| 【Background】Sunitinib(SNT)is a small-molecule,multi-targeted tyrosine-kinase inhibitor,and widely used as an effective anti-cancer drug for the treatment of metastatic renal cell carcinoma,gastrointestinal stromal tumor and advanced pancreatic neuroendocrine tumors.Unfortunately,current strategies based on SNT were shown to be associated with serial severe adverse effects,such as the development of hypertension and cardiac dysfunction,which would significantly contribute to poor long-term prognosis of such patients.However,there is still a lack of effective treatment for SNT-induced cardiovascular dysfunction.Sodium-glucose co-transporter 2(SGLT2)inhibitors have been shown to decrease the adverse cardiac events and risks of cardiovascular mortality among heart failure patients with or without diabetes,which has made these drugs promising treatment options for patients with chronic heart failure.However,it is not clear whether SGLT2 inhibitors have cardiovascular benefits in patients with cancer chemotherapy-related cardiac dysfunction.We aimed to determine whether empagliflozin(EMPA),an SGLT2 inhibitor,has a protective role against SNT-induced cardiac dysfunction in a mouse model.So as to find a potential cardioprotective approach for prevention and treatment of SNT-induced cardiotoxicity in clinic.PART 1:Manifestations and Mechanisms of Sunitinib-induced Cardiotoxicity【Objective】1.A mouse model of SNT-induced cardiotoxicity was established to evaluate the effects of SNT on cardiovascular functions of mice.2.To explore the underlying mechanism of cardiotoxicity induced by SNT.【Method】1.Adult male C57BL/6J wild-type mice aged 6 to 8 weeks were randomly divided into two groups: Vehicle group and SNT group.SNT cardiotoxicity mouse model was established by intragastric intervention with vehcile(5% DMSO)or SNT(40 mg/kg/d)for 28 days,respectively.After the intervention,the effects of SNT on blood pressure(BP),cardiac function and coronary microvscular function were evaluated by non-invasive blood pressure measurement system,cardiac ultrasound and pulse Doppler flow detector.At the study ending point,the plasma myocardial injury markers were detected by ELISA.The effects of SNT on myocardial apoptosis,myocardial fibrosis,oxidative stress,microangiogenesis and myocardial mitochondria were evaluated by histopathological staining and transmission electron microscopy(TEM).2.H9c2 cardiomyocytes were cultured in vitro and treated with different concentrations of SNT(range from 1-20 μM)for 24 h,48 h or 72 h,respectively. The effects of SNT on proliferation and cell viability of cardiomyocytes,apoptosis, autophagy and its related pathways were evaluated by CCK-8 cell viability assay, Western Blot analysis and transfection of dual-fluorescence labeled LC3 adenovirus(Ad-m RFP-GFP-LC3),respectively.The role of autophagy in SNT cardiotoxicity was investigated using the autophagy agonist Rapamycin(Rapa)or autophagy inhibitor bafilomycin A1(Baf A1).【Results】1.SNT induced BP elevation,cardiac function impairment and coronary microvascular dysfunction.(1)The systolic BP(SBP),diastolic BP(DBP)and mean BP(MBP)of mice were significantly increased in SNT group compared with those of Vehicle group.(2)Compared with Vehicle group,the left ventricular ejection fraction(LVEF)%, fractional shortening(FS)% and mitral valve flow E/A ratio(MV E/A ratio)were significantly decreased in SNT group,while mitral valve flow deceleration time(DT)and isovolumetric relaxation time(IVRT)were significantly prolonged.(3)The coronary flow reserve(CFR)of SNT group was significantly decreased compared with that of Vehicle group,2.SNT impaired myocardial mitochondrias and enhanced oxidative stress.(1)Compared with Vehicle group,the DHE red fluorescence intensity of myocardium in SNT group was significantly enhanced.(2)The number of damaged mitochondrias in the myocardium of mice in SNT group was significantly increased compared with that of Vehicle group.3.SNT triggered apoptosis and decreased cell viability of cardiomyocytes.(1)Plasma c Tn T and NT-pro BNP contents of mice in SNT group were significantly increased compared with that of Vehicle group.(2)Compared with Vehicle group,the number of TUNEL positive cells in the myocardium of mice in SNT group was significantly increased.(3)SNT significantly reduced the cell viability of cardiomyocytes in a concentration and time-dependent manner compared with that of Control group.(4)The death rate of cardiomyocytes in SNT group was significantly higher than that in Control group.4.SNT blocked the autophagic flux of cardiomyocytes.(1)The LC3-II/LC3-I ratio and P62 protein expression in SNT group were significantly increased compared with Control group.(2)Compared with Control group,the number of autophagosomes in SNT group was significantly increased,while the number of autophagolysosomes did not change significantly,when compared with those of Control group.(3)The cell viability of cardiomyocytes pretreated with Rapa,an autophagy agonist, was higher than that treated with SNT alone.However,the cell viability of cardiomyocytes pretreated with Baf A1,an autophagy inhibitor,was significantly lower than that treated with SNT alone.5.SNT triggered cardiomyocyte autophagic inhibition via inducing dysfunction of AMPK-m TOR signaling pathway.Western Blot analysis showed that the ratio of p-AMPK/AMPK in SNT group was down-regulated,while the ratio of p-m TOR/m TOR was up-regulated,when compared with Control group.【Conclusion】SNT can cause autophagic dysfunction of cardiomyocytes by inhibiting AMPK-m TOR-autophagy signaling pathway,and ultimately lead to cardiotoxicity, which showed as BP elevation,cardiac function impairment and coronary microvascular dysfunction.PART 2:Effect of SGLT2 Inhibitor Empagliflozin on Sunitinib-induced Cardiotoxicity【Objective】1.To determine whether EMPA could alleviate SNT-induced cardiotoxicity.2.To investigate whether the effect of empagliflozin on SNT-induced cardiotoxicity affects tumor growth and the anti-tumor efficacy of SNT.【Method】1.The grouping and intervention of C57BL/6J WT mice were the same as in the first part.After the intervention,BP,cardiac function and coronary microvascular function were evaluated by non-invasive blood pressure measurement system, cardiac ultrasound and pulse Doppler flow detector.At the study ending point,the plasma myocardial injury markers were detected by ELISA.The changes of cardiac oxidative stress and myocardial mitochondria were assessed by DHE staining and transmission electron microscopy in histopathological sections.2.Human renal cell adenocarcinoma cells(ACHN cells)were cultured in vitro and treated with different concentrations of SNT(range from 1 to 20 μM)or EMPA (range from10 to 1000 n M)for 24 h,48 h and 72 h,respectively.The effect of SNT or EMPA on the proliferation and viability of ACHN cells was evaluated by CCK-8 cell viability assay.3.ACHN cells were cultured in vitro and inoculated subcutaneously into 5-6-week-old M-NSG severely immunodeficient mice to construct human cell-derived tumor xenograft(CDX)mouse model.When the subcutaneous tumor grew to about 50 mm3,M-NSG mice were randomly divided into: Control group, EMPA group,SNT group and SNT + EMPA group.The intervention of M-NSG mice were the same as C57BL/6J WT mice.The changes of body weight and subcutaneous tumor volume were measured and recorded during the intervention.After intervention,cardiac function was evaluated by echocardiography.At the study ending point,the subcutaneous tumor weight in different group was measured and recorded.【Results】1.EMPA significantly attenuated SNT-induced cardiotoxicity without affecting body weight and blood glucose.(1)There was no difference in body weight and blood glucose among the groups.(2)Compared with Control group,the BP of mice in SNT group increased graduallywith the extension of intervention time;However,the BP of mice in SNT+EMPA group were significantly decreased when compared with that of mice in SNT group.(3)Compared with Control group,the LVEF %,FS %,MV E/A ratio and CFR of mice in SNT group were decreased,while DT and IVRT were significantly prolonged;However,these echocardiographic datas were significantly improved when co-treated with EMPA.2.EMPA ameliorated SNT-induced myocardial injury. Compared with Control group,plasma c Tn T and NT-pro BNP of mice in SNT group were significantly increased;However,these plasma myocardial injury markers were significantly decreased when co-treated with EMPA.3.EMPA attenuated the enhancement of cardiac oxidative stress and myocardial mitochondrial damage induced by SNT.(1)The DHE red fluorescence intensity of the myocardial tissue of mice was significantly enhanced in SNT group,while this fluorescence intensity was decreased by co-treated with EMPA.(2)Compared with Control group,the number of damaged mitochondrias in the myocardium of SNT group was significantly increased;However,compared with SNT group,the number of damaged mitochondrias in the myocardium of the SNT + EMPA group was significantly reduced.4.EMPA alleviated the cardiotoxicity of SNT without affecting tumor growth and the anti-tumor efficacy of SNT(1)Compared with Control group,the cell viability of ACHN cells in SNT group was significantly decreased,while that of EMPA group showed no significant difference.(2)No significant difference was observed in body weight among all groups.Compared with Control group,EMPA did not affect the tumor growth,while the growth rate of tumor volume was significantly slowed down by SNT.Compared with SNT group,there was no significant difference in tumor volume of mice in SNT + EMPA group.(3)There was no significant difference in the tumor weight between Control group and EMPA group,but the tumor weight of mice in SNT group was significantly decreased when compared with that of Control group.There was no significant difference in tumor weight of mice in SNT + EMPA group compared with SNT group.(4)Compared with Control group,there was no statistical difference in various cardiac ultrasound indexes of mice in EMPA group,while the LVEF %,FS % and MV E/A ratio of mice in SNT group were significantly decreased.However,these enchocardiographic indexes were signifcantly improvede by co-treated with EMPA.【Conclusion】EMPA significantly alleviated the cardiotoxicity induced by SNT.This cardioprotective effect of EMPA did not induce any changes in body weight or blood glucose,nor did it affect tumor growth or the anti-tumor efficacy of SNT.PART 3:The Mechanism of SGLT2 Inhibitor Empagliflozin in Alleviating Sunitinib-induced Cardiotoxicity【Objective】To investigate the molecular mechanism of EMPA in alleviating sunitinib-induced cardiotoxicity.【Method】1.The grouping and intervention of C57BL/6J WT mice were the same as in the first part.The protein expression levels of apoptosis and autophagy related pathways in each group were detected by Western Blot analysis.Apoptosis of myocardial cells in each group was evaluated by TUNEL staining in cardiac pathological sections.2.H9c2 cardiomyocytes were cultured in vitro and treated with SNT(5 μM)or SNT(5 μM)combined with EMPA(50 ~ 1000 n M)at different concentrations for 48 h. The effects of different interventions on the proliferation and viability of cardiomyocytes were detected by CCK-8 cell viability assay.Hoechst 33258 staining or TUNEL staining was used to detect the apoptosis of H9c2 cells. Western Blot was used to detect the protein expression levels of apoptosis and autophagy related pathways after different interventions.Ad-m RFP-GFP-LC3 transfection and confocal microscopy were used to observe the effects of different interventions on cardiomyocyte autophagic flux.The role of AMPK-m TOR-autophagy signaling pathway in EMPA’s alleviation of SNT cardiotoxicity was investigated using AMPK inhibitor(Compound C,CC)or autophagy inhibitor bafilomycin A1(Baf A1).【Results】1.EMPA attenuated SNT-induced myocardial injury both in vivo and in vitro.(1)Compared with Control group,the number of TUNEL positive cells in SNT group was significantly increased,while this increasing was significantly decresed by co-treated with EMPA.(2)Compared with Control group,the cell viability of cardiomyocytes in SNT group was significantly decreased.However,SNT-induced cell viability loss was ameliorated by co-treated with EMPA.(3)The number of apoptotic cells in SNT group was significantly increased compared with Control group.Co-treated with EMPA significantly attenuated SNT-induced apoptosis.2.EMPA reversed SNT-induced cardiomyocyte autophagic inhibition and improved the autophagic function of cardiomyocytes.(1)The LC3-II/LC3-I ratio and P62 protein expression of cardiomyoctes in SNT group were significantly higher than those in Control group,both in myocardial tissue and cardiomyocyte.However,the LC3-II/LC3-I ratio and P62 protein expression in SNT + EMPA group were significantly down-regulated when compared with those in SNT group.(2)Compared with Control group,the number of autophagosomes in SNT group was significantly increased,while the number of autophagolysosomes was not significantly changed.Compared with SNT group,the number of autophagosomes in SNT + EMPA group was significantly decreased,while the number of autophagolysosomes was increased.(3)There was no significant difference in LC3-II/LC3-I ratio and P62 protein expression between Baf A1 + SNT group and SNT group.Compared with SNT + EMPA group,there was no significant difference in LC3-II/LC3-I ratio and P62 protein expression in Baf A1 + SNT + EMPA group.3.EMPA restored SNT-induced dysfunction of AMPK-m TOR signaling pathway both in vitro and in vivo.Compared with Control group,SNT down-regulated the ratio of p-AMPK/AMPK and up-regulate the ratio of p-m TOR/m TOR both in vitro and in vivo.However,the ratio of p-AMPK/AMPK was increased,while the ratio of p-m TOR/m TOR was decreased,when co-treated with EMPA.4.Inhibition of AMPK or autophagy blocked EMPA-conferred protection against SNT-induced cardiotoxicity in cardiomyocytes.(1)There was no significant difference in cell mortality and cell viability amongSNT + EMPA + CC group,SNT + EMPA + Baf A1 group and SNT group.(2)There was no significant difference in the changes of autophagy marker proteins and AMPK-m TOR pathway proteins among SNT + EMPA + CC group,SNT + EMPA + Baf A1 group and SNT group.【Conclusion】EMPA could ameliorate SNT-induced cardiotoxicity via regulating cardiomyocyte autophagy,which was mediated by AMPK-m TOR signaling pathway. |
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