db mice,n=6,received 0.3 mL of PBS daily by oral gavage);and(3)db/db+hirudin(db/db+hirudin mice,n=6,received 200 mg/kg hirudin in 0.3 mL of PBS daily by oral gavage).At 24 h after the last treatment,the mice were sacrificed;they were anesthetized with chloral hydrate administered intraperitoneally.Immortalized mouse podocytes were maintained in RPMI-1640.The cells were treated with varying doses of hirudin or high glucos,to detect the expression levels of cytoskeleton related proteins nephrin,synaptopodin,podocin and TRPC6in podocytes.Results The 24-hour urinary protein level of db/db mice treated with hirudin decreased in a time-dependent manner,while the levels of Scr and BUN also decreased,and the pathological changes of kidney were improved.In db/db mice treated with hirudin,the levels of nephrin,podocin,synaptopodin and TRPC6 recovered significantly.Compared with db/m group,F-actin in podocytes of db/db mice decreased significantly,while hirudin treatment could maintain the stability of F-actin and protect the cytoskeleton of kidney cells of db/db mice.Compared with the high glucose group,hirudin treatment reversed the expression levels of nephrin,synaptopodin,podocin and TRPC6 to some extent.Conclusion The effect of hirudin in reducing proteinuria in db/db mice is partly due to RhoA-TRPC6 signaling,resulting in the alleviation of podocyte injury.

关 键 词：糖尿病肾病 水蛭素 足细胞 

分 类 号：R587.2[医药卫生—内分泌]