黄芪甲苷通过调控肾素原受体-自噬途径改善嘌呤霉素诱导肾足细胞骨架损伤的作用研究  

Functional research of Astragaloside IV ameliorating puromycin induced cytoskeleton injury of nephropodocytes by regulating renin receptor autophagy pathway

作　　者：曾又佳[1] 贺良平 张冰[3] ZENG You-jia;HE Liang-ping;ZHANG Bing(Shenzhen Traditional Chinese Medicine Hospital,Guangdong Shenzhen 518033,China;Chongqing Jiulongpo District Hospital of traditional Chinese Medicine,Chongqing 400000,China;The Fourth Clinical Medical College,Guangzhou University of Chinese Medicine,Guangdong Shenzhen 518033,China)

机构地区：[1]深圳市中医院,广东深圳518033 [2]重庆九龙坡中医院,重庆400000 [3]广州中医药大学第四临床医学院,广东深圳518033

出　　处：《中国中医基础医学杂志》2020年第10期1479-1483,共5页JOURNAL OF BASIC CHINESE MEDICINE

基　　金：国家自然科学基金资助项目(81202671)-基于肾素原受体双通路调控探讨益气活血法治疗嘌呤霉素诱导肾足细胞病变的作用机制;广东省自然科学基金资助项目(2017A030313518)-GLCCI1-GR/HSP27/Rho家族信号通路在肾足细胞糖皮质激素应答修复中的作用和益气活血法的促敏机制。

摘　　要：目的:探讨肾素原受体(prorenin-receptor,PRR)及自噬在嘌呤霉素诱导的肾足细胞骨架损伤中的作用及黄芪甲苷(astragaloside IV,AS-IV)的改善机制。方法:体外培养人肾足细胞株AB8/13,30μg/ml嘌呤霉素(puromycin,PAN)孵育0~48 h后,免疫印迹法检测足细胞PRR蛋白表达。PRR siRNA干扰足细胞48 h,PAN干预24 h,检测PRR及自噬蛋白(LC3II,P62)表达,免疫荧光法检测细胞骨架。分化后足细胞分为对照组、PAN组(30μg/ml)、PAN(30μg/ml)+不同剂量AS-IV干预组(5μg/ml,25μg/ml,50μg/ml)孵育48 h,免疫荧光法检测各组细胞骨架蛋白F-actin、synaptopodin表达,免疫印迹法检测各组synaptopodin、PRR、LC3II、P62蛋白表达。结果:PRR在PAN刺激早期表达升高,24 h达峰,48 h显著下降。PRR小RNA干扰后,足细胞PRR表达下调、LC3II表达下调,但自噬抑制相关蛋白P62显著升高,骨架损伤提前发生;低中高剂量AS-IV均可改善嘌呤霉素诱导的足细胞变构,其中低、中剂量(5μg/ml,25μg/ml)AS-IV改善最明显,AS-IV干预后较PAN组PRR蛋白显著升高,LC3II蛋白表达上调,P62表达下调,synaptopodin表达上调。结论:PRR可能通过介导自噬在PAN诱导的肾足细胞损伤中发挥保护作用,低中剂量AS-IV可能通过提高PRR、稳定自噬发挥肾足细胞保护作用。Objective:To explore the role of prorenin-receptor(PRR)and autophagy in puromycin induced podocyte cytoskeleton injuries and to explain the potential mechanism of astragaloside IV’s renal podocyte protection in this model.Methods:Podocytes(AB8/13)were incubated in 30μg/ml puromycin for 0,3,6,12,24,36,48 hours,respectively.PRR protein was detected by Western Blot assay(WB).PRR siRNA and negative control siRNA(GAPDH siRNA)were transfected into podocytes,respectively.Then puromycin was used to treat podocyte for 24 h.WB was used to test PRR protein,and autophagy related proteins(LC3II and P62).Immunofluorescence(IF)was used to detect the cell cytoskeleton related proteins(F-actin,synaptopodin)expression.Differentiated podocytes were incubated in puromycin,puromycin and different dose of astragaloside IV(5μg/ml,25μg/ml,50μg/ml).IF and WB were used to detect cell cytoskeleton related protein,PRR protein and autophagy related proteins.Results:PRR protein expression was upregulated in the early stage,but downregulated in the late stage of stimulation.24 h was the peak of PRR protein expression.After PRR siRNA interference,the PRR protein was downregulated significantly,the LC3II expression was also downregulated,but autophagy inhibition related protein-P62 protein expression increased.Meanwhile,the podocyte cytoskeleton injuries occurred prior to that in control siRNA group.Low,moderate and high dose astragaloside IV all obviously reduced the puromycin induced podocyte cytoskeleton change.Meanwhile astragaloside IV could increase PRR protein and LC3II protein expression,down-regulate P62 protein expression in this model.Conclusion:Astragaloside IV plays a renal protective role in puromycin induced podocyte cytoskeleton injuries,partly by regulating PRR-autophagy pathway.

关 键 词：黄芪甲苷 肾素原受体 自噬 足细胞 嘌呤霉素 

分 类 号：R285.5[医药卫生—中药学]