免疫相关GTP酶1通过PI3K/AKT/mTOR信号通路调控巨噬细胞自噬  

Effect of immunity-related GTPase 1(IRGM1)on Macrophage autophagy mediated by the PI3/AKT/mTOR pathway

作　　者：张春福[1] 张文娟[1] 肖雅 陈勇[1] 吴源泉[1] 颜云峰[1] 魏胜梅 潘春球 吐尔地卡日-牙生 ZHANG Chun-fu;ZHANG Wen-juan;XIAO Ya;CHEN Yong;WU Yuan-quan;YAN Yun-feng;WEI Sheng-mei;PAN Chun-qiu;TU Erdikari-yasheng(Department of Emergency Intensive Care Unit(EICU),First People′s Hospital of Kashi,Kashi,Xinjiang Uygur Autonomous Region 844000,China;Department of General medicine,The Second People′s Hospital of Kashi,Kashi,Xinjiang Uygur Autonomous Region 844000,China)

机构地区：[1]喀什地区第一人民医院EICU,844000 [2]喀什地区第二人民医院全科医学,844000

出　　处：《现代消化及介入诊疗》2023年第3期311-316,共6页Modern Interventional Diagnosis and Treatment in Gastroenterology

基　　金：喀什地区应用技术研究与开发计划项目(KS202110493);自治区科技支疆项目计划(2022E02040);新疆维吾尔自治区自然科学基金面上项目(2021D01C010);新疆维吾尔自治区自然科学基金面上项目(2021D01C649);喀什地区第一人民医院“珠江学者·天山英才”合作专家工作室创新团队计划(KDYY202021)。

摘　　要：目的探讨免疫相关GTP酶1(IRGM1)调控PI3K/AKT/mTOR通路对巨噬细胞自噬活动的影响。方法小鼠RAW264.7细胞常规传代培养,siRNA-IRGM1序列通过Lipofectamine 2000转染RAW264.7细胞制备siRNA-RAW246.7细胞;利用脂多糖(LPS,20 ug/mL)诱导RAW246.7构建细胞自噬模型,分为四组:RAW246.7组,siRNA-RAW246.7组,RAW246.7+LPS组,siRNA-RAW246.7+LPS组;Western blot检测各组IRGM1及自噬相关分析分子(LC3-Ⅱ/Ⅰ、P62)、AKT及mTOR磷酸化水平;qRT-PCR检测各组IRGM1及P62表达水平;利用流式细胞学比较各组细胞凋亡率和ELISA检测各组IL-6炎症因子表达情况。结果Western blot结果显示:与RAW246.7组、siRNA-RAW246.7组比较,LPS诱导巨噬细胞(RAW246.7+LPS组、siRNA-RAW246.7+LPS组)自噬相关基因LC3-Ⅱ/LC3-Ⅰ比例蛋白表达升高(P<0.05),P62表达明显下降(P<0.05)。其中与RAW246.7+LPS组比较,siRNA-RAW246.7+LPS组自噬活动显著降低(P<0.05),同时流式细胞检测提示siRNA-RAW246.7+LPS组细胞凋亡率显著低下(P<0.05);说明沉默IRGM1抑制了巨噬细胞自噬活动。与RAW246.7+LPS组比较,siRNA-RAW246.7+LPS组的P-AKT及P-mTOR磷酸化水平低(P<0.05)。ELISA检测结果提示:与RAW246.7+LPS组比较,siRNA-RAW246.7+LPS组的IL-6表达水平低(P<0.05)。结论IRGM1与细胞自噬活动有关,并且可能通过PI3K/AKT/mTOR信号通路调控巨噬细胞自噬。Objective To investigate the effect of immunity-related GTPase 1(IRGM1)on Macrophage autophagy mediated by the PI3/AKT/mTOR pathway.Methods Mouse RAW264.7 cells were routinely subcultured.siRNA-IRGM1 sequence was transfected into RAW264.7 cells by Lipofectamine 2000 to prepare siRNA-RAW246.7 cells.RAW246.7 were induced by lipopolysaccharide(LPS,20 ug/mL)to established the autophagy model.They were divided into four groups:RAW246.7 group,siRNA-RAW246.7 group,RAW246.7+LPS group and siRNA-RAW246.7+LPS group.Western blot detected the phosphorylation levels of IRGM1,autophagy-related molecules(LC3-Ⅱ,LC3-Ⅰ,p62),AKT,mTOR,respectively;The qRT-PCR was employed to detect the expression of IRGM1 and p62 in each group.Flow cytometry was used to compare the cell apoptosis rate and ELISA was used to detect the expression of IL6 inflammatory factors in each group.Results Western blot showed that the expression of LC3-Ⅱ/LC3-Ⅰratio protein significantly increased and P62 decreased significantly in RAW246.7+LPS group and siRNA-RAW246.7+LPS group,compared with other groups.Meanwhile,flow cytometry showed that the apoptosis rate in siRNA-RAW246.7+LPS group was significantly lower than that in RAW246.7+LPS group(P<0.05);It is suggested that silent IRGM1 inhibits the autophagy activity of macrophages.The phosphorylation levels of P-AKT and P-mTOR in siRNA-RAW246.7+LPS group were lower than those in RAW246.7+LPS group(P<0.05).ELISA detection showed that the expression level of IL-6 in siRNA-RAW246.7+LPS group significantly decreased than that in RAW246.7+LPS group.Conclusion IRGM1 may regulate macrophage autophagy through PI3K/AKT/mTOR signal pathway.

关 键 词：IRGM1 自噬 脓毒血症 巨噬细胞 

分 类 号：R36[医药卫生—病理学]