| Objective:Utilizing rat ischemia/reperfusion injury (IRI) model to observe the effects ofischemia, emulsified isoflurane and pinacidil postconditioning on cardiac function, and toexplore the activation mechanism of Nrf2-ARE pathway and the protective effect onisolated rat hearts using ischemia/pharmacological postconditioning; explore themechanisms of the cardioprotective effects of different concentration of emulsifiedisoflurane and pinacidil postconditioning, and the correlation between pharmacologicalpostconditioning produced ROS level and its myocardial protective effect.Methods:1. The grouping and obvervational indexes of the activation mechanism of the Nrf2-AREpathways.1.1Grouping.72male Sprague-Dawley rats were randomly divided into9groups (n=8), i.e. normalgroup (Group N), control group (Group Con), ischemia postconditioning group (GroupIPO), emulsified isoflurane postconditioning group (Group EI2;1.68mM), intralipidpostconditioning group (Group FAT), pinacidil postconditioning group (Group P50), N-(2-mercaptopropionyl)-glycine (MPG;2mM)+IPO group (Group M+IPO), MPG+EI2group (Group M+EI2), MPG+P50group (Group M+P50).1.2The solution of perfusion:Rat hearts were perfused with Krebs-Henseleit (K-H) buffer for20minutes forequilibration. Subsequently,(1) Group N: was perfused with K-H buffer for100minutesafter equilibration;(2) Group Con: was perfused with4℃ST.Thomas solution to stop theheart beating after equilibration, then the hearts were underwent40minutes globalischemia under32℃, and followed by the K-H solution for60minutes;(3) Group IPO:after global ischemia period, the hearts were subjected to six10-seconds cycles of ischemia/reperfusion at the beginning of reperfusion, then were reperfused for58minutes;(4) Group EI2: after global ischemia, rat hearts were perfused with K-H buffer containingemulsified isoflurane (1.68mM) for2minutes before reperfusion;(5) Group FAT: afterglobal ischemia, hearts were then perfused with EI2carrier---FAT for processing2minutes before reperfusion.(6) Group P50: after global ischemia, rat hearts were perfusedwith K-H buffer containing pinacidil (50μM) for2minutes before reperfusion;(7) GroupM+IPO: after global ischemia, the hearts were subjected to perfuse with K-H buffercontaining MPG (2mM) for3minutes, and then underwent six10-seconds cycles ofischemia/reperfusion before reperfusion;(8) Group M+EI2: after global ischemia, thehearts were perfused with K-H buffer containing MPG (2mM) for3minutes, and thenunderwent with K-H buffer containing concentrations of emulsified isoflurane (1.68mM)for2minutes before reperfusion;(9) Group M+P50: after global ischemia, the heartswere perfused with K-H buffer containing MPG (2mM) for3minutes, and then subjectedto perfuse with K-H buffer containing pinacidil (50μM) for2minutes before reperfusion.1.3Observational objects.(1) Cardiac function indexes (such as HR, LVDP, LVEDP, and the Max dp/dt) at the endpoint of equilibration and reperfusion were observed and recorded, and left ventricularmyocardial tissue were collected at the end of reperfusion.(2) The ultrastructure of myocardial tissue was observed by electron microscopy and themitochondrial Flameng score was calculated.(3) RT-PCR and western-blot were applied to detect the gene transcription and proteinexpression of HO-1, NQO1, SOD1, and Nrf2in left ventricular myocardial tissue afterreperfusion.2. Testing the effects of three kinds of postconditioning way on the level of ROS.2.1Grouping.60male Sprague-Dawley rats were randomly divided into10groups (n=6, each group),i.e. the normal group (Group N), the control group (Group Con), the ischemiapostconditioning group (Group IPO), the different concentrations of emulsified isoflurane(0.84,1.68,2.52mM) postconditioning group (Group EI1, EI2, EI3), the intralipidpostconditioning group (Group FAT), and the different concentrations of pinacidil (10,30,50μM) postconditioning group (Group P10, P30, P50).2.2The solution of perfusion with the same as1.2 2.3Testing on the ROS level of cardiac muscle tissue: Each group of ROS level at twotime points were detected, namely tested on the ROS level of left ventricle tissue at5minutes of reperfused and at the end of reperfused (u/ml).Results:1. The cardiac function indexes: There is no significant difference in the cardiac functionamong each group at the point of equilibrium; The HR, LVDP and+dp/dtmax at the end ofreperfusion: The cardiac function indexes are lower among each group compared withgroup N; group IPO, EI2and group P50are better than group Con (P<0.05); Comparedwith group IPO, there are no significant difference in group EI2and group P50, but groupM+IPO is obviously decreased (P <0.05); The cardiac function indexes are lower ingroup FAT and group M+EI2as compared with group EI2(P <0.05); Compared withgroup P50, group M+P50index is decreased significantly (P <0.05). The LVEDP at theend of reperfusion: The LVEDP is higher than that among each group as compared withgroup N, which is significantly increased in group Con and group FAT (P<0.05);Compared with group Con, there are significant increased in group IPO, EI2and groupP50(P <0.05); Compared with group IPO,there is no significant difference in group P50and group EI2, but group M+IPO is significantly increased (P <0.05); The LVEDP ishigher in group FAT and group M+EI2compared with group EI2; Compared with groupP50, the group M+P50is obviously decreased (P <0.05).2. The ultrastructure of myocardial tissue and the mitochondrial Flameng score2.1The ultrastructure of myocardial tissue: The ultrastructure of myocardial tissue ingroup N is mostly normal; The ultrastructure of myocardial tissues in group Con presenceserious damage; The ultrastructure damage of myocardial tissue is improved in group IPO,EI2and group P50as compared with that in group Con, and the ultrastructure damage ofmyocardial tissue in group EI2is improved than group FAT; the ultrastructure damage ofmyocardial tissue in group M+IPO is more serious than group IPO, the ultrastructuredamage of myocardial tissue in group M+EI2is more serious than group EI2, theultrastructure damage of myocardial tissue in group M+P50is more serious group P50.2.2The mitochondrial Flameng score: The mitochondrial Flameng score is higher among each group as compared with group N (P <0.05); The mitochondrial Flameng score islower in group IPO, EI2and group P50as compared with group Con and correspondingnonblocking group (M+IPO, M+EI2, M+P50)(P <0.05); There is no obviously differenceof the mitochondrial Flameng score in group EI2, P50and in group IPO.3. The mRNA expressions of Nrf2, HO-1, NQO1, and SOD1: The mRNA expressionsamong each group are lower as compared with group N; Compared with those in groupCon, the mRNA expressions in group IPO,EI2and group P50are obviously increased (P<0.05), and there is no obviously difference of those in group EI2, P50and in group IPO;The mRNA expressions in group EI2,P50and in group IPO are higher than those in groupadding active oxygen scavenger (MPG)(P <0.05); The mRNA expressions are lower ingroup FAT as compared with those in group EI2(P <0.05).4. The protein expressions of Nrf2, HO-1, NQO1, and SOD1: The protein expressions arelower among each group as compared with those in the group N (P <0.05), and the proteinexpressions are lowest in group Con; The protein expressions are increased in group IPO、EI2and in group P50as compared with those in group Con (P <0.05), there is no differentof protein expressions in group IPO、EI2and in group P50, Moreover, the proteinexpressions are obviously increased in group IPO、EI2and in group P50as compared withthose adding active oxygen scavenger (MPG)(P <0.05); The protein expressions in groupEI2are obviously increased than in group FAT (P <0.05).5. The effect of three kinds postconditioning methods on the ROS level of myocardialtissue5.1The ROS level of myocardial tissue in group N at5minutes of reperfusion and at theend of reperfusion is lowest than other groups, Contrary, The ROS level of myocardialtissue in group Con is highest than other groups, and The ROS level of myocardial tissuehave obviously different in group IPO and in group EI2and in group P50at5minutes ofreperfusion as compared with corresponding group at the end of reperfusion (P <0.05);The ROS level of myocardial tissue is significantly increased in group IPO and in groupEI2and in group P50at5minutes of reperfusion as compared with group N at5minutesof reperfusion (P <0.05), but the ROS level of myocardial tissue in group IPO,EI2and group P50at5minutes of reperfusion is obviously decreased as compared with group Conand group FAT (P <0.05), and that in group EI2and P50,5minutes of reperfusion haveno different compared with group IPO; The ROS level of myocardial tissue in group IPO,EI2and group P50is significantly decreased at the end of reperfusion than in group Conand FAT (P <0.05).5.2The ROS level of myocardial tissue at5minutes of reperfusion among differentconcentration groups of EI is significant difference (P <0.05), and the group EI2ismoderate amount of ROS among different concentration groups of EI; Compared withdifferent concentration groups of EI at5min reperfusion, the ROS level of myocardialtissue in corresponding concentration group is obvious decreased at the end of reperfusion(P <0.05); Moreover, the ROS level of myocardial tissue among different concentrationgroups of EI is significant variation at the end of reperfusion (P <0.05), and that in groupEI2is lowest, the ROS level of myocardial tissue among different concentration groups ofEI is significantly decreased at the end of reperfusion than in group Con (P <0.05). TheROS level of myocardial tissue at the end of reperfusion is in high negative correlationwith the expression of Nrf2mRNA and protein at the end of reperfusion.5.3The ROS level of myocardial tissue at5minutes of reperfusion among differentconcentration groups of pinacidil is significant difference (P <0.05), and the group P50ishighest among different concentration groups of pinacidil; The ROS level of myocardialtissue in group P10and P50is significant difference at the end of reperfusion as comparedwith that in group P10and P50at5min reperfusion (P <0.05); Moreover, the ROS levelof myocardial tissue among different concentration groups of pinacidil is incompletedifference at the end of reperfusion, but that in group P50is the lowest, and the ROS levelof myocardial tissue among different concentration groups of pinacidil is significantlydecreased at the end of reperfusion than in group Con (P <0.05). The ROS level ofmyocardial tissue at the end of reperfusion is in high negative correlation with theexpression of Nrf2mRNA and protein at the end of reperfusion.Conclusion:1. Ischemic postconditioning, pinacidil and emulsified isoflurane postconditioning have protective effect of myocardial tissue from ischemia reperfusion injury, while improve themyocardial tissue ultrastructure and cardiac function index. The cardiac protective effect ofthree kinds postconditioning methods involved the ROS in early reperfusion，whichactivate the Nrf2-ARE pathway, and up-regulate the expression downstream antioxidantprotein and phase II detoxifying enzyme, ultimately reduce a large amount of ROS releaseduring the reperfusion period.2. Different concentrations of two drugs (pinacidil and emulsified isoflurane)postconditioning methods playing different roles in the cardioprotective effects, that is dueto the different level of ROS in early of reperfusion and activation of the Nrf2-AREpathway. Among them, the most appropriate ROS level of myocardial tissue in the earlyreperfusion is in group P50(50μM) and in group EI2(1.68mM) compared with othergroups. Therefore, the myocardial protection effect in group P50(50μM) and in group EI2(1.68mM) are optimum. |
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