The Protective Effects of <i>N</i>-n-butyl Haloperidol Iodide on Myocardial Ischemia-Reperfusion Injury in Rats by Inhibiting Egr-1 Overexpression

Abstract

Aims: Our previous studies have shown that N-n-butyl haloperidol iodide (F₂) can antagonize myocardial ischemia/reperfusion (I/R) injury by blocking intracellular Ca²⁺ overload. The present study is to test the hypothesis that the protective effects of F₂ on myocardial I/R injury is mediated by downregulating Egr-1 expression. Methods: The Sprague–Dawley rat myocardial I/R model and cardiomyocyte hypoxia/reoxygenation (H/R) model were established. With antisense Egr-1 oligodeoxyribonucleotide (ODN), the relationship between Egr-1 expression and myocardial I/R injury was investigated. Hemodynamic parameters, myeloperoxidase (MPO), cardiac troponin I (cTnI) and tumor necrosis factor-α (TNF-α) were measured to assess the degree of injury and inflammation of myocardial tissues and cells. Egr-1 mRNA and protein expressions were examined by Northern-blot and Western-blot analyses. Results: Treatment with antisense Egr-1 ODN significantly reduced Egr-1 protein expression and attenuated injury of myocardial tissues and cells. Meanwhile, treatment with F₂ significantly inhibited the overexpression of Egr-1 mRNA and protein in myocardial tissues and cells. Consistent with downregulation of Egr-1 expression by F₂, inflammation and other damages were significantly relieved evidenced by the amelioration of hemodynamics, the reduction in myocardial MPO activity as well as the decrease in leakage of cTnI and release of TNF-α from cardiomyocyte. Conclusions: These results suggested that the overexpression of Egr-1 was causative in myocardial I/R or H/R injury, and F₂ could protect myocardial tissues and cells from I/R or H/R injury, which was largely due to the inhibition of Egr-1 overexpresssion.

The Protective Effect of Egr-1 Antisense Oligodeoxyribonucleotide on Myocardial Injury Induced by Ischemia-reperfusion and Hypoxia-reoxygenation

“…As reported previously [13], FITC labeled Egr-1 AS-ODN was found in the cytoplasm and nuclei of myocardial tissues, while mainly in the nuclei of cultured cardiomyocytes (data not shown), indicating that Egr-1 AS-ODN had successfully been uptaken up.…”

“…In order to elucidate the cause-effect relationship between Egr-1 overexpression and myocardial I/R injury, we further studied the effects of Egr-1 AS-ODN on changes of enzymetic activities induced by I/R or H/R [13]. In above-mentioned study, we found that because of its direct inhibitory effect on Egr-1 protein Egr-1 phosphorothioate AS-ODN attenuated the myocardial I/R injury in cytomembrane structure as evidenced by the reduction of CK and LDH leakage, and in oxidative stress caused by reactive oxygen species (ROS) as evidenced by the increase of superoxide dismutase (SOD) activity and the decrease of malondialdehyde (MDA) yield [13].…”

“…Adult male SpargueDawley rats (weighing 200-250g, Grade ΙΙ, Certificate 2007A024) were provided by the Experimental Animal Center of Medical College, Shantou University (Shantou, China). Myocardial I/R model of rat was performed as previously described [13]. In brief, after anesthetization, thoracotomy and pericardiotomy, the left anterior descending coronary artery (LADCA) was occluded reversibly by a silk suture.…”

“…Primary culture of neonatal rat ventricular myocytes and H/R model were carried out by previously described methods [13]. In short, the isolated cells from 1-4 days neonatal rats were purified by preplating 30 min at 37°C and adding 5-Bromo deoxyuridine into the medium for the first 3 d to eliminate non-cardiomyocytes [14].…”

“…Our preliminary study had shown that Egr-1 antisense oligodeoxynucleotide AS-ODN could relieve myocardial injury as evidenced by the inhibition in leakage of CK and LDH, and the reduction in oxidative stress by the inhibition of Egr-1 protein expression [13]. The purpose of this study is to investigate further the effects of Egr-1 AS-ODN on morphology, function and inflammation of myocardial tissues or cultured cardiomyocytes in I/R model in vivo and in vitro, which will elucidate the central role of Egr-1 in occurrance and development of myocardial I/R injury.…”