| Nowadays, the incidence and mortality of limb's explosion injury remain at a high level in war time, owing to the changes of war-tactics and extensive application of explosive weapon. Because of the severity of explosion injury, woundeds need to be emergency aid. Because of the poor medical condition at war, it is so difficult to offer effective medical evacuation and emergency aid in time that the state of the injury aggravates, furthermore, severe organ complications occur. Therefore, it is the emphasis on taking some effective measures for stabilizing the injury condition and preventing the occurence and development of severe organ complications.The discovery of endogenous gas signaling molecules, nitrogen monoxidum (NO) and carbon monoxide (CO), now including hydrogen sulfide (H2S), offer an original expect for the prevention and cure of organ complications in decades. Previous studies certify that endogenous H2S involves in the regulation of many physiological and pathophysiological functions in mannalian tissue, but it remain unclear to the role of H2S in lung injury after limb's explosion injury. Therefore, the present study was designed to explore the possible effects of H2S on lung injury after limb's explosion injury in rats firstly, and then isolate and culture rat pulmonary microvascular endothelial cells (PMVECs), observe the role of low concentration H2S in PMVECs injury induced by TNF-αand the possible mechanisms in this procedure at last. Thereby, this study will offer experimental supports and theoretical bases for the treatment of lung injury after limb's explosion injury.The present study includes three parts, the main results and conclusions are as follow:PART I Change of CSE/H2S system in lung after limb's explosion injury in ratsI The establishment of limb's explosion injury modelWe fixed detonator (80mg diazodinitrophenol) on medial surface skin of middle- inferior segment tibiofibula in left posterior limb with adhesive tape and then exploded. The results showed that the explosion leaded to not only smashed wound of distal segment of left posterior limb, but also severe secondary lung injury showing congestion, oedema and inflammatory cell infiltration so on, lung water content increased markedly (p<0.05). The animal model can meet the demands for following experiments.II Change of CSE/H2S system in lung injury induced by limb's explosion injury in ratsBase on experiment I, we observed the changes of inflammatory reaction, peroxidatic reaction and cystathionine-gamma-lyase/hydrogen sulfide system (CSE/H2S) in lung tissue. The results showed that TNF-α, IL-6, IL-10, MDA concentrations and MPO activity in plasma and lung homogenate increased markedly, but SOD activity decreased markedly after limb's explosion injury in rats (p<0.05 or p<0.01); Furthermore, CSE activity in lung homogenate and H2S concentration in plasma also decreased markedly(p<0.05), and the level of CSE/H2S system was negative correlation with lung water content and the width of alveolar septum so on (p<0.05 or p<0.01). The results imply that CSE/H2S system involves in the regulation of secondary lung injury after limb's explosion injury.PART II Role of CSE/H2S system in lung injury after limb's explosion injury in ratsI Acute toxicological effects of sodium hydrosulfide on healthy ratsThe results of part I imply that CSE/H2S system involves in the regulation of secondary lung injury after limb's explosive injury. In this part, we observed the effects of CSE/H2S system on lung injury by administration exogenous donator of H2S (NaHS) or CSE inhibitor (PPG). In view of toxicity of NaHS, we observed the toxicological effects of NaHS on healthy rats to choose suitable administration dosage of NaHS. The results showed that acute toxicity symptoms occur when the administration dosage of NaHS was≥30mg/kg, and the symptoms became more and more serious, even fulgurate died, when the dosage gradually increased. But when the dosage was≤10mg/kg, acute toxicity didn't occur. Therefore, administration H2S with low dosage (≤10mg/kg, ip) can be used to exogenous donator of H2S in following experiments.II Effects of CSE/H2S system on lung injury after explosion injury in free fieldIn this experiment, we observed the effects of CSE/H2S system on secondary lung injury after limb's explosion injury in free field by injecting exogenous donator of H2S (NaHS) or CSE inhibitor (PPG). After the rats were injected NaHS (1,5,10mg/kg, ip), TNF-α, IL-6, IL-10, MDA concentrations and MPO activity in plasma and lung homogenate decreased markedly (p<0.05 or p<0.01), but SOD activity increased markedly compared with explosion injury group (p<0.05), and lessened the extent of lung congestion, pneumonedema and inflammatory cell infiltration; But the effects of PPG were converse (p<0.05 or p<0.01). The results imply that CSE/H2S system plays protective role in secondary lung injury after limb's explosion injury in free field.III Role of CSE/H2S in lung injury induced by explosion injury in enclosed spaceIn this experiment, we observed the effects of CSE/H2S system on lung injury induced by limb's explosion injury in enclosed space by injecting exogenous donator of H2S (NaHS) or CSE inhibitor (PPG). The results showed that CSE activity in lung and H2S concentration in plasma markedly decreased after explosion injury, and the level of CSE/H2S system was negative correlation with lung water content and the width of alveolar septum(p<0.05 or p<0.01); Pneumorrhagia and disorganization of alveoli pulmonum occurr firstly, and then gradually showed pneumonedema and inflammatory cell infiltration. After injected NaHS in group S'(5mg/kg, ip), TNF-α, IL-6, IL-10, MDA concentrations and MPO activity in plasma and lung homogenate decreased markedly, but SOD activity increased markedly compared with group E'(p<0.05 or p<0.01). The results imply that CSE/H2S system plays protective role in lung injury (stacked effects for primary blast lung injury and secondary lung injury) induced by limb's explosion injury in enclosed space.PART III Effects of low concentration H2S on rat PMVECs injury induced by TNF-αI Culture and identification of rat pulmonary microvascular endothelial cellsThe results above imply that CSE/H2S system plays protective role in lung injury after limb's explosion injury in not only free field but also enclosed space. In view of the importance of rat pulmonary microvascular endothelial cells (PMVECs) in lung injury, we observed the role of low concentration H2S in rat PMVECs injury induced by TNF-αand the possible mechanisms in this part. In this experiment, we isolated and cultured PMVECs by tissue piece method and identified by factorⅧrelated antigen (FⅧ-Ag) with indirect immunofluorescence. The results showed that PMVECs crawled out about 24h after cultured; At the beginning, the cells were single diffused distribution or small cell mass. About 48~60h after cultured, cell mass monolayer grew by adherence to dish liking cobblestones. The cultured cells showed positive fluorescence of FⅧ-Ag in immunofluorescence. The results imply that the cultured cells are PMVECs, and the cell purity can meet the demands of following experiments.II Effects of low concentration H2S on rat PMVECs injury induced by TNF-αWe observed the effects of low concentration H2S on PMVECs injury induced by 20ng/ml TNF-αwith adding 100 or 300μM NaHS into culture medium in this experiment. The results showed that PMVECs expressed and secreted much more ICAM-1 and IL-6 after stimulated with 20ng/ml TNF-α, MDA concentration, LDH activity and iNOS/NO level in the medium supernatant also increased markedly, but SOD activity decreased remarkedly (p<0.05 or p<0.01); Furthermore, the nuclear translocation of Nuclear factor- E2-related factor 2 (Nrf 2) increased markedly (p<0.01). Low concentration NaHS (100, 300μM) could decrease the levels of ICAM-1 and IL-6 expressed and secreted by PMVECs; MDA concentration, LDH activity and iNOS/NO level also decreased markedly, but SOD activity increased remarkedly (p<0.05 or p<0.01, vs T ); Furthermore, the nuclear translocation of Nrf 2 increased markedly compared with PMVECs stimulated with TNF-α(p<0.01). The results imply that low concentration H2S plays protective role in PMVECs injury induced by TNF-α, and Nrf 2 and iNOS/NO pathways are possible to involve in the procedure of protective effects of low concentration H2S.The results above suggest thatI. Limb's explosion injury in free field in rats can lead to severe inflammatory reaction and peroxidatic reaction, showing congestion, oedema and inflammatory cell infiltration so on, result into severe secondary lung injury.II. Lung injury after limb's explosion injury in enclosed space is stacked effects between primary blast lung injury and secondary lung injury; Pneumorrhagia and disorganization of alveoli pulmonum are more severe than which in free field.III. CSE/H2S system in lung plays a protective role in lung injury induced by limb's explosion injury in rats, in not only free field but enclosed space. Down-regulation of CSE/H2S system involves in the procedure of lung injury. Administrating low dosage H2S (1~10mg/kg, NaHS) in the morning can relieve lung injury markedly.IV. Low concentration H2S (100~300μM) can inhibit the activity of PMVECs induced by TNF-α, suppress inflammatory reaction and peroxidatic reaction, relieve cell injury and play protective effects in this procedure; Furthermore, Nrf2 and iNOS/NO are possible to involve in the protective effects of low concentration H2S.
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