氢气通过调节HO-1和HMGB1的释放对严重脓毒症小鼠肠损伤起保护作用  

Hydrogen plays a protective role in intestinal injury of mice with severe sepsis by regulating the release of heme oxygenase-1 and high mobility group protein B1

作　　者：殷玲[1] 杨曼 于洋[1] 谢克亮[1] 于泳浩[1] Yin Ling;Yang Man;Yu Yang;Xie Keliang;Yu Yonghao(Department of Anesthesiology,Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院麻醉科,天津300052

出　　处：《中华危重病急救医学》2020年第11期1372-1377,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81671888)。

摘　　要：目的探讨核因子E2相关因子2(Nrf2)通过调控血红素加氧酶-1(HO-1)/高迁移率族蛋白B1(HMGB1)通路在氢气(H_(2))治疗脓毒症小鼠肠损伤中的保护作用及机制。方法选择6~8周龄雄性野生型(WT)和Nrf2基因敲除型(Nrf2-KO)ICR小鼠各140只,体重20~25 g。按随机数字表法分别将WT小鼠及Nrf2-KO小鼠分为假手术组(Sham组)、H_(2)对照组(Sham+H_(2)组)、盲肠结扎穿孔术(CLP)致脓毒症模型组(CLP组)及H_(2)治疗组(CLP+H_(2)组),每组35只。其中Sham+H_(2)组及CLP+H_(2)组分别于术后1 h及6 h各吸入2%H_(2)60 min,Sham组和CLP组仅吸入空气。每组取20只小鼠观察术后7 d存活情况;剩余小鼠于术后24 h处死,收集腹腔灌洗液(PLF)测量细菌菌落数从而确定细菌负荷,用蛋白质免疫印迹试验(Western blotting)检测肠组织中HO-1及HMGB1蛋白表达;用免疫组化法测定肠组织HO-1表达,用免疫荧光法测定肠组织HMGB1表达。结果WT脓毒症小鼠及Nrf2-KO脓毒症小鼠7 d存活率均为0;在WT小鼠中,CLP+H_(2)组术后7 d存活率较CLP组明显升高(45%比0%,P<0.05),而在Nrf2-KO小鼠中,CLP+H_(2)组术后7 d存活率与CLP组比较差异无统计学意义(0%比0%,P>0.05)。与Sham组比较,WT及Nrf2-KO脓毒症小鼠PLF中细菌菌落数均显著增加。与CLP组相比,2%H_(2)处理能显著提高WT脓毒症小鼠的细菌清除率〔菌落数(CFU,×10^(3)):34.7±6.3比74.2±8.1,P<0.01〕,但对Nrf2-KO脓毒症小鼠无显著影响〔菌落数(CFU,×10^(3)):73.3±7.5比75.8±8.6,P>0.05〕。Western blotting、免疫组化或免疫荧光结果显示,与Sham组相比,WT和Nrf2-KO脓毒症小鼠肠组织HO-1及HMGB1表达均明显升高。与CLP组比较,2%H_(2)处理后WT脓毒症小鼠HO-1表达进一步升高〔HO-1蛋白表达(HO-1/β-actin):0.716±0.035比0.460±0.045,HO-1阳性表达(积分A值):0.703±0.135比0.430±0.116,均P<0.01〕,HMGB1表达明显降低〔HMGB1蛋白表达(HMGB1/β-actin):0.052±0.038比0.082±0.008,HMGB1阳性表达(积分A值):24.530±9.317比41.830Objective To investigate the protective effect of the hydrogen(H_(2))inhalation on intestinal injury in severe sepsis mice and its relationship with nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/high mobility group protein B1(HMGB1)pathway.Methods The 280 male wild-type(WT)and Nrf2 knockout(Nrf2-KO)ICR mice(6-8 weeks old,20-25 g weight)were assigned into 8 groups randomly(WT and Nrf2-KO mice,4 groups respectively,n=35):Sham group,H_(2) inhalation group(Sham+H_(2) group),sepsis model group which was induced by cecal ligation and puncture(CLP group)and H_(2) treatment group(CLP+H_(2) group).In Sham group,except cecal ligation and perforation,the same surgery was used.The mice in Sham group and CLP group inhaled only air for 1 hour and 6 hours,respectively,while the mice in Sham+H_(2) group and CLP+H_(2) group inhaled 2%H_(2) for 60 minutes.After modeling,20 mice randomly in each group were selected to measure 7-day natural survival rates;the remaining mice(15 mice)were killed in 24 hours after modeling,and the bacterial load was determined by collecting the peritoneal lavage fluid(PLF)of each mouse to measure the colony forming unit(CFU);and the intestinal tissue was taken after perfusion,and the intestinal tissue of 3 mice was used to extract protein for HO-1 and HMGB1 levels by using Western blotting.Intestinal tissue of 3 mice was extracted to prepare histomorphology sections for the identification of HO-1.Intestinal tissue of 3 mice was used to detect the expression of HMGB1 by immunofluorescence.Results The 7-day survival rate of WT and Nrf2-KO mice in CLP group was zero;in WT mice,the 7-day survival rate of CLP+H_(2) group was significantly higher than that of CLP group(45%vs.0%,P<0.05),while no difference of the 7-day survival rate in Nrf2-KO mice was noted between CLP and CLP+H_(2) groups(0%vs.0%,P>0.05).Compared with Sham group,the CFU in the PLF of both mice in CLP group increased significantly.Compared with CLP group,2%H_(2) treatment could significantly improve the bacterial clearance

关 键 词：肠损伤 脓毒症 氢气 核因子E2相关因子2 

分 类 号：R459.7[医药卫生—急诊医学]