Experimental Study On Preventive Cardioprotection Of EPO And HBSP Against Anthracycline-induced Cardiotoxicity

Anthracycline antibiotic(ANT)was first discovered in 1950.With the development of cancer therapy,ANT has become a landmark class of drugs.In the past 30 years,ANT has been widely used in clinic.Combination therapy based on ANT is the first-line treatment for breast cancer,leukemia,malignant lymphoma and other malignant tumors.ANT significantly improves the survival rate and prognosis of cancer patients.However,ANT can cause dose-dependent cardiotoxicity,including cardiomyopathy and congestive heart failure,which severely limits its clinical application.At present,color echocardiography is used to detect heart lesions in patients receiving ANT chemotherapy.In recent years,scientists have also focused on the corresponding cardioprotective drugs.More and more studies have confirmed that EPO is a multipotent cytokine.Its main biological functions include reducing ischemia-induced apoptosis,promoting inflammatory cell apoptosis,antioxidant and other functions.Pre-clinical and clinical studies have confirmed that EPO can play a cardioprotective role in acute myocardial infarction,myocardial ischemia/reperfusion,diabetic cardiomyopathy and other diseases.In clinic,large dose or long-term use of EPO is needed to exert the protective effect of multiple organs and tissues,but it can cause excessive stimulation of bone marrow hematopoietic system,leading to side effects such as hypercoagulability,thrombosis,hypertension,pure red blood cell dysfunction anemia and so on.Therefore,HBSP,an EPO-derived peptide with little side effects but multiple organ protective effects,has become a hotspot of current research.Studies have confirmed that cardiotoxicity induced by ANT is associated with increased apoptosis of cardiac myocytes and oxidative stress.Therefore,we consider that EPO and its derivative peptide HBSP may also play a protective role in ANT-induced myocardial injury.In this study,we will explore the effective way to establish rat model of ANT-induced myocardial injury,and further explore whether EPO and its derivative HBSP have protective effect on ANT-induced myocardial injury and the possible mechanism.Part I Establishment of a rat model of epirubicin-induced myocardial injuryObjective:In this study,rats with moderate body size and tenacious vitality were selected to study the best model of epirubicin-induced myocardial injury in rats,so as to provide a good animal model on subsequent experiments.Methods:We established two models of cardiotoxicity induced by Epirubicin in Wistar rats.Rats were injected with EPI at a dose of 2.5mg/kg intravenously weekly and feeding for 11 weeks for EPIiv model,2.5mg/kg intraperitoneally weekly for 11 weeks for a EPIip model.Two groups of model group were set up corresponding control group,namely CONiv group(n=8),CONip group(n=8).The control group of two groups was given the same amount of saline.The general condition and weight changes of rats in each group were observed.HE stains and immunohistochemical staining were performed on paraffin sections of rat hearts after execution.Results:In this experiment,the weight of the rats in the two groups of models was slower than that of the control group,and the general state showed significant changes,namely,mental wilt,hair loss,hair disorder,diet and water intake.However,the tail vein administration group showed an irritating state,and the tail was ulcerated.Considering the local strong stimulation caused by epirubicin injection leakage,the general state of the rat were worsened and death was caused.In the pathological changes and morphology of myocardial tissue,light microscopy showed that the cardiomyocytes of EPIiv group and EPIip group showed significant changes,namely cardiomyocyte edema,vacuolar degeneration,myofibril rupture and Disappearance,interstitial hyperemia.Further pathological scores were obtained according to Billingham semi-quantitative criteria.There was no significant difference in myocardial cell pathological scores between EPIiv group and EPIip group.Qualitative analysis of immunohistochemical sections also confirmed this view.Conclusions:Epirubicin intermittent small doses intermittent administration(2.5mg/kg),cumulative dose of 15mg/kg,feeding to eleven weeks of dosing regimen,can successfully establish myocardial injury caused by anthracyclines in rats,which is the first choice for related experiments.Part II EPO and HBSP Protects Against Epirubicin-induced Cardiomyopathy in Rat ModelObjective:This study was designed to explore the protective effect of erythropoietin(EPO)on epirubicin-induced cardiomyopathy in rat model and related possible mechanisms.Methods:Four sets of modeling methods were set up.Rats were injected with EPI 2.5mg/kg intraperitoneally once a week for 6 weeks without EPO or HBSP for EPI model.Rats were injected with EPI 2.5mg/kg intravenously once a week for 6 weeks with EPO(5000IU/kg)or HBSP(60?g/kg)intraperitoneal pretreatment three times a week,respectively,two days before the administration of EPI and the third day after the administration of EPI.At the 11th week,the rats were sacrificed for blood smear and color echocardiography.The immunohistochemical sections of rat myocardial tissue were analyzed by semi-quantitative analysis of Caspase3 and Bcl-xl.Results:In the EPI group,the rats were generally in poor condition,and hair unkempt erection,abdominal hair loss,significant reduction in diet and drinking water,and weight loss were significantly different from those in the CON group.The general state of the rats in the EE group and the EH group was slightly improved compared with the EPI group,and the general condition of the rats in the EH group were also slightly better than that in the EE group.The weight of rats in EPI group,EE group and EH group was significantly lower than that in CON group.After statistical analysis,there was no significant difference in body weight between EPI group,EE group and EH group.Significant myelosuppression occurred in EPI group,EE group and EH group,and red blood cells,white blood cells and platelets were significantly lower than CON group.The red blood cells of the rats in the EE group were significantly increased,but the white blood cells and platelets were significantly lower than the control group.The number of red blood cells,white blood cells and platelets in the EH group were not significantly different from those in the EPI group.The results of cardiac color Doppler ultrasound showed that the left ventricular end diastolic diameter(LVIDd)and left ventricular end systolic diameter(LVIDs)in the CON group was significantly lower than those in the EPI group of the end of the 11th week,and the EF and FS values were significantly higher than those in the EPI group.In this experiment,there were no statistical differences in LVIDd,LVIDs,EF or FS between the three groups in the CON group,the EE group and the EH group.In the EPI group,the myocardial tissue was dissected after the anatomy on the eleventh week.It was found that the myocardial fibers were obviously swollen,vacuolated,sparsely arranged,and some myocardial myofibrils dissolved.Compared with the EPI group,the EE group and the EH group showed significant improvement in the above-mentioned lesions,and the myofibrils were arranged neatly,and the myocardial fibers were mildly edematous compared with the CON group.Myocardial histopathological scores were performed under light microscopy according to Billingham semi-quantitative criteria.Further statistical analysis showed:pathological scores in EPI group>EE group>EH group>CON group.The content of proapoptotic protein Caspase3 and anti-apoptotic protein Bcl-xl in cardiomyocytes were determined by immunohistochemistry.Image J software was used for image analysis and mean optical density(MOD)was determined.The results showed that compared with the CON group,the MOD of Caspase 3 in EE group,EH group and EPI group increased significantly(P<0.05),while the MOD of Bcl-xl decreased significantly(P<0.05).The MOD of Caspase 3 in rat myocardium was significantly different among EE group,EH group and EPI group(P<0.05).The MOD of Caspase 3 in myocardium of four groups was EPI group>EE group>EH group>CON group.The MOD of Bcl-xl in myocardium of rats in EE group was not significantly different from that in EH group(P>0.05),but it was significantly different from that in EPI group(P<0.05).The MOD of Bcl-xl in myocardium of the four groups was CON group>EE group=EH group>EPI group.Conclusions:EPO and HBSP have protective effects against EPI-induced myocardial damage,including improving left ventricular function,inhibiting left ventricular dilatation,reducing myocardial cell lesions,and inhibiting cardiomyocyte apoptosis.HBSP has a stronger protective effect of EPI-induced myocardial damage than EPO.The mechanism of action of both may be related to up-regulation of anti-apoptotic proteins and inhibition of pro-apoptotic protein expression.