大黄酸调控mTOR信号通路活性抑制肾小管上皮细胞自噬蛋白表达的分子机制

目的:探讨大黄酸抑制饥饿诱导的肾小管上皮(NRK-52E)细胞自噬蛋白的作用和分子机制。方法:用Hank’s平衡盐溶液(Hank’s balanced salt solution,HBSS)诱导NRK-52E细胞产生饥饿状态,在干预后的各时间点(0,0.5,1,2,6 h),首先,检测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的表达水平;其次,检测细胞哺乳动物类雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)表达及其磷酸化水平(phosphorylated-m TOR Ser2448,p-m TOR S2448);然后,以大黄酸(5 mg·L-1)、HBSS(1 m L)以及m TOR抑制剂雷帕霉素(100 nmol·L-1)单独或联合干预,分别检测其LC3 I/II,m TOR,p-m TOR S2448蛋白表达水平的变化。结果:HBSS诱导NRK-52E细胞LC3II蛋白高表达、p-m TOR S2448蛋白低表达;大黄酸与HBSS联合干预可以逆转HBSS诱导的NRK-52E细胞LC3 II和p-m TOR S2448蛋白表达水平;雷帕霉素与大黄酸、HBSS联合干预可以恢复HBSS诱导的NRK-52E细胞LC3 II蛋白表达水平。结论:HBSS抑制m TOR信号通路活性而诱导肾小管上皮细胞发生自噬;大黄酸调控m TOR信号通路活性而抑制肾小管上皮细胞自噬蛋白的表达,这可能就是其干预细胞自噬的作用和分子机制。

Molecular mechanism of rhein on inhibiting autophagic protein expression in renal tubular epithelial cells via regulating mTOR signaling pathway activation

Objective: To explore the effects and molecular mechanisms of rhein on reducing starvation-induced autophagic protein expression in renal tubular epithelial(NRK-52E)cells. Method: Hank's balanced salt solution(HBSS)was used to induce NRK-52E cells to be in the state of starvation. After the intervention of HBSS for 0,0.5,1,2 and 6 hours,firstly,the protein expression of microtubule-associated protein 1 light chain 3(LC3 I/II),which is a key protein in autophagy,was detected. Secondly,the protein expressions of mammalian target of rapamycin(mTOR)and phosphorylated-mTOR Ser2448(p-mTOR S2448)were examined. And then,after the co-treatment of rhein(5 mg·L^-1)and HBSS(1 mL)without or with mTOR inhibitor,rapamycin(100 nmol·L^-1),the protein expressions of LC3 I/II,mTOR and p-mTOR S2448 were tested,respectively. Result: HBSS could induce the up-regulation of LC3 II and the down-regulation of p-mTOR S2448 at protein expression level in NRK-52E cells. The co-treatment of rhein and HBSS could reversely regulate the protein expressions of LC3 II and p-mTOR S2448 in NRK-52E cells significantly. The co-treatment of rapamycin,rhein and HBSS could recover the level of LC3 II protein expression in HBSS-intervened NRK-52E cells. Conclusion: HBSS induces autophagy in renal tubular epithelial cells by inhibiting mTOR signaling pathway activation. Rhein reduces the autophagic protein expression in renal tubular epithelial cells through regulating mTOR signaling pathway activation,which is the possible effects and molecular mechanisms.