Sirt1对创伤弧菌脓毒症中巨噬细胞凋亡的影响及机制  

Effects of Sirt1 on macrophage apoptosis in Vibrio vulnificus sepsis

作　　者：周慧男 吴成林 刘剑飞 张陈 周丽君[1] 钦可为 ZHOU Huinan;WU Chenglin;LIU Jianfei;ZHANG Chen;ZHOU Lijun;QIN Kewei(The Second School of Clinical Medicine,Southern Medical University,Guangzhou 510515,China;Central Laboratory,the Sixth Medical Centre,Chinese PLA General Hospital,Beijing 100048,China;Senior Department of Otolaryngology Head and Neck Surgery,the Sixth Medical Centre,Chinese PLA General Hospital,Beijing 100048,China;School of Medicine,South China University of Technology,Guangzhou 510006,China)

机构地区：[1]南方医科大学第二临床学院,广州510515 [2]解放军总医院第六医学中心,中心实验室,北京100048 [3]解放军总医院第六医学中心,耳鼻咽喉头颈外科医学部研究所,北京100048 [4]华南理工大学医学院,广州510006

出　　处：《军事医学》2024年第8期601-607,共7页Military Medical Sciences

基　　金：北京市自然科学基金项目(7242135);国家自然科学基金项目(82202382)。

摘　　要：目的在创伤弧菌感染体内外模型探究沉默调节蛋白1(Sirt1)对巨噬细胞凋亡的调控作用及其分子机制。方法通过遗传霉素G418筛选构建Sirt1稳定过表达的小鼠RAW264.7巨噬细胞;采用CCK-8法检测Sirt1过表达在创伤弧菌细胞溶素(VVC)对RAW264.7细胞损伤效应中的调控作用;Western印迹法检测VVC处理RAW264.7细胞中Sirt1过表达及Sirt1激活剂白藜芦醇(RSV)预处理对凋亡相关蛋白聚腺苷二磷酸核糖聚合酶(PARP)、裂解型-PARP(cleaved-PARP)、胱天蛋白酶3(caspase3)、裂解型胱天蛋白酶3(cleaved-caspase3)表达的影响;建立小鼠创伤弧菌脓毒症模型并腹腔注射RSV,12 h后取各组小鼠肺、脾、肝组织,采用免疫组织化学法检测凋亡蛋白cleaved-caspase3的表达,同时收集小鼠腹腔巨噬细胞并用Western印迹法检测凋亡通路蛋白PARP、cleavedPARP、caspase3、cleaved-caspase3及乙酰化p53蛋白的表达变化。结果过表达Sirt1能够降低VVC诱导小鼠巨噬细胞RAW264.7细胞损伤;过表达Sirt1及RSV预处理可降低VVC刺激下RAW264.7细胞中凋亡相关蛋白的表达;在小鼠创伤弧菌脓毒症模型中,RSV预防性给药能降低肺、脾、肝等组织中凋亡标志物cleaved-caspase3的表达,同时降低小鼠腹腔巨噬细胞中凋亡相关蛋白cleaved-PARP、cleaved-caspase3及乙酰化p53的表达。结论Sirt1能抑制p53的乙酰化并减少小鼠巨噬细胞的凋亡,在创伤弧菌脓毒症中起保护作用。Objective To investigate the role of silencing regulatory protein 1(Sirt1)in the regulation of Vibrio vulnificus sepsis-induced macrophage apoptosis and the molecular mechanisms.Methods Mouse RAW264.7 macrophages which stably overexpressed Sirt1 were constructed and screened by genistein G418.CCK-8 analysis was used to detect the proliferation of cells in the control group and Sirt1-Flag group.The changes of expression levels of apoptosis-associated protein poly ADP-ribose polymerase(PARP),cleaved-PARP,caspase3,cleaved-caspase3 and acetylated p53 in different treatment groups were detected via Western blotting.A Vibrio vulnificus sepsis model in mice was established,and the expression levels of apoptosis-associated protein cleaved-caspase3 in the lung,spleen and liver of mice of different treatment groups were detected by immunohistochemistry.Results Overexpression of Sirt1 reduced VVC-induced RAW264.7 cell damage.Overexpression of Sirt1 as well as RSV pretreatment lowered the expression of apoptosis-associated protein cleavedPARP,cleaved-caspase3 and acetylated p53 in VVC-stimulated RAW264.7 cells and mouse peritoneal macrophages.In the mouse model of Vibrio vulnificus sepsis,therapeutic administration of RSV reduced the expression of apoptosis-associated protein marker cleaved-caspase3 in lung,spleen and liver tissues.Conclusion Sirt1 can inhibit p53 acetylation and reduces apoptosis in mouse macrophages,which helps protect against Vibrio vulnificus sepsis.

关 键 词：创伤弧菌 白藜芦醇 聚腺苷二磷酸核糖聚合酶 胱天蛋白酶3 沉默调节蛋白1 细胞凋亡 巨噬细胞 

分 类 号：R378.3[医药卫生—病原生物学]