Breviscapine Regulates Mitochondrial Autophagy To Protect Doxorubicin Study On Induced Myocardial Injury

Objective:Anthracyclines are the most widely used first-line chemotherapy agents against solid tumors and leukemia,but their adverse cardiac effects and even heart failure seriously limit their clinical application.Breviscapine is a flavonoid compound with a wide range of pharmacological effects such as antioxidant,anti-inflammatory,anti-tumor,and so on.The purpose of this study was to investigate the protective effect of breviscapine on anthracycline-induced cardiotoxicity and its mechanism.Methods:In vivo:48 C57 mice were randomly divided into control group,doxorubicin group,high/medium/low dose breviscapine group,and dexrazoxane group.UHPLC-Q-TOF/MS was used to detect the biomarkers and metabolic pathways in the plasma of each group.Ejection fraction（EF）,short axis shortening rate（FS）,and heart rate（HR）were measured by echocardiography.Serum levels of heart failure biomarkers NT-pro BNP and c Tn I were detected by Elisa.Hematoxylin-eosin（HE）staining and Masson staining were used to detect the pathological changes in myocardial tissue.Immunohistochemistry was used to detect the expression of IL-1βin myocardium.In vitro:H9c2 cells were randomly divided into control group,doxorubicin group,doxorubicin+dexrazoxane group,and doxorubicin+breviscapine group.The effective concentrations of breviscapine and doxorubicin were detected by cck8method.SOD,GSH,NADH,LDH activities,and MDA content were detected by spectrophotometry.ROS intensity and apoptosis rate were detected by flow cytometry.Jc-1mitochondrial membrane potential,Tunel cell apoptosis,and Ca²⁺probe were detected by fluorescence microscopy.The structure of mitochondria was detected by electron microscopy.Apoptosis-related proteins AMPK,rho A,and Bcl-2 were detected by WB method.Expression levels of autophagy-related proteins LC3,P62,Beclin1,Pink1,parkin,VDAC1 and Tom20;Protein interactions between Beclin1 and Bcl-2,parkin,and Tom20 were detected by Co-IP method.Mitotracer expression was detected by immunofluorescence staining.In addition,ATP and ROS were detected by combined mitochondrial autophagy inhibitor Mdivi-1.Finally,doxorubicin combined with breviscapine stimulated Mc F-7 cells,and the cell survival rate was detected by cck-8 method to observe whether breviscapine could reduce the anti-tumor activity of Mc F-7 cells while alleviating the toxic and side effects of doxorubicin.Results:Compared with doxorubicin group,EF%,FS%and HR increased in breviscapine group.HE and Masson staining showed that compared with doxorubicin group,breviscapine could improve myocardial structural disorder,muscle fiber breakdown,and myocardial fibrosis.Immunohistochemical results showed that breviscapine effectively improved inflammatory cell infiltration in the heart of mice induced by doxorubicin,and significantly increased serum NT-pro BNP and c Tn I levels.Metabolomics analysis showed that breviscapine was mainly involved in regulating unsaturated fatty acid metabolites and amino acid metabolic pathways.Breviscapine could improve H9c2 cell viability,improve doxorubicin-induced mitochondrial membrane potential reduction,and improve the level of intracellular oxidative stress and apoptosis.Electron microscope results showed that breviscapine could significantly ameliorate the mitochondrial structure damage caused by doxorubicin.WB results showed that breviscapine significantly increased the expression levels of Pink1 and parkin.When combined with Mdivi-1,the improvement effect of breviscapine was significantly reduced.Finally,cck8results showed that combined breviscapine stimulation of MCF-7 cells could improve the killing rate of doxorubicin to tumor cells.Conclusion:Breviscapine can effectively improve left ventricular dilation induced by doxorubicin,improve cardiac function,and ameliorate cardiomyopathic structural damage induced by doxorubicin.Mechanically,breviscapine can not only clear damaged mitochondria by regulating myocardial energy metabolism,reducing lipid oxidation,and improving energy production rate and breviscapine regulates mitochondrial autophagy by regulating Pink1/parkin-related signaling pathways and maintains mitochondrial homeostasis of cardiomyocytes.In addition,breviscapine combined with doxorubicin did not affect its antitumor activity.