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

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 mL） and bafilomycin A1 （10 nmol · L^-1） with or without emodin, were observed through fluorescence microscopy in NRK-52E cells transient transfected by RFP-LC3 plasmid. With the intervention of mammalian target of rapamycin mTOR inhibitor rapamycin （100 nmol · L^-1 ） , the effect of blocking mTOR signaling pathway on autophagic inhibition of emodin was observed. Finally, the effect of mTOR signaling pathway on autophagic inhibition of emodin was further evaluated through the over-expression of endogenous mTOR inhibitory protein DEP domain-containing mTOR-interacting protein （DEPTOR）. Result： HBSS hunger could induce high protein expression of LC3Ⅱ in NRK-52E cells, and the intervention of emodin could reverse the unregulated protein expression of LC3 Ⅱ 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 LC3 Ⅱ protein expression restored in NRK-52E 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 LC3 Ⅱ protein expression and the activation of autophagy in NRK-52E cells, and the effect of blocking autophagy may be mediated through mTOR signaling pathway.