Study On The Mechanism Of Heart Failure Induced By Doxorubicin In Rats Based On Metabolomics And Proteomics

Heart failure is a disease with extremely complex pathophysiological causes,which is difficult to diagnose and treat,and cannot completely prevent the disease from progressing,leading to a high mortality.Therefore,the discovery of new key targets and pathways of heart failure is still a hotspot,which is not only conducive to multi-target treatment,but also a key breakthrough in the treatment of heart failure.In this study,the heart failure model of rats was established by doxorubicin administration,and the non-targeted metabolomics research based on UPLC-Q-TOF/MS technology to obtain the differential metabolites,meanwhile the quantitative proteomic study based on TMT technology were carried out to obtain differential proteins.Then,the two groups of data were integrated and analyzed to find the differential proteins and small molecular metabolites that play a role in bridging heart failure.The key targets and metabolic pathways were screened out and Western bolt was used to verify the nodulin,the main conclusions are as follows:1.The heart failure model of rats was established by doxorubicin.The ultrasonic index,biochemical index of serum and pathological section of heart were used to evaluate the rat model,so as to ensure the establishment of heart failure model.Compared with the NS group,the EF,FS,LVPW,s of the model group were significantly lower?p<0.01?,LVPW,d were lower?p<0.05?,IVID,d and IVID,s significantly increased?p<0.01?,indicating that doxorubicin caused serious damage to the cardiac systolic function and cardiac structure of rats.Biochemical results showed that LDH,CK and CK-MB in model group were significantly increased?p<0.01?,indicating that the cardiac function of rats was seriously damaged.Histopathological observation showed that the cardiac tissue and cardiac structure of rats in the model group were seriously damaged.Therefore,the above results can confirm the establishment of doxorubicin induced heart failure model in rats.2.In order to obtain the differential metabolites,UPLC/Q-TOF-MS technology was used to analyze the rat plasma samples.After the validation of the method,data preprocessing and other operations,the data were analyzed by multivariate statistical analysis,and 21 potential biomarkers of heart failure,such as tryptophan,tyrosine,glutamine,?-ketoglutarate,were finally screened.In this experiment,21 biomarkers were analyzed by ROC curve and cluster analysis.And the results show that these 21 biomarkers have a good accuracy in diagnosing heart failure.Finally,based on the analysis of these markers and their changes in Met PA database,it is speculated that heart failure mainly involves energy metabolism,amino acid metabolism,fatty acid metabolism and glycerophospholipin metabolism,which provides a reliable basis for the mechanism research of heart failure.3.Based on TMT and LC-MS/MS technology,278 proteins with significant changes were identified and quantified.118 of them were up-regulated and 160 were down regulated.The results of cluster analysis showed that differential protein could effectively separate the two groups,indicating the rationality of differential protein screening.Then GO analysis shows that heart failure was closely related to the production of metabolites and energy,ATP metabolism,purine ribonucleotide metabolism,respiratory electron transport chain,glucose catabolism and other important biological processes.In addition,the enrichment analysis of KEGG pathway showed that glutathione metabolism,glycolysis,myocardial contraction and other signaling pathways play an important role in heart failure.Through the analysis and discussion of the above pathways,it is found that heart failure is closely related to oxidative stress,energy metabolism disorder and myocardial cell Ca²⁺imbalance,which lays a foundation for the subsequent integration analysis.4.Based on the results of metabolomics and proteomics,25 differential proteins,such as acaa2 and atp5j,are closely related to the differential metabolites of heart failure.The pathways involved are mainly fatty acid metabolism,glycolysis,amino acid metabolism,glycerophosphate metabolism,glutathione metabolism and myocardial contraction.It is speculated that the important mechanism of heart failure is lipid The abnormality of fatty acid metabolism and sugar metabolism interferes with TCA circulation and leads to the insufficiency of myocardial energy supply,which leads to the accumulation of ROS in the heart and the influence of calcium homeostasis on the systolic function of the heart.Most of these proteins are located in the downstream of the pathway,so it is very important to find the upstream key target proteins.In addition,the protein prediction function of string database was used to select the proteins that play an important role in the proteomic results and the known doxorubicin target protein to jointly predict the upstream protein,and finally the key target protein PTP1B,which provides a new idea for the effective treatment of heart failure.5.Based on the potential target protein PTP1B and its subsequent pathway,Western blot was used to detect the expression of IRS,HIF-1?,Nrf2 and HK-2.The results showed that compared with the blank group,the expression level of IRS protein in the model group increased significantly,while the expression level of p-IRS1,HIF-1?,Nrf2 and HK-2 protein decreased significantly,which indicated that PTP1B played an important role in the cardiac dystrophy caused by the insufficiency of energy supply and oxidative stress,which could be an important target for the treatment of heart failure.This not only provides a new therapeutic target for heart failure,but also provides a new strategy for the follow-up new drug research and development.However,in this study,only preliminary verification has been carried out,so further functional verification of the target protein from the cell and animal level is needed.