The Role And Signal Transduction Of Cardiotrophin-1 In Myocardial Ischemia-Reperfusion Injury

Background and Research StrategyIt has been proved by plenty of basic and clinical researches that the injury of ischemic cardiomyocyte as well as cardic dysfunction, metabolic abnormality and ultrastructual change aggravate after reperfusion, which is called ischemic-reperfusion (I/R) injury. The mechanism of I/R injury is complicated, multiple pathways could be referred and oxygen free radical(OFR) which is a key factor to cell senescence and death plays an important role in the mechamism of I/R injury. Cardiomyocyte apoptosis has a close relationship with I/R injury and mitochondrial permeability transition pole-mPTP regulates cardiomyocyte apoptosis. Decrease of mitochondrial tansmembrane potentialΔψm is considered as the earliest occurence in the process of cell apoptosis caused by multiple stimulating factor of cell apoptosis,it occurs before the characteristic of cell nucleus apoptosis including chromatin condensation and fragmentation of DNA.Once mitochondrial DYmt collapsed,the cell apoptosis or death would occur because of Caspases activator and cell electrochemistry redox state caused by more OFR and less ATP ,which is caused by the pause of electron transfer chain and oxidative phosphorylation.Those cardiomyocytes which are close to apoptosis could be saved by stabilizing mitochondrial membrane potentialΔψm and preventing the change of mitochondrial permeability .Therefore, the research of protective factors is a hot topic nowdays .Cardiotrophin-1(CT-1) is a cytokine cloned in embryonic stem cell in rats recently,which is expressed in cardiocyte and cardiac myofibroblast and classified to IL-6 family.It regulates the normal physiological process in heart including facilitating normal development of heart and cell survival.As a member of IL-6 family,it has IL-6 -like function and synthesize acute reponse protein to show myocardial preservation.It is proved that more CT-1 can be seen in ischemic myocardium after acute myocardial infarction.It can reduce death of myocyte cells and help those residual cells survive .Besides it help other fibroblast move to make the infarction area recover and protect cardiac function.It is found that CT-1 can help myocardial cell survive in serum -free medium in vitro.CT-1 can cause heat shock protein hsp70,hsp90 to overexpress,which has protective funtion in cultivating neonatal myocyte cells.All above illustrates that CT-1 has a close relationship with myocardial injury and cardiac function.Therefore, we made a hypothesis that CT-1 also plays an central role in cardiomyocyte metabolism and function in acute I/R injury in rats and lightens CT-1 as an endogenious protective substrate against I/R injury. In our previous study,our results showed that the proliferation of cardiac fibroblasts induced by CT-1 is essentially mediated by JAK/STAT3 signalling pathway , independent of PI3-K and the action is negatively regulated by ERK1/2 signalling pathway.However, It has not been illustrated how CT-1 performs the protective effect in I/R injury as well as which signalling pathway would be referred.All above need researches to further confirm.Therefore, our research contains the following aspects to explain:First,To investigate whether CT-1 has the direct effect on myocardial injury in ischemic-reperfusion heart and probe into its mechanism from isolated organ level through observing the effect of CT-1 on reperfusion myocardial infarctin area, reperfusion arrhythmia ,cardial function and myocardial pathological change and myocardial eNOSmRNA and iNOSmRNA expression in rats.Second,To verify the role of CT-1 in I/R injury and its mechanism by observing the effects of CT-1 in cardiomyocyte survival rate ,apoptosis and myocyteΔψm,ROS in acute hypoxia -oxygeonation model in cultured rat cardiocytes exposed to simulated ischaemia followed by reoxygenation .To further elucidate the underlying mechanism and signalling pathways involved in CT-1 cardioprotection against ischaemic-reperfusion injury through the intervention of signalling pathway blockers and suppressor of cytokine signaling 1(SOCS1)- antisense oligonucleotides;To explore how does CT-1 prevent I/R injury and protect cardiomyocyte by its signal pathway through detecting differentiating and apoptosis-associated gene from celluar and molecular level.Part I : To investigate the cardioprotection of CT-1 in preventing I/R injury and the underlying mechanismObjective:Cardiac ischemia-reperfusion injury was evaluated through the exam of infarct size, arrhythmia,creatine kinase and cardiac function by the Langendorff technique in rat I/R model. The effects of CT-1 were evaluated when CT-1 was administered either prior to simulated ischaemia or at reperfusion .Besides,to investigate the underlying mechanism through detecting the concentration of myocardial expression of iNOSmRNA and iNOSmRNA .To probe into whether CT-1 has protective effect in preventing I/R injury as well as its mechanism from ex vivo whole heart level . Methods:All the rats were divided into 6 groups(n=10)(1)control group;(2)ischemia-reperfusion group(I/R group);(3)CT-1 group 1(pre-ischemia with CT-110μg/l );( 4 ) CT-1 group2 ( post-ischemia with CT-110μg/l );( 5 )CT-1+LY294002 group ;(6)CT-1+PD98059 group.LY294002,PD98059 are the blocker of PI3K /Akt,ERK1/2 signalling pathway respectively, which were dissolved in 0.1M NaOH solution and PH was adjusted to 7.4 by phospphate buffer solution(PBS),the perfusion concentration was 5μM and 10μM respectively。Langendorff perfusion was used in the rats heart ex vivo with the following methods:following baseline perfusion with Kerbs buffer for 20min,then ischemia 30min ,and reperfusion 60min.Heart rate,heart rhythm,left ventricular systolic pressure(LVSP),the rate of rise of left ventricular pressure(dp/dtmax),curve P-V and coronary flow were recorded respectively in multileads physiologic recorder.Coronary resistance and left ventricular compliance were evaluated by coronary flow and curve P-V respectively .The concentration of LDH and CK were detected in perfusate. The infarct size was measured by (TTC)staining method after reperfusion. iNOSmRNA and eNOSmRNA expression of myocardium were measured by reverse transcription-polymerase chain reaction( RT-PCR).Results:1,Arrhythmia:Premature, paroxysmal tachycardia and even malignant arrhythmia such as ventricular fibrillation occured during ischemia period while heart rate decreased, frequent premature and tachycardia occured during reperfusion period in I/R group. Arrhythmia occured later both in pre-ischemia and post-ischemia with CT-1 preconditioning before reperfusion,only a few premature occured besides, frequency of premature and tachycardia decreased obviouly in addition to its duration( P<0.05 ) .Occurences of arrhythmia including premature ,tachycardia and ventricualr fibrillation increased after adding signal pathway blockers LY294002(Akt/PI3K blockers )or PD98059(ERK1/2 blockers).2,Myocardial injury:38.6%±4.62% of infarct size found in I/R group while 22.5%±3.15% and 23.7%±2.75% in CT-1 group1 and CT-1 group2 respectively,which were obvioulsy less than I/R group(P<0.01).There was not statistically significant about LDH,CPK baseline value in every group before I/R.LDH,CPK except those in CT-1 group 1 increased 30 minutes post-ischemia .LDH,CPK increased furthuerly 60 minutes post -reperfusion ,which were significant difference comparing with those in pre-reperfusion.LDH,CPK decreased in two CT-1 groups after 60 minutes reperfusion comparing with those in I/R group,which still increased after 30 minutes ischemia .All above illustrate that CT-1 could apparently reduce I/R injury whenever it's processed before or after ischemia.However, part of cardioprotection of CT-1 could be eliminated after giving LY294002 and PD98059 respectively.3,Cardiac function :LVSP and +dp/dtmax,-dp/dtmax decreased apparently after 30 minutes ischemia , left ventricular diastolic stiffness increased and the compliance decreased in I/R group comparing with those in pre-ischemia.All the parameters above- mentioned impaired furtherly after 30 minutes reperfusion,although those turned better later,LVSP and +dp/dtmax,-dp/dtmax were still less than those before ischemia and left ventricular diastolic stiffness was higher than that pre-ischemia after 60 minutes reperfusion.LVSP and +dp/dtmax,-dp/dtmax improved apparently and left ventricular diastolic stiffness decreased comparing with those in I/R group whenever it's processed with CT-1 before or after ischemia,however,this kind of effect of CT-1 could be blocked by those two pathway blockers.4,Coronary flow:There was not statistically significant about coronary flow and coronary resistance before I/R ex vivo.All the coronary flow except that in CT-1 group 1 decreased apparently while coronary resistance increased obviously after 30 minutes ischemia.Athough coronary flow in I/R group recovered to some extent after 60 minutes reperfusion,however, it decreased apparently with those of pre-ischemia(8.1±0.7VS 10.8±0.9ml/min).There was not statistically significant in two CT-1 groups after 60 minutes reperfusion.Coronary flow was more than those in I/R group and coronary resistance decreased apparently.It is concluded that no matter CT-1 was processed before or after ischemia ,blood supply of ischemia-reperfusion heart could be improved a lot.5,Expression of myocardial iNOSmRNA and eNOSmRNA:Expression of myocardial iNOSmRNA up-regulated apparently(1.892±0.056VS0.425±0.032,P<0.05) while Expression of eNOSmRNA down-regulated obviously comparing with control group after ischemia-reperfusion (0.579±0.046VS 1.973±0.047,P<0.05).Expression of myocardial iNOSmRNA down-regulated apparently while Expression of eNOSmRNA up-regulated apparently comparing with I/R group no matter CT-1 was given before or after ischemia(1.892±0.062 VS 0.579±0.046;1.845±0.069 VS 0.579±0.046,p<0.01) ; Expression of iNOSmRNA increased apparently while eNOSmRNA decreased comparing with two CT-1 groups after giving pathway blockers LY294002 and PD98059.However,there was no significant difference between auxiliary solvent DMSO group and CT-1 group.It's showed that CT-1 can decrease the expression of myocardial iNOSmRNA and increase that of eNOSmRNA.However, expression of iNOSmRNA in LY294002 and PD98059 groups was obvioulsy more than those in CT-1 group, which was reverse to eNOSmRNA expression. Discussion and ConclusionIt is well known that ischemia can cause myocardial injury.Reperfusion has double sides,it can reduce infarct size through improving myocardial oxygeon supply and restricting cardiomyocyte"autolysis",however,it can reduce the therapy effect to some extent since it can impair myocardial injury through causing disequilibrium of cardiac cytokines and over-expression of some cytokine and adhesion molecules.Meanwhile ,some non-specifically protective protein and endogenious substrates can be released to cope with the myocardial injury caused by damaging stimulus.CT-1 which mainly comes from ventricular myocardial cells .As a member of IL-6 family,it can not only promote heart normal development and cell survival,but also synthesize acute response protein to perform myocardial preservation. It is found in the research with Langendorff equipment that left ventricular systolic pressure,left ventricular myocardial contractility impaired from reperfusion than those in ischemia period,in addition to decreasing coronary flow and increasing coronary resistance.I/R injury can be lightened apparently after processing CT-1.Incidence of reperfusion arrhythmia especially those malignant arrhythmia such as ventricular tachycardia as well as infarct size reduced,coronary resistance decreased while coronary flow increased.Besides,myocardial conctractility and left ventricular diastolic stiffness can be improved.The protective effect of CT-1 can be abolished by ERK1/ERK2 and PI3K signal pathway blockers.All above indicates that CT-1 can prevent I/R injury as a remedial protein.In addition,it can inhibit myocaridal injury through regulating synthesis of NO by up-regulating expession of eNOSmRNA and decreasing oxygeon free radical .Part 2 The mechanism and Signal transduction of CT-1 in protecting cardiomyocyte from acute hypoxia-reoxygeonation injuryObjective : Through the observation of cardiomyocyte survival rate,apoptosis,CK,cardiomyocyte mitochondrial membrane potential (Δψm ) , myocyte ROS,and nox-1 protein expression–subunit of NADPH oxidase complexs etc, to futher investigate the effect and mechanism of CT-1 in preventing I/R injury in cultured rat cardiocytes exposed to simulated ischaemia followed by reoxygenation when CT-1 was administered at reoxygenation . To investigate the signalling pathways involved in the protective effect of CT-1 in prevent cardiomyocyte I/R injury from cellular and molecular aspects,and the signalling pathway contains PI3K/Akt/ GSK-3β,PI3K/Akt/eNOS,ERK1/2 and SOCS1/STAT3 .To expound how CT-1 use its signalling pathway to exert a protective effect against the damaging effects of simulated ischaemia/ reoxygenation through detecting expression of gene- related to apoptosis.Method:1,Culture of neonatal rat myocyteCardiomyocytes from the hearts of 1～3-day-old neonatal rats (Sprague–Dawley), were prepared by a modification of a previously published protocol. The cells were dispersed in a series of incubations at 37℃in a nominally calcium free, HEPES-buffered salt solution containing pancreatin. The dispersed cells were preplated for 1 h to allow contaminating fibroblasts to attach. The myocytes free within the culture media were plated in 35mm culture dish at a density of 1×106/cm2 after purification with different speed adherence method.2,Preparation of hypoxia -reoxygeonation modelThe experiment start from cardiomyocytes converging to synchronous pulsation. To simulate ischaemia in vitro, cultured cardiocytyes were incubated in simulated ischaemia buffer (NaH2PO4mmol/L, NaHCO3mmol/L, CaCl2 1.8mmol/L, MgSO41.2mmol/L,HEPES20 mmol/L,NaCl98.5 mmol/L, KCl10.0 mmol/L, sodium lactate 40 mmol/L PH6.5)for 3 h in an atmosphere of 5% CO2 and 95% argon at 37℃. For analysis of the protective effect of CT-1 added at the time of reoxygenation, cardiaomyocyte cultures were exposed 3 h of lethal ischaemia then incubated in a normoxic environment at (37℃humidified atmosphere of 5% CO2 , 21% O2 for 3 h (reoxygenation). At the point of reoxygenation, the ischaemic buffer was removed and replaced with 2 ml minimal media with and without the addition of 10ng/ml of CT-1 for 3 h.3.1 Experiment grouping:Section 1 Protective effect of CT-1 in preventing acute cardiac myocyte hypoxia-oxygeonation injury mediated by ERK1/2 , PI3K/Akt/ GSK-3β,PI3K/Akt/eNOS pathways.There were six groups according to experiment scheme:①control group: cultivated normally in CO2 incubation after replacing medium;②hypoxia-re oxygeonation group: hypoxia 3h and cultivation with reoxygeonation for 3h ;③CT-1 group:CT-1(10ng/ml) was given after 3h hypoxia and cultivation with reoxygeonation for 3h ;④LY294002 group (PI3K/akt blocking agent):LY294002 with final concentration 10μmol/l was given after 3h hypoxia,CT-1(10ng/ml) was given 10min later,then processed with reoxygeonation for 3h;⑤PD98059 group (ERK1/2 blocking agent):PD98059 with final concentration 20μmol/L was given after 3h hypoxia,CT-1(10ng/ml) was given 10min later,then processed with reoxygeonation ;⑥DMSO group(auxiliary solvent--dimethyl sulfide group) :DMSO was given after 3h hypoxia,CT-1(10ng/ml) was given 10min later,then processed with reoxygeonation for 3h.3.2 Experiment grouping: Section 2, Protective effect of CT-1 in preventing acute cardiomyocyte hypoxia- reoxygeonation injury mediated by supressor of cytokine signaling 1--SOCS1/JAK pathwayTwo distinct antisense oligonucleotides (ASODN) complementary to sequences that encompass the translation initiation site of SOCS1 protein gene were designed and transfected into cardiomyocyte with lipofectamine. In addition, one sense ODN (SODN) and one scrambled ODN (ScODN) were designed to act as compare sequence. There were seven groups in the experiment :①control group:cultivated with DMEM for 6h;②hypoxia-reoxygeonation group:hypoxia for 3h and then reoxygeonation for 3h;③CT-1 group:CT-1(10ng/ml) was added after 3h hypoxia ,then reoxygeonation for 3h;④ASODN group1 and group2: antisense oligonucleotides group(two antisense SOCS1 groups,final concentration was 20μmol/L): after antisense oligonucleotides SOCS1 for 24h, was given 3h hypoxia , then CT-1(10ng/ml) was given and processed with reoxygeonation for 3h ;⑤SODN group(sense oligonucleotides SOCS1 group,final concentration was 20μmol/L):after sense oligonucleotides SOCS1 for 24h, was given 3h hypoxia , then CT-1(10ng/ml) was given and processed with reoxygeonation for 3h.⑥ScODN group(scrambled oligonucleotides SOCS1 group,final concentration was 20μmol/L):after scrambled oligonucleotides SOCS1 for 24h, was given 3h hypoxia , then CT-1(10ng/ml) was given and processed with reoxygeonation for 3h.4,Parameters for detection(1) myocyte morphous and beating frequency, rhythm detected with contrast phase microscope.(2)cardiomyocyte survival rate measured with MTS method,cardiomyocyte apoptosis detected by flow cytometry,LDH and CPK in supernatant analysed with automatic biochemistry analyzer.(3)cardiomyocyte mitochondrial membrane potentialΔΨm detected with 5,5',6,6'- tetrachloro- 1,1',3,3 '-tetrethyl benzimidalyl carbocyanine iodide (JC1)fluorescence microscope and flow cytometry ;cardiomyocyte reactive oxygeon species(ROS)detected with Diacetatedichlorofluorescein (DCFH CA)flow cytometry. (4)SOCS1 mRNA, eNOSmRNA,iNOSmRNA,BadmRNA and Id3mRNA of cardiomyocyte detected by RT-PCR method.(5)PI3-K phosphorylated PI3-K protein,ERK1/2 protein and phosphorylated ERK1/2 protein,GSK-3βprotein and phosphorylated GSK-3βprotein, SOCS1 protein, Subunit of NADPH oxidation enzyme complex (nox-1protein), detected by Western blot.ResultsSection 11,Frequency and rhythm of cardiomyocyte beat:Cardiomyocyte beated regularly and the beating frequency was about 148 beats/min in each group before hypoxia,there was no significant difference.The cardiomyocyte beated slowly ,weakly and irregularly after hypoxia-reoxygeonation.The cardiomyocyte beats in CT-1 group was similar to those in control group as well as those before hypoxia-reoxygeonation .However,it beated slowly ,weakly and irregularly after processing with blockers LY294002 and PD98059,while DMSO could not affect the role of CT-1.Therefore, it indicates that LY294002 and PD98059 could particularly block the effect of CT-1 in protecting cardiomyocyte from hypoxia-reoxygeonation injury.2,Cardiomyocyte injury:The survival and apoptosis rates were 96.2±3.2% and 2.8±0.4% respectively in control group. The survival rate decreased apparently to 76.8±4.6% and apoptosis rate increased obviously to 19.4±2.3% in hypoxia-reoxygeonation group,there was significantly different(P<0.05) between two groups.LDH and CK-MB increased significantly comparing with those in control group.The survival rate in CT-1 group increased apparently comparing with that in hypoxia-reoxygeonation group (89.8±8.3%VS 78.8±4.6%, P<0.05).Besides,LDH and CK-MB decreased and apoptosis was inhibited obviously in CT-1 group. However, those effects of CT-1 could be blocked obviously by signaling pathway blocker LY294002 and PD98059, becasue it's found that survival rate decreased and apoptosis rate increased after processing with LY294002 and PD98059.3,Cardiomyocyte ROS and expression of nox-1 protein:Cardiomyocyte ROS and expression of nox-1 protein in hypoxia -reoxygeonation group were obviously more than those in control group(14.28±1.42VS 3.54±0.16;1.127±0.107VS 0.215±0.018,P<0.01).Comparing with those in hypoxia -reoxygeonation group ,endocellular ROS and the level of nox-1 protein decreased after processing with CT-1(6.73±0.21VS 14.28±1.42, P<0.01),which were close to those in control group.Endocellular ROS and expression of nox-1 protein increased apparently after processing with blockers LY294002 and PD98059. However ,endocellular ROS and expression of nox-1 protein in DMSO group were close to those in CT-1 group.The results indicate that CT-1 can inhibit synthesis of ROS ,which have something to do with the inhibition of nox-1 protein expression.4,Cardiomyocyte mitochondrial membrane potential(Δψm):Results of flow cytometry:cardiomyocyteΔψm in hypoxia-reoxygeonation group was significantly lower than that in control group.(86.28±7.15 VS 40.55±4.25,p<0.01),cardiomyocyteΔψm of CT-1 group was higher than that in hypoxia-reoxygeonation group (69.13±6.84 VS 40.55±4.25, p<0.05),while myocyteΔψm was lower than that in CT-1 group after processing with LY294002 and PD98059 blocker respectively(50.13±5.82VS 69.13±6.84, 54.51±6.62 VS 69.13±6.84, p<0.05),but myocyteΔψm in DMSO group was close to that in CT-1 group.Results of fluorescence microscope showed:JC1 can not concentrate in myocyte mitochondria and it produces green color fluorescence in intracytoplasm as monosomatous form when mitochondrial membrane potentialΔψm was ruined.Fluorescence intensity was directly related to injury ofΔψm.JC1 produces intensive red color fluorescence as aggregate in normal cell. Cardiomyocyte green color fluorescence in hypoxia-reoxygeonation group was stronger than that in control group, while red color fluorescence was weaker than that in control group apparently,which could indicate that mitochondrialΔψm had been ruined. JC1 green color fluorescence intensity decreased apparently but red color fluorescence intensity increased after processing with CT-1.However,JC1 green color fluorescence intensity increased apparently but red color fluorescence intensity decreased after processing with blocker LY294002 and PD98059 ,meanwhile cardiomyocyteΔψm decreased. 4,Expression of cardiomyocyte iNOSmRNA and eNOSmRNA:The expression of iNOSmRNA was low in control group,but it increased apparently after hypoxia-reoxygeonation injury as well as expression of eNOSmRNA down-regulation ,there was significant difference(0.932±0.088 VS 0.117±0.053;0.347±0.023 VS 0.965±0.063,P<0.05 respectively ) .Comparing wiht those in hypoxia-reoxygeonation group, expression of iNOSmRNA decreased apparently while expression of eNOSmRNA up-regulated after processing with CT-1(0.359±0.027VS 0.932±0.088 ; 0.756±0.059VS 0.347±0.023,P<0.05 respectively),Expression of iNOSmRNA increased but eNOSmRNA decreased after processing with LY294002 and PD98059,there was significant difference comparing with those in CT-1 group.However,there was no significant difference between DMSO group and CT-1 group(0.379±0.016VS 0.359±0.027,P>0.05).5,PI3K protein and phosphorylated PI3K protein in cardiomyocyte :There was no significant difference among PI3K protein of groups. Phosphorylated PI3K protein increased in hypoxia-reoxygeonation group comparing with control group(0.475±0.032 vs0.197±0.018,P<0.05)and those in CT-1 group increased apparently comparing with hypoxia-reoxygeonation group (0.986±0.093 vs0.475±0.032,P<0.05). Phosphorylated PI3K protein decreased obviously after processing with PI3K/Akt blockers ( 0.325±0.029 vs0.986±0.093,P<0.05 ),however,there was no obvious difference between DMSO group ,PD98059 group and CT-1 group(0.963±0.048 vs0.986±0.093, P>0.05),which indicate that CT-1 had activated PI3K pathway ,besides, PI3K/Akt blockers had nothing to with ERK1/2 pathway.6,Phosphorylated ERK1/2 protein in cardiomyocyte :There was not statistically significant about ERK1/2 protein in each group after hypoxia-reoxygeonation.However, phosphorylated ERK1/2 protein in hypoxia-reoxygeonation group increased somewhat comparing with that in control group, but there was no statistical difference(0.495±0.043 vs0.249±0.016, p>0.05).While those in CT-1 group increased obviously comparing with those in hypoxia-reoxygeonation group (0.975±0.062 vs0.495±0.043,P<0.05) , Phosphorylated ERK1/2 protein decreased apparently after processing with ERK1/2 blocker(0.127±0.011 vs0.975±0.062;P<0.05).There was no significant difference between auxiliary solvent group and CT-1 group.(0.974±0.063 vs0.975±0.062, P>0.05);There was not obvious difference between PI3K/Akt blocker group and CT-1 group (0.969±0.059 vs0.975±0.062;0.969±0.059 vs0.975±0.062, P>0.05),it indicates that CT-1 can promote myocyte ERK1/2 protein phosphorylation in myocyte with hypoxia -reoxygeonation.7,Expression of GSK-3βprotein and phosphorylated GSK-3βprotein in cardiomyocyte:There was not statistical difference about expression of GSK-3βprotein in each group after 3h hypoxia and reoxygeonation.But expression of phosphorylated GSK-3βprotein in hypoxia-reoxygeonation group decreased apparently comparing with control group while that in CT-1 group increased obviously comparing with hypoxia-reoxygeonation group . Phosphorylated GSK-3βprotein was significantly lower than that in CT-1 group after processing with PI3K/Akt blockers while there no obvious difference between auxiliary solvent group and CT-1 group,which indicates that CT-1 can induce GSK-3βprotein phosphorylation in cardiomyocyte with hypoxia-reoxygeonation.PI3K/Akt blocker can block the effect of CT-1 in causing GSK-3βprotein phosphorylation while ERK1/2 blocker PD98059 does not have this function.8,Expression of BadmRNA and Id3mRNA in cardiomyocyte:The expression of BadmRNA in hypoxia-reoxygeonation group was apparently more than that in control group((0.986±0.035 VS 0.325±0.023,P<0.01)), while Id3mRNA had no obvious change.The expression of BadmRNA in CT-1 group down regulated comparing with that in hypoxia-reoxygeonation group, and expression of Id3mRNA somewhat upregulated but no significantly difference .Both signal pathway blockers LY294002 and PD98059 could block the effect of CT-1 apparently.Section 21,The role of CT-1 in cardiomyocyte hypoxia-reoxygeonation injury mediated by supressor of cytokine signaling 1 --SOCS1:The cardiomyocyte beat rate was about 150 beats/min and there was not significantly different in each group on baseline. Comared with baseline,cardiomyocyte beats slowly and irrgularly after hypoxia-reoxygeonation .Meanwhile, survival rate decreased and apoptosis rate increased apparently. However, there was no obvious difference between CT-1 group and hypoxia-reoxygeonation group. Cardiomyocyte beats regularly and was close to that in control group. Cardiomyocyte beat slowly,weakly and irregularly after interventing with antisense SOCS1; Comparing with those in CT-1 group ,cardiomyocyte survival rate decreased and apoptosis rate increased.(87.8±7.5% VS 74.8±5.2%;4.8±1.5% VS 8.8±2.2%,P<0.05 respectly ),LDH and CK-MB increased,however,SODN and ScODN could not intervent the effect of CT-1.2,Effect of CT-1 on myocardial cell ROS and mitochondrial membrane potentialΔψm mediated by SOCS1:Myocardial cell ROS increased markedly after hypoxia-reoxygeonation( 14.28±1.42VS 3.54±0.56, P<0.001),while that in CT-1 group decreased comparing with hypoxia-reoxygeonation group(4.73±0.48VS 14.28±1.42, P<0.01)and was close to that in control group. ROS increased obviously after antisense SOCS1 infection ,but SODN and ScODN infection were not found to have this effect.Results in flow cytometry showed that : myocardial cellΔψm in hypoxia-reoxygeonation group was obviously lower than that of control group(69.13±6.84 VS 40.55±4.25, p<0.05), and myocardial cellΔψm in CT-1 group increased comparing with hypoxia- reoxygeonation group(51.32±5.75VS 69.13±6.84, p<0.05).Δψm was lower than those in CT-1 group after giving antisense SOCS1 infection ,but it was similar to that in CT-1 group after sense SOCS1 or scrambled SOCS1 infection.Results in fluorescence microscope indicated that:myocyte green fluorescence intensity in hypoxia-reoxygeonation group was obviously stronger than that of control group,which indicates mitochondrial permeability transition pore(mPTP )has been ruined.Myocardial cell JC1 fluorescence green intensity decreased apparently after processing with CT-1 ,but it strengthened and myocardial cell mPTP decreased after antisense SOCS1 infection .However,results in sense SOCS1group and scrambled SOCS1group were similar to that in CT-1 group. 3,The effect of antisense oligonucleotides on expression of SOCS1mRNA and protein induced by CT-1 :After hypoxia-reoxygeonation ,Expression of SOCS1 mRNA and SOCS1 protein increased somewhat comparing with control group ,but there no significant difference.However,expression of both SOCS1 mRNA and SOCS1 protein in CT-1 group increased apparently comparing with hypoxia-reoxygeonation group (0±0.065 0.986±0.065 vs0.326±0.063;2.668±0.219 vs1.539±0.127,P<0.05). It indicates that CT-1 can induce overexpression of myocyte SOCS1.Both SOCS1mRNA and protein decreased apparently after two sections of SOCS1 antisense oligonucleotides transfection comparing with that in CT-1 group.however, there was no significant difference between sense oligonucleotides group or scrambled oligonucleotides group and CT-1 group.The results show that the transinfections of SOCS1 antisense oligonucleotides can effectively inhibit expression of cardiomyocyte SOCS1 induced by CT-1 .4,The effect of CT-1 on myocardial phosphorylated STAT3 mediated by SOCS1 :There was no significant difference about non-phosphorylation STAT3 in each group.But expression of phosphorylated STAT3 in hypoxia-reoxygeonation group increased obviously comparing with control group (0.672±0.034 vs0.147±0.012, P<0.05), while that in CT-1 group decreased apparently comparing with hypoxia-reoxygeonation group (0.425±0.035 vs0.672±0.031,P<0.05),Phosphorylated STAT3 increased obviously after processing with SOCS1 antisense oligonucleotides (0.593±0.048 vs0.425±0.035,P<0.05),while there was no significant difference between sense oligonucleotides group or scrambled oligonucleotides group and CT-1 group.This illustrates that SOCS1 can inhibit STAT3 phosphorylation induced by CT-1.Conclusion1,The effect of CT-1 on cardiomyocyte injury:CT-1 can protect cardiac cells against hypoxia -reoxygenation injury, which includes improving cell survival rate and reducing apoptosis as well as release of myocardial damaging enzyme.besides,it can help myocardial cell beat regularly and powerfully.2,The mechanism of CT-1 in alleviating myocardial injury induced by hypoxia-reoxygeonation: (1) decrease myocardial cell reactive oxygeon species( ROS ) through inhibiting expression of nox-1mRNA ,subunit of NADPH oxidase complex ;(2) through increase expession of eNOSmRNA and reduce expession of iNOSmRNA,regulate synthesis of NO to inhibit myocyte apoptosis;(3) mitigate injury of myocardial cell mitochodrial membrane potentialΔψm,and improve function of myocardial cell mitochondrial membrane transport .3,The signaling pathways involved in the cardioprotection of CT-1 as follows:(1) activating PI3K/Akt/GSK-3βpathway;(2) activating PI3K/Akt/eNOS pathway;(3) activating extracellular signal-regulated kinase ERK1/2 pathway;(4) activating signal suppressor SOCS1 pathway to inhibit phosphorylation of STAT3.4,CT-1 inbibits expression of BadmRNA-apoptosis advancing gene to reduce myocyte apoptosis induced by hypoxia-reoxygeonation through activating all above–mentioned signalling pathways.