The Expression And Function Of AOC3 Gene In Hemorrhagic Shock

Background:Shock is an antient topic.From the beginning of 17 th century people began to explore the treatment of shock.In the course of 300 years,the invention of the effective drug and the increase idea of treatment had great progress,but for severe hemorrhagic shock was still in a high mortality rate,because of not fully understanding its pathophysiology.Hemorrhagic shock is one of the most common shock.Effective blood perfusion decrease and then cause the tissue oxygenation and dysfunction is the foundation of shock incidence of joint channel and the pathology.In clinic,the pathophysiological basis of hemorrhagic shock is cycling capacity reduction,tissue oxygen supply reduction,metabolites the accumulation of microcirculation failure,then induce the microcirculation failure.This microcirculation failure will not recover because of the big circulation recovery in a short time,but will appear and look forward to the pathophysiological changes of the opposite.The long time obstruction of microcirculation will bring chang to the cells and organs.It’s result is the failure of function and the death of body.There are some hypotheses such as oxygen metabolism,proinflammatory cytokine hypothesis,microcirculation hypothesis.Because of the means of study become gradually rich,these hypothesis gradually integrate together.Decrease of circulation blood;failure of the microcirculation;oxygen metabolism disorder and proinflammatory cytokine release too much;circulatory failure aggravation,these pathophysiological processes are not only independent but interrelated.This link is not a single linear contact but links,increase or alleviate joint of circulatory failure are not known to us.After the treatment of severe shock,even if the blood pressure has been restored to near normal,but still in the high flow resistance phenomenon.The reason of the high blood flow resistance is the leukocyte adhesion and obstruction in the microvasculature not from blood vessels.In shock stage,the number of white blood cell adhesion to venule on the wall is 16-20 times of the normal circumstances,leukocyte adhesion to venule leads to increase resistance to flow,with reduced blood flow resistance increases and the driving pressure,blood flow in micro vein decreased rapidly and lead to the occurrence of low perfusion.Leukocyte sticking will further the formation of leukocyte impaction and lead to no reflow phenomenon(no-reflow).Finding a treatment for leukocyte impaction and no flow is very important in irreversible shock.Through blood transfusion,infusion,boosting drugs,anti shock treatment,blood pressure dropped to rebound calls refractory hypotension.The reason of refractory hypotension is arteriolar smooth muscle cells(ASMCs)in responsiveness to endogenous or exogenous vasoconstrictors.To investigate the mechanism of severe shock,vascular reactivity decreased becomes the focus of study.According to the existing literature,it may be related to such mechanismn as oxygen free radicals that endogenous catecholamine inactivation,adrenergic receptor desensitization,accumulation of metabolites and energy depletion,cytokines(NO,ET).The development of modern molecular biology progress provided the possibility for molecular biological treatment of hemorrhagic shock.Through the pre-experiment on hemorrhagic shock rats gene screening,our experimental group found a gene AOC3 in hemorrhagic shock rats,which expression showed obvious difference in shock and recovery.VAP-1(vascular adhesion protein-1)is an amine oxidase(copper-containing)comprised of 763 amino acids,and is also a member of semicarbamizide-sensitive amine oxidases(SSAOs)family.This kind of protein is mainly expressed in vascular endothelial cells,fat cells and smooth muscle cells of human and a variety of mammals.As a kind of ecto-enzymes,VAP-1 on the vascular endothelial cell surface may fall off into the plasma to form sVAP-1(soluble VAP-1)through crack reaction of soluble protein,which will catalyze aromatic and aliphatic primary amines in blood into corresponding aldehydes and produce H2O2 and NH3.The dual functions of VAP-1 allow leukocytes to patrol throughout the whole body.Therefore,previous studies of VAP-1 mainly focused on inflammation.In chronic liver disease,chronic kidney diseases,sespic shock and other diseases,plasma sVAP-1 concentration was increased significantly.For disease models of peritonitis,hepatitis,colitis,skin inflammations,stroke,sepsis,ischemia-reperfusion injury and allograft rejection,anti-VAP-1 mAbs or VAP-1 enzyme inhibitors can relieve further development of inflammation by preventing the migration of leukocytes due to inflammation.In addition,since the VAP-1 mediates leukocyte migration and has the characteristic of adhesion to vascular endothelium,it is also closely related to the occurrence mechanisms in terms of cardiovascular and cerebrovascular diseases such as dissemination of tumor cells and ischemic stroke.Therefore,VAP-1 always has good application prospects no matther whether it is used as a prognostic biomarker or a potential drug target.But the role of VAP-1 for the proceeding of hemorrhagic shock has not been reported.Our group expect a break in this area to provide a means for the treatment of severe hemorrhagic shock.Objective:1.To identify the expression of AOC3 gene and its encoded protein VAP-1 in human sinusoidal endothelial cells(HHSEC)under hypoxia.2.To identify the function of inducing leukocyte adhesion of AOC3 gene in human sionusoidal endothelial cells(HHSEC)under hypoxia.3.To identify the variation rules of VAP-1 concentration and SSAO activity in serum in the process of constant pressure hemorrhagic shock and recovery in patients.Methods:1.We had constructed AOC3 over-expression adenovirus vector,and then used it to transfect HHSEC.The expressive levels of AOC3 gene and VAP-1 of the cells(including normal or adenovirus transfected HHSEC under normoxic or hypoxic conditions(95%N2 and 5%CO2 for 24 hours)were determined.2.The leukocyte-endothelium adhesion assay kit was used to detect the adhension between human sinusoidal endothelial cells(HHSEC)and leucocyte under normoxic or hypoxic conditions while using SSAO inhibitor or not and normal or adenovirus transfected HHESC.3.We selected 20 hemorrhagic shock patients cause of liver spleen rupture;massive hemorrhage of gastrointestinal tract;massive hemorrhage of Obstetrical and gynecological diseases and 20 nomarl persons,and decteted the levels of sVAP-1 and SSAO activity in shock and recovery process by using methods of ELISA and spectrophotometer.Results:1.The AOC3 mRNA levels of the group under hypoxic conditions were significantly increased compared with the group under normoxic conditions(P<0.001),and the difference was more significantly obvious in the AOC3 transgenic HHSEC(P<0.001).The variation of VAP-1 protein levels were positive correlation with the AOC3 gene.2.Under hypoxic conditions,the capacity of inducing leukocyte adhesion for the VAP-1 was obviously increased compared with group under normoxic conditions(P<0.05),and SSAO inhibitor(2-BEA)could obviously inhibit the VAP-1-induced leukocyte adhesion whether in the normal HHSEC(P=0.01)or in the AOC3 gene adenovirus transfected HHSEC(P<0.001).3.In the process of constant pressure hemorrhagic shock and recovery in patients,ELISA quantitative results showed that sVAP-1 levels in serum of shock group was significantly higher than in recovery group and the control group,and that in the recovery group was significantly higher than in the control group.The changes of SSAO activity levels were consistent with sVAP-1.Conclusions:1.The results showed that,in vitro,the expression level of AOC3 were increased significantly under hypoxic conditions.2.The degree of VAP-1-induced leukocyte adhesion was obviously enhanced under hypoxic conditions,but the uptrend of adhesion could be inhibited with SSAO inhibitor(2-BEA).3.In vivo experiments we confimed that in the shock course in patients the levels of VAP-1 in serum were increased,and the SSAO activity was enhanced,and the levels of VAP-1 and SSAO activity in serum were decreased after recovery.