PI3K/Akt/mTOR信号通路在脂多糖诱导的大鼠肝星状细胞自噬中的作用

Role of PI3K/Akt/mTOR Signaling Pathway in Autophagy of Rat Hepatic Stellate Cells Induced by Lipopolysaccharide

该文探讨了磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在脂多糖(LPS)诱导的大鼠肝星状细胞-T6(HSC-T6)自噬中的作用。体外培养HSCT6细胞,随机分为对照组、LPS组、雷帕霉素(Rapamycin,Rapa)组、LPS+Rapa组、LY294002组、LPS+LY294002组,SC79组、LPS+SC79组,各组经相应处理后,单丹磺酰尸胺(MDC)染色法观察自噬溶酶体变化;细胞免疫荧光法检测各组微管相关蛋白轻链Ⅱ(LC3Ⅱ)表达;Western blot检测各组通路蛋白p-Akt、p-mTOR、Akt、mTOR及自噬相关蛋白LC3Ⅱ、Beclin1的表达;qRT-PCR检测各组LC3Ⅱ和Beclin1 mRNA的表达。结果显示,LPS+Rapa组、LPS+LY294002组较LPS组的自噬溶酶体、LC3Ⅱ荧光亮点含量无明显差异(P>0.05),LPS+SC79组较LPS组的自噬溶酶体、LC3Ⅱ荧光亮点含量明显减少(P<0.05);Western blot显示,LPS+Rapa组、LPS+LY294002组较LPS组LC3Ⅱ、Beclin1、p-Akt、p-mTOR蛋白表达水平无明显差异(P>0.05),LPS+SC79组较LPS组LC3Ⅱ、Beclin1含量明显减少,p-Akt、p-mTOR蛋白表达水平明显增加(P<0.05);qRT-PCR显示LPS+Rapa组、LPS+LY294002组较LPS组LC3Ⅱ、Beclin1 mRNA含量无明显差异(P>0.05),LPS+SC79组较LPS组LC3Ⅱ、Beclin1 mRNA含量明显减少(P<0.05)。该项研究结果表明,LPS可能通过抑制PI3K/Akt/mTOR信号通路促进HSC-T6细胞自噬。

This work was to investigate the role of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway in the lipopolysaccharide(LPS)-induced autophagy of rat hepatic stellate cells.HSC-T6 cells were cultured in vitro and randomly divided into control group,LPS group,Rapa group,LPS+Rapa group,LY294002 group,LPS+LY294002 group,SC79 group and LPS+SC79 group.After treatment,the changes of autophagic lysosome were observed by mono-dansylcadaverine(MDC)staining;the expression of microtubule-associated protein light chainⅡ(LC3Ⅱ)was detected by immunofluorescence assay;the expressions of p-Akt,p-mTOR,Akt,mTOR,LC3Ⅱand Beclin1 in each group were detected by Western blot;the expressions of LC3Ⅱand Beclin1 mRNA in each group were detected by qRT-PCR.The results showed that there were no significant differences in the levels of autophagic lysosome and LC3Ⅱfluorescent spots in comparison between LPS group and LPS+Rapa group,or LPS+LY294002 group(P>0.05),but the levels of autophagic lysosome and LC3Ⅱfluorescent spots particles in LPS+SC79 group were significantly lower than those in LPS group(P<0.05).Western blot showed that there were no significant differences in the levels of LC3Ⅱ,Beclin1,p-Akt and p-mTOR in comparison between LPS group and LPS+Rapa group,or LPS+LY294002 group(P>0.05),but the levels of LC3Ⅱand Beclin1 in LPS+SC79 group were significantly lower than those in LPS group,and the levels of p-Akt and p-mTOR protein were significantly increased(P<0.05).qRT-PCR showed that there were no significant differences in the expressions of LC3Ⅱand Beclin1 mRNA in comparison between LPS group and LPS+Rapa group,or LPS+LY294002 group(P>0.05),but the expressions of LC3Ⅱand Beclin1 mRNA were significantly decreased in LPS+SC79 group compared with LPS group(P<0.05).It is possibly that LPS promote autophagy in HSC-T6 cells by inhibiting PI3K/Akt/mTOR signaling pathway.