| Apoptosis and necrosis are two different forms of cell death in myocardial ischemia/reperfusion injury(IRI). Apoptosis is a genetically determined, active process with typical morphological and biological changes, these changes are regulated by the actions of a series of genes. So there is important significance to study apoptosis in myocardial ischemia /reperfusion injury. Prostaglandin EI has the effects of dilating vessel, ameliorating microcirculation, inhibiting platelet aggregation, and lessening myocardial infarct area. Previous study has demonstrated that PGEi has evidently protective effects on myocardial ischemia/reperfusion injury induced by ligation of coronary artery, but there are no reports so far about whether PGEi can inhibit cardiomyocyte apoptosis. The purpose of this study is to investigate the effects of PGEi on hypoxia/reoxygenation apoptosis in cultured neonatal rat cardiomyocytes.MethodsThe hearts were isolated from l~3-day-old Wistar rats and the ventricles were minced in phosphate buffer solution. The myocardial cells were dissociated with 0.125% pancreatin. After dispersed, the cells were collected, purified and incubated in 25ml culture bottles(the cells density was adjusted to 5x105cells ml-1 with Dulbecco's modified eagle culture medium consisting of 15% fetal calf serum). The models ofhypoxia/reoxygenation were made with the first generation of cultured cardiomyocytes. The normal cultured medium was replaced by hypoxia solution, the cells were cultured hermetically for 30min to induce hypoxia, then the hypoxia solution was replaced by reoxygenation solution, the cells were cultured for 40min to induce reperfusion. Cultured cardiomyocytes of neonatal rats were divided into five groups: control group(c); hypoxia/reoxygenation(H/R) group; hypoxia/reoxygenation±low dose of PGE1(5ug L-1) group; hypoxia/reoxygenation±medium dose of PGE1(15ug L-1) group; hypoxia/reoxygenation±high dose of PGE1(45ug L-1) group. The effects of PGE1 on hypoxia/reoxygenation apoptosis in cultured neonatal rat cardiomyocytes were studied at morphological, biochemical and molecular biological levels.ResultsBeing injured by hypoxia and reoxygenation, refracting power of the cells declined, pseudo-podium became shortened or disappeared, cell beat became weak or stopped. Most cells of H/R group by electron microscopy appeared as cytoplasmic concentration, nuclear chromatin condensation and margination. PGE^Sug-L'^lSug-L'^Sug-L"1) relieved the injury. Most cells approached to normal, only few cells showed above changes.The results of DNA electrophoresis in the H/R group showed the typical DNA ladder and the DNA ladder decreased gradually as the dose of PGE1 increased. The TUNEL staining showed that the sum of apoptotic cells in the H/R group was larger than that in PGE1(45u g L-1) group.The results of in situ hybridization showed that the integral light density of bcl-2 mRNA in PGE,(5ug L-1, 15ug L-1 ,45ug L-1) groups(3. 406 ±0.405, 4.505±0.687, 8.559 ±0.602) was significantly higher than that in H/R group(l. 989 ±0.522) (P < 0.01 ). The integral light density of bax mRNA in PGE1(5ug L-1, 15ug L-1, 45ug L-1) groups(6.418± 0.792, 4.353±0.767, 3.171 ±0.728) was significantly lower than that in H/R group(8.547 ±0.852) (P < 0.01) . The same results were also demonstrated by the method of immunohistochemical staining.ConclusionsThe results suggest that H/R injury can induce cardiomyocyte apoptosis, PGE1 has obviously anti-apoptotic effects on cardiomyocyte and the mechanisms are possibly by inhibiting the expression of bax and increasing the expression of bcl-2. |
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