脂多糖通过p38／MAPK通路对巨噬细胞自噬的调控作用

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

【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+m TOR抑制剂(rapamycin)组。将前期构建的载体pc DNA3.1-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。q RT-PCR方法检测各组中与细胞自噬相关的基因Atg5,Atg7,LC3-Ⅱ和Bnip3 m RNA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;q RT-PCR检测到自噬相关基因Atg5,Atg7,LC3-Ⅱ,和Bnip3 m RNA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m TOR通路之外,p38/MAPK通路是其抑制通路之一。

[ Objective ] To detect the role of P38/MAPK signaling pathways in the activation of autophagy in macrophages caused by lipoP0lysaccharide. [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 peDNA3.1-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 AtgS, 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＋ rapamyein group were significantly increased, qRT-PCR detected that autophagy-related genes Atg5, Atg7, LC3- I1 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- I] expression 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.