Protective Effects And Potential Mechanisms Of Cyclovirobuxine D Against Doxorubicin-induced Cardiotoxicity

Doxorubicin （DOX）, one of the most effective anticancer drugs, whichhas been wildly used in clinic for treating a variety kinds of haematologicalmalignancies and solid tumors such as carcinomas, soft tissue sarcomas,multiple myeloma, non-Hodgkin’s lymphomas, and Hodgkin’s disease. Butbeing with serious cumulative cardiotoxic side effects brings DOXlimitations of its clinical application. At present there is no any ideal drugcould prevent and treat DOX-induced cardiotoxicity. Cyclovirobuxine D（Cvb-D）, an active monomer extracted from traditional Chinese medicineBuxus microphylla, is a pregnane derivatives with molecular formulaC₂₆H₄₆N₂O. Cvb-D is widely used in China for treating many cardiovasculardiseases, such as arrhythmia, angina, coronary heart disease and heartinsufficiency. Therefore, this research was undertaken to investigate theprotecting effect of Cvb-D to DOX on cardiac toxicity and to explore itspossible mechanisms to provide new ideas and methods for preventing andtreating cardiac toxicity of DOX.A DOX-induced cardiomyopathy model of C57/BL mice was established and the mice were randomly divided into four groups. Pretreatment withCvb-D （1mg/kg） had been kept for four days for Mice. DOX wasadministered as a single dose at15mg/kg intraperitoneally on day5. Afterthe administration of DOX, the mice were daily weighted.4days after DOXadministration, noninvasive echocardiography technique was used for theassessment of cardiac function. After myocardial tissue sections werestained by HE and Masson, microscope had been used to observe themorphological changes of myocardial tissue. To collect samples by drawingorbital blooding, and then to determine creatine kinase in serum （CK） andlactate dehydrogenase （LDH） level. To feature evaluation of Cvb-D onDOX induced toxicity damage and myocardial superoxide anion （O·-2） wasmeasured by xanthine oxidation method. Nitric oxide （NO） content ofmyocardial tissue was detected by griss method for evaluating the effect ofCvb-D on DOX-induced mice myocardial oxidative state.Detect oxidation and protein carbonylation level than lipid myocardial tissueand evaluate Cvb-D on DOX induced myocardial oxidative injury in miceby separately using the TBARS and the DNPH colorimetric.At the same time to evaluate the Cvb-D effect of myocardial antioxidantsystem changes induced by DOX, determine the activity of Mn-superoxidedismutase by xanthine oxidase （Mn-SOD） method, detect reduced/oxidizedglutathione peroxidase （GSH/GSSG） ratio of myocardial tissue by cyclicresponse of the DNTB; In order to evaluate the effect of Cvb-D on mitochondrial pathway in cardiomyocytes apoptosis induced by DOX inmice,TUNEL method was used to detect myocardial apoptotic cells incardiac tissue which were counted as apoptosis index （AI）. Detect theexpression of Bcl-2and Bax protein in tissue homogenates and thecytoplasm/mitochondria （Cytochrome C） distribution by Western blotmethod to express Caspase-3in myocardial tissue. Detect the expression ofPGC-1α, Nrf-1protein in myocardial tissues. Determine the copy numberof mitochondrial genes（mtDNA） by QPCR, which can be used to evaluatethe effect of Cvb-D on DOX induced mitochondrial damage in myocardialtissue of mice.The results show that, compared with the control group, Cvb-D had noobvious effect on mouse; Cvb-D can improve heart function of DOXinduced changes, decrease left ventricular end-diastolic diameter caused byDOX （LVIDD） and increase left ventricular end systolic diameter （LVIDD）,improve left ventricular shortening fraction （FS,%） and left ventricularejection fraction （EF,%） and other parameters; the inhibition of weight andheart wet weight ratio reduction caused by DOX induced, relievesmyocardial swelling, vacuolization of histomorphology changes andmyocardial fibrosis; decrease LDH and CK level in serum; Cvb-D couldreduce myocardial oxidative tissue damage caused by DOX, includingreducting the damage of MDA content to lipid peroxidation of myocardialtissue and protein carbonylation, increasing GSH/GSSG levels, decreasing levels of superoxide anion and nitric oxide. As results showed above, Cvb-Dhas obvious protective effect on DOX heart toxicity, and its mechanism maybe related to Cvb-D antioxidant, free radicals may play an important role inthis process; Cvb-D can inhibit the increase of myocardial cell apoptosisinduced by DOX, improving the expression and activity of Caspase-3inmyocardial tissue and the ratio of Bcl-2/Bax protein expression; Cvb-D canimprove the distribution change of cytochrome C in mitochondria andimprove mitochondrial reducted by DOX, increase DNA copy number andpromote the expression of PCG-1α, Nrf-1gene which is closely related tothe mitochondria function.These results above indicate that CVB-D has a significant protective effecttowards DOX cardiotoxicity. Its mechanism may be related CVB-Danti-oxidation. Scavenging of free radical may play an important role in thisprocess, at the same time, the CVB-D can alleviate mitochondrial damageof DOX-induced, inhibit apoptosis of cardiac myocyte caused by caspase-3activation which is induced by DOX, specalate the protective effect ofCVB-D DOX cardiotoxicity may be related to the inhibition ofmitochondrial-dependent apoptosis pathway..