Exploring The Mechanism Of Doxorubicin-induced Heart Failure Based On Gut Microbiota And Metabolomics

Objective:The aim of this study was to explore the changes of intestinal microbiota and serum metabolites in mice during DOX-induced heart failure,to clarify the correlation between intestinal flora and serum metabolites,to screen the markers,and to provide new targets and ideas for diagnosis and treatment of DOX-induced heart failure.Methods:1.Grouping: 52 C57BL/ 6J mice aged 6-8 weeks were selected and randomly divided into Control group,Doxorubicin treatment one week group(1W-DOX group),Doxorubicin treatment three week group(3W-DOX group),and Doxorubicin treatment five week group(5W-DOX),13 mice in each group;2.Model establishment: control mice were injected intraperitoneally with saline at a concentration of 5 mg/kg once a week for 5 weeks,with a cumulative dose of 25 mg/kg.1WDOX group: once a week for 1 week,cumulative dose of 5mg/kg;3W-DOX group: once a week for 3 weeks,cumulative dose of 15mg/kg;5W-DOX group: once a week for 5 weeks,cumulative dose of 25mg/kg;3.To perform cardiac echocardiography to measure and record left ventricular diastolic and end-systolic internal diameters,left ventricular ejection fraction,left ventricular shortaxis shortening rate and other indicators to assess cardiac function;4.Mice were executed and myocardial tissues were taken for hematoxylin-eosin staining,and histopathological changes in each group of hearts were observed under light microscope;5.Mice fecal samples were collected for 16 S amplicon sequencing analysis,and mice serum samples were collected for LC-MS/MS non-targeted metabolomics analysis.6.Multi-omics correlation analysis to detect the correlation between intestinal flora and serum metabolites in mice.Results:1.Echocardiography test results: compared with the control group and 1W-DOX group,LVEF and LVFS were significantly decreased and LVIDd and LVIDs were significantly increased in the 3W-DOX and 5W-DOX groups.The LVEF and LVFS in the 5W-DOX group decreased significantly compared with the 3W-DOX group,and the LVIDd and LVIDs increased significantly compared with the 3W-DOX group.2.Histological test results: the distribution of cardiomyocytes in control group was regular and tight,the cell nuclei and cytoplasm morphology were normal,and no abnormal changes were observed in myocardial fibers;the myocardial plasma eosinophil staining was enhanced in 1W-DOX group;the myocardium of mice in 3W-DOX group was mildly bruised,the arrangement of myocardial cells was partially disordered,edema was seen,and plasma and fibrin exuded from the veins;the myocardium of mice in 5W-DOX group was severely bruised,the myoplasm was lysed,and the texture of the myocardium was significantly increased.In the 5W-DOX group,the myocardium was severely stagnated,the myoplasm was lysed,the texture of the myocardium was not clear,and swollen,degenerated or necrotic myocytes were visible.3.16 S amplicon sequencing results: The composition and structure of intestinal flora of control and 5W-DOX mice were significantly different.At the phylum level,a total of eight phylum sequences of fecal flora were found in the control and 5W-DOX groups,mainly including Firmicutes,Bacteroidetes,Proteobacteria and Actinobacteria.Compared with the control group,the abundance of Firmicutes decreased,the abundance of Bacteroidetes increased,and Firmicutes / Bacteroidetes(F/B)ratio decreased in the 5W-DOX group.At the genus level,12 differential genera were screened in the 5W-DOX group compared to the control group,Bifidobacterium,Romboutsia,Escherichia,Mycoplasma,Turicibacter,Clostridium＿XVIII,Butyriciminas,Clostridium＿sensu＿stricto and Gemmiger with significantly increased abundance;The abundance of Roseburia,Asaccharobacter and Peptococcus was significantly decreased.4.Metabolomic results: there were significant differences in serum metabolites between control and 5W-DOX groups.A total of 58 differential metabolites were screened using VIP≥1,FC≥1.2 or≤0.83,q-value<0.05 and Level≤3.The bile acids and their derivatives(Cholic acid,Deoxycholic acid,Taurocholic acid),steroids and its derivatives(Aldosterone),amino acids and peptides(Cystine,N-acetyl-l-phenylalanin,L-histidine),phenols and their derivatives(4-methylphenol),steroid lipids(Lithocholic acid taurine conjugate),and Trimethylamine n-oxide(TMAO)were enriched in the 5W-DOX group.Among them,Lhistidine is involved in amino acid biosynthesis;Taurocholic acid is involved in primary bile acid biosynthesis,bile secretion and aldosterone metabolism;Cholic acid is involved in primary bile acid biosynthesis and bile secretion;Aldosterone is involved in steroid hormone biosynthesis,aldosterone synthesis and secretion and Aldosterone is involved in steroid hormone biosynthesis,aldosterone synthesis and secretion,and aldosterone-regulated sodium reabsorption;Deoxycholic acid is involved in bile secretion.Differential metabolites with predictive value for HF include Cholic acid(AUC=0.95),Lithocholic acid taurine conjugate(AUC=0.95),Aldosterone(AUC =0.90),Deoxycholic acid(AUC=0.87),TMAO(AUC = 0.93),Taurocholic acid(AUC = 0.9),L-histidine(AUC = 0.89),Glycocholic acid(AUC=0.92),Cystine(AUC=0.86),N-acetyl-l-phenylalanin(AUC=0.96).5.Results of multi-omics analysis: Escherichia and Turicibacter in the intestinal flora were positively correlated with the differential metabolite TMAO(r=0.70 and r=0.61,P< 0.05);while Roseburia in the intestinal flora was negatively correlated with the differential metabolite TMAO(|r|=0.58,P<0.05).Escherichia and Turicibacter in the intestinal flora were positively correlated with the differential metabolites Cholic acid,Deoxycholic acid and Taurocholic acid(r>0.5,P<0.05);while Roseburia in the intestinal flora was positively correlated with the differential metabolites Cholic acid,Deoxycholic acid and Taurocholic acid were negatively correlated(|r|<0.5,P<0.05).Conclusion:This study revealed significant differences in intestinal flora composition and serum metabolic profiles in DOX-induced heart failure mice by 16 S amplicon technology and metabolomics.Firmicutes / Bacteroidetes ratio was reduced in the intestine of heart failure mice,and Roseburia was significantly reduced,leading to decreased butyrate production;Escherichia and Turicibacter were greatly and significantly increased.TMAO,L-histidine,Cholic acid,Deoxycholic acid,Taurocholic acid were significantly increased in metabolites of heart failure mice and could be used as markers of DOX-induced heart failure.Escherichia and Turicibabcter in the intestinal flora were positively correlated with TMAO,Cholic acid,Deoxycholic acid,Taurocholic acid.cholic acid,Deoxycholic acid,Taurocholic acid are involved in primary bile acid biosynthesis and bile secretion pathways,and since these pathways are involved in the pathophysiological process of HF development,it is suggested that Escherichia and Turicibabcter may play a role in the development of DOXinduced HF through their related metabolites and metabolite downstream pathways.This study provides new targets and new ideas for the diagnosis and treatment of DOX-induced HF.