The Role Of Endothelin-1, Vascular Endothelial Growth Factor, Hypoxia Inducible Factor-1 In Pulmonary Hemorrhage In Newborn Rats

Part 1The establishment of pulmonary hemorrhage model in different phases in newborn rats induced by hypothermia-hypoxia/rewarming-reoxygenatingObjective To establish pulmonary hemorrhage model in different phases in newborn rats induced by hypothermia-hypoxia/rewarming- reoxygenating and to investigate the gross anatomical and histological changes in lungs under different conditions of hypoxia,hypothermia- hypoxia,hypothermia-hypoxia/ rewarming-reoxygenating with normoxia and hypothermia-hypoxia/rewarmingreoxygenating with hyperoxia.Methords 58 Wister newborn rats were randomly divided into 5 groups: control group(group C,n=10)and four experimental groups:group H(n=12), group HH(n=12),group HHR(n=12),group HHRO₂(n=12).Group C were exposed to normothermia and normoxia and,group H to hypoxia(5～6%O₂) for 6 hours,group HH to hypothermia(10±1℃)and hypoxia for 6 hours,group HHR to hypothermia and hypoxia for 6 hours and then to hyperthermia(37℃) and normoxia for 2 hours,group HHRO₂ to hypothermia and hypoxia for 6 hours and then to hyperthermia and hyperoxia(＞95%O₂)for 2 hours.The gross anatomical and histological changes(HE staining)in lungs were observed.There were 5 degrees in the changes of gross anatomical in lungs.Results(1)13 rats in experimental groups died while none in group C and the mortality rate of experimental groups was 27.08%.(2)Lungs of group H represented edema and,markedly stagnated blood,spotty pulmonary hemorrhage,local pulmonary hemorrhage could be observed in group HH and,more severe pulmonary hemorrhage were observed in groups HHR and HHRO₂,in which 2 cases of group HHR and 4 cases of group HHRO₂ suffered diffuse pulmonary hemorrhage and diffuse pulmonary hemorrhage occured mostly in the phase of rewarming/reoxygenating.There were significan differences of gross anatomical changes between group C and every experimental group and there were significant differences between every two experimental groups except for groups HHR and HHRO₂.(3)Histological changes in experimental groups included pulmonary alveoli and interstitial edema,spacer breaking,pulmonary alveoli dilating,fusion and pulmonary alveoli hemorrhage.Conclusion Pulmonary hemorrahge model can be established successfully in new born rats subjected hypothermia-hypoxia/rewarming -reoxygenating.Pulmonary hemorrhage developed from pulmonary edema to spotty pulmonary hemorrhage,local pulmonary hemorrhage and diffuse pulmonary hemorrhage.Diffuse pulmonary hemorrhage occured mostly in the phase of rewarming/reoxygenating.Hypoxia,hypothermia-hypoxia and rewarming/reoxygenating are factors contributing to lung injury.Hyperoxia may induce more severe lung injury.Part 2Expressions of endothelin-1,vascular endothelial growth factor, hypoxia inducible factor-1 in pulmonary tissue of newborn rats developing pulmonary hemorrhageObjective To investigate the expressions and effects of endothelin-1 (ET-1),vascular endothelial growth factor(VEGF),hypoxia inducible factor-1 (HIF-1)in pulmonary tissue of newborn rats developing pulmonary hemorrhage Methods 58 Wister newborn rats were randomly divided into 5 groups: control group(group C,n=10)and four experimental groups:group H(n=12), group HH(n=12),group HHR(n=12),group HHRO₂(n=12).Pulmonary hemorrhage models were established in different phases as described in part1.Expressions of preproendothelin-1mRNA(ppET-1mRAN),VEGFmRNA, HIF-1αmRNA were studied by reverse transcription-polymerase chain reaction (RT-PCR).Contents of ET-1 in pulmonary tissues were measured by radioimmunoassay.Expressions of VEGF were studied by Western Blot and immunohistochemical method.Results(1)Expressions of ppET-1mRAN increased significantly in groups H,HH and HHRO₂(P＜0.01)and contents of ET-1 elevated significantly in all experimental groups especially in groups H and HH(P＜0.01).There were significant positive correlations between the expressions of ppET-1mRAN and contents of ET-1 in groups H and HH(r=0.72,P＜0.05 and r=0.67,P＜0.05, respectively).(2)Expressions of VEGF188mRNA increased significantly in groups H,HH(P＜0.01)while VEGF increased significantly in all experimental groups especially in groups HR and HHRO₂(P＜0.05 in groups H and HH,P＜0.01 in groups HHR and HHRO₂).(3)There were significant positive correlations between the expressions of ET-1 and VEGF in groups H and HH (r=0.72,P＜0.05 and r=0.66,P＜0.05,respectively).(4)There was no significant difference in expressions of HIF-1αmRNA between group C and experimental groups.Conclusion(1)ET-1 and VEGF may contribute to the development of pulmonary hemorrhage in every phase.(2)ET-1 may promote the expression of VEGF.(3)HIF-1,as a transcription factor,may participate the pathological process by regulates the expressions of ET-1 and VEGF.Hypoxia regulates the expression of HIF-1αby a post-transcriptional mechanism. Part 3Effect of dual endothelin-1 receptor antagonist on prevention of pulmonary hemorrhage in newborn ratsObjective To study if dual endothelin-1 receptor antagonist,bosentan,can decrease the incidence of pulmonary hemorrhage in newborn rats induced by hypothermia-hypoxia/rewarming-reoxygenating and alter the expression of VEGF in pulmonary tissue and to evaluate the effect of bosentan on prevention of pulmonary hemorrhage.Methods 106 Wister newborn rats were randomly divided into 3 groups: control group(group C,n=10),experimental group(n=48)and bosantan group (n=48).Pups in experimental and bosentan groups were randomly divided into four subgroup:subgroup H(n=12),subgroup HH(n=12),subgroup HHR(n=12), subgroup HHRO₂(n=12).Rats in bosentan group accepted intragastric administration with bosentan(150mg/kg).2 hours later,the pulmonary hemorrahge models were established in experimental group and bosentan group as described in part 1.The gross anatomical changes in lungs were observed and the expressions of VEGF were studied by Western Blot method.Results(1)5 rats died in bosentan group while 13 in experimental group.There was a significant difference of mortality rate between experimental group and bosentan group(X²=3.35,P＜0.05).(2)Gross anatomical findings in experimental groups including edema and pulmonary hemorrhage as described in part 1.Attenuated gross anatomical changes could be observed in bosentan group:spotty pulmonary hemorrhage in subgroup HH and local pulmonary hemorrhage in subgroup HHR and subgroup HHR0₂ could be found,no diffuse pulmonary hemorrhage could be found in any subgroup.There were significant differences of gross anatomical changes between subgroups HHR,HHR0₂ in experimental group and their match subgroups in bosentan group(P＜0.05 and P＜0.01,respectively).(3)There was no significant difference of VEGF expression between group C and bosentant group(P＞0.05).Conclusion Dual endothelin-1 receptor antagonist,bosentan,can attenuate the pulmonary hemorrhage in newborn rats induced by hypothermia-hypoxia/ rewarming-reoxygenating and the mechanism may correlate with ET-1 blockade and downregulation of the expression of VEFG.