中国中药杂志

2015, v.40(10) 1965-1970

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大黄素抑制HBSS诱导的肾小管上皮细胞自噬的分子机制
Molecular mechanism of emodin on inhibiting autophagy induced by HBSS in renal tubular cells

胡浩;孙伟;顾刘宝;涂玥;刘红;
HU Hao;SUN Wei;GU Liu-bao;TU Yue;LIU Hong;Research Institute of Kidney Disease,Nanjing University of Chinese Medicine;Department of Nephrology,Jiangsu Provincial Hospital of Chinese Medicine,The Affiliated Hospital of Nanjing University of Chinese Medicine;Department of Endocrinology,Jiangsu Province Official Hospital;

摘要(Abstract):

目的:探讨大黄素(emodin)对饥饿诱导的大鼠肾小管上皮细胞(NRK-52E)自噬的调节作用及其可能机制。方法:首先,用Western blot检测大黄素对Hank's平衡盐溶液(Hank's balanced salt solution,HBSS)饥饿诱导的细胞自噬标志性蛋白——哺乳动物同族物微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)Ⅰ/Ⅱ表达水平的影响;其次,用红色荧光蛋白-微管相关蛋白1轻链3(redfluorescent protein-microtubule associated protein light chain3,RFP-LC3)质粒瞬时转染NRK-52E细胞,以HBSS(1 m L)联合巴弗洛霉素A1(10 nmol·L-1)处理,予大黄素(10μmol·L-1)干预后,荧光显微镜下观察RFP-LC3荧光颗粒的变化;然后,以哺乳动物类雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)抑制剂雷帕霉素(100 nmol·L-1)干预,观察阻断m TOR信号通路对大黄素抑制细胞自噬作用的影响;最后,通过诱导内源性m TOR抑制蛋白DEPTOR(DEP domain-containing m TOR-interacting protein)过表达,进一步评估m TOR信号通路对大黄素抑制细胞自噬的影响。结果:HBSS饥饿可诱导NRK-52E细胞LC3Ⅱ蛋白高表达,大黄素干预后可逆转HBSS诱导的LC3Ⅱ蛋白表达增加;HBSS联合巴弗洛霉素A1处理后可引起RFP-LC3荧光颗粒数目增多,大黄素干预后可抑制其数目的增加;雷帕霉素与大黄素、HBSS联合干预后,雷帕霉素可以恢复HBSS诱导的NRK-52E细胞LC3Ⅱ蛋白表达水平;而过表达内源性m TOR抑制蛋白DEPTOR同样可以阻断大黄素对LC3Ⅱ蛋白表达的抑制作用。结论:大黄素可以抑制HBSS诱导的肾小管上皮细胞LC3Ⅱ蛋白表达和自噬活化,其阻断自噬的作用可能是通过m TOR信号通路介导的。
Objective: To explore the regulative effects and possible mechanisms of emodin on autophagy induced by starvation in rat' s renal tubular epithelial cells( NRK-52E). Method: Firstly,Hank's balanced salt solution( HBSS) was used to induce starvation and the protein expression of microtubule-associated protein 1 light chain 3( LC3) Ⅰ / Ⅱ,an autophagic marker of mammalian congener,was detected by Western blot with or without the treatment of emodin. Secondly,the changes of redfluorescent protein-microtubule associated protein light chain3( RFP-LC3) fluorescent particles,treated by HBSS( 1 m L) and bafilomycin A1( 10 nmol·L-1) with or without emodin,were observed through fluorescence microscopy in NRK-52 E cells transient transfected by RFP-LC3 plasmid. With the intervention of mammalian target of rapamycin m TOR inhibitor rapamycin( 100 nmol·L-1),the effect of blocking m TOR signaling pathway on autophagic inhibition of emodin was observed. Finally,the effect of m TOR signaling pathway on autophagic inhibition of emodin was further evaluated through the over-expression of endogenous m TOR inhibitory protein DEP domain-containing m TOR-interacting protein( DEPTOR). Result: HBSS hunger could induce high protein expression of LC3Ⅱ in NRK-52 E cells,and the intervention of emodin could reverse the unregulated protein expression of LC3Ⅱ induced by HBSS. The number of RFP-LC3 fluorescent particles was increased after the co-treatment of HBSS and bafilomycin A1,and this increase was inhibited by emodin. After the co-treatment of rapamycin,emodin and HBSS,the LC3Ⅱ protein expression restored in NRK-52 E cells,compared with the treatment of HBSS. Over-expression of DEPTOR could also block the inhibitive effect of emodin on LC3 Ⅱ protein expression. Conclusion: Emodin could inhibit HBSS-induced LC3Ⅱ protein expression and the activation of autophagy in NRK-52 E cells,and the effect of blocking autophagy may be mediated through m TOR signaling pathway.

关键词(KeyWords): 大黄素;自噬;肾小管上皮细胞;微管相关蛋白1轻链3;雷帕霉素
emodin;autophagy;renal tubular epithelial cells;microtubule-associated protein 1 light chain 3;rapamycin

Abstract:

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基金项目(Foundation): 国家自然科学基金面上项目(81373607);; 江苏高校优势学科建设工程资助项目

作者(Author): 胡浩;孙伟;顾刘宝;涂玥;刘红;
HU Hao;SUN Wei;GU Liu-bao;TU Yue;LIU Hong;Research Institute of Kidney Disease,Nanjing University of Chinese Medicine;Department of Nephrology,Jiangsu Provincial Hospital of Chinese Medicine,The Affiliated Hospital of Nanjing University of Chinese Medicine;Department of Endocrinology,Jiangsu Province Official Hospital;

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