| Research Background:Chemotherapy is a primary method for tumor therapy,but most of the chemotherapeutic agents(including cis-platinum,anthracyclines,anti-cell microtubules,alkylating agents,antimetabolites and molecular targeted drugs)have serious side effects,especially the cardiotoxicity.It occurred with the following symptoms such as chest tightness,palpitation,precardiac discomfort,shortness of breath,and even heart failure in cardiac myocytes.Electrocardiogram examination can show T waves or S-T waves changes as well.Several agents(including cis-platinum and adriamycin)have toxic effect and selectivity on myocardium,accompanied with abnormal electrocardiogram,such as changes of heart rhythm and rate,premature systole,ST-T waves rise or decrease.Long-term cardiac damage can lead to acute or chronic heart failure,which is difficult to recover and associated with a high mortality rate,.Apelin is a kind of bioactive peptide hormone secrated by adipocytes.Human Apelin gene locates at chromosome X q25-26.3,the molecular weight is 12876,including 2 introns and 3 exons,and its RNA length is 3125bp.The receptor of Apelin(APJ)belong to G-protein-coupled receptors,which is constituted of 380 amino acid and includes 7 spiral transmembrane structure.Apelin has the cardiovascular effect of positive inotropic action,heart protection and blood pressure decrease.Additionally,Apelin also possess the founction of regulation of behavior,tempreture,appetite,immunity,water-salt balance,and adipo-insulin endocrine axis.Platinum-based chemotherapy is an effective method against human cancer,in which cis-platinum was applied widely due to the advantages of high efficiency and broad spectrum.But along with severe side effect,especially the cardiotoxicity.Therefore,it is urgent to decrease the side effect.In this study,the effect of Apelin-13 to relieve the cardiotoxicity caused by cis-platinum was estimated through H9c2 cell line and C57 mice model.The results showed that Apelin-13 can alleviated the decrease of H9c2 myocardial activity and significantly relieved the cardiotoxicity induced by cis-platinum via inhibiting the myocardial apoptosis.At the same time,Apelin-13 also improved the development of cardiac vessels.Objective:Considering the cardiotoxicity caused by cis-platinum and the positive potential of Apelin-13 in heart protection and antihypertension,this study was conducted to explore the protection of Apelin-13 against the myocardial damage and evaluate the potential of Apelin-13 to relieve the cardiotoxicity induced by cis-platinum in cell and mice models.Methods:1?To estimate the effect of Apelin-13 to relieve the cardiotoxicity induced by cis-platinum,H9c2 myocardial activity changes was detected by MTT method before/after Apelin intervention.Firstly,H9c2 rat myocardial cells were treated with different concentrations of Apelin-13 or cisplatin to observe whether they had toxic effects on the cells and the effects on cell viability,respectively.Finally,the corresponding concentrations of Apelin-13 and cisplatin were co-administered to observe the changes in the viability of H9c2 rat cardiomyocytes and to evaluate the effect of Apelin-13 on cisplatin-induced cardiotoxicity.2.Flow cytometry was used to analyze the H9c2 myocardial apoptosis before/after Apelin-13 intervention and to investigate the role of Apelin-13 in cisplatin-induced cardiomyocyte apoptosis.3.The caspase-3 activity in rat cardiomyocytes treated with Apelin-13 and cisplatin was detected by immunofluorescence.The activities of Caspase-3,Caspase-7,Caspase-9 and PARP expression,were detected by Western blotting.Hence,possible mechanism of platinum-induced cardiotoxicity was found in these changes.4.Finally,the model of cisplatin challenge in mice was established and randomly divided into control group,cisplatin group(20mg/kg),Apelin-13 group(10?g/kg),and cisplatin+Apelin-13 group(20mg/kg cisplatin+l0?g/kg Apelin-13),according to the established schedule by given intraperitoneal injection for 14 days,the mice were sacrificed to collect the specimens.The body weight and heart weight of the mice were measured.In the mice model,histopathology was applied to evaluate the myocardial damage before/after Apelin intervention.The effect of Apelin-13 on ameliorating cisplatin-induced cardiotoxicity was further evaluated from the mouse body.Results:1.Apelin-13 was proved nontoxic when used alone in H9c2 cell,and the concentration of 50-600 nM can effectively improve the cell growth with a rise of cell activity.Cis-platinum can inhibit H9c2 cell growth with a dose-dependent manner,as reflected by the decrease of cell activity.At the same time,cells exhibited apoptosis feature,such as shrinkage,decline in cell number,and appearance of apoptotic body.Pretreatment of Apelin-13 significantly relieved the inhibition of H9c2 cell induced by cis-platinum,as well as the improvement in cellular morphology.2.Treatment of cis-platinum induced significant myocardial apoptosis with increase of Sub-Gl peak,increase of caspase-3 activity,PARP clevage,activation of caspase-3,7,9.Whereas,pre-treatment of Apelin-13 significantly alleviated H9c2 cell apoptosis caused by cis-platinum with the decrease of Sub-Gl peak and attenuation of PARP cleavage and activation of caspase-3,7,9.3.Treatment of cis-platinum decreased the weight of the mice hearts,triggered the myocardial apoptosis,and inhibited the development of blood vessels.The indicators showed increase of caspase-3 activity and decrease of CD34 expression.Intervention of Apelin-13 significantly inhibited the decrease of the mice hearts weight,relieved myocardial apoptosis,and improved the development of blood vessels in the mice heart.Conclusion1.Cis-platinum can induce mitochondria-mediated apoptosis in myocardial cells,inhibit the growth of H9c2 myocardial cells in vitro.Apelin-13 pre-treatment inhibited the apotosis and relieved the cardiotoxicity caused by cis-platinum.2.Apelin-13 inhibited myocardial cells apoptosis in vivo,and improved the development of blood vessels in the heart.Research Background:Platinum is a broad-spectrum medicine and have significant effect against tumor.Cis-platinum-based chemotherapy is one of the most effective methods to treat human tumor.But at the same time,cis-platinum also has significant cardiotoxicity,and the detailed mechanism is still unclear.The side effect caused by cis-platinum,including cardiac insufficiency,bradycardia,angina,acute myocardial infarction and arrhythmia,not only restrict application of cis-platinum but also increase the risk of patients suffering from cardiovascular disease.Several researches indicated that oxidative stress plays an important role in the pathological process of cardiotoxicity caused by cis-platinum;cis-platinum can induce the ROS generation,promote the formation of ROS and MDA,as well as aggravate oxidative stress.It is reported that Apelin-13 posseed the ability to eliminate the free radical and regulate cardiovascular system.Through the previous study we found that pretreatment of Apelin-13 can alleviate ROS and superoxide anion producing,inhibit DNA damage and PARP pyrolysis,as well as caspase activation.Further study showed that Apelin-13 inhibited H9c2 cell apoptosis induced by cis-platinum through regulating MAPKs and PI3K/Akt signal pathway.In this study,treatment of cis-platinum significantly induced accumulation of ROS and superoxide anion in H9c2 cell,indicating that oxidative stress is closely related to cisplatin cytotoxicity.Firstly,MAPKs and PI3K/Akt pathway were explored to examine the effect of Apelin-13 on protecting myocardial cell from the damage of cai-platinum,and then we investigated the effect of Apelin-13 on inhibiting DNA damage mediated by oxidative stress through H9c2 cell line and protecting myocardial cell from cardiotoxicity by regulating MAPKs and PI3K/Akt pathway.ObjectiveIn the previous study,it had been confirmed that Apelin-13 possess the effect of relieving the myocardial cell apoptosis and damage,and to further explore the potential mechanism,in this study,the detailed mechanism of Apelin-13 resisting DNA damage from oxidative stress was evaluated,MAPKs and PI3K/Akt pathway was detected regarding the effect of Apelin-13 alleviating the damage of myocardial cell.Therefore,this study can provide a theoretical basis to reveal the protection against cis-platinum cytotoxicity and application of Apelin-13 on clinical practice.Methods1.To further explore the mechanism of Apelin-13 relieving myocardial cell apoptosis and damage caused by cis-platinum,our study first detected oxidative stress mediated by ROS and superoxide anion,DNA damage,as well as the indicators concerning apoptosis.2.After giving Apelin-13 and cisplatin stimulation,comet assay was applied to detect DNA damage caused by single-strand breaks to observe whether Apelin-13 may prevent cisplatin-induced DNA damage by inhibiting the accumulation of ROS.3.H9c2 rat cardiomyocytes were stimulated by cisplatin and Akt upstream inhibitor(ly2294002)and ERK inhibitor(U0126),then MTT were used to detect toxicity and cell activity before/after intervention of drugs.The role of Akt and ERK in cisplatin-induced cardiotoxicity was evaluated through MTT.4.Apelin,APJ,ATM,ATR,Total-p53,serl5-p53,H2A,Akt,JNK,p38,ERK and other related protein expression were detected by Western Blot after Apelin-13,cisplatin,Akt upstream inhibitor(ly2294002)and ERK inhibitor(U0126)stimulation,finally,the indicators of MAPKs/Akt pathway were analyzed to estimate the mechanism of Apelin-13 alleivating myocardial cell damage challenged by cis-platinum.Results1.Cis-platinum can result in the producing of intracellular ROS and superoxide anion,and then caused DNA damage,manifested as activation of the concerned pathway(including ATM,ATR,ser15-p53,H2A)and DNA single strand breakage.Pretreatment of Apelin-13 significantly inhibited the producing of ROS and superoxide anion induced by cis-platinum and relieved DNA damage and single strand breakage,manifested as decrease of DNA damage signal molecule phosphorylation.2.Cis-platinum significantly inhibited the expression of Apelin and APJ,and interfered MAPKs and Akt pathway,manifested as activation of p-p38 and p-JNK,and inactivation of p-ERK and p-Akt.Whereas pretreatment of Apelin-13 effectively inhibited the inhibition of Apelin and APJ expression caused by cis-platinum and relieved the effect of cis-platinum on MAPKs and Akt pathway.Conclusion1.Cis-platinum can induce the generation of reactive oxide species,cause DNA damage pathway,and result in myocardial cytotoxicity by regulation of MAPKs and Akt pathway.2.Apelin-13 can relieve myocardial cytotoxicity caused by cis-platinum through inhibiting ROS-mediated DNA damage and regulating MAPKs and Akt pathway.3.This study provides a theoretical basis for the clinical application of Apelin-13 as a new cardio-protective drug and may be an effective strategy to alleviate side effects of chemotherapy in the future. |
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