The Effects And Mechanism Of GLP-1on Injury Of Neonatal Mice Cardiomyocytes Induced By Hypoxia-reoxygenation

Objective:1.To culture neonate rat cardiomyocytes in vitro.2.To study the effect of glucagons-like peptide-1(GLP-1) on cardiomyocytes injury induced by hypoxia-reoxygenation(H/R) in rats to verify weather GLP-1could protect the heart and the possible mechanisms.3.To supply more theory foundation for the security and validity of GLP-1and correlated drug in clinical application.Method:1.The cultured neonate rat cardiomyocytes were characterized by microscope observation and immunohistochemistry method.2.Two steps:The first step, to verify weather GLP-1could protect cardiomyocytes injury induced by H/R in rats, primary cultured cardiomyocytes were randomly divided into three groups:Group A:normal control group; Group B:H/R group (hypoxia for16hours, reoxygenation for4hours); Group C H/R+GLP-1group.The second step:to observe the effect of LY294002(PI3K inhibitor) on GLP-1’s action, primary cultured cardiomyocytes were randomly divided into five groups:Group A:normal control group; Group B:H/R group (hypoxia for16hours, reoxygenation for4hours,); Group C:H/R+GLP-1group Group D:H/R+GLP-1+LY294002group; Group E:H/R+LY294002group.3.The activity of lactate dehydrogenase in the culture medium was detected by test kit.4.Stained with propidium iodide, cardiomyocytes apoptosis rate was detected by flow cytometry.5.Caspase-3activity was detected by test kit.Result:1.Cardiomyocytes were confirmed and these purity was more than90%.2.GLP-1can protect cardiomyocytes injury induced by H/R in rats.Hypoxia for16hours, reoxygenation for4hours, then LDH activity, cardiomyocytes apoptosis rate and Caspase-3activity were measured, the result display that compared with Group A (normal control group), LDH activity, cardiomyocytes apoptosis rate and Caspase-3activity all increased significantly in Group B (H/R group)(P<0.01). This indicated that the model of H/R injury has been builded successfully; Compared with Group B, LDH activity, cardiomyocytes apoptosis rate and Caspase-3activity all decreased significantly in group C (H/R+GLP-1group)(P<0.01). And this indicated that GLP-1can reduce cardiomyocytes injury induced by H/R in rats.3.LY294002(PI3K inhibitor) can weaken GLP-1’s action.To observe the effect of LY294002(PI3K inhibitor) on GLP-1’s action, primary cultured cardiomyocytes in Group D (H/R+GLP-1+LY294002group) received LY294002+GLP-1at the beginning of hypoxia, the result display that compared with Group C, LDH activity, cardiomyocytes apoptosis rate and Caspase-3activity all increased significantly in Group D (P<0.05, P<0.01, P<0.01respectively), This indicated that LY294002can weaken GLP-1’s protection on cardiomyocytes injury induced by H/R in rats. In addition, the distinction of above indexes between Group B and Group E (H/R+LY294002group) has no statistical significance.Conclusion:It was demonstrated in our vitro study that GLP-1can directly act on cardiomyocytes and protect them from H/R injury mainly by inhibiting their apoptosis, the mechanism of which may be concerned with GLP-1’s effects on antiapoptotic may be through PI3K/AKT signaling pathways.