The Protection And Mechanism Of N-n-butyl Haloperidol Iodide On Yocardial Ischemia-Reperfusion Injury In Rats And Cardiomyocyte Exposure To Ypoxia-Reoxygenation Induces Egr-1 Expression Involving Ca2+/PKC Pathway

Early growth response-1 (Egr-1), a transcription factor and an immediate early gene, can beinduced in a transient manner by a variety of extracellular stimuli. The over expression of Egr-1was the crucial mechanism in heart graft rejective reaction and lung ischemia-reperfusion injury.But the mechanism of Egr-1 in I/R remains unclear. Use antisense oligonucleotides(AS) in vivoand in vitro, found that Egr-1 was the main mechanism in heart I/R injury. N-n-butylHaloperidol Iodide (F₂) could supress the Egr-1 mRNA and protein expression. Does all theprotection of F₂ on myocardial I/R injury through suppressing Egr-1 expression. Previous studyhas found that Ca²⁺,PKC participate in the regulation of Egr-1.But the mechanism of Egr-1expression in I/R remains unclear. So this research focuses on two problems.â‘ Use AS, AS+ F₂in cultured cardiomyocytes, view the Egr-1 protein expression and the heart function.â‘¡Useverapamil, PKC specific inhibitor H7, BIM in cultured cardiomyocytes after H/R to investigateCa²⁺/PKC Pathway.Methods1. The rat experimental models in vivo (I/R) were established by 60 min left anteriordescending coronary artery (LAD) occlusion followed by 180 min of reperfusion. All ratswere randomly assigned into one of five groups: Control, I/R, PEG, AS and AS+ F₂.Hemodynamics was monitored with BL420 system. The expression levels of Egr-1 proteinin myocardium were examined by Western-blot. Levels of CK,LDH in the serum and MPOin the myocardium were mesured by ehromatometry. Levels of MDA in the myocardium were mesured by thiobarbituric acid method. Levels of SOD in the myocardium weremesured by xanthine oxidase method.2. Primary culture of neonatal rat cardiomyocytes and preparation of H/R model: After beingreplaced the initial culture medium with hypoxic buffer, the cardiomyocytes were incubatedin an air-tight chamber gassed with pure N₂ for 3 h of hypoxia. The buffer was then replacedwith fresh oxygenated culture medium and the dishes were transferred into a normoxicincubator for 1 h of reoxygenation. The cardiomyocytes were randomly divided into one offollowing groups: Control, H/R, DMSO, EGTA, VER, H7 or BIM. The expression levels ofEgr-1 mRNA in cultured cardiomyocytes were examined by RT-PCR. The expression levelsof Egr-1 protein in cultured cardiomyocytes were examined by Western-blot.Results1. The effect of AS, AS+ F₂ on Egr-1 protein expression in myocardiumRelative to the Control group, Ischemia-reperfusion induced a significantly increase inEgr-1 protein expression in myocardium(P＜0.05). Compared with the I/R group, levels ofEgr-1 protein measured by Western blot analysis in myocardium in the group AS and AS+F₂ were significantly decreased at the end of experiment(P＜0.05). Compare to the AS group,These changes in levels of Egr-1 protein were not altered by F2 (P＞0.05).2. The effect of AS, AS+ F₂ on MPO, SOD and MDA in myocardiumRelative to the I/R group, levels of MPO, MDA in myocardium in the group AS and AS+F2were significantly decreased(P＜0.05 ). levels of SOD were increased(P＜0.05 ). Compare tothe AS group, these changes in levels of levels of MPO, MDA, SOD were strengthened.3. The effect of AS, AS+F₂ on CK, LDH activities in serumIschemia-reperfusion induced a significantly increase in CK, LDH activities in serum(P＜0.05). Compared with I/R group, AS group and AS+F₂ group reduced CK and LDHactivity(P＜0.05). Compared with AS group, AS+F₂ group reduced CK and LDH activity(P＜0.05).4. The effect of AS, AS+F₂ on hemodynamicsValues of HR, SP, DP, LVSP,±dp/dtmax were significantly lower in I/R hearts during ischemia period. Compared with I/R group, values of HR, SP, DE LVSP,±dp/dtmax werehigher in AS and AS+F₂ groups, But there was no significant difference between the twogroups. Compared with AS group,Values of SP, DP, LVSP, +dp/dtmax during reperfusionwere higher in AS+F₂ groups.5. The effect of VER, EGTA, H7 or BIM on Egr-1 expression in cultured cardiomyocytes5.1 Relative to the Control group, levels of Egr-1 rnRNA measured by RT-PCR analysisin cultured cardiomyocytes in the H/R group were significantly increased at the end ofexperiment (P＜0.05). These changes in levels of Egr-1 mRNA were not altered by DMSO,but significantly reduced when VER, EGTA, H7 or BIM was added into cultured cells (P＜0.05).5.2 Consistent with changes in levels of Egr-1 mRNA in the H/R group, Egr-1 proteinmeasured by Western blot analysis in cultured cardiomyocytes were significantly increasedrelative to the Control group (P＜0.05). These changes in levels of Egr-1 protein were notaltered by DMSO, but significantly reduced when treated with VER, EGTA, H7 or BIM (P＜0.05).Conclusions(1) F₂ can protect myocardium from ischemia-reperfusion by inhibiting Egr-1 overexpression,but that is not the whole mechanisms of F₂.(2) Ca²⁺/PKC sigal pathway involved in hypoxia-reoxygen induced Egr-1 gene expression incultured cardiomyocytes.