Objective: To investigate the effect of determine whether the CO-releasing molecules-liberated CO could attenuate leukocytes sequestration and the inflammatory response in the lung of thermally injured mice.
Methods: Thirty-six C57BL/6 mice were randomly divided into 3 groups: burn group, burned with hot water on the back skin with an area as large as 15% of the total body surface area with the hair shed so as o cause full-thickness thermal injury, CORM-2 group, undergoing the same thermal injury and then receiving intravenous injection of CORM-2 immediately, and sham operation group, undergoing sham thermal injury. Twenty-four hours later the mice were killed. The myeloperoxidase enzyme (MPO) level in lung tissue was detected. Evans blue test and lung wet/dry weight ratio were used to examine the lung edema degree. Bronchoalveolar lavage (BAL) fluid was collected to undergo ELISA to detect the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. Activation of nuclear factor (NF)-kappaB was detected with electrophoretic mobility shift assay. The expression level of intercellular adhesion molecule-1, ICAM-1) in the lung was assessed by Western blotting. Whole blood samples were collected from the left ventricles. Serum was isolated and used to stimulated lung endothelial cells for 4 h. Polymorphonuclear neutrophilic leukocytes (PMNs) were isolated from mice bone marrow, labeled with Na(51)CrO(4), cultured, and added with the murine lung endothelial cells (MLECs) stimulated by serums from the mice of the 3 groups so as to measure leukocyte adhesion.
Results: The MPO activity of the CORM-2 group was (42 +/- 7) U/g tissue, significantly lower than that of the burn group [(87 +/- 11) U/g tissue, P < 0.05]. The Evans blue extraction level of the CORM-1 group was (53.1 +/- 4.6), not significantly different from that of the burn group [(55.1 +/- 3.8), P > 0.05], however, still significantly higher than that of the sham group [(8.8 +/- 1.3), P < 0.05]. The wet/dry weight ratio of the CORM-2 group was 4.80 +/- 0.11, significantly higher than that of the sham group (3.20 +/- 0.07, P < 0.05), but not significantly different from that of the burn group (4.70 +/- 0.18, P > 0.05). The TNF-alpha and IL-1beta levels of the CORM-2 group were (92 +/- 4) pg/ml and (27.2 +/- 2.9) pg/ml respectively, both significantly higher than those of the sham group [(24 +/- 4) pg/ml and (6.6 +/- 1.0) pg/ml respectively, both P < 0.05], but significantly lower that those of the burn group [(160 +/- 9) pg/ml and (27.2 +/- 2.9) pg/ml respectively, both P < 0.05]. The A value for the lung ICAM-1 protein level of the CORM-1 group was (2.4 +/- 0.4), significantly higher than that of the sham group [(1.4 +/- 0.6)], however, significantly lower than that of the burn group [(3.5 +/- 1.1), P < 0.05]. The lung NF-kappaB activity of the CORM-1 group was significantly lower than that of the burn group. The PMN adhesion to the MLECs stimulated by the CORM-2-treated thermally injured mice serum was (25.4 +/- 5.6)%, significantly lower than that of the burn group [(46.5 +/- 8.5)%, P < 0.05]. Also, CORM-2 markedly decreased the production of inflammatory mediators in BAL fluid without suppressing the permeability of pulmonary microcirculation.
Conclusion: CORM-released CO attenuates the inflammatory response in the lung of thermally injured mice by decreasing leukocyte sequestration and interfering with NF-kappaB activation, protein expression of ICAM-1, thus suppressing endothelial cells pro-adhesive phenotype.