| ObjectiveThe aim of this study was to explore the mechanism of Epac-Rap1 signaling pathway on myocardial ischemia/reperfusion injury in rats,and to investigate the protective effect and the mechanism of vitexin on myocardial ischemia/reperfusion(I/R)injury in rats,in order to provide evidence for clinical application of vitexin.Methods1.56 male Sprague-Dawley(SD)rats were randomly divided into 7 group: sham group,Ischemia/Reperfusion(I/R)1h,2h,4h,8h,16 h,24h group.Myocardial ischemia reperfusion injury model was established by ligating left anterior descending coronary for 1h,and followed by reperfusion for 1h,2h,4h,8h,16 h,24h,and recording the electrocardiogram.After ischemia-reperfusion,cardiac function was measured to assess myocardial injury.The histological change of heart was investigated by hematoxylin and eosin(HE)staining.The corresponding index of Epac-Rap1 signal channel was examined by using Western Blotting and immunofluorescence(IF).mRNA encoding gene was determined by RT-PCR.The blood samples were collected to measure the serum levels of cAMP.2.66 male Sprague-Dawley(SD)rats were randomly divided into 11 group: sham group,MIRI group,8-CPT group,ESI-09 group,6-BNZ group,H-89 group,VT+8-CPTgroup,VT+ESI-09 group,VT group,VT+6-BNZ group,VT+H-89 group.Myocardial ischemia reperfusion injury model was established by ligating left anterior descending coronary for 1h,and followed by reperfusion for 4h,and recording the electrocardiogram.After ischemia/reperfusion,cardiac function was measured to assess myocardial injury.The histological change of heart was investigated by hematoxylin and eosin(HE)staining.The corresponding index of Epac-Rap1 signal channel was examined by using Western Blotting and immunofluorescence(IF).m RNA encoding gene was determined by RT-PCR.The blood samples were collected to measure the serum levels of cAMP.Results1.Compared with Sham group,MIRI groups(reperfusion after 1h,2h,4h,8h,16 h and 24h)significantly increased the elevation of the ST segment at ischemia 1h,and decreased the elevation of the ST segment at ending reperfusion in time-based dependency.HE staining results show that myocardial tissues were even staining and arranged regularly,the normal cellular structure frame was observed in the Sham group.And in the MIRI group,the large numbers of myocardial fibers showed disordered structures and accumulated.Myocardial matrix exhibited infiltration of inflammatory cells,where a large number of scar tissue was exhibited after reperfusion 4h.According to the ELISA results,the MIRI group(from 1h to 24 h)increased the levels of cAMP in serum in time-based dependency.Western blot analyses revealed that the I/R group from1 h to 24 h increased identically Epac1,Rap1,CAMKⅡ expression and p-ERK phosphorylation comparing to Sham group.Meanwhile,immunofluorescence showed that MI/R groups strengthened Epac1 and Rap1 responses to fluorescence and with the extension of reperfusion time,the ability of strengthened fluorescence were aggrandized step and step.At the same period detected myocardial tissue mRNA Epac1 and Rap1 were statistically significant increased at each time point.2.Compared with control group,the elevations of the ST segment weresignificantly increased at the ischemia 1h and reperfusion 4h.Compared with MIRI group,the agonist of Epac significantly increased the elevation of the ST segment,the inhibitor of Epac,Vitexin,the union of 8-CPT,ESI-09 and vitexin significantly decreased the elevation of the ST segment.HE staining results show that that myocardial tissues were even staining and arranged regularly,the normal cellular structure frame was observed in the Sham group.And in the MIRI group,the large numbers of myocardial fibers showed disordered structures and accumulated.Myocardial matrix exhibited infiltration of inflammatory cells,where a large number of scar tissue was exhibited.Compared with MIRI group,the agonist of Epac and PKA significantly increased apparently myocardial damage.The inhibitor of Epac and PKA significantly decreased apparently myocardial damage.According to the ELISA results,the MIRI group significantly increased the levels of cAMP in serum,Epac and PKA agonist increased the serum of cAMP,Epac inhibitor,PKA inhibitor and vitexin significantly decreased cAMP levels in serum.Western blot analyses revealed that the levels of Epac1,Rap1,and CaMKⅡand p-ERK were significantly higher than the Sham group,The Epac agonist significantly increased the levels of Epac1,Rap1,CaMKⅡand the phosphorylation of p-ERK,which were attenuated by vitexin treatment.Meantime,the Epac inhibitor significantly decreased the levels of Epac1,Rap1,CaMKⅡand the phosphorylation of p-ERK.The PKA agonist significantly increased the levels of CaMKⅡand the phosphorylation of p-ERK,the PKA inhibitor significantly decreased the levels of CaMKⅡand the phosphorylation of p-ERK,and it did not affect the Epac1 and Rap1 levels.IF results show that MIRI groups strengthened Epac1 and Rap1 responses to fluorescence.Compared with MIRI group,the ability of strengthened fluorescence were aggrandized for Epac agonist,and the ability of strengthened fluorescence were weaken for Epac inhibitor and vitexin in myocardial tissue,PKA agonist and inhibitor had no obvious effect in the myocardial tissue.The PCR results show that the MIRI group detected myocardial tissue m RNA Epac1 and Rap1 were statistically significant increased,Epac agonist significantly increased themRNA content of Epac1 and Rap1,Epac inhibitors and vitexin significantly decreased the mRNA content of Epac1 and Rap1,PKA agonist and inhibitor had no obvious effect in the myocardial tissue.ConclusionsThe research results showed that the MIRI elevated the levels of cAMP in myocardial tissue,also up-regulated the activation of Epac in myocardial intracellular signal and the Rap1 activation of its downstream,while the myocardial damage were aggravated.Epac1 agonist(8-CPT)promoted Epac1 overexpression and downstream Rap1 activation of myocardial cell,and then aggravated myocardial injury;Epac1inhibitors(ESI-09)reduced cardiomyopathy rational damage according to suppressing the Epac1 activation and overexpression of myocardial cell in the MIRI,suppressing the Rap1 activation of its downstream,suppressing the gene expression of Epac1 mRNA and Rap1 mRNA,also inhibiting CaMKⅡ expression and phosphorylation of ERK of its downstream;vitexin(3mg/kg)inhibited significantly myocardial tissue injury on MIRI in rats,inhibited the Epac1 activation and overexpression of myocardial cell in the MIRI,inhibited the Rap1 activation of its downstream,inhibited the gene expression of Epac1 m RNA and Rap1 mRNA,also inhibiting CaMKⅡ expression and phosphorylation of ERK,and then Epac1 inhibitors(ESI-09)with vitexin have synergy;According to these results,it revealed that Epac-Rap1 signaling pathway may play a important role in myocardial ischemia reperfusion injury in rats,Meantime,the significant protective effect against myocardial ischemia reperfusion injury in rat which exerted by vitexin may occur through modulating Epac-Rap1 signaling pathway. |
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