Research purpose:Clinical studies suggest that the incidence rate and mortality of complications caused by anti-tumor treatment are increasing year by year,among which cardiovascular disease is one of the most common adverse reactions.The anthracycline drug Doxorubicin(Dox)is currently one of the most effective broad-spectrum chemotherapy drugs in clinical use.However,the risk of death caused by Dox-induced cardiotoxicity(DIC)has surpassed the risk of cancer recurrence,severely limiting clinical application.Dexrazoxane(Dex)is the only antianthracycline heart attenuater approved by the US FDA,but its use is restricted by the European Food and Drug Administration due to severe side effects.Clinical single antioxidant protection schemes have also failed.At present,the pathogenesis and therapeutic drugs of DIC urgently need to be revealed and developed.This study aims to investigate the mechanism of action of estrogen-related receptor α(ERRα)mediated-mitochondrial bioenergy in DIC,and further compare the pharmacological effects of the best small molecule selected by the research group from the traditional Chinese medicine compound Qishen granules,which has been proven to have significant therapeutic effects on tumor heart disease,with similar small molecules.Finally,the pharmacological mechanism of Formononectin(For),which ranks first,will be studied,to provide new intervention links and protective drugs for the treatment of tumor heart disease.Research methods:1.Construction and evaluation experiment of DIC miniature pig model:Male miniature pigs were divided into two groups:the control group and the Dox group.The Dox group of small pigs underwent coronary artery microcatheter injection using the Dox method every two weeks for 5 consecutive modeling sessions.1)Cardiac ultrasound and magnetic resonance imaging were performed at weeks 0,2,4,6,10,12,and 14 to evaluate cardiac function;serum myocardial injury markers were detected;plasma non-targeted metabolomics sequencing was performed.2)At 14 weeks,the left ventricle was collected for transcriptome sequencing and targeted metabolomics sequencing;RT-qPCR was used to detect the expression levels of genes of myocardial fatty acid oxidation(FAO),mitochondrial oxidative phosphorylation(OXPHOS)pathway and Esrra,Esrrb and Esrrg genes;Western Blots method was used to detect the expression of total myocardial ERRa protein and the amount of nuclear translocation.2.Research on the mechanism of overexpression of Esrra gene therapy for curing DIC:Male C57BL/6 mice were divided into four groups:AAV9 control+saline group(AAV9+Saline),AAV9 control+Dox group(AAV9+Dox),AAV9-Esrra+saline group(AAV9Esrra+Saline),and AAV9-Esrra+Dox group(AAV9-Esrra+Dox).Cardiac specific overexpression of Esrra gene was achieved by injecting cTNT promoter driven adenoassociated virus 9 into the tail vein.Inject Dox into the tail vein to create the model,and inject the same amount of saline into the control group.Detect the expression of Esrra,FAO,and OXPHOS pathway genes in myocardial tissue;perform cardiac ultrasound;Hematoxylin eosin staining(HE staining)was used to detect myocardial pathological changes;detect serum cTnT,NT proBNP levels,and myocardial ATP content.3.Drug screening experiment of evaluating isoflavones molecules based on doxorubicin-induced cardiomyocyte injury model:In the early stage of our research group,the DIC zebrafish model was applied to high-throughput screening of the effective components of Qishen granules.It was found that on the basis of ranking first in For,10 isoflavone small molecules with cardioprotective effects were added as candidate components,and the Doxinduced cardiomyocyte model was used to rank the efficacy.Cell viability(CV)was measured using cell proliferation assay;detect of ATP content;apoptosis rate(AR)was detected by flow cytometry;the mitochondrial membrane potential(MMP)was measured.Molecules were ranked according to the combined scores of CV,ATP,AR and MMP.4.Efficacy evaluation experiment of Formononetin in animal models:Male C57BL/6 mice were divided into 6 groups:Control,Dox,Dox+For low-dose group,Dox+For mediumdose group,Dox+For high-dose group,and Dox+positive drug Dex group.Inject Dox into the tail vein for modeling,and administer for 28 days.Small pigs were divided into four groups:Control,Dox,Dox+For,Dox+Dex,and administered for 4 weeks.Perform cardiac ultrasound and magnetic resonance imaging to evaluate cardiac function;detect serum myocardial injury markers;HE staining was used to detect myocardial pathological changes;observe myocardial mitochondrial structure under electron microscopy;TUNEL method was used to detect cell apoptosis;determination of myocardial reactive oxygen species using DHE method;detect total protein expression and nuclear translocation of myocardial ERRα;perform mRNA-seq sequencing to analyze mitochondrial bioenergy pathway gene expression.5.In vitro pharmacological research experiment of Formononetin:Myocardial cells are divided into four groups:Scramble shRNA group(sc shRNA),Scramble shRNA+For group(sc shRNA+For),ERRα shRNA group(ERRa shRNA),ERRα ShRNA+For group(ERRa shRNA+For).Detect total protein expression and nuclear translocation of ERRa;Seahorse method was used to detect the oxygen consumption rate(OCR)of cells;Detect the expression of FAO and OXPHOS pathway genes;mitochondrial membrane potential and apoptosis rate were detected by flow cytometry;MitoSOX and MitoTracker fluorescence co-staining were used to detect the content of superoxide in mitochondria.double Luciferase reporter gene was used to detect the effect of For on the transcriptional activation of ERRa.Research findings:1.DIC pig model was successfully constructed,and mitochondrial bioenergy deficiency is an important pathological mechanism(1)The ultrasound results showed that compared to the beginning of the experiment(week 0),the Dox group of small pigs showed a significant decrease in left ventricular ejection fraction(LVEF)and left ventricular short axis shortening(LVFS)at week 14,with an increase in left ventricular end diastolic dimension(LVEDD)and left ventricular end systolic dimension(LVESD),suggesting that a small pig model of tumor heart disease was successfully constructed at 14 weeks.(2)According to the patterns of changes in plasma metabolites at different stages,they are clustered into 5 categories.The patterns of changes in metabolites in cluster 5 are significantly correlated with the patterns of changes in cardiac function,and are enriched in the lipid metabolism pathway.This suggests that the disorder of circulating lipid metabolism runs through the entire process of tumor heart disease,and its specific patterns of changes may be a potential marker feature of early myocardial injury.(3)Targeted metabolomics results showed a decrease in long-chain acylcarnitine content in the myocardium of the Dox group compared to Control,indicating a decrease in fatty acid oxidation levels.(4)Transcriptome sequencing results showed that compared with Control,Dox down-regulation genes were significantly enriched in the mitochondrial bioenergy pathway,especially the expression of FAO and OXPHOS genes was significantly reduced,suggesting that mitochondrial bioenergy,especially mitochondrial fatty acid oxidation metabolism was reduced.(5)Compared with Control,the expression of Esrra gene in the myocardium of the Dox group was significantly reduced,especially with a significant decrease in the translocation of ERRa protein into the nucleus.2.Gene therapy based on Esrra overexpression improves mitochondrial bioenergy in the treatment of tumor-related heart diseaseCompared with the AAV9+Dox group,the AAV9-Esrra+Dox group significantly increased the expression of the Esrra gene in the myocardium of mice,accompanied by a significant increase in cardiac function,manifested as a significant increase in LVEF and LVFS,while a significant decrease in LVEDD and LVESD.HE staining and serum biochemical results showed that compared to the AAV9+Dox group,the AAV9-Esrra+Dox group mice had orderly arrangement of myocardial tissue and reduced muscle fiber dissolution;the levels of cTnT and NT proBNP in serum were significantly reduced.The above results suggest that overexpression of the Esrra gene significantly reduces DIC.Further detection of myocardial ATP content and expression of oxidative metabolism genes showed that compared to the AAV9+Dox group,the AAV9-Esrra+Dox group showed an increase in myocardial ATP content,while the expression of OXPHOS and FAO pathway genes(including Cox5b,Ndufs8,Atp5a,Cpt1b,Acadm,Acsl1,Mtcol,Sdhb,etc.)was significantly upregulated.In summary,overexpression of the Esrra gene may improve DIC by increasing mitochondrial oxidative metabolism.3.Among the 11 isoflavones small molecules evaluated by the doxorubicin-induced cardiomyocyte injury model,Formononetin ranks first in terms of efficacyThe comprehensive scores of Formononetin,Resveratrol,Daidzein,Calycosin,Genistein,Puerarin,Coumestrol,Osthol,and Chickpea sprout A were 1.5,2.5,4,4.5,4.5,5.25,6.75,and 7.25 respectively,suggesting that For ranked first.4.Formononetin can significantly improve the cardiac function of DIC mice/miniature pigsUltrasound and magnetic resonance imaging results showed that compared with the Dox group,the For treatment group significantly improved cardiac function,showed no delayed myocardial enhancement,and myocardial edema subsided.For treatment significantly reduced serum BNP,ANP,CK-MB,and LDH,and increased SOD.HE staining showed that in the For treatment group,there was a significant decrease in muscle fiber lysis and a relatively neat arrangement of myocardial tissue.Electron microscopy showed that For treatment significantly reduced mitochondrial damage and vacuolization caused by Dox,as well as significantly reduced cell apoptosis and ROS accumulation caused by Dox.The WB results showed that compared to the Dox group,the For treatment group significantly increased the total protein expression and nuclear translocation of ERRα.The results of mRNA-seq transcriptome sequencing showed that,compared with Dox group,the For treatment group significantly recalled mitochondrial bioenergy related pathways,especially the gene expression of FAO and OXPHOS pathways.In summary,For significantly improves cardiac function in tumor heart disease model animals and enhances mitochondrial bioenergy mediated by ERRα.5.Formononetin alleviates doxorubicin-induced myocardial damage by enhancing ERRα-mediated mitochondrial bioenergyCompared with the sc shRNA group,the total ERRα protein expression and nuclear translocation of cardiomyocytes in the ERRa shRNA group were significantly reduced,accompanied by decreased OCR and ATP content,decreased FAO and OXPHOS pathway gene expression,decreased mitochondrial membrane potential,increased apoptosis and ROS content.Compared to ERRa shRNA group,the total protein expression and nuclear translocation of ERRa in ERRa shRNA+For group,were significantly up-regulated,accompanied by increased OCR and ATP content,increased gene expression of FAO and OXPHOS pathways,recovery of mitochondrial membrane potential,decreased apoptosis rate and ROS content.The Luciferase reporter gene experiment showed that For enhanced the transcriptional activity of ERRα,which was expressed by the increased transcription of target genes Acadm and Atp5a.In summary,For enhances its transcriptional activation ability by promoting ERRa expression and entry into the nucleus,enhancing mitochondrial bioenergy and alleviating DIC.Research conclusions:1.Mitochondrial bioenergy deficiency,especially a decrease in fatty acid oxidation levels,is an important pathological mechanism of tumor heart disease;2.Gene therapy with cardiac specific overexpression of Esrra can promote mitochondrial oxidative metabolism gene expression,improve mitochondrial bioenergy,and alleviate DIC;3.Mangostein significantly improves the cardiac function of tumor heart disease mice/miniature pigs,reduces mitochondrial oxidative damage,promotes gene expression of FAO,OXPHOS,and TCA cycle pathways,and enhances mitochondrial bioenergy mediated by ERRα;4.Formononetin enhances the transcription activation of mitochondrial oxidative metabolism genes by promoting the expression and nuclear translocation of ERRα,thereby enhancing mitochondrial bioenergy and treating tumor heart disease.In summary,this study provides a theoretical basis and clinical guidance for the revelation of new links in the treatment of tumor heart disease and the development of new therapeutic drugs. |