The Study On The NGF-TrKA Induced Protection On Ischemia/Reperfusion Rat Hearts With A Mechanism Related To Endoplasmic Reticulum Stress

Objective To investigate the characteristic of endogenous NGFexpression in the early period following ischemia reperfusion in a in vivorat heart model with regional ischemia-reperfusion; to investigate the effectand dose-effect relationship of exogenous NGF on the recovery of cardiacfunction, myocardial injury, myocardial apoptosis following ischemia andreperfusion in an isolated rat heart model; to investigate the effect of NGFon the myocardial apoptosis, endoplasmic reticulum stress related proteinexpression and the intracellular concentration of calcium ion in the culturedH9C2cardiac myoblasts undergoing hypoxia and reoxygenation, and toevaluate the relationship between modulation of endoplasmic reticulumstress and anti-apoptosis by NGF, as well as the potential mechanism.MethodsPart One24adult SD rat hearts were randomly assigned to one ofthe four groups (n=6). Sham-operation group, a silk was passed through theleft anterior coronary (LAD) without ligation of the LAD. Ischemia group (I group), LAD was ligated for30minutes. Ischemia-reperfusion group (I/Rgroup), LAD was ligated for30minutes followed120minutes ofreperfusion. K252a group, the TrkA receptor inhibitor K252a50μg/kg wasadministered subcutaneously30minutes before ischemia,30minutes ofischemia and120minutes of reperfusion was followed. Heart rate (HR) andmean arterial pressure (MAP) were recorded before LAD ligation,30minutes after ligation and120minutes after reperfusion. Blood sampleswere collected at the end of the treatment for determination of the serumconcentration of lactate dehydrogenase and creatine kinase-MB. Themyocardial tissues in the apex were removed for examination of NGFmRNA by RT-PCR and NGF protein expression by western-blot analysis.The myocardial tissues in the left ventricular and right ventricular wereremoved for the examination of NGF expression by immunohistochemicalanalysis.Part Two Isolated adult SD rat hearts were randomly assigned to oneof the six groups (n=8). Sham-operation group, ischemia-reperfusion group(I/R group), NGF Ⅰ~Ⅲ group, and NGF+K252a group. All the heartswere perfused by Krebs-Henseleit (KH) buffer with a langendorff perfusionsystem. Hearts in the Sham group were perfused with KH buffer for225minutes. I/R group, the hearts were subjected to an equilibration period ofKHB perfusion for75min followed by30min of global ischemia and120min of reperfusion. NGF Ⅰ~Ⅲ group, after a45min equilibration period with KHB perfusion, the hearts were perfused with KHB contained with10,100and1000ng/ml of NGF for30min followed by30min of globalischemia and120min of reperfusion. NGF+K252a group, after15minutesof equilibration perfusion, the hearts were perfused with KHB containedwith100nM of K252a for30minutes, followed with NGF perfusion in aconcentration of100ng/ml, then ischemia and reperfusion was continued asI/R group. LVDP, LVEDP,±dp/dt and HR were continuously measured andrecorded at the end of the equilibration period (as baseline level) and5,30,60, and120minutes during reperfusion. Coronary effluent samples at theend of the baseline perfusion and at5min of reperfusion were collected tomeasure the myocardial-specific enzymes (CK-MB, LDH). At the end ofreperfusion, hearts were quickly removed from the perfusion system.Myocardium tissues in the left ventricle were cut into2-mm thickspecimens for determination of myocardial apoptosis by using the terminaldUTP nick-labeling (TUNEL) assay.Part Three Cultured H9C2myoblasts were assigned to one of thefollowing groups according to the treatment. Control group, the cells werecultured with normal medium and incubated in a humidified atmospherecontaining95%air and5%CO2at37℃for8hours. Hypoxia andreoxygenation group, the cells were incubated in a three-gas incubator filledwith95%N2and5%CO2with―ischemia buffer‖for4hours and followedby4hours of reoxygenation, which is similar to the treatment as the control group. NGF Ⅰ~Ⅲ group, NGF was added to the culture medium atdifferent time points. The concentration of NGF in the medium was100ng/ml. NGF1~4group, NGF was added to the culture medium at thebeginning of reoxygenation, the concentration of NGF in the medium was1ng/ml、10ng/ml、100ng/ml、1000ng/ml respectively. NGF+LY294002group and NGF+K252a group, LY294002or K252a was added to theculture medium with NGF, with a concentration of100nM and50μM inthe medium. The cell viability was quantitatively evaluated with a cellcount kit-8after the treatment. Cell apoptosis was determined by flowcytometry analysis. Expression of GRP78, CHOP, Caspase-12, P-Akt andAkt after reperfusion was evaluated by western-blot analysis. Thefluorescence intensity of the intracellular free calcium ions was assessed byfluorescene staining.ResultsPart one (1) Compared with the Sham group, heart rate of I group,I/R group and K252a group was significantly increased at30minutes ofischemia and120minutes after reperfusion（P<0.0001）. MAP of I group,I/R group and K252a group was lower than that of S group at30minutes ofischemia, the level of K252a was lower the other two groups.(2) Comparedwith S group, LDH and CK-MB concentration in I group, I/R group andK252a group were significantly increased at the end of treatmen（tP<0.01）.The concentrations of the two cardiac enzymes in the K252a group were the lowest among the three groups, the level of I/R group was higher than thatof I group（P<0.01）.(3) In the heart undergoing ischemia and reperfusion,NGF expression in the ischemia area was significantly higher than that ofthe none ischemia area（P<0.01）.(4) Compared with S group, the level ofNGF mRNA and NGF protein expression in I group, I/R group and K252agroup was significantly increased（P<0.0001）.Among the three groups, thelevel of I/R group and K252a group was higher than that of I group（P<0.0001）, while no intergroup differences were found between the twogroups（P>0.05）.Part two (1) HR，LVDP and±dp/dtmaxin the I/R group, NGFⅠ/Ⅱ/Ⅲ group, NGF+K252a group were all decreased. LVDP in NGFⅠand Ⅱgroup was higher than that of NGF Ⅲgroup（P<0.01）,±dp/dtmaxin NGFⅡgroup was higher than that of NGF Ⅲgroup（P<0.01）. LVEDP in the I/Rgroup, NGFⅠ/Ⅱ/Ⅲgroup, NGF+K252a group were all higher than that ofSham group. NGF Ⅱhas a lowest level of LVEDP among the groupstreated with NGF（P<0.01）.(2) LDH、CK-MB concentration and apoptosisrat in the I/R group, NGFⅠ/Ⅱ/Ⅲgroup, NGF+K252a group were allincreased after ischemia and reperfusion compared with Sham group（P<0.01）, NGF Ⅱgroup has the lowest level of the cardiac enzymes andapoptosis rate among the rest groups（P<0.01）. NGFⅢgroup has a highestapoptosis rate among the three groups treated with NGF.Part three (1) Compared with I/R group, groups treated with NGF may increase the cell viability whenever it was added to the culture medium,and the viability reached the highest level in NGFⅢ group. In addition,NGF treatment with a concentration of100ng/ml showed better effects onincreasing the cell viability compared with other concentrations（P<0.01）.(2) NGF may significantly decrease the apoptosis rate when added to theculture medium during reoxygenation, this effect may completely inhibitedby K252a and LY294002.(3) NGF may promote the P-Akt expression afterhypoxia reoxygenation（P<0.0001）, while with a negative effect on theexpression of GRP78, CHOP and Caspase-12. The effect of NGF on theseproteins express may be completely inhibited by K252a and LY294002.(4)The changes of GRP78, CHOP and Caspase-12expression were negativelycorrelated with the cell apoptosis, while the expression of P-Akt waspositively correlated with the apoptosis.(5) No differences were found onthe fluorescence intensity of the intracellular free calcium ions among thegroups undergoing hypoxia and reoxygenation（P>0.05）.Conclusion(1) High expression of NGF may be activated by ischemia as anearly protective reaction, and reached to a higher level during reperfusion.During the early stage of reperfusion, the ischemia area shows a mostdramatic expression of NGF, which implies an endogenous protectivemechanism in myocardium induced by ischemia.(2) Exogenous NGF pretreatment may exert none neurogenic protections to the ischemic heart in a dose dependent way. Excessive NGFtreatment may aggravate myocardial ischemia-reperfusion injury.(3) PI3K-Akt pathway mediated the protection of NGF on thehypoxia induced myocardial apoptosis, the mechanism may be related tothe modulation of endoplasmic reticulum stress. The modulation ofintracellular calcium concentration may not be involved in the NGFinduced anti-apoptosis on the hypoxic cardiac myocytes.