Study Of The Effect And Mechanisms Of Nitrogen Monoxide In Experimental Pulmonary Thromboembolism

Part I Effect of the NO on pulmonary thromboembolismObjective To observe the functional changes of pulmonary vascular endotheliumafter pulmonary thromboembolism(PTE), and explore the effect of nitrogen monoxide (NO) on PTE.Methods Rats were randomly divided into control group(C-group), model group(M-group),L-arginine-treated group (L-group),L-NAME-treated group (N-group). PTE was established by intravenouw injection of auto-blood clots. All rats were sacrificed on 1hour, 3hour, 1day, 3day, 5day, 7day after treatment respectively. 1 ml vascular blood was withdrawn from each animal to measure blood gas analysis and lung removed for histopathologic with microscope. The plasma level of NO and von Willebrand factor (vWF) were analyzed by biochemistry and enzyme-linked immunosorbent assay (ELISA) respectively. The level of endothelin-1 (ET-1) protein and endothelial nitric-oxide synthase (eNOS) protein in lung tissue was evaluated by immunohistochemical method while the expression of eNOS mRNA was detected by in situ hybridization.Results In the C-group, the notable embolism in pulmonary arteries, inflammatory cellinfiltration around alveoli could be found by microscopy. PaO₂ and PaCO₂ were significantly lower at 1 hour after embolization (P<0.01), and PaCO₂ was gradually increased to the normal. The plasma level of NO, the expression of eNOS protein andmRNA were lower after embolization, whereras the plasma level of vWF and expression of ET-1 in lung were increased significantly (P<0.05). L-Arg treated rats showed emblism, hemorrhage, inflammatory reaction relative lighten, but blood gas analysis was not obviously improved. By contraries, L-NAME treated rats were deteriorated. In the L-group, the plasma level of NO, the expression of eNOS protein and mRNA in lung tissue were elevated whereras the plasma level of vWF and expression of ET-1 in lung tissue were decreased significantly than the M-group (P<0.05). But in the N-group, The plasma level of NO, the expression of eNOS protein and mRNA in lung tissue were more decreased whereras the plasma level of vWF and expression of ET-1 in lung tissue were more increased significantly than the other groups (P<0.05).Conclusion PTE may break pulmonary vascular endothelial function and induce inflmmation reaction. Anti-inflammatory therapy is important to PTE. L-arginine can protect vascular endothelium, ameliorate vascular endothelial function and alleviate lung inflammatory injury.Part IIEffect of the NO on inflammatory factors and NF- κB in experimental pulmonary thromboembolismObjective To discuss changes of inflammatory factors (TNF-α , IL-1β , IL-6) andNF- κB in lung tissue in PTE and the effect of NO and explore the mechanism of inhibiting inflammatory function of NO.Methods Rat PTE model, grouping and measurement method of NO were same aspart I . By using RT-PCR method to evaluate the expression of TNF- α , IL-1β, IL-6 mRNA in lung tissue and expression of NF- κB protein in lung tissue was detectedby Western blot method.Results The plasma level of NO was the same as part I . In M-group, theexpression of TNF- α , IL-1β , IL-6 mRNA in lung tissue, compared with C-group, were markedly elevated at 1 hour after embolization(P<0.01), then gradually reached its peak at 5 day after embolization. Untill 7 day after embolization, the expression of TNF- α , IL-1β , IL-6 mRNA in lung tissue began to decreased, but still the higher than that of C-group. The expression of NF-κ B protein in lung tissue increased gradually with the days passing by(.P<0.0l). In the L-group, compared with M-group and C-group, the expression of TNF- α , IL-1β, IL-6 mRNA and NF- κ B protein in lung tissue were decreased markedly(P<0.05). In the N-group, the expression of TNF- α , IL-1β, IL-6 mRNA and NF- κB protein in lung tissue were higher than the other groups(P<0.05). Conclusion In PTE, NF-κB is activated and TNF-α , IL-1β, IL-6 mRNA were largely expressed. NO may inhibit lung inflammation injury because it can inhibit expression of inflammatory factors TNF- α , IL-1β, IL-6 mRNA by depressing expression of NF- κB protein.Part IIIEffect of the NO on HIF-1 and ERK in experimental pulmonarythromboembolismObjective To discuss changes of hypoxia-inducible factor-1α (HIF-1α ) mRNA,extracellular signal-regulated kinase(ERK) protein in lung tissue in PTE and the effect of NO and explore the mechanism of treatment PTE of NO.Methods Rat PTE model, grouping and measurement method of NO were same as part I . By using RT-PCR method to evaluate the expression of HIF-1α mRNA inlung tissue and expression of ERK1/2 protein was detected by Western blot method. Results The plasma level of NO was the same as part I . In M-group, the expressionof HIF-1α mRNA and ERK1/2 protein in lung tissue increased gradually with the dayspassing by(P<0.01). In the L-group, compared with M-group and C-group, theexpression of HIF-1α mRNA and ERK1/2 protein in lung tissue were decreasedmarkedly(P<0.05). In the N-group, the expression of HIF-1α mRNA and ERK1/2protein in lung tissue were higher than the other groups(P<0.05).Conclusion In PTE, because of anoxemia, the expression of HIF-1α mRNA wasup-regulated. NO may inhibit CTEPH formation because it can down-regulatedexpression of HIF-1α mRNA by depressing expression of ERKl/2 protein.