The Effects Of N-n-butyl Haloperidol Iodide On Egr-1 Expression And Cellular Injury In Rat Cardiac Microvascular Endothelial Cells Exposed To Hypoxia And Reoxygenation

Early growth response gene 1 (Egr-1) is one of the immediate-early genes in response to stimulation, and its rapid up-regulation in mRNA, protein and nuclear binding activity have been demonstrated in various cells after ischemia/reperfusion (I/R). Egr-1 is poorly expressed in the normal vascular wall but is activated by the stimulation of hypoxia or I/R, and plays a pivotal role in the coordinated transcription of multiple inflammatory and coagulant genes. The expressions of these genes mediate coagulation, inflammation and vascular permeability, which are the main pathological changes of I/R injury. Thus, Egr-1 was designated as having a significant role in the pathogenesis of I/R injury.Recently, the endothelium has been shown to play a key role in the injury suffered after I/R. An early event during reperfusion is the development of endothelial dysfunction. When rendered hypoxic and then reoxygenated, endothelial cells become activated to express proinflammatory properties that include the induction of leukocyte-adhesion molecules, procoagulant factors and vasoconstrictive agents that increase vasomotor tone. These changes may contribute to the no-reflow phenomenon by promoting endothelial edema, neutrophil and platelet plugging, and microthrombosis.N-n-butyl haloperidol iodide (F₂) is a new compound that has cardioprotective effects. However, we do not know whether these protective effects elicited by F₂ are also related to a modulation of Egr-1 expression, a central role in the pathogenesis of myocardial I/R injury. It is well recognized that the endothelial cells are highly sensitive to I/R in the myocardial tissue. Here, we test the hypothesis that the effect of F₂ on myocardial I/R injury is associated with attenuation of inflammatory reaction and damage of endothelial cell by inhibiting the expression of Egr-1. A hypoxia/reoxygenation (H/R) model of cultured rat cardiac microvascular endothelial cells (CMECs) was served as the model of I/R in vitro for futher study. The purpose of the study is to investigate the effects of F₂ on Egr-1 mRNA transcription and protein expression in cultured endothelial cells after H/R, and research the relationship between the changes of Egr-1 and endothelial H/R injury, which is helpful to elucidate the new mechanisms of F₂.Methods1. Isolation of neonatal rat CMECs: CMECs were isolated from 1- 4 d old Sprague-Dawley rat hearts and identified immunochemically by treatment with antibodies against human factor VIII-related antigen. All experiments were carried out with cells at passage 3-5.2. Preparation of H/R model: After replaced the initial culture medium with hypoxic buffer, the CMECs were incubated in an air-tight chamber gassed with pure N2 for 3 h of hypoxia. The buffer was then replaced with fresh oxygenated culture medium and the dishes were transferred into a normoxic incubator for 2 h of reoxygenation.3. Groups: The CMECs were randomly divided into one of five groups: Control, H/R, H/R+vehicle (polyethylene glycol 400, PEG), H/R+F₂ (1×10^-6mol/L) and H/R+ Positive control drug (Verapamil, VER, 1mg/L). F₂, VER or equal volumn of PEG were added to the hypoxic buffer and reoxygenated medium. The cells of Control group were incubated with fresh culture medium in normoxic condition for 5 h.4. The expression levels of Egr-1 mRNA in cultured CMECs were examined by RT-PCR.5. The expression levels of Egr-1 protein in cultured CMECs were examined by Western-blot and immunocytochemistry, by which the subcellular localization of Egr-1 protein expression in cultured CMECs was also detected.6. Levels of tumor necrosis factor-α(TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in the medium were mesured by sandwich enzyme-linked immunoabsorbent assay (ELISA) method to assess the degree of inflammation of cultured CMECs.7. Neutrophil accumulation was determined by measuring myeloperoxidase (MPO) activity.8. The interactions between platelets and endothelial cells were observed with a scanning electron microscopy (SEM).9. The leakage of lactate dehydrogenase (LDH) in the culture medium, activity of superoxide dismutase (SOD) and level of malondialdehyde (MDA) in the cultured CMECs were measured by colorimetric method to assess the degree of injury.Results1. The cultivation and identification of CMECs.Endothelial cells were exhibited with the "cobblestone" on morphology and identified by positive staining for factor VIII-related antigen.2. The effect of F₂ on Egr-1 expression in cultured CMECs.Relative to the Control group, levels of Egr-1 mRNA measured by RT-PCR in cultured CMECs in the H/R group were significantly increased at the end of experiment. These changes in levels of Egr-1 mRNA were not altered by PEG, but significantly reduced when F₂ or VER was added into cultured cells.Consistent with changes in levels of Egr-1 mRNA in the H/R group, levels of Egr-1 protein measured by Western blot and immunocytochemistry in cultured CMECs were significantly increased relative to the Control group. Egr-1 protein in H/R group was mainly localized in the nucleus. These changes in levels of Egr-1 protein were not altered by PEG, but significantly reduced when treated with F₂ or VER.3. The effects of F₂ on levels of TNF-α, IL-1β, IL-6 and ICAM-1 in culture medium.Compared with the Control group, H/R caused TNF-α, IL-1β, IL-6 and ICAM-1 secretion from CMECs. Treatment with F₂ or VER, but not PEG, can significantly reduce TNF-α, IL-1β, IL-6 and ICAM-1 secretion from CMECs.4. The effect of F₂ on adherence of neutrophils to CMECs in vitro.Compared with the Control group, H/R caused significant adhesion of neutrophils to CMECs. The increased adherence to H/R-conditioned CMECs was significantly diminished by F₂ or VER. PEG had no effect on leukocyte adherence.5. The effect of F₂ on interactions of platelets and CMECs in vitro.The analysis performed on several random fields revealed that H/R induced the formation of micro thrombosis consisting of platelets. Unstimulated platelets did not aggregate and exhibited round, discoid form (Control groups). The aggregates were significantly diminished by F₂ or VER, but were not changed by PEG.6. The effects of F₂ on changes of enzymatic activities in culture medium or in CMECs.After reoxygenation, the levels of LDH and MDA increased and the activity of SOD decreased significantly, as compared with the respective Control group. However, administration of F₂ or VER could maintain the activitiy of SOD, reduce MDA, and minimize the leakage of LDH out of CMECs caused by H/R. There were no differences between H/R and PEG groups.Conclusions1. Endothelial dysfunction was mediated by Egr-1 and resulted in a series of cardiovascular injuries related to inflammatory reaction during H/R.2. F₂ inhibits the overexpression of Egr-1 mRNA and protein in cultured CMECs caused by H/R. Meanwhile, F₂ attenuates the inflammatory reaction by inhibiting the overexpression of Egr-1-inducible genes such as TNF-α, IL-1β, IL-6 and ICAM-1. F₂ could protect endothelial function by inhibiting the overexpression of Egr-1, which might be the protective mechanisms of F₂ on myocardial I/R injury.3. Endothelial cell is one of the target cells for action of F₂.