Protective Effects Of Prostaglandin E₁ On Hypoxia/reoxygenation Injury In Cultured Neonatal Rat Myocytes

Myocardial ischemia/reperfusion injury (IRI) is one of the most important complications in opening therapy of coronary artery. But the real mechanisms of the phenomenon have been subjected to debate. Prostaglandin E, has the effects of dilating vessel, maintaining stability of cell membrane, inhibiting platelet aggregation and excessive calcium entry. This paper is to study the protective effects of prostaglandin E, on hypoxia/reoxygenation injury in cultured neonatal rat myocytes.The hearts were isolated from l~3-day-old Wistar rats and the ventricles were minced in phosphate buffer solution. The myocardial cells were dispersed with 0.125% pancreatin. The cell suspensions from each digest were collected and centrifuged. After being purified, the cells density was adjusted to 3 X 105 - ml-1 with Dulbecco's modified Eagle culture medium consisting of 15% fetalcalf serum o For biochemical assays, the myocytes were planted in 25ml culture bottles. For MTT assay, the myocytes were planted directly on plastic plates. Cells were incubated at 37℃ in 5%CO2 atmosphere. The models of hypoxia/reoxygenation were made with the first generation of cultured myocytes. The normal cultured medium was replaced by hypoxic solution to induce hypoxia. Then the hypoxia solution was replaced by reoxygenation solution to induce reperfusion. Cultured myocytes of neonatal rats were divided into five groups: control group(c); hypoxia/reoxygenation (H/R) group; hypoxia/reoxygenation+low dose of PGE, (5ng ?ml"1) group; hypoxia/reoxygenation+medium dose of PGE, (15ng · ml"1) group; hypoxia/reoxygetionhigh dose ofPGE,(45ng · ml"1) group.The photos of all groups were taken. The activities of plasma superoxide dismutase (SOD) were measured by the method of xanthine oxidase and the contents of serum malonicaldehyde (MDA) by the method of thiobarbituric acid after 60min of hypoxia and 30min of reoxgynation. The activities of dehydrogenase in mitochondria were detected by MTT assay after 2h of hypoxia and 0.5h of reoxgynation. The activities of LDH in culture were evaluated after 30min of hypoxia and 40min of reoxygenation. Ultrastructural features of cardiomyocytes were observed by electron microscopy.Results: Being injured by hypoxia and reoxgynation, refracting power of the cells mass declined. The processes were shortened. Cellbeat became weak or stopped. The activities of SOD and dehydrogenase in H/R group obviously decreased (P<0.01, compared with the control group). The contents of LDH and MDA in H/R group were higher than those in any other groups (P<0.01). PGE1 (5ng · ml-1, 15ng · mr',45ng · ml-1) relieved the injury: the morphologic appearance of cells comparatively approached normal, the activities of SOD and dehydrogenase were significantly higher, LDH and MDA contents were significantly lower compared with H/R group. Most cells of H/R group by electron microsope appeared as the early stage of apoptosis, such as cytoplasmic concentration, nuclear chromatin condensation and margination. Some were in advanced stage of apoptosis.The results showed that the model of hypoxia/reoxygenation injury with cultured neonatal rat cells may well build up the myocardium ischemic/reperfusion injury. The model is dependable and reproducible. The experiment showed PGE, have protective effects on the cultured neonatal rat myocardial cells injured by hypoxia/reoxygenation, the mechanisms are related to its antioxidation effects.