The Role And Regulatory Mechanism Of SOCS3 In The Process Of Cardiomyocytes Hypoxia Reoxygenation

BackgroundIschemic heart disease(IHD)is a common and frequently occurring disease that seriously endangers human health.The number of deaths was as high as 7 million400 thousand in 2012.Besides,IHD costs a large number of medical resources.Through thrombolysis and PCI striking treatment as soon as possible to restore coronary blood supply is the key to save the lives of patients,but some studys have found that myocardial ischemia lasts for a period of time after the reperfusion will bring new injury to the heart tissue,casuing myocardial stunning,decreasing heart function and malignant arrhythmia,thereby cause onset of secondary damage of myocardial ischemia-reperfusion injury(IRI).To study the mechanism of IRI,and to explore the effective measures of IRI myocardial protection has long been a dream pursued by cardiovascular physicians.A large number of studies have found that IRI is connected with myocardial mitochondrial energy metabolism disorder,intracellular calcium overload,oxygen free radical release and apoptosis.Suppressor of cytokine signaling(SOCS)is a cytoplasmic protein family family with characteristic of the SOCS box in the SH2 domain as the core structure.It end C has 40 amino acids conserved module.The family can be expressed or induced in a variety of cells.It is found that the family has members of CIS(the cytokine-inducible SH2 domain-containing),SOCS1-7,and other 8members.SOCS has a competitive JAK catalytic site,thereby inhibiting JAK signaling pathway.The SOCS1 and SOCS3 homology with the strongest,most closely related to cardiovascular disease.Previous studies have found that the SOCS1 is competed with cardiotrophin-1(CT-1)catalytic site signaling pathways downstream of JAK,thereby inhibiting JAK-STAT3 signal transduction pathway,leading to myocardial cell apoptosis and myocardial infarction area expanded.Micro RNA(miRNA)is a kind of endogenous,and the length about 18 ~ 25 nucleotides RNA.It has several important roles in the cell through the degradation or translation inhibition in the expression of negative regulating target m RNA,It isfound that miRNA is involved on of target genes transcription of target m Rin the pathophysiology of heart development,cardiac hypertrophy and remodeling,arrhythmia,heart failure and myocardial cell apoptosis.In cardiac fibroblasts,miR-19 a by regulating the TGF-β R II beta inhibition of autophagy.Besides,it also has been found that miR-19 is related to myocardial apoptosis.Recently,it has been found that miR-19 b attenuates apoptosis in cardiomyocytes via targeting PTEN.Given the miR-19 a and miR-19 b with gene cluster is belong to miR-17-92,miR-19 a very likely to be involved in cardiac ischemia-reperfusion injury.Recently,research has found that miR-19 a targeted in liver cells prevent the transcription of SOCS3 expression increase JAK-STAT and promoting liver cell proliferation.So we speculate that in the process of ischemia-reperfusion injury,miR-19 a may targete SOCS3 and attenuate myocardial cell apoptosis.In summary,we speculate that there may be such a regulation in myocardial IRI process: myocardial protective cytokine IRI(such as CT-1)expression,JAK-STAT3 activity increased,STAT3 activity increased phosphorylation can upregulate the expression of SOCS3,in turn SOCS3 expression can inhibit JAK,and antiapoptotic effect of offset part of STAT3 on myocardial cell.If miR-19 a could inhibit the expression of IRI,the activity of JAK-STAT3 could be increased indirectly and the antiapoptotic effect of SOCS3 could be enhanced.In order to confirm this conjecture.This study is as follows:First,we will observe vitro experiments on SOCS3 ischemia and reperfusion injury in apoptosis.we will establish the models of myocardial hypoxia reoxygenation,and then examine the cell viability and biochemical tests in vitro experiments to confirm the relationship between SOCS3 and apoptosis protein Bim.Secondly,we will investigate the mechanism of miR-19 a targeting in SOCS3 from molecule and cell to vitro.The expression of SOCS3 protein was detected by miR-19 a overexpression or inhibition,and the effect on the apoptosis of myocardial cells.Thirdly,we need to determine the relationship between SOCS-3 and acute myocardial infarction.In order to analysis of whether high expression SOCS3 ofplasma can predict the major adverse cardiac events,the study confirmed acute myocardial infarction patients as the research object.The first part : To investigate the Relationship between SOCS3 and Bim in Hypoxia Reoxygenation Cardiomyocytes and its Effect on Cell ApoptosisObjective: This study was to establish models of myocardial hypoxia reoxygenation by developing H9c2 cardiomyocytes in vitro,and then to measure SOCS3 expression;SOCS3 expression was determined by RNA interference in the cell;after to observe the SOCS3 expression of Bim,the determination of cardiomyocytes apoptosis,which could acknowledge that how SOCS3 to effect on the expression of Bim in myocardial hypoxia reoxygenation and its effect on apoptosis.Method:1)H9c2 cardiomyocytes.2)The model of hypoxia reoxygenation(Hypoxia reoxygenation,HR)was established by simulating ischemia reperfusion injury,and the expression of SOCS3 was detected by Western-blot.3)Design and synthesis of si RNA(SOCS3-si RNA)targeting SOCS3 gene of 3pairs,Western-blot screening the best SOCS3-si RNA.4)The best effect of SOCS3-si RNA was transfected into H9c2,hypoxia reoxygenation was carried out.The experiment was divided into 4 groups:Blank control group(Control group),HR group,HR+Negative Control group,HR+SOCS3-si RNA group;5)Using CCK8 method to detect the proliferation of cells,flow cytometry was used to detect the apoptosis rate,Western-blot technique was used to detect the expression of SOCS3,apoptosis related genes Bim,Bax and Bcl-2.Result:1)The expression of SOCS3 was significantly up-regulated during hypoxia reoxygenation.2)Compared with the blank control group,the survival rate of HR group was significantly reduced(P<0.05),the survival rate of H9c2 in SOCS3-si RNA group was significantly increased(P<0.05),and the apoptosis rate was significantly decreased(P<0.05);3)Compared with HR group,the expression of Bim was down regulated in SOCS3-si RNA+HR group(P<0.01);4)Compared with HR group,the expression of Bcl-2 was up-regulated in SOCS3-si RNA+HR group,the expression of Bax was down regulated,and the ratio of Bcl-2/Bax was increased(P<0.05).Conclusion:1)The expression of SOCS3 was significantly increased in the model of myocardial ischemia and hypoxia in vitro;2)The expression of Bim and Bax can be down regulated by silencing SOCS3 in myocardial hypoxia reoxygenation model;3)The expression of Bcl-2 can be up-regulated by silencing SOCS3 in myocardial hypoxia reoxygenation modelThe second part: The Effects of SOCS3 on the JAK-STAT3 Signal Transduction Pathway and the Regulation of miR-19 a on SOCS3 in Hypoxia Reoxygenation CardiomyocytesObjective: This study intends to establish the in vitro HR H9c2 cardiomyocytes model,measure the expression of miR-19 a in HR in the process of determination,mimics or miR-19 a inhibitor transfected miR-19 a regulate the expression of miR-19 a in H9c2,the determination of apoptosis rate and apoptosis related gene protein expression of miR-19 a in hypoxic heart muscle cell complex effect on apoptosis of SOCS3 cells and the mechanisms of regulation HR mediated oxygen.Method:1)HR model was established by simulating ischemia reperfusion injury,andmiR-19 a expression was detected by q RT-PCR.2)miR-19 a mimics and miR-19 a inhibitor were transfected into H9c2 cells by using Lipofectamine3000,respectively.3)the experiment was divided into 6 groups: Blank control group(group Control),HR,HR+mimics Negative Control,HR+miR-19 a mimics group,HR+Mirco RNA inhibitor NC group,HR+miR-19 a inhibitor group;4)using the CCK8 method to detect the cell proliferation activity,flow cytometry to detect the apoptosis rate,SOCS3,p-STAT3,STAT3,Caspases,Bim,Bax and Bcl-2 were detected by Western-blot.Result:1)The expression of miR-19 a was down regulated during HR.2)Compared with the control group,the survival rate in HR group decreased significantly(P<0.05),miR-19 a H9c2 cell survival rate in mimics group was significantly increased(P<0.05),the apoptosis rate was significantly decreased(P<0.05);miR-19 a H9c2 cell survival rate in inhibitor group was significantly decreased(P<0.05),the apoptosis rate increased significantly(P<0.05);3)Overexpression of miR-19 a significantly inhibited the expression of SOCS3,Caspases,Bim and Bax,enhanced the activity of p-STAT3,increased the expression of Bcl-2,and inhibited cell apoptosis.4)Inhibition of miR-19 a significantly up regulate the expression of SOCS3,Caspases,Bim and Bax protein,,inhibit the activity of p-STAT3,down regulate the expression of Bcl-2,promote the apoptosis.Conclusion:1)The expression of miR-19 a was significantly decreased in the model of hypoxia reoxygenation in vitro;2)Mi R-19 a may play an important role in anti apoptosis by regulating the expression of SOCS3 in cardiomyocytes;The third part:Upregulation of Plasma SOCS-3 is associated with Poor Prognosis of Acute Myocardial InfarctionObjective To explore the correlation of upregulation of human plasma SOCS-3and poor prognosis of acute myocardial infarction.Methods Sixty-eight patients with AMI were enrolled,who were admitted to the our Hospial from March 2016 to October 2016.At the same time,20 healthy young volunteers Without cardiovascular diseases and match such as age,gender,were enrolled as control group.Enzyme-linked immunosorbent assay(ELISA)was used to examine the expression level of plasma SOCS-3,c Tn I,CK-MB in AMI patients and healthy controls.Then the potential clinical value of plasma SOCS-3and hypertension,left ventricular ejection fraction(LVEF),e GFR,Troponin I,CK-MB,NT-pro BNP,number of coronary artery stenosis,Killip classification and major adverse cardiac events(MACE)were evaluated.ResultsOur results showed plasma SOCS-3 was significantly increased in AMI compared with the healthy controls.In addition,plasma SOCS-3 was able to discriminate AMI patients with with an optimal sensitivity and specificity of 70.0and 85.5 respectively(AUC=0.856)Moreover,plasma SOCS-3 was significantly associated with a number of clinicopathological parameters including hypertension,left ventricular ejection fraction(LVEF),e GFR,Troponin I,CK-MB,NT-pro BNP,number of coronary artery stenosis and Killip classification.AMI patients with higher plasma SOCS-3 had a higher risk for suffering poorer major adverse cardiac events(MACE)and 6 month overall sur-vival.Furthermore,plasma SOCS-3 was an independent risk factor for MACE.Conclusion Plasma SOCS-3 was a valuable prognostic biomarker for patients with AMI.In conclusion,through the study of this project,we will confirm that inhibiting the expression of SOCS3 can reduce myocardial IRI,so as to explore the mechanism of myocardial protection of down-regulation SOCS3.