Therapeutic Effect Of Ulinastatin On Seawater And Freshwater Aspiration-induced Lung Injury

Drowning is one of the important human accidental death causes,and it is the second largest cause of death in child injury.After drowning,the first be damaged organ is lung,there are mainly include two aspects: One is the seawater or freshwater directly damage lung,on the other hand is to further lung injury caused by the hypoxia,metabolic acidosis and inflammation.The main mediums of drowning are seawater and freshwater,due to the differences between seawater and fresh water in physical and chemical properties,the degree and mechanism of acute lung injury caused by seawater and freshwater is different.Vascular endothelial growth factor(VEGF)is a heparin binding glycoprotein.It can promote lung development,promote the synthesis of pulmonary surfactant and secretion,increase vascular permeability,promote angiogenesis.Some studies have indicated that hypoxia was the main factor in VEGF expression.Hypoxia inducible factor-1?(HIF-1?)is the upstream regulator of VEGF,and it also plays an important role in the process of VEGF expression induced by hypoxia.Ulinastatin(human urinary trypsin inhibitor)is a protease inhibitor extracted from human urine and it is also one of the human body endogenous anti inflammatory substances.It has a role in fighting inflammation and reducing oxidative stress injury.At present,ulinastatin is widely used in clinical in curing critically ill patients and protecting the organ after organ transplantation.A number of experimental studies have confirmed that ulinastatin could attenuate acute lung injury caused by oleic acid and sepsis.In addition,ulinastatin can relieve the inflammation in lung injury caused by freshwater and seawater drowning.But the specific mechanism is still unknown.In order to clarify the effect of HIF-1? / VEGF pathway in seawater and freshwater aspiration-induced lung injury and whether the HIF-1? / VEGF pathway is involved in the protective effect of ulinastatin in seawater and freshwater aspiration-induced lung injury.Firstly,observing the changes of expression of HIF-1? and VEGF after seawater and freshwater drowning.Then observing the changes of expression of HIF-1? and VEGF in lung tissue by using ulinastatin,combined with 1ung permeability index and inflammatory marks.To investigate whether the HIF-1?/VEGF pathway is engaged in the protective effect of ulinastatin on seawater and freshwater aspiration-induced lung injury.Part ?The expression of HIF-1? and VEGF in seawater and freshwater aspiration-induced lung injuryObjective:To observe the changes of the expression of hypoxia inducible factor-1?(HIF-1?)and vascular endothelial growth factor(VEGF)in seawater and freshwater aspiration-induced lung injury,explore the possible mechanism involved in lung injury.Methods:Eighteen rabbits were randomly assigned to 3 groups(6 in each group): the control group,the seawater group and the freshwater group.Rabbits in the latter 2 groups were given 3ml/kg seawater and 18ml/kg freshwater intratracheally to induce the models of acute lung injury respectively.Arterial partial oxygen pressure and lung wet/dry weight(W/D)ratio were detected.Protein contents in bronchoalveolar lavage fluid(BALF)and serum,as well as tumor necrosis factor(TNF)-?,interleukin(IL)-1?,and IL-6 levels in BALF,were also determined.HIF-1? and VEGF expressions were measured via quantitative real-time PCR and Western blot.Changes in the histopathology of lung specimens were evaluated through hematoxylin–eosin staining and the lung injury scores were calculated by the method of Smith scores.Results:Seawater and freshwater aspiration dramatically decreased PaO 2/FiO2 in the both groups compared with the control group.Furthermore,the values of TNF-?,IL-6 and IL-1? were significantly increased in BALF.HIF-1? and VEGF expressions were markedly activated,histopathologic changes were more serious and the Smith scores were significantly increased compared with the control group.In addition,seawater aspiration induced dramatically higher values of IL-6,expressions of HIF-1? and VEGF,significantly serious histopathologic changes and higher Smith scores compared with the freshwater group.Conclusion:The expression of HIF-1? and VEGF were increased in seawater and freshwater aspiration-induced lung injury.These results indicated that the HIF-1?/VEGF pathway may play an important role in seawater and freshwater aspiration-induced lung injury.Because of the different mechanisms of seawater and freshwater aspiration-induced lung injury,the HIF-1?/VEGF pathway may has different physiological function in the two forms of lung injury.Part ? Therapeutic effect of different doses of ulinastatin on seawater and freshwater aspiration-induced lung injuryObjective:To observe the possible mechanism of ulinastatin cure seawater and freshwater aspiration-induced lung injury,explore the best treatment dose.Methods:This part of the experiment was divided into two aspects,in the first part we observed therapeutic effect of different doses of ulinastatin on seawater aspiration-induced lung injury.Thirty rabbits were randomly assigned to 5 groups(6 in each group): the control group(Group C),the seawater group(Group S),the small dose(25000U/kg)ulinastatin seawater group(Group SU),the medium dose(50000U/kg)ulinastatin seawater group(Group MU),the large dose(100000U/kg)ulinastatin seawater group(Group LU).Rabbits in the latter 4 groups were given 3ml/kg seawater intratracheally to induce the models of acute lung injury respectively.In the second part we observe therapeutic effect of different doses of ulinastatin on freshwater aspiration-induced lung injury.Thirty rabbits were randomly assigned to 5 groups(6 in each group): the control group(Group C),the freshwater group(Group F),the small dose(25000U/kg)ulinastatin freshwater group(Group SU),the medium dose(50000U/kg)ulinastatin freshwater group(Group MU),the large dose(100000U/kg)ulinastatin freshwater group(Group LU).Rabbits in the latter 4 groups were given 18ml/kg freshwater intratracheally to induce the models of acute lung injury respectively.After the model were successfully established,given the two parts intravenously UTI.Arterial partial oxygen pressure and lung wet/dry weight(W/D)ratio were detected.Protein contents in bronchoalveolar lavage fluid(BALF)and serum,as well as tumor necrosis factor(TNF)-?,interleukin(IL)-1?,and IL-6 levels in BALF,were also determined.HIF-1? and VEGF expressions were measured via quantitative real-time PCR and Western blot.Changes in the histopathology of lung specimens were evaluated through hematoxylin–eosin staining and the lung injury scores were calculated by the method of Smith scores.Results:1.Therapeutic effect of different doses of ulinastatin on seawater aspiration-induced lung injury.(1)Group S and Group LU aspiration dramatically decreased PaO 2/FiO2 in the both groups compared with the Group C at the T0,T0.5,T1 and T3.(2)Compared with Group C and Group LU,Group S increased the wet/dry weight(W/D)ratio and LPI obviously(p<0.05).Group LU's LPI was lower than Group SU's(p<0.05).(3)The values of TNF-?,IL-6 and IL-1? were significantly increased in BALF in Group S than in Group C and Group LU(p<0.05).Group LU's TNF-?,IL-6 and IL-1? were lower than Group SU's(p<0.05).(4)HIF-1? and VEGF mRNA expressions in Group S were higher than in Group C,Group MU and Group LU(p<0.05).Group LU could reduce the HIF-1? and VEGF mRNA expressions more obvious than Group SU(p <0.05).(5)HIF-1? and VEGF protein expressions in Group S were higher than in Group C,Group LU(p<0.05).Group LU could reduce the HIF-1? and VEGF protein expressions more obvious than Group SU(p<0.05).(6)The Group S had the severest histopathologic changes,so its Smith scores were the highest.Compared with Group SU,Group LU could reduce the Smith scores more obvious(p<0.05).2.Therapeutic effect of different doses of ulinastatin on freshwater aspirationinduced lung injury.(1)Group F and Group LU aspiration dramatically decreased Pa O 2/FiO2 in the both groups compared with the Group C at the T0,T0.5,T1.(2)Compared with Group C and Group LU,Group F increased the wet/dry weight(W/D)ratio and LPI obviously(p<0.05).(3)The values of TNF-?,IL-6 and IL-1? were significantly increased in BALF in Group F than in Group C and Group LU(p<0.05).(4)HIF-1? and VEGF mRNA expressions in Group F were higher than in Group C,and Group LU(p<0.05).Group LU could reduce the HIF-1? and VEGF mRNA expressions more obvious than Group SU,Group MU(p<0.05).(5)HIF-1? and VEGF protein expressions in Group F were the highest in these 5 group(p<0.05).(6)The Group F had the severest histopathologic changes,so its Smith scores were the highest.Compared with Group SU and Group MU,Group LU could reduce the Smith scores more obvious(p<0.05).Conclusion:Ulinastatin can relieve seawater and freshwater aspiration-induced lung injury,the HIF-1?/VEGF pathway was engaged in this protective effect of ulinastatin.Regulated the expression of HIF-1?,thereby affected the expression of VEGF protein.