Induction of acute lung inflammation in mice with hemorrhagic shock and resuscitation: role of HMGB1

Abstract

Background: Hemorrhagic shock and resuscitation (HS/R) can induce multiple organ failure which is associated with high mortality. The lung is an organ commonly affected by the HS/R. Acute lung injury is a major cause of dysfunction in other organ systems. The objective of this study is to test the hypothesis that HS/R causes increased gut permeability which results in induction of high mobility group box1 protein (HMGB1) and further leads to the development of acute lung inflammation.

Materials and methods: A mouse model of HS/R was employed in this study. Gut permeability and bacterial translocation were assessed with circulating FD4 and lipopolysaccharide (LPS). Circulating HMGB1 was determined with ELISA. Acute lung inflammation (ALI) was determined with lung myeloperoxidase (MPO) activity and pulmonary protein leakage.

Results: HS/R induced intestinal barrier dysfunction as evidenced by increased circulating FD4 and LPS at 30 min and 2 hrs after resuscitation, respectively. In addition, circulating HMGB1 levels were increased in mice with HS/R as compared with sham animals (p < 0.05). HS/R resulted in ALI (increased lung MPO activity and pulmonary protein leakage in mice with HS/R compared with sham mice, p < 0.05). Inhibition of HMGB1 (A-box and TLR4(-/-)) attenuated the ALI in mice with HS/R. However, inhibition of HMGB1 did not show protective effect on gut injury in early phase of HS/R in mice.

Conclusions: Our results suggest that induction of HMGB1 is important in hemorrhagic shock and resuscitation-induced acute lung inflammation.

Keywords: Acute lung inflammation; Gut injury; HMGB1; Hemorrhagic shock.

Figure 1

Hemorrhagic shock and resuscitation results in increased mouse gut permeability and bacterial translocation into mouse circulation. Mice were subjected to either sham procedure or hemorrhagic shock and resuscitation (HS/R). A. Gut permeability was assessed 30 min after completion of resuscitation. Five mice in each group, *p < 0.05 compared with sham group. B. Blood samples from sham or HS/R mice were collected at times indicated. Plasma levels of circulating LPS were determined with a Limulus Amebocyte Lysate (LAL) endotoxin assay kit. Six mice per group, *p < 0.05 compared with sham group.

Figure 2

HMGB1 mediated HS/R-induced acute lung injury (ALI). Mice were subjected to sham surgery, HS/R, or HS/R plus A-Box (300 μg, i.p., given at the beginning of the transfusion of shed blood). A. Mouse blood was collected 24 hrs after resuscitation, plasma was obtained by centrifugation and circulating HMGB1 was determined with a HMGB1 ELISA kit. Five mice per group, *p < 0.05 vs sham group. B and C. Twenty-four hours after resuscitation, mouse ALI was assessed using tissue MPO activity (B) and pulmonary protein leakage (C). Six mice per group for MPO activity experiments; Five mice in each group for pulmonary protein leakage experiments, *p < 0.0 vs. sham; ^#p < 0.05 vs. HS/R.

Figure 3

Deficiency of TLR4 attenuates HS/R-induced acute lung inflammation. Wild type (WT) and TLR4^−/− mice were subjected to either sham or HS/R and mouse circulating HMGB1 (A), lung MPO activity (B) and pulmonary protein leakage (C) were determined 24 hrs after resuscitation. A. Five mice in each group, *p < 0.05 vs. WT sham; +p < 0.05 vs. TLR4^−/− sham; no difference between WT HS/R and TLR4−/− HS/R; B and C. Five mice in each group. *p < 0.05 vs WT sham, ^#p < 0.05 vs WT HS/R.

Figure 4

Inhibition of HMGB1 does not show protection to gut injury in the early phase of HS/R in mice. A. Mice were subjected to sham surgery, HS/R, or HS/R plus A-Box (300 μg, i.p.) given at the beginning of the transfusion of shed blood). Gut permeability and bacteria translocation were determined at 30 min and 2 hrs after completion of resuscitation by measuring circulating FD4 or circulating LPS, respectively. Five mice in each group, *p < 0.05 vs sham. B. Wild type (WT) and TLR4^−/− mice were subjected to either sham or HS/R. Gut permeability and bacteria translocation were determined at 30 min and 2 hrs after completion of resuscitation by measuring circulating FD4 or circulating LPS, respectively. Five mice in each group, *p < 0.05 vs related sham group.