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
周慧男 1吴成林 2刘剑飞 2张陈 3周丽君 4钦可为5
作者信息
- 1. 南方医科大学第二临床学院,广州 510515;解放军总医院第六医学中心,中心实验室,北京 100048
- 2. 解放军总医院第六医学中心,中心实验室,北京 100048;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
- 3. 解放军总医院第六医学中心,中心实验室,北京 100048
- 4. 南方医科大学第二临床学院,广州 510515
- 5. 解放军总医院第六医学中心,中心实验室,北京 100048;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;华南理工大学医学院,广州 510006
- 折叠
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,12h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、cleaved-PARP、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.
Abstract
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 cleaved-PARP,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Key words
Vibrio vulnificus/resveratrol/poly ADP-ribose polymerase/caspase-3/Sirt1/apoptosis/macrophage引用本文复制引用
基金项目
北京市自然科学基金项目(7242135)
国家自然科学基金项目(82202382)
出版年
2024
国家科技期刊平台
NSTL
万方数据