EPO Protects Cardiac Function In Diabetic Cardiomyopathy Rats By Inhibiting Cardiomyocyte Apoptosis

PART 1:Establishment of rat models of diabetic cardiomyopathy(DCM).Objective:The aimed of this study was to investigate the formation time of diabetic cardiomyopathy rats.Methods:(1)35 male SD rats were randomly divided into control group(n=5)and diabetic group(n=30).The rats in the diabetic group were intraperitoneally injected with 60 mg/kg streptozotocin at a time.Then the diabetic rats were randomly divided into 3 groups:diabetes mellitus 4-week group,diabetes mellitus 8-week group and diabetes mellitus 12-week group,10 rats in each group.The changes of body weight,fur,activity and drinking water were observed and the blood glucose and weight of rats were measured weekly.(2)The left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),left ventricular end-diastolic dimensionand(LVDd)and left ventricular end-systolic dimension(LVDs)were measured by ultrasound.(3)The heart tissues were collected and use Hematoxylin and eosin(HE)staining and electron microscopy myocardial morphological changes.(4)The cardiomyocyte apoptosis were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling.(5)Real time fluorescence quantitative polymerase chain reaction(RT-PCR)were used to detect the mRNA of Bcl-2 and Bax.(6)The expression of caspase-3 protein was detected by Western blot.Results:(1)The blood glucose in DM 4-week group,DM 8-week group and DM 12-week group were significantly higher than the normal control group(p<0.05),and the body weight were lower than the normal control group(p<0.05).(2)The heart function in DM 4-week group,DM 8-week group and DM 12-week group were significantly decreased compared with the normal control group(p<0.01).(3)HE staining showed that the myocardial cells arranged neatly,no cell degeneration and edema in control group.But in DM 4-week group,DM 8-week group and DM 12-week group,the structure of the myocardial tissue were very disorganized,and the morphological changed more and more serious along with the prolongation of the time of diabetes.(4)Transmission electron microscopy revealed that the control group had clear arrangement of myocardial cells,and the bands were clearly visible.The nuclei were not collapsed or ruptured.The mitochondria were oval in shape,The mitochondrial crista were completed without breakage and vacuolization.But in DM 4-week group,DM 8-week group and DM 12-week group,the myocardial cells arranged disorder,the nucleus deformation and failure,the mitochondria deformed and the mitochondrial crista vacuolation and fracture were visible.(5)The results of the TUNEL assay demonstrated that the apoptosis index(AI)of cardiac myocytes was(1.72 ± 0.74)%in the control group,in DM 4-week group,AI was(32.81+2.80)%,which was significantly higher than the control group(p<0.001).The AI in DM 8-week group((47.27 ± 8.03)%)was significantly higher than the DM 4-week group(p<0.001),and lower than the DM 12-week group((68.52±8.69)%)(p<0.001).(6)RT-PCR:Compared with the control group,the levels of myocardial cell Bcl-2 mRNA expression in DM 4-week group,DM 8-week group and DM 12-week group were decreased significantly(p<0.05),and the decrease was time-dependent.But no significant differences were observed in the levels of Bax mRNA expression in each group(p>0.05)?(7)Western blot:Compared with the control group,the caspase-3 protein levels in DM 4-week group,DM 8-week group and DM 12-week group were increased significantly(p<0.05),and the increase was time-dependent.Conclution:These results indicate that intraperitoneal injected with streptozotocin(60 mg/kg)at a time could establish a stable rat model of diabetic cardiomyopathy.PART 2:EPO protects cardiac function in diabetic cardiomyopathy rats by inhibiting cardiomyocyte apoptosis.Objective:In order to investigate the mechanism of recombinant human erythropoietin on myocardium of diabetic cardiomyopathy rats,and put forward the feasible theoretical basis for the future treatment of diabetic cardiomyopathy.Methods:(1)25 male SD rats were randomly divided into 3 groups:normal control group(n=5),diabetic cardiomyopathy without treatment group(DCM,n=10),diabetic cardiomyopathy with rhEPO treatment group(DCM+EPO,n=10).The diabetic cardiomyopathy models were established according to the first part.The rats in the diabetic cardiomyopathy with rhEPO treatment group were treated with rhEPO 2000U/kg by intraperitoneal injection per week for 8 weeks.Then the blood glucose and weight of rats were measured weekly.(2)Echocardiography,HE staining,electron microscopy,TUNEL,Western blot and PCR were the same as the first part.Results:(1)Compared with the control group,the blood glucose were significantly increased in DCM and DCM with rhEPO treated group(p<0.05).Compared with the control group,the body weight were significantly decreased in diabetic cardiomyopathy group and diabetic cardiomyopathy with rhEPO treated group(p<0.05).There were no significant difference in body weight and the blood glucose level in DCM and DCM with rhEPO treated group(p>0.05).(2)The heart function were lower in DCM and DCM with rhEPO treated group than control group(p<0.05).Compared with DCM group,the heart function in DCM with rhEPO treated group were significant increased(p<0.05).(3)HE staining showed that the cardiomyocytes in control group were arranged orderly.But in DCM group,the myocardial cells arranged in disorder,a large number of cardiomyocytes degeneration,edema and inflammatory cell infiltration.The DCM with rhEPO treated group was significantly better than the DCM group.(4)Transmission electron microscopy revealed that the control group had clear arrangement of myocardial cells,and the bands were clearly visible.The nuclei were not collapsed or ruptured.The mitochondria were oval in shape,The mitochondrial crista were completed without breakage and vacuolization.But in DCM group,the mitochondria in myocardium cells revealed structural disorders with vacuolar degeneration.The myocardial ultrastructures in rhEPO-treated rats was attenuated compared with the untreated diabetic group.(5)TUNEL:In control group,the AI was(1.72 ± 0.74)%,In DCM group,the AI was(68.52 ± 8.69)%,which was significantly higher than the control group(p<0.001).In DCM with rhEPO treated group,the AI was(37.20 ± 11.16)%,which was significantly higher than the control group(p<0.001),but significantly lower than the DCM group(p<0.001).(6)RT-PCR:Compared with the control group,the levels of myocardial cell Bcl-2 mRNA expression in DCM and DCM with rhEPO treated group were significantly decreased(p<0.05),Compared with the DCM group,the level of myocardial cell Bcl-2 mRNA expression in DCM with rhEPO treated group was significantly increased(p<0.05).There were no significant difference in the levels of myocardial cell Bax mRNA expression in the control and DCM group(p=0.273).Compared with the control and DCM group,the levels of myocardial cell Bax mRNA expression in DCM with rhEPO treated group were significantly decreased(p<0.05).(7)Western blot:Compared with the control group,the caspase-3 protein level in DCM group was increased significantly(p<0.05),while rhEPO could reduced the level of caspase-3(p<0.05).Conclutions:(1)EPO can improve the cardiac function in diabetic cardiomyopathy rats;(2)EPO can protect the cardiac function of diabetic cardiomyopathy rats by inhibiting the apoptosis of cardiomyocytes.