脂多糖通过PI3K/Akt/mTOR通路调控巨噬细胞自噬

LPS regulates macrophage autophagy through PI3K/Akt/mTOR pathway

目的:观察脂多糖对巨噬细胞自噬活化的影响及相关信号通路的探讨。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、单纯脂多糖(LPS)刺激组、LPS+PI3K抑制剂(hVps34)组和LPS+mTOR抑制剂(雷帕霉素)组。构建荧光真核表达载体pcDNA3.1-GFP-LC3,转染巨噬细胞,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Atg5、Atg7、LC3-II和Bnip3 mRNA表达水平的改变。利用Western blotting检测LC3-II、p-Akt和p-mTOR蛋白在各组中的表达情况,以评价LPS激活巨噬细胞自噬的分子通路。结果:成功构建稳定表达GFP-LC3的巨噬细胞,在荧光显微镜下可以观察到自噬在饥饿状态组、LPS+hVps34组和LPS+雷帕霉素组均有明显增强;qRT-PCR检测到Atg5、LC3-II和Bnip3 mRNA的表达在饥饿状态组、LPS+hVps34组和LPS+雷帕霉素组均有明显增强,而在LPS组中略微下降;Western blotting检测发现p-Akt在饥饿状态组、LPS组和LPS+雷帕霉素组中表达明显升高;p-mTOR在饥饿状态组、LPS+hVps34组和LPS+雷帕霉素组表达明显下降;LC3-II的表达在饥饿状态组、LPS+hVps34组和LPS+雷帕霉素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,其可能的信号通路为PI3K/Akt/mTOR通路,但仍存在其它有效的调控通路。

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 santophagy group, LPS group, LPS ＋ PI3K inhibitor （hVps34） group and LPS ＋ roTOR 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 deter- mined by Western blotting, so as to evaluate the molecular pathways of autophagy in LPS-activated macrophages. RE- SULTS： The macrophages stably expressing GFP-LC3 were successfully established, which were used to observe the auto- phagy 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 AtgS, 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, Lt~ 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 thePI3K/Akt/mTOR pathway, but there are some other effective regulatory pathways.