脂多糖通过p38/MAPK通路对巨噬细胞自噬的调控作用

摘 要:【目的】 观察脂多糖(LPS)对巨噬细胞自噬的影响及p38/MAPK通路在其中的作用?【方法】 体外培养巨噬细胞株RAW264.7,分为对照组?饥饿状态激活自噬组?单纯LPS刺激组?LPS+P38抑制剂(SB203582)组和LPS+mTOR抑制剂(rapamycin)组?将前期构建的载体pcDNA3.1-GFP-LC3,转染RAW264.7,通过荧光显微镜观察各组细胞中自噬体形成情况?qRT-PCR方法检测各组中与细胞自噬相关的基因Atg5,Atg7,LC3-Ⅱ和Bnip3 mRNA表达水平的改变?利用Western blotting检测LC3-Ⅱ?p-P38?P38蛋白在各组中的表达情况,以评价LPS激活巨噬细胞自噬过程中p38/MAPK通路的作用?【结果】 在荧光显微镜下可以观察到自噬在饥饿状态组?LPS+SB203582组和LPS+rapamycin组有明显增强;qRT-PCR检测到自噬相关基因Atg5,Atg7,LC3-Ⅱ,和Bnip3 mRNA的表达在饥饿状态组?LPS+SB203582组和LPS+rapamycin组有明显增强;Western blotting 检测发现p-P38在饥饿状态组?LPS组和LPS+rapamycin组中表达明显升高; LC3-Ⅱ的表达在饥饿状态组?LPS+SB203582组和LPS+rapamycin组中表达要高于对照组和LPS组?【结论】 LPS参与巨噬细胞自噬的调控,除经典mTOR通路之外,p38/MAPK通路是其抑制通路之一?

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

Abstract: 【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+mTOR inhibitors (rapamycin) group. Fluorescent expression vector pcDNA3.1-GFP-LC3 constructed in previous work, was transfected into macrophages, and the fluorescence microscopy was used to detect the autophagosome formation in each group. qRT-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. qRT-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+rapamycin was 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 PI3K/Akt/mTOR pathway.