| Objective To investigate the protective effect of nerve growth factoron Ischemia-reperfusion injury in isolated rat hearts, to determine if nervegrowth factor attenuate the endoplasmic reticulum stress pathway ofapoptosis induced by ischemia-reperfusion in isolated rat heart and the roleof PI3K/Akt signaling pathway.MethodsPart I: Forty-eight isolated rat hearts were randomly divided into6groups after20min of equilibration (n=8each):sham operation group(S),ischemia (I),ischemia/reperfusion group (I/R), NGF0.025ng/ml,0.1ng/ml,0.4ng/ml for20min followed by10min washout before ischemia.Except for S group, all hearts were subjected to30min of global ischemiafollowed by120min of reperfusion with Kerbs-Henseleit buffer.S Group: rat hearts were continuously perfused with K-H for180min. I/R Group: after30min perfusion with K-H, the rat hearts were subjectedto30min global ischemia and120min reperfusion.NGF Group: the rathearts were perfused with K-H contained0.025ng/ml,0.1ng/ml,0.4ng/mlNGF for20min preischemia and for10min preischemia with K-H, theother with the same of I/R group.Determination the expression of NGF and expression of NGF mRNAin myocardial of these groups (by Western-blot and RT-PCR) in the formthree groups.Eight hearts in each group were slected and HR,left ventricu-lar developed pressure (LVDP),left ventricular end-diabetic pressure(LVEDP),+dp/dtmaxwere at the end of equilibration, the end of perfusionand at5,30,60,120min of reperfusion.The activities of creatine kinase(CK-MB) and lactate dehydrogenase (LDH) in coronary effluent weremeasured at the end of10min equilibration (balance), and at5,30,60and120min of reperfusion. Myocardial tissues were obtained at the end ofreperfusion for determination of apoptosis (by TUNEL).Part II: Thirty-two isolated rat hearts were completely randomlyassigned to4different groups:ischemia/reperfusion group (I/R), NGFgroup (N),LY294002group (L) and NGF+LY294002group (N+L).Theisolated rat hearts were perfused on a Langendorff apparatus with K-H.Allthe hearts were subjected to30min global ischemia and followed by120min reperfusion.(1) I/R Group: after60min perfusion with K-H, the rat hearts were subjected to30min global ischemia and120min reperfusion.(2) N Group:after20min stabilized perfusion with K-H contained0.1ng/ml NGF, the rat hearts were perfused with K-H for40min washoutbefore ischemia, the other with the same of I/R group.(3)L group:after20min stabilized perfusion with K-H contained1.5mg/kg LY294002,the rathearts were perfused with K-H for40min washout before ischemia, theother with the same of I/R group.(4) N+L group:after20min stabilizedperfusion with K-H contained1.5mg/kg LY294002,the rat hearts wereperfused with K-H for10min washout.Then20min stabilized perfusionwith K-H contained0.1ng/ml NGF, the rat hearts were perfused with KHfor10min washout before ischemia, the other with the same of I/R group.Eight hearts in each group were slected and HR,left ventriculardeveloped pressure (LVDP),left ventricular end-diabetic pressure (LVEDP),+dp/dtmaxwere at the end of equilibration,the middle of perfusion,the endof perfusion and at5,30,60,120min of reperfusion.The activities of lactatedehydrogenase (LDH) and creatine kinase (CK-MB) in coronary effluentwere measured at the end of20min equilibration (balance), the middle ofperfusion and at5,30,60and120min of reperfusion. Myocardial tissueswere obtained at the end of reperfusion for determination ofapoptosis (byTUNEL). In the end,Determination the expression of Akt,pAkt,GRP78andmRNA in myocardial of these groups (by Western-blot and RT-PCR). ResultsPart I: Compared with group S, group I and I/R the expression ofNGF and NGF mRNA in myocardial were significantly decreased (P＜0.05). Compared with group I, group I/R the expression of NGF and NGFmRNA in myocardial were decreased (P＜0.05). Compared with groupI/R,LVDP,+dp/dtmaxand HR were significantly increased in group N0.1anddecreased in group N0.025and group N0.4,(P＜0.05).Compared with groupI/R, group N0.1the CK-MB and LDH activities in coronary effluent andapoptosis index in myocardial were significantly decreased;group N0.4theCK-MB and LDH activities in coronary effluent were up-regulated,(P＜0.05). Compared with group N0.1, the CK and LDH activities in coronaryeffluent a were significantly increased in group N0.4(P＜0.05).Part II:1. The parameter of heart functionThe hemodynamic parameters were no significant difference beforeischemia in each group (P>0.05). Compared with group I/R, LVDP, HR and+dp/dtmaxwere decreased significantly, LVEDP were significantly increasedin N group (P<0.05).There were no significant difference between I/Rgroup and L group.Compared with I/R group,+dp/dtmaxwere increased,LVEDP were lower in N+L group in T3(P <0.05). There were no significantdifference in other Time (P <0.05). 2. The LDH and CK-MB concentrationCompared with group I/R, the LDH and CK-MB concentration weresignificantly decreased in N group (P<0.05).There were no significantdifference between I/R group and L group.Compared with I/R group, LDHand CK-MB were lower In N+L group in T3(P <0.05).There were nosignificant difference in other Time (P <0.05).3. The apoptotic indexCompared with group I/R, the apoptotic index was lower in N group(P<0.05). There were no significant difference between group I/R,L.4. The expressions of Akt,pAkt,GRP78and Akt mRNA, GRP78mRNACompared with I/R group, the expressions of Akt,pAkt,GRP78and AktmRNA were significantly increased, GRP78mRNA were no significantdifference in N group (P<0.05). There were no significant differencebetween group I/R and L.In N+L group, above of all thing is adverse.Conclusions1. The expression of NGF and NGF mRNA in myocardial is related tomyocardial ischemia-reperfusion injury.2.0.1ng/ml NGF pretreatment can attenuate myocardial I/R injury.While0.4ng/ml NGF pretreatment could aggravate cardiac injury. 3. NGF pretreat could attenuate myocardial apoptosis by activatingendoplasmic reticulum stress pathway in ischemia reperfusion injury inisolated rat hearts,which partly through PI3K/Akt signaling pathway. |
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