Preliminary Study of p38 / MAPK Pathway in LPS Regulated Macrophages Autophagy

【Objective】 To detect the role of P38 / MAPK signaling pathways in the activation of autophagy in macrophages caused by lipopolysaccharide. 【Methods】 The macrophage cell line RAW264.7 cultured in vitro, and was divided into five groups according culture environment, including normal culture group, starvation activates autophagy group, simple LPS group, LPS +P38 inhibitor(SB203582) group and LPS+m TOR inhibitors(rapamycin) group. Fluorescent expression vector pc DNA3.1-GFP-LC3 constructed in previous work, was transfected into macrophages, and the fluorescence microscopy was used to detect the autophagosome formation in each group. q RT-PCR was used to detect autophagy associated genes Atg5, Atg7, LC3-Ⅱ and Bnip3 expression levels in each group.Western Blot was used to test LC3-Ⅱ, p-P38, P38 expression in each group, so as to evaluate LPS activated macrophages autophagy molecular pathways. 【Results】 We successfully got the stably expressing GFP-LC3 macrophages, which can be used to observe the autophagy under a fluorescence microscope. The autophages in starvation group, LPS stimulation +SB203582 group and LPS stimulation+ rapamycin group were significantly increased. q RT-PCR detected that autophagy-related genes Atg5, Atg7, LC3-Ⅱ and Bnip3 expression levels were significantly increased in starvation group, LPS stimulation +SB203582 group and LPS stimulation +rapamycin group. Western Blot showed that p-P38 in starvation group, LPS group and LPS stimulation +rapamycin group was significantly increased. LC3-Ⅱexpression level in starvation group, LPS stimulation+SB203582 group and LPS stimulation+rapamycinwas higher than control group and LPS group. 【Conclusions】 LPS can regulate macrophage autophagy, and p38 / MAPK pathway is one of its down-regulated pathways besides the classic PI3 K / Akt / m TOR pathway.