转录因子EB增强巨噬细胞自噬功能及逆转内毒素耐受的作用  

Transcription factor EB enhances macrophage autophagy and reverses endotoxin tolerance

作　　者：杨婷 刘鑫 蒋青松[1,2] 王宇洁 时新惠 杨雄 刘思佳 李小丽 YANG Ting;LIU Xin;JIANG Qingsong;WANG Yujie;SHI Xinhui;YANG Xiong;LIU Sijia;LI Xiaoli(Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology,Faculty of Pharmacology,College of Pharmacy,Chongqing Medical University,Chongqing;Clinical Research Center,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,China;不详)

机构地区：[1]重庆医科大学药学院药理学系,重庆高校生物化学与分子药理学重点实验室,重庆 [2]陆军军医大学(第三军医大学)第一附属医院临床研究中心,重庆 [3]不详

出　　处：《陆军军医大学学报》2025年第8期794-806,共13页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(82172133);重庆市自然科学基金面上项目(CSTB2022NSCQ-MSX0214)。

摘　　要：目的 研究转录因子EB(transcription factor EB,TFEB)在内毒素耐受巨噬细胞中的作用。方法 采用脂多糖(LPS)诱导建立内毒素耐受细胞模型,将RAW264.7细胞分为空白组(Medium组)、5ng/mLLPS组(LPS5组)、100ng/mLLPS组(LPS100组)、5ng/mLLPS+100ng/mLLPS组(Tolerance组)。采用ELISA法检测炎症因子TNF-α和IL-6的释放水平;Western blot和免疫荧光法检测自噬相关蛋白LC3和P62以及TFEB在细胞质和细胞核中的分布;通过慢病毒过表达TFEB或siRNA敲低TFEB后,观察耐受细胞的自噬水平和细菌清除能力的变化。结果 与LPS 100组相比,Tolerance组细胞炎症因子TNF-α和IL-6的释放水平以及细菌清除能力显著降低(P<0.01);LC3水平降低,P62水平升高,细胞质和细胞核中TFEB的水平均显著降低(P<0.01);过表达TFEB可显著上调Tolerance组细胞中LC3水平,降低P62水平,并增强细菌清除能力(P<0.01)。siRNA敲低TFEB对Tolerance组LC3和P62水平以及细菌清除能力无明显影响。结论 过表达TFEB能恢复内毒素耐受细胞的自噬功能,增强细菌清除能力,从而改善脓毒症的免疫抑制状态,有望成为脓毒症防治的潜在靶点。Objective To investigate the role of transcription factor EB(TFEB)in endotoxin-tolerant macrophages.Methods The RAW264.7 cells were divided into blank group(DMEM medium),LPS 5 group(5 ng/mL LPS treatment for 4 h),LPS 100 group(100 ng/mL LPS treatment for 4 h),and tolerance group(5 ng/mL LPS for 12 h followed by 100 ng/mL LPS for 4 h).The releases of inflammatory factors TNF-αand IL-6 were measured using ELISA.Western blotting and immunofluorescence assay were used to evaluate the distribution of autophagy-related proteins LC3 and P62,as well as TFEB in the cytoplasm and nucleus.Lentiviral overexpression of TFEB or siRNA-mediated knockdown of TFEB were performed to observe the changes in autophagy levels and bacterial clearance ability in the tolerant cells.Results The cells in the tolerance group had significantly lower contents of TNF-αand IL-6,as well as reduced bacterial clearance ability(P<0.01),down-regulated LC3 expression while up-regulated P62 level,and decreased expression of TFEB in both the cytoplasm and nucleus(P<0.01)when compared with the cells of the LPS 100 group.Overexpression of TFEB significantly increased LC3 level,reduced P62 level,and enhanced bacterial clearance ability in the endotoxin-tolerant cells(P<0.01).In contrast,siRNA-mediated knockdown of TFEB had no significant impacts on LC3 and P62 expression levels or bacterial clearance ability.Conclusion Overexpression of TFEB can restore the autophagy of endotoxin-tolerant cells and enhance their bacterial clearance capacity,thereby alleviating the immunosuppressive state of sepsis.These findings suggest that TFEB holds promise as a potential therapeutic target for the prevention and treatment of sepsis.

关 键 词：脓毒症 免疫抑制 内毒素耐受 转录因子EB 自噬 

分 类 号：R392.3[医药卫生—免疫学] R3945.3[医药卫生—基础医学] R631.2