The Role Of HMGB1 In Acute Lung Injury After Hemorrhage

Objective:To copy the animal models of acute lung injury(ALI) induced by hemorrhagic shock and investigate the role of high mobility group box1 protein(HMGB1) in ALI after hemorrhage.Method:Forty-two male BALB/c mices,8-12wk of age,28±3g/mice,were divided into seven groups randomly,including the group control,group of ALI induced by hemorrhagic shock(including 4h,16h,24h and 48h these four observed points after ALI) and group of anti-HMGB1 antibody blockade(including 4h after blockade and 24h after blockade),n=6.The control mice had cardiac puncture but wasn't removed any blood. Cardiac puncture was used to remove 30%of the calculated total blood volume(0.27 ml/10g body wt) over 60s,then mice suffered from hemorrhagic shock.In previously study,it has been showed that ALI occured 1h after the induction of hemorrhagic shock in animal models of mice.4h,16h,24h and 48h after ALI,mice was killed and the lung samples were collected.Evens blue dye(EBD) solution(5mg/ml,200ul) was injected through a tail vein 1h before killed and the pulmonary vasculature was flushed free of blood by gentle infusion of 10ml of PBS into the beating right ventricle.Monoclonal mouse anti-HMGB1 antibody(200ug/mouse) was injected into the peritoneum 1h after the induction of hemorrhagic shock.The therapeutic effects of antibidy were evaluated at 4h (for MPO assay in the lungs) and at 24h(for the lung leak by EBD assay).Western Blot was used to determine HMGB1 levels in the lungs.ELISA was used to determine IL-1βand TNFαlevels in the lungs.MPO level in the lungs was determined using MPO kits. EBD level in the lungs was measured by optical density(OD) rate.Result:Destruction of the pulmonary cell walls,migration of inflammatory cells(polymorphonuclear leucocytes mainly) and pulmonary hemorrhages were present in the lungs of mice from 4h after ALI induced by hemorrhagic shock to 48h after ALI.The pathologic transformation of lungs was most serious at 24h after ALI.The accumulation of neutrophils and the destruction of the pulmonary cell walls was attenuated in anti-HMGB1 antibody blockade group.The IL-1βand TNFαlevels in the lungs at ALI 4h group(IL-1β333.83±31.18pg/ml;TNFα776.67±103.67pg/ml) were higher than control group(IL-1β284.83±30.49pg/ml;TNFα584.17±181.17 pg/ml)(P＜0.05),and the other time groups didn't show any difference against control group.The MPO levels in the lungs at ALI 4h group(38.33±3.88U/g of lung),ALI 16h group(25.17±1.94U/g of lung), ALI 24h group(20.33±2.42U/g of lung) and ALI 48h group(9.66±2.16U/g of lung) were significantly higher than contral group(8.00±0.89U/g of lung)(P＜0.01).The peak of MPO level in the lungs was at 4h after ALI induced by hemorrhagic shock.The EBD level in the lungs at ALI 4h group(10.87±1.34g/ml/g of lung),ALI 16h group (14.60±1.31 g/ml/g of lung),ALI 24h group(20.05±2.79g/ml/g of lung) and ALI 48h group (8.98±2.71g/ml/g of lung) were significantly higher than control group(4.63±0.43g/ml/g of lung)(P＜0.01).The peak of EBD level in the lungs was at 24h after ALI induced by hemorrhagic shock.Compared with ALI groups,the peak levels of MPO and EBD in the lungs significantly decreased under administration of anti-HMGB1 antibody(MPO 25.67±1.63U/g of lung at 4h after antibody blockade;EBD 5.68±0.53g/ml/g of lung at 24h after antibody blockade)(P＜0.01).The rate of HMGB1/β-actin in the lungs at ALI 16h after hemorrhage(0.99±0.16),ALI 24h after hemorrhage(1.00±0.32) and ALI 48h after hemorrhage(0.90±0.0.27) was significantly higher than control group(0.50±0.18) (P＜0.05).The peak level of HMGB1 in the lungs was at about 24h after ALI induced by hemorrhagic shock.Conclusion:HMGB1 and cytokine expression were significantly increased in the lungs in animal models of ALI induced by hemorrhagic shock.The HMGB1 levels in the lungs increased from about 16h afer ALI,reached to the peak level at about 24h after ALI and remained to 48h after ALI.While the IL-1βand TNFαlevel in the lungs increased at 4h after ALI only and then decreased to normal level.Blockade by anti-HMGB1 antibody attenuated hemorrhage-induced lung leak and neutrophil accumulation.The MPO and EBD levels in the lungs significantly decreased at anti-HMGB1 antibody blockade groups.These results demonstrate that HMGB1 acts as a late mediator of hemorrhagic shock-induced ALI.Administration of anti-HMGB1 antibody can attenuate the pathophysiologic effects of ALI after hemorrhage.