Effect Of Erythropoietin In Combination With Granulocyte-Colony Stimulating Factor On Treatment Of Acute Myocardial Infarction

Objective To evaluate the potential protective effects of EPO in combination with G-CSF on left ventricular function and remodeling after acute myocardial infarction(AMI)and the possible molecular mechanism by the investigation of hypoxia cardiomyocytes and the structural and functional changes of heart in AMI rats,so as to provide new ideas for AMI treatments.Methods Left ventricular cardiomyocytes were isolated from neonate rat,the air in the cultural medium was changed by 95%N₂ and 5%CO₂ for hypoxia cardiomyocytes.The protective effects of EPO and G-CSF in different concentrations were evaluated.Under the optimized concentration,the protective effects of EPO in combination with G-CSF were investigated.The survival, apoptotic and necrotic rates of cardiomyocytes were assessed by flow cytometry.Cardiomyocytes were divided into five groups,normal cardiomyocytes, hypoxia cardiomyocytes,hypoxia cardiomyocytes treated with EPO,hypoxia cardiomyocytes treated with G-CSF,and hypoxia cardiomyocytes treated with EPO in combination with G-CSF.Twenty four hours after hypoxia, cardiomyocytes were collected.Bcl-2,Bax,Caspase-3 mRNA level were determined by Northern-blot analysis and cytochrome C,STAT-3,Caspase-3 protein level were determined by Western-blot analysis in each group.Rats underwent permanent left anterior descending(LAD)coronary artery ligation.The experimental design consisted of five groups of rats:sham, myocardial infarction(MI),MI treated with EPO,MI treated with G-CSF,MI treated with EPO and G-CSF.Blood samples were collected at the 1st,7th,and 28th day after MI for blood routine and blood biochemics examinations.Heart specimens were collected at the 1st and 7th day after MI for Northern-blot and Western-blot experiments.Apoptosis of cardiomyocytes were measured by TUNEL staining,morphologic of cardiomyocytes were measured by hematoxylin/eosin(HE)staining.At the 28^thday after MI,heart specimens were collected for HE staining,Masson trichrome staining,scarlatinum staining,andâ…§agent staining to evaluate the survival,scar collagen deposition,and angiogenic effects.Cardiac structure and function were assessed by echocardiography at the 7^thand 28^thday after MI.Cardiac function was evaluated at the 28^thday by haemodynamic examination.Results Cardiomyocytes were isolated from neonate rat successfully.Model of hypoxia cardiomyocytes was established successfully.The mortality and the rate of apoptotic cell to total necrotic cell were higher in hypoxia cardiomyocytes than normal cells significantly(26.73%vs.5.63%,70.05%vs.37.83%,P＜0.001).At the concentration of 1U/ml of EPO and 6ng/ml of G-CSF,the protective effects occurred,5U/ml,25U/ml,and 125U/ml of EPO were similar,150ng/ml of G-CSF was the most effective,30ng/ml and 750ng/ml were similar.The apoptotic rate and total necrotic rate of hypoxia cardialmyocytes were lower significantly in EPO,G-CSF and combination groups than in the hypoxia group(18.73%vs. 6.01%vs.7.08%vs.4.57%,P＜0.001,26.73%vs.9.54%vs.11.6%vs.8.15%, P＜0.001),the protective effects of EPO and G-CSF were similar(P＞0.05),and the combination group was much better than EPO and G-CSF groups(P＜0.01).Compared to the hypoxia group,the expression of Bcl-2 mRNA was upregulated and Bax mRNA,Caspase-3 mRNA and Caspase-3 protein were downregulated significantly in group EPO and G-CSF(P＜0.01),no difference was found between the two group(P＞0.05),the beneficial effects in combination group was better than in EPO and G-CSF group.Cytochrome C was a little downregulate in EPO group(P＞0.05),and much downregulate in G-CSF and combination group(P＜0.05),STAT-3 expression was upregulated in combination group(P＜0.01).There were no significant difference among five groups at the 7^thand 28^th day after MI in blood test,creatinine and blood urea nitrogen(P＞0.05).The levels of ALT,AST,CK,CK-MB,and TNT were decreased in EPO and G-CSF group (P＜0.01),especially in combination group(P＜0.01)at the 1st day after MI.CK, CK-MB and TNT were decreased in EPO and G-CSF group(P＜0.01),especially in combination group(P＜0.01)at the 7th day after MI.At the 1st day after MI,the expression of Bcl-2 mRNA and STAT-3 protein were upregulated and Bax mRNA,Caspase-3 mRNA,Caspase-3 protein and Cytochrome C protein were downregulated significantly in EPO and G-CSF group(P＜0.01),the change in combination group were obviously than EPO and G-CSF group(P＜0.01).At the 7th day after MI,the expression tendency of Bcl-2,Bax and Caspase-3 mRNA, Cytochrome C,STAT-3 and Caspase-3 protein were similar with the 1st day after MI,but the levels of expression decreased significantly(P＜0.01). Echocardiography indicated LVEF and FS improved in all treated groups at the 7th day after MI,combination group was much significant,LVESD and LVESV reduced obviously(P＜0.01).At the 28th day after MI,LVEF,FS,LVESD, LVEDD,LVESV and LVEDV improvements still could be seen in all treated groups,especially in combination group.At the 28th day after MI,haemodynamic examination demonstrated,LVEDP was decreased and LVSP,+dP/dt_maxwere increased in all treated groups,combination group was improved the most.The 1st day after MI,there were many apoptotic cells in MI group,much higher than EPO and G-CSF group,especially combination group(P＜0.01).The 7th day after MI, the apoptotic cell number tendency was similar with the 1st day after MI,but the number was lower obviously(P＜0.01).The 28th day after MI,the number of new vessels increased in the EPO and G-CSF group,more significantly in combination group.A large reduction of the scar and collagen deposition in infarcted area and peri-infarcted area were observed in EPO and G-CSF group and even more significant in combination group when compared to MI group.Conclusions The optimized concentration for EPO is 5U/ml and for G-CSF is 30ng/ml.The protective effects of EPO in combination with G-CSF are better than EPO or G-CSF alone.EPO upregulates expression of Bcl-2 and STAT-3 and downregulates expression of Bax,and Caspase-3,hypoxia cardiomyocyte is protected by activating JAK-STAT signaling pathway.G-CSF upregulates expression of Bcl-2 and STAT-3 and down regulates expression of Bax, cytochrome C and Caspase-3,hypoxia cardiomyocyte is protected mainly by activating chondriosome signaling pathway and partly by activating JAK-STAT signaling pathway.EPO in combination with G-CSF upregulates expression of Bcl-2 and STAT-3 and downregulates expression of Bax,cytochrome C and Caspase-3 obviously,hypoxia cardiomyocyte is protected by activating both chondriosome and JAK-STAT signaling pathways.EPO in combination with G-CSF treatment is safe and well tolerated in rat models of acute myocardial infarction,provides anti-apoptotic effect by activating both chondriosome and JAK-STAT signaling pathways.The effects are more obviously in combination group indicate that the two signaling pathways are cooperated and amplified each other.Combination treatment prevents left ventricular remodeling and improves cardiac systolic and diastolic function by inhibiting cardiomyocyte apoptosis, reducing tissue collagen deposition and inducing neovascularisation.