LPS regulates macrophage autophagy through PI3K/Akt/mTOR pathway

AIM:To detect the activation of macrophage autophagy caused by lipopolysaccharide(LPS) and the possible related signaling pathways.METHODS:The macrophage cell line RAW264.7 cultured in vitro was divided into 5 groups according to the culture environment,including normal culture group,starvation-activated sautophagy group,LPS group,LPS + PI3K inhibitor(hVps34) group and LPS + mTOR inhibitor(rapamycin) group.Fluorescent expression vector pcDNA3.1-GFP-LC3 constructed in previous work was transfected into the macrophages.The fluorescence microscopy was used to detect the formation of autophagosome.The mRNA expression of autophagy-associated genes Atg5,Atg7,LC3-II and Bnip3 in the macrophages was detected by qRT-PCR.The protein levels of LC3-II,p-Akt and p-mTOR were determined by Western blotting,so as to evaluate the molecular pathways of autophagy in LPS-activated macrophages.RESULTS:The macrophages stably expressing GFP-LC3 were successfully established,which were used to observe the autophagy under fluorescence microscope.Compared with normal culture group,the autophagy in starvation group,LPS + hVps34 group and LPS + rapamycin group was significantly increased.The mRNA expression levels of Atg5,LC3-II and Bnip3 were significantly increased in starvation group,LPS + hVps34 group and LPS + rapamycin group,while in LPS group,those decreased slightly.The protein level of p-Akt in starvation group,LPS group and LPS + rapamycin group was significantly increased,while p-mTOR in starvation group,LPS + hVps34 group and LPS + rapamycin group significantly declined.LC3-II expression level in starvation group,LPS + hVps34 group and LPS + rapamycin group was higher than that in control group and LPS group.CONCLUSION:LPS regulates macrophage autophagy,and its possible pathway is the PI3K / Akt / mTOR pathway,but there are some other effective regulatory pathways.