摘要
目的探讨高迁移率族蛋白B1(HMGB1)在内毒素脂多糖(LPS)诱导脓毒症大鼠肠黏膜损伤中的作用及机制。方法用腹腔注射LPS构建脓毒症大鼠模型;用HMGB1抑制剂EP溶液40 mg/kg干预来观察HMGB1在脓毒症中的作用,同时设磷酸盐缓冲液(PBS)对照组。制模后72 h后取各组大鼠腹主动脉血,用酶联免疫吸附试验(ELISA)检测黏膜屏障通透性血浆标志物D-乳酸和二胺氧化酶(DAO)水平;光镜下观察小肠组织黏膜病理学改变并进行肠黏膜损伤评分(Chiu评分),透射电镜下观察小肠上皮超微结构改变;用实时定量反转录-聚合酶链反应(RT-qPCR)和蛋白质免疫印迹试验(Western Blot)分别检测小肠组织中紧密连接蛋白封闭蛋白(Occludin)、炎性因子HMGB1及其下游信号分子核转录因子-κB p65(NF-κB p65)的mRNA与蛋白表达水平。结果小肠组织病理学及超微结构观察结果显示,LPS组小肠组织黏膜明显水肿,部分腺体不完整,白细胞浸润增多,微绒毛缺失,排列紊乱,紧密连接数量较PBS对照组明显减少;LPS组黏膜屏障通透性血浆标志物D-乳酸和DAO水平、炎性因子HMGB1及其下游信号分子NF-κB p65的mRNA与蛋白表达水平均较PBS对照组明显升高,小肠组织中Occludin的mRNA和蛋白表达水平较PBS对照组明显降低,提示脓毒症大鼠小肠组织肠黏膜屏障损伤,通透性增加,且结构受损。而给予HMGB1抑制剂EP干预后,LPS诱导的小肠组织肠黏膜屏障损伤得到明显改善,表现为:EP干预组小肠组织Chiu评分及血浆D-乳酸和DAO水平均较LPS组明显降低〔Chiu评分(分):1.60±0.48比3.40±0.48,D-乳酸(mmol/L):3.30±0.22比5.30±0.16,DAO(U/L):23.66±0.97比30.47±1.11,均P<0.05〕,且小肠组织中Occludin的mRNA和蛋白表达水平较LPS组明显升高〔Occludin mRNA(2-ΔΔCt):0.82±0.05比0.37±0.08,Occludin蛋白(Occludin/β-actin):1.04±0.09比0.75±0.11,均P<0.05〕,而炎性因子HMGB1及其下游信号分子NF-κB p65的mRNA和蛋白表达水平较
Objective To investigate the role and mechanism of the high mobility group box 1 (HMGB1) in intestinal mucosal barrier injury in rat with sepsis induced by endotoxin lipopolysaccharide (LPS).Methods The rats were given intraperitoneal injection of LPS to reproduce a model of sepsis. The effect of HMGB1 inhibitor EP solution (40 mg/kg) on sepsis was observed, and phosphate buffer (PBS) control group was set up. Seventy-two hours after modeling, abdominal aortic blood was obtained, and enzyme-linked immunosorbent assay (ELISA) was used to measure the plasma levels of D-lactic acid and diamine oxidase (DAO) of mucosal barrier permeability. The pathological changes of the intestinal mucosal were observed with light microscope and the Chiu score was recorded. The intestinal mucosal ultrastructural changes were observed with electron microscopy. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot were used to measure the mRNA and protein expressions of Occludin, inflammatory factor HMGB1 and its downstream signal molecule nuclear transcription factor-κB p65 (NF-κB p65) in the rat small intestine.Results The results of histopathology and ultrastructure of the small intestine showed that in the LPS group, the intestinal mucosa tissue swelled obviously, part of the glands were incomplete, the infiltration of neutrophils increased, themicrovillus cells were absent, arranged indisorder, and the number of tight connections significantly reduced compared with the PBS control group. The levels of D-lactic acid and DAO indicating mucosal barrier permeability, the levels of inflammatory factor HMGB1 and its downstream signaling molecule NF-κB p65 mRNA and protein expressions in the LPS group were significantly higher than those in the PBS control group, and the mRNA and protein expression of Occludin in the small intestine was significantly lower than that in the PBS control group, suggesting that the intestinal mucosal barrier function in septic rats was damaged, permeability inc
作者
陈思如
修光辉
周娟
刘萍
陈献忠
孙洁
凌斌
Chen Siru;Xiu Guanghui;Zhou Juan;Liu Ping;Chen Xianzhong;Sun Jie;Ling Bin(Department of Critical Care Medicine,the Fourth Affiliated Hospital of Kunming Medical University(the Second People's Hospital of Yunnan Province),Kunming 650021,Yunnan,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2020年第7期803-807,共5页
Chinese Critical Care Medicine
基金
国家自然科学基金(81901950,81360289)
云南省科技厅-昆明医科大学应用基础研究联合专项基金(2017FE467-194,2017FE468-180,2018FE001-078,2019FE001-009)
云南省基础研究计划项目(2019FB099)
云南省高层次卫生计生技术人才培养项目(H-2017060)
徐俊专家工作站(2017IC025)。
