The Mechanism Of Ursolic Acid In Preventing Doxorubicin Induced Cardiac Toxicity In Mice

ObjectiveTo explore the mechanism of ursolic acid to weaken doxorubicin induced cardiac toxicity,We established the mouse model of doxorubicin-induced cardiotoxicity with ursolic acid intervention.Furthermore,Finding a way that antagonizes doxorubicin induced cardiac toxicity without affecting its anti-tumor activity.MethodsA total of 45 mice were randomly divided in 3 groups,including Sham,Doxorubicin and Doxorubicin+Ursolic acid treatment groups(n=15 per group).Doxorubicin was dissolved in the vehicle DMSO(2%).Sham group,DOX group,subcutaneous injecting of 2% DMSO saline 0.1ml/day;UA + DOX group,subcutaneous injecting of ursolic acid(80mg/kg/day)0.1ml/day.After a week,the cardiac function parameters of three groups were detected by echocardiography.Adriamycin 15 mg/kg was intraperitoneally injected into DOX group and UA + DOX group.While,the Sham group were intraperitoneally injected with 2% DMSO saline.The 7th and 28 th days after injection were selected as early and late doxorubicin cardiac injury time point.Echocardiography was performed on each group of mice at each of the two injury times induced by doxorubicin to assess cardiac function.On the 7th day after doxorubicin injection,10 mice were sacrificed in each group,and heart samples and blood samples were taken for myocardial enzyme CK-MB determination,TUNEL staining and Western blotting to detect apoptosis-related proteins,which react the effect of ursolic acid on the 7ths.To assess the effect of ursolic acid on late doxorubicin induced cardiac injury,the remaining 5 mice in each group were sacrificed on 28 after doxorubicin injection,and heart samples were obtained and paraffin embedded.The H&E and Masson’s staining methods were performed.Results 1.Ursolic acid preserves cardiac function in mice treated with doxorubicinWe found that at both 7 and 28 days,administration of ursolic acid significantly improved FS and EF,compared with the DOX group(P<0.01).2.Ursolic acid decreases CK-MB levels in the heart after doxorubicin treatmentTo evaluate the effects of ursolic acid on CK-MB production in the heart after treatment with doxorubicin,mice in each group were sacrificed and heart specimens were harvested at 7 days after doxorubicin treatment.The results showed that ursolic acid significantly decreased CK-MB level in the heart after doxorubicin treatment,compared with the DOX group(P<0.05).3.Ursolic acid decreases apoptosis of cardiac cells in mice treated with doxorubicinUrsolic acid significantly decreased cardiac cell apoptosis in mice treated with doxorubicin compared with the DOX group(P<0.01).Meanwhile,Ursolic acid significantly decreased cleaved caspase-3 levels in the hearts of mice,compared with doxorubicin(P<0.01).4.Ursolic acid inhibits inflammation and fibrosis in the mouse heart after treatment with doxorubicinUrsolic acid overtly decreased inflammatory cell infiltration compared with Sham group;Compared with the DOX group,the UA+DOX group significantly reduced the degree of inflammatory cell infiltration(Fig.4A);Furthermore,Ursolic acid significantly decreased fibrosis compared with the DOX group(P<0.01).5.Ursolic acid increases AKT and eNOS phosphorylation levels in the heart of mice treated with doxorubicinEffects of ursolic acid on AKT and p-AKT : doxorubicin significantly decreased AKT phosphorylation compared with sham(P<0.05),while ursolic acid significantly increased AKT phosphorylation compared with doxorubicin(P<0.05).Effects of ursolic acid on P-eNOS: doxorubicin significantly decreased eNOS phosphorylation compared with sham(P<0.01),while ursolic acid significantly increased eNOS phosphorylation compared with doxorubicin(P<0.01).6.Ursolic acid downregulates NOX4 and inhibits eNOS uncoupling in the mouse heart after doxorubicin treatmentEffect of ursolic acid on eNOS: doxorubicin significantly increased eNOS expression compared with sham(P<0.05),while ursolic acid significantly increased eNOS expression compared with doxorubicin(P<0.01).Effect of ursolic acid on Nox4: doxorubicin significantly increased NOX4 expression compared with sham(P<0.01),while ursolic acid significantly decreased Nox4 expression compared with doxorubicin(P<0.01).ConclusionUrsolic acid improved Fractional shortening(FS)and Ejection fraction(EF)of the heart,and decreased cardiac apoptosis in mice treated with doxorubicin.Mechanistically,ursolic acid increased AKT and eNOS phosphorylation levels,and enhanced eNOS expression,while inhibiting doxorubicin induced eNOS uncoupling through NOX4 downregulation.These effects of ursolic acid resulted in heart protection from doxorubicin induced injury.