Role Of PI3K/Akt/mTOR Signaling Pathway In Autophagy Of Rat Hepatic Stellate Cells Induced By Lipopolysaccharide

Objective:To investigate the role of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/ mammalian target of rapamycin(mTOR)signaling pathway in the autophagy of rat hepatic stellate cells induced by lipopolysaccharide(LPS).Methods:(1)HSC-T6 cells were cultured in vitro and randomly divided into control group,LPS group,rapamycin group,LPS+rapamycin group,LY294002 group,LPS+LY294002 group,SC79 group and LPS+SC79 group.(2)After treatment,the changes of autophagy lysosome were observed by mono-dansylcadaverine(MDC)staining.(3)the expression of microtubule-associated protein light chain?(LC3?)was detected by immunofluorescence assay.(4)the expressions of p-Akt,p-mTOR,Akt,mTOR,LC3?and Beclin1 in each group was detected by Western blot.(5)the level of LC3?and Beclin1 mRNA in each group was detected qRT-PCR.Results:(1)The autophagy lysosome of LPS group,Rapa group,LPS+Rapa group,LY294002 group and LPS+LY294002 group increased significantly,and SC79 group decreased significantly(P <0.05).There was no significant difference in autophagic lysosome content between LPS+Rapa group and LPS+LY294002 group compared with LPS group(P>0.05),and the autophagic lysosome content was significantly reduced between LPS+SC79 group and LPS group(P <0.05).(2)The LC3?green stained granules of LPS group,Rapa group,LPS+Rapa group,LY294002 group and LPS+LY294002 group increased significantly,and SC79 group decreased significantly.Compared with LPS group,LPS+Rapa group and LPS+LY294002 group had no significant difference in the content of green-stained particles(P>0.05),while LPS+SC79 group had significantly lower content of fluorescent spots than LPS group(P <0.05).(3)Western blot showed that the levels of LC3?and Beclin1 in Rapa group and LY294002 group were significantly increased than those in control group,the levels of p-mTOR in Rapa group were significantly lower than those in control group,the levels of p-Akt and p-mTOR in LY294002 group were significantly lower than those in control group,the levels of LC3?and Beclin1 in SC79 group were significantly lower than those in control group,the levels of p-Akt and p-mTOR in SC79 group were significantly increased than those in control group(P<0.05).(4)Western blot showed there were no significant differences in the expression levels of LC3?,Beclin1,p-Akt and p-mTOR between LPS+Rapa group and LPS+ LY294002 group compared with LPS group(P>0.05).The levels of LC3?and 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).(5)qRT-PCR showed that the levels of LC3?and Beclin1 in Rapa group and LY294002 group were significantly increased than those in control group,the levels of LC3?and Beclin1 in SC79 group were significantly lower than those in control group(P<0.05).(6)qRT-PCR showed that there was no significant difference in LC3?and Beclin1 mRNA between LPS+Rapa group and LPS+LY294002 group compared with LPS group(P>0.05).LC3?and Beclin1 mRNA levels were significantly decreased in LPS+SC79 group compared with LPS group(P<0.05).Conclusion:(1)LPS promotes autophagy in HSC-T6.(2)PI3K/Akt/mTOR signaling pathway may be involved in the autophagy regulation of HSC-T6 cells,and the level of autophagy increase in HSC-T6 cells by inhibiting this pathway,.(3)LPS may promote autophagy in HSC-T6 cells by inhibiting PI3K/Akt/mTOR signaling pathway.