Objective To investigate the role of high mobility group box 1 (HMGB1) in the signaling pathway of mouse intestinal ischemia-reperfusion injury.Methods Twenty-four Specific Pathogen free male C57BL / 6 mice were randomly divided into three groups (n =8):the sham operation group (sham),the control group(control) and the HMGB1 antibody group (anti-HMGB1).The vehicle alone or anti-HMGB1 antibody(1 mg/kg,0.025％) was injected respectively via the caudal vein 30 min prior to ischemia in the control group or the anti-HMGB1 group.All mice were anesthetized,opened abdominal wall and exposed arteria mesenterica superior.The control group and the anti-HMGB1 group underwent 60 min of mesenteric ischemia and 60 min of reperfusion and the sham group were merely opened abdominal wall for 120 min without ischemia-reperfusion.The levels of NF-κB p65,IL-6 and TNF-α in plasma and the activity of MPO in lung and liver and the morphological changes of lung and intestinal tissue were measured.The mRNA levels of HMGB1 and NF-κB were evaluated using real-time quantitative PCR and the protein levels of HMGB1 and NF-κB were evaluated using Western blot.The experimental data was analyzed using one-way analysis of variance.Results The levels of IL-6,TNF-α and NF-κB p65 in plasma was significantly higher in the control group and the anti-HMGB1 group compared with the sham group (the sham group vs.the control group vs.the anti-HMGB1 group,NF-κB p65,104.64 ± 11.89:228.53 ± 24.85:145.00 ± 33.63,F=38.036,P＜0.05;IL-6,50.02 ±6.33:104.91 ±31.18:62.28 ± 6.73,F =49.763,P ＜ 0.05;TNF-α,43.79 ± 4.18:70.81 ± 6.97:52.76 ± 5.71,F =34.571,P ＜ 0.05).The increasing degree in the antiHMGB1 group was significantly reduced compared with the control group(P ＜ 0.05).The activity of MPO of liver and lung in the control group and the anti-HMGB1 group was significantly higher than those in the sham group(P ＜0.05).Compared with the sham group,the degree of tissue injury in jejunum,ileum and lung was serious in the control group,and that in the anti-HMGB1 group was significantly lower than the control group.The expression of HMGB1 mRNA and NF-κB mRNA in the lung and the ileum in the sham group and the control group were all higher than the sham group(HMGBl mRNA in lung:sham group 1.04 ± 0.19 vs.control group 2.25 ± 0.18 vs.anti-HMGB1 group 1.89 ± 0.18,F =66.203,P ＜ 0.05 ; in ileum:1.14 ± 0.54 vs.6.26 ± 0.60 vs.4.93 ± 0.55,F =133.427,P ＜ 0.05; NF-κB mRNA in lung:1.03 ± 0.21 vs.2.04±0.29 vs.1.42±0.23,F =26.229,P ＜0.05;ilenm:1.03 ±0.23 vs.3.71 ±0.53 vs.2.23±0.55,F =50.477,P ＜ 0.05).Subjected to intestinal ischemia-reperfusion injury,the protein expression of HMGB1 and NF-кB in the lung,jejunum and ileum in the control group and the anti-HMGB1 group increased compared with the sham group(P ＜ 0.05),but that was significantly lower in the anti-HMGB1 group than the control group (P ＜ 0.05).Conclusion The administration of anti-HMGB1 antibodies may reduce the damage caused by ischemia-reperfusion effectively.%目的 探讨高迁移率族蛋白1(HMGB1)在小鼠肠道缺血再灌注损伤信号转导通路中的作用.方法 SPF级雄性C57BL/6小鼠24只,随机分为3组:假手术组、对照组和HMGB1抗体组(anti-HMGB1),每组8只.对照组和anti-HMGB1组在行肠道缺血手术前30 min分别经尾静脉注射磷酸盐缓冲液和HMGB1抗体.所有小鼠均麻醉,开腹,对照组和anti-HMGB1组用无创血管夹夹闭肠系膜上动脉60 min后松夹再灌注60 min,假手术组仅开腹,不进行夹闭.光镜下观察3组小鼠肺脏、空肠和回肠组织形态学变化;分别应用髓过氧化物酶(MPO)试剂盒、酶联免疫吸附试验、real-timePCR和Western blot检测MPO活力、血浆核因子(NF)-κB p65、白细胞介素(IL)-6和肿瘤坏死因子(TNF)-α浓度、空肠、回肠及肺脏中HMGB1和NF-κB mRNA和蛋白表达水平.所得数据采用单因素方差分析进行统计学检验.结果 对照组和anti-HMGB1组小鼠血浆炎性因子IL-6、TNF-α、NF-κBp65均较假手术组升高(假手术组、对照组、anti-HMGB1组的NF-κB p65分别为104.64±11.89,228.53±24.85、145.00±33.63,F=38.036,P＜0.05:IL-6分别为50.02±6.33、104.91±31.18、62.28±6.73,F=49.763,p＜0.05:TNF-α分别为43.79 ±4.18、70.81 ±6.97、52.76±5.71,F=34.571,P＜0.05).anti-HMGB1组3种炎性因子表达水平均低于对照组(p值均＜0.05).对照组和anti-HMGB1组小鼠肝脏和肺脏组织的MPO活力均较假手术组升高.与假手术组相比,对照组小鼠空肠、回肠和肺脏组织的损伤严重,anti-HMGB1组小鼠的损伤程度小于对照组.对照组和anti-HMGB1组小鼠肺脏和回肠的HMGB1 mRNA和NF-κB mRNA的表达均高于假手术组(假手术组、对照组、anti-HMGB1组肺脏HMGB1 mRNA分别为1.04 ±0.19、2.25 ±0.18、1.89±0.18,F=66.203,P＜0.05;回肠HMGB1 mRNA分别为1.14±0.54、6.26±0.60、4.93 ±0.55,F=133.427,P＜0.05;肺脏HMGB1 mRNA分别为1.03 ±0.21、2.04±0.29、1.42±0.23,F=26.229,P＜0.05;回肠NF-κBmRNA分别为1.03±0.23、3.71 ±0.53、2.23±0.55,F=50.477,P＜0.05).肠道缺血再灌注损伤后,对照组和anti-HMGB1组的肺脏和空肠、回肠组织中HMGB1和NF-κB蛋白表达较假手术组增加,但anti-HMGB1组较对照组降低.结论 阻断HMGB1与TLR4结合可有效减轻小鼠肠道缺血再灌注造成的组织损伤.
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