氢气对野生型及Nrf2基因敲除型脓毒症小鼠肺损伤的影响:与Nrf2/HO-1/HMGB1通路的关系  

Effects of hydrogen on lung injury in wild-type and Nrf?2 gene knockout mice: relationship with Nrf?2/HO-1/HMGB1 pathway

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

机构地区：[1]天津医科大学总医院麻醉科,天津市麻醉学研究所,300052

出　　处：《中华危重病急救医学》2019年第7期862-866,共5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(Nrf 2)通过调控Nrf 2/血红素加氧酶-1(HO-1)/高迁移率族蛋白B1(HMGB1)通路在氢气(H2)治疗脓毒症小鼠肺损伤中的关键作用.方法雄性野生型(WT)和Nrf 2基因敲除型(Nrf 2-KO)ICR小鼠各120只,分别按随机数字表法分为假手术组(Sham组)、H2对照组(Sham+H2组)、盲肠结扎穿孔术(CLP)致脓毒症模型组(CLP组)及H2干预组(CLP+H2组),每组30只.采用CLP制备脓毒症模型,Sham组和Sham+H2组除盲肠结扎和穿孔外,均采用相同的手术方式.Sham+H2组及CLP+H2组小鼠分别于术后1 h、6 h吸入2% H2各1 h,Sham组和CLP组仅吸入空气.制模后每组各取20只小鼠观察7 d存活情况;其他小鼠于制模后24 h处死取肺组织,采用酶联免疫吸附试验(ELISA)检测超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性及丙二醛(MDA)含量,采用蛋白质免疫印迹试验(Western Blot)检测HO-1、HMGB1表达,采用免疫荧光法检测HO-1阳性表达.结果与Sham组相比,WT及Nrf 2-KO小鼠CLP组7 d存活率均明显降低〔WT小鼠:0%(0/20)比100%(20/20),Nrf 2-KO小鼠:0%(0/20)比100%(0/20),均P<0.05〕;WT小鼠中CLP+H2组7 d存活率较CLP组明显升高〔40%(8/20)比0%(0/20),P<0.05〕,而Nrf 2-KO小鼠中CLP+H2组与CLP组7 d存活率相比差异无统计学意义〔0%(0/20)比0%(0/20),P>0.05〕.在WT小鼠中,与Sham组相比,CLP组肺组织SOD和CAT活性均明显降低〔SOD(kU/g):131.30±28.21比251.00±22.84,CAT(kU/g):13.43±1.52比20.76±1.63,均P<0.01〕,MDA含量及HO-1、HMGB1表达均明显升高〔MDA(μmol/g):6.26±1.18比 4.16±0.58,HO-1/β-actin :0.160±0.045 比 0.023±0.005,HMGB1/β-actin :0.656±0.055 比 0.005±0.001,均P<0.05〕;与CLP组相比,CLP+H2组肺组织SOD、CAT活性及HO-1表达均明显升高〔SOD(kU/g):220.32±35.06 比 131.30±28.21,CAT(kU/g):18.95±2.49 比 13.43±1.52,HO-1/β-actin :0.376±0.025 比0.160±0.045,均P<0.01〕,MDA含量及HMGB1表达均明显降低〔MDA(μmol/g):4.26±0.75比6.26±1.18, HMGB1/β-actin :0.343±0.040比0.656±0.Objective To investigate the key role of nuclear factor E2-related factor 2 (Nrf?2) in the treatment of lung injury in sepsis mice by regulating Nrf?2/heme oxygenase-1 (HO-1)/high mobility group protein B1 (HMGB1) pathway. Methods 120 male wild type (WT) and 120 Nrf?2 knockout (Nrf?2-KO) ICR mice were randomly divided into Sham group, H2 control group (Sham+H2 group), cecal ligation and puncture (CLP) induced sepsis model group (CLP group) and H2 intervention group (CLP+H2 group), with 30 mice in each group. The sepsis model was reproduced by CLP. The same operation was done in Sham group and Sham+H2 group except CLP. The mice in Sham+H2 group and CLP+H2 group were challenged by 2% H2 for 1 hour at 1 hour and 6 hours after operation respectively, while the mice in Sham group and CLP group only inhaled air. Twenty mice in each group were collected to observe the 7-day survival. The other mice were sacrificed at 24 hours after the reproduction of model, and the lung tissues were harvested. The activities of superoxide dismutase (SOD) and catalase (CAT) and malondialdehyde (MDA) contents were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of HO-1 and HMGB1 were determined by Western Blot, and the positive expression of HO-1 was also detected by immunofluorescence. Results Compared with Sham groups, the 7-day survival rates of WT and Nrf?2-KO mice in CLP groups were significantly lowered [WT: 0%(0/20) vs. 100%(20/20), Nrf?2-KO: 0%(0/20) vs. 100%(0/20), both P < 0.05];the 7-day survival rates of CLP+H2 group in WT mice were significantly higher than those of CLP group [40%(8/20) vs. 0%(0/20), P < 0.05], but there was no significant difference between CLP+H2 group and CLP group in Nrf?2-KO mice [0%(0/20) vs. 0%(0/200), P > 0.05]. In WT mice, compared with Sham group, the activities of SOD and CAT in lung tissue of CLP group were decreased significantly [SOD (kU/g): 131.30±28.21 vs. 251.00±22.84, CAT (kU/g): 13.43±1.52 vs. 20.76±1.63, both P < 0.01], the MDA content, the expressions of HO-1

关 键 词：肺损伤 脓毒症 氢气 核因子E2相关因子2基因敲除 

分 类 号：R45[医药卫生—治疗学]