Nrf2在氢气治疗严重脓毒症肠损伤中的作用

The role of Nrf2 in the hydrogen treatment for intestinal injury caused by severe sepsis

目的 探讨Nrf2在氢气治疗严重脓毒症肠损伤中的作用.方法 将152只雄性ICR小鼠按随机数字表法分为假手术组、氢气对照组、脓毒症组和氢气治疗组4组,每组38只.采用盲肠结扎穿孔术（CLP）制备脓毒症模型,假手术组和氢气对照组不进行CLP,其他操作相同.氢气对照组和氢气治疗组于假手术或CLP后1h、6h分别吸入2％氢气1h.每组取20只小鼠,观察术后7d内的生存率.每组剩余18只小鼠,分别于术后6、12和24 h各处死6只,取部分肠组织,用蛋白质免疫印迹试验（Western Blot）检测Nrf2、高迁移率族蛋白B1 （HMGB1）的蛋白表达;用逆转录-聚合酶链反应（RT-PCR）检测Nrf2 mRNA表达;于术后24 h取部分中段空肠,行苏木素-伊红（HE）染色,光镜下观察肠组织损伤程度.结果 假手术组和氢气对照组各指标差异均无统计学意义.脓毒症组小鼠7d生存率较假手术组明显降低（0比100％,P＜0.05）;氢气治疗组小鼠7d生存率较脓毒症组明显升高（55％比0,P＜0.05）.与假手术组比较,脓毒症组术后6、12和24 h肠组织Nrf2的蛋白表达（灰度值）和mRNA表达均明显升高（Nrf2蛋白6 h：1.973±0.350比1.000±0.000,t=4.411,P=0.002; 12 h：2.367±0.186比1.000 ±0.000,t=10.210,P=0.000; 24 h：2.517±0.280比1.000±0.000,t =9.521,P=0.000;Nrf2 mRNA 6 h：1.606±0.271比1.000 ±0.000,t=3.631,P=0.002; 12 h：1.692±0.399比1.000±0.000,t=3.233,P=0.005;24 h：1.784±0.341比1.000±0.000,t=3.894,P=0.001）,术后24 h肠组织HMGB1表达（灰度值）明显升高（1.507±0.220比1.000±0.000,t=3.948,P=0.004）.与脓毒症组比较,氢气治疗组术后6、12和24 h肠组织Nrf2的蛋白和mRNA表达明显升高（Nrf2蛋白6 h：2.583±0.395比1.973±0.350,t=2.765,P=0.024; 12 h：2.725±0.235比2.367±0.186,t=2.674,P=0.028; 24 h：2.930±0.212比2.517±0.280,t=2.595,P=0.032; Nrf2 mRNA 6 h：2.008±0.400比1.606±0271,t=2.405,P=0.029;12 h：2.188 ±0.475比1.692±0.399,t=2.317,P=0.034;24 h：2.333±0.406比1.784±0.341,t=2.728,P=0.015）,术后24 h肠组织HMGB1表达明显降低（1.147±0.152比1.507±0.220,t=2.805,P=0.023）.HE染色结果显示,脓毒症组出现明显的肠组织损伤;氢气治疗组肠组织损伤较脓毒症组有所减轻.结论 氢气可以通过激活Nrf2-抗氧化反应元件（ARE）通路,使脓毒症小鼠肠组织中Nrf2蛋白表达增加,HMGB1水平降低,最终对严重脓毒症小鼠肠组织起到保护作用,并可明显提高脓毒症小鼠的生存率.

Objective To investigate the role of Nrf2 on hydrogen treatment for intestinal injury caused by severe sepsis.Methods 152 male ICR mice were randomly divided into four groups：sham operation group,hydrogen control group,sepsis group,and hydrogen treatment group,each n=38.Sepsis model was reproduced by cecal ligation and puncture （CLP）.The mice in sham operation group and hydrogen control group did not receive CLP,and the operative procedure was the same as follows.The mice in hydrogen control group and hydrogen treatment group received 1-hour inhalation of 2％ hydrogen 1 hour and 6 hours after sham operation or CLP.Twenty animals in each group were selected and observed for 7-day survival rate.Eighteen animals in each group were selected and sacrificed at 6,12 and 24 hours after CLP.The intestinal tissues were obtained to determine the expression of Nrf2 and high mobility group B1 （HMGB1） protein by Western Blot,and the expression of Nrf2 mRNA by reverse transcription-polymerase chain reaction （RT-PCR）.The middle portion of jejunum was obtained to evaluate the degree of septic injury by light microscope after hematoxylin and eosin （HE） staining.Results There was no statistical signifieance in variables between sham operation group and hydrogen control group.Compared with sham operation group,the 7-day survival rate was significantly decreased in sepsis group （0 vs.100％,P＜0.05）; compared with sepsis group,the 7-day survival rate was significantly increased in hydrogen treatment group （55％ vs.0,P＜0.05）.Compared with sham operation group,the expression of Nrf2 protein （gray value） and Nrf2 mRNA were up-regulated in sepsis group at 6,12 and 24 hours after CLP （Nrf2 protein 6 hours：1.973 ± 0.350 vs.1.000 ± 0.000,t=4.411,P=0.002; 12 hours：2.367 ± 0.186 vs.1.000 ±0.000,t=10.210,P=0.000; 24 hours：2.517 ±0.280 vs.1.000 ±0.000,t=9.521,P=0.000; Nrf2 mRNA 6 hours：1.606 ± 0.271 vs.1.000 ± 0.000,t=3.631,P=0.002; 12 hours：1.692 ± 0.399 vs.1.000 ± 0.000,t=3.233,P=0.005; 24 hours：1.784 ± 0.341 vs.1.000 ± 0.000,t=3.894,P=0.001）,and it was also the expression of HMGB1 （gray value） at 24 hours after CLP operation （1.507 ± 0.220 vs.1.000 ± 0.000,t=3.948,P=0.004）.Compared with sepsis group,the expression of Nrf2 protein and Nrf2 mRNA in intestines were up-regulated at 6,12 and 24 hours after CLP in hydrogen treatment group （Nrf2 protein 6 hours：2.583 ± 0.395 vs.1.973 ± 0.350,t=2.765,P=0.024; 12 hours：2.725 ± 0.235 vs.2.367 ± 0.186,t=2.674,P=0.028; 24 hours：2.930 ± 0.212 vs.2.517 ± 0.280,t=2.595,P=0.032; Nrf2 mRNA 6 hours：2.008 ± 0.400 vs.1.606 ± 0.271,t=2.405,P=0.029; 12 hours：2.188 ± 0.475 vs.1.692 ±0.399,t=2.317,P=0.034; 24 hours：2.333 ±0.406 vs.1.784 ±0.341,t=2.728,P=0.015）.Compared with sepsis group,the expression of HMGB1 was down-regulated significantly at 24 hours after CLP in hydrogen treatment group （1.147 ± 0.152 vs.1.507 ± 0.220,t=2.805,P=0.023）.HE staining showed that there was significantly aggravated intestinal pathological injury in the mice of sepsis group; compared with sepsis group,the pathology was significantly less marked in hydrogen treatment group.Conclusion Through activation of Nrf2-antioxidant response element （ARE） pathway,hydrogen may increase the level of Nrf2,which is a kind of protective protein,in the intestine of mice,thus decreases the level of late pro-inflammatory factor,HMGB1,and it may protect the intestinal tissues in septic mice and increase the survival rate significantly.