摘要
目的探讨PI3K/AKT通路在巨噬细胞糖酵解中的作用,拟从代谢重编程的角度阐明巨噬细胞M1极化的机制。方法以脂多糖(LPS)处理RAW264.7细胞建立巨噬细胞M1极化模型;使用2-脱氧-D-葡萄糖(2-DG)抑制糖酵解过程;使用LY294002抑制PI3K/AKT活性;Seahorse XF糖酵解速率试剂盒检测巨噬细胞糖酵解水平;q-PCR方法检测巨噬细胞M1型标记(诱导型一氧化氮合酶、肿瘤坏死因子α、白细胞介素6和白细胞介素1β),葡萄糖酵解相关基因(Glut1、HK2、Idha和Aldoa);Western blot方法检测p-p38、p38、p-ERK、ERK、p-p65、p65、IκB、p-AKT、AKT、p-mTOR、mTOR、缺氧诱导因子1α(HIF-1α)和GAPDH水平。结果在LPS诱导的RAW264.7细胞M1极化模型中,p-mTOR和p-AKT水平升高,HIF-1α表达水平升高,糖酵解相关基因水平升高,细胞外酸化率升高,表明LPS诱导细胞糖酵解水平升高;2-DG抑制糖酵解,降低M1极化相关因子的mRNA水平;LY294002抑制PI3K/AKT活性,降低p-mTOR水平和HIF-1α的蛋白水平,降低糖酵解相关基因水平,进而抑制RAW264.7糖酵解水平和M1极化过程。结论在RAW264.7细胞中PI3K/AKT信号通路通过激活mTOR信号通路,激活糖酵解,从而激活巨噬细胞M1极化过程。
Objective To expolre the role of PI3K/AKT pathway in macrophage glycolysis,and to elucidate the mechanism of macrophage M1 polarization from the perspective of metabolic reprogramming.Methods RAW264.7 cells were treated with lipopolysaccharide(LPS)to establish a macrophage M1 polarization cell model;2-Deoxy-D-Glucose(2-DG)was used to inhibit glycolysis;LY294002 was used to inhibit PI3K/AKT activity;Seahorse XF assay kit was used to detect glycolysis levels in macrophages;q-PCR was used to detect macrophage M1-type markers(inducible nitric oxide synthase,tumor necrosis factor alpha,interleukin 6 and interleukin 1 beta),glycolysis-related genes(Glut1,HK2,Idha and Aldoa);p-p38,p38,p-ERK,ERK,p-p65,p65,IκB,p-AKT,AKT,p-mTOR,mTOR,hypoxia inducible factor 1 alpha and GAPDH levels were detected by Western blot.Results In the LPS-induced M1 polarization model in RAW264.7 cells,the levels of p-mTOR and p-AKT,the protein level of HIF-1α,the levels of glycolysis-related genes and the level of glycolysis was all increased.2-DG inhibited glycolysis and decreased the mRNA level of M1 polarization-related genes.LY294002 inhibited PI3K/AKT activity,decreased p-mTOR levels and protein levels of HIF-1α,decreased glycolysis-related gene levels,and inhibited RAW264.7 glycolysis levels and M1 polarization process.Conclusions In mouse macrophages,the PI3K/AKT signalling pathway may activate glycolysis by activating the mTOR signalling pathway in RAW264.7 cells,thereby activating glycolysis and promoting macrophage M1 polarization process.
作者
马佳睿
徐芳芷
左惠演
满永
张曦月
窦琳
唐蔚青
刘进
黄秀清
黎健
Ma Jiarui;Xu Fangzhi;Zuo Huiyan;Man Yong;Zhang Xiyue;Dou Lin;Tang Weiqing;Liu Jin;Huang Xiuqing;Li Jian(Beijing Hospital,Beijing Institute of Geriatrics,The Key Laboratory of Geriatrics,National Center of Gerontology,National Health Commission,Institute of Geriatric Medicine,Chinese Academy of Medical Science,Beijing 100730,P.R.China;Beijing Key Laboratory of Gene Resource and Molecular Development,Laboratory of Neuroscience and Brain Development,College of Life Sciences,Beijing Normal University,Beijing 100875,China)
出处
《中国心血管杂志》
2023年第2期145-151,共7页
Chinese Journal of Cardiovascular Medicine
基金
国家自然科学基金(81770858、81600618)。
