The Anti-apoptosis Effect Of Erythropoietin In Neonatal Rat Cardiacmyocytes Exposed To High-glucose

Objective:To investigate the effects of erythropoietin(EPO) on the apoptosis in neonatal rat cardiac myocytes exposed to high-glucose, and evaluate the mechanism of endoplasmic reticulum stress(ERS) involved in it.Methods:Cardiomyocytes were isolated from new-born Sprague-Dawley rats and were identified by immunohistochemistry staining of a-actin. Cardiomyocytes were cultured in normal condition for 72 hours, then divided into 4 groups:normal-glucose group, high-glucose group, the monnitor group, different concentrations of EPO (5 U/mL,10U/mL,20U/mL,40U/mL). Apoptosis ratio was measured by bivariate flow cytometry. Selected the best concentration.ERS inhibitor was employed to detect the underlying mechanism by which EPO inhibits cardiomyocyte apoptosis;The cell culture was divided into five groups:normal glucose(NG),high glucose(HG),4-phenylbutyrate acid+high glucose(PBA+HG),EPO+normal glucose(EPO+NG), EPO+high glucose(EPO+HG). Apoptosis ratio experiments were performed by bivariate flow cytometry. Intracellular calcium level was measured by confocal microscopy in conjunction with the calcium indicator Fluo-3. Real-time PCR(RT-PCR) was used to detect the mRNA level of EPOR、GRP78 and Serca2a, and Western blot was used to detect the protein level of EPOR、GRP78 and Serca2a.Results:After 24 hours, some cultured single myocytes began to beat spontaneously. Cells were confirmed as cardiomyocyte by immunohistochemistry staining of a-actin. Compared with the NG, apoptosis ratio significantly increased in HG group [(41.36±10.49)% vs (16.08±3.97)%, P<0.05],and the monnitor group have no effect on apoptosis. The apoptosis ratio in different concentrations of EPO (5U/ml,10U/ml,20U/ml,40U/ml)were (36.59±19.94)%,(29.63±16.4)%,(20.97±10.55)%, (24.08±8.3)% respectively, EPO inhibited the increase of apoptosis induced by high-group (P<0.05), and the best effective concentration was 20U/mL. There was no significant differences between the NG group and the monitor group in apoptosis ratio (P>0.05). PBA inhibited the increase of apoptosis induced by high-group (P<0.05). Compared with the NG group, calcium fluorescence intensity was increased, the expression of GRP78 was up-regulated, and the expression of EPOR and Serca2a were down-regulated in the HG group (P<0.05).There was no significantly difference between NG group and EPO+NG group.Compared with the HG group, the increase of calcium fluorescence intensity were inhibited, the mRNA and protein expression of GRP78 were decreased, the mRNA and protein level of EPOR and Serca2a were up-regulated in the EPO+HG and PBA group (P<0.05). There was no significant difference in apoptosis, calcium fluorescence intensity, mRNA level and the protein expression of GRP78, EPOR and Serca2a between the EPO+HG group and the PBA+HG group (P>0.05).Conclusion:1.Hyperglycemia induce the expression of endoplasmic reticulum stress in cadiomyocytes and intervene the balance of calcium ion,which results in the apoptosis of cardiomyocytes.2.EPO can inhibite the endoplasmic reticulum stress and maintain balance of calcium ion,then reduce apoptosis ratio in neonatal rat cardiomyocytes exposed in high glucose.