Based On Omics And Network Pharmacology, The Mechanism Of Action Of Kangxian Yixin Recipe In The Treatment Of Dilated Cardiomyopathy Was Explored

Objective To establish a Dilated Cardiomyopathy,(DCM)rat model of furazolidone-induced dilated cardiomyopathy,comprehensively use proteomics,metabonomics and network pharmacology to study the mechanism of Kangxian Yixin Decoction in the treatment of DCM,identify and screen the expression of differential proteins and metabolites in myocardial tissue of DCM,and carry out bioinformatics analysis.Combined with the analysis of the potential action targets of Kangxian Yixin Decoction in the treatment of DCM,the mechanism of Kangxian Yixin Decoction in the treatment of DCM was comprehensively discussed,so as to provide more basis for the clinical promotion and application of Kangxian Yixin Decoction.Methods1.The furazolidone-induced DCM rat model was prepared.After the intervention of Kangxian Yixin Decoction,the cardiac function was evaluated by cardiac ultrasound,the pathological changes of myocardial tissue were observed by HE staining,the degree of myocardial fibrosis was observed by Masson staining,and the ultrastructure of myocardial tissue was observed by transmission electron microscope.2.Combined with the research methods of proteomics and metabonomics,the myocardial tissues of DCM rats and rats after intervention of Kangxian Yixin Decoction were analyzed and screened for related differential proteins and differential metabolites,and bioinformatics analysis was carried out to explore the possible mechanism of Kangxian Yixin Decoction in the treatment of DCM,and further analyze its mechanism through the data combination between the two groups.3.Based on the technology of network pharmacology,the potential targets of Kangxian Yixin Decoction and its different compatible components in treating DCM were screened.Through the functional enrichment analysis of potential targets and protein interaction analysis in Metascape and String database,the characteristics of Kangxian Yixin prescription were expounded,and the possible mechanism of its treatment of DCM was revealed.4.The expression of the selected key differential proteins was verified by Western Blot to explore the possible mechanism of Kangxian Yixin Decoction in the treatment of DCM.Results1.The DCM rat model was made with furazolidone.The results of echocardiography showed that the left ventricular end-systolic diameter(LVESD)and left ventricular end-diastolic diameter(LVEDD)in model group were significantly increased,while the left ventricular shortening fraction(LVFS)and left ventricular ejection fraction(LVEF)were significantly decreased(P < 0.01).After the intervention of Kangxian Yixin Decoction,the above-mentioned indexes were improved(P < 0.01).Through the observation of pathological sections,Masson staining and ultrastructure of cardiomyocytes,it was found that the myocardial tissue of DCM was severely damaged,the arrangement of nuclei was irregular,some nuclei pyknotic and fragmented,the arrangement of muscle fibers was loose and disordered,the fibrillation of cardiac muscle was enhanced,and the pattern paste of myofilament junction,local myofilament rupture,disordered arrangement of mitochondria,swelling of mitochondria with vacuole-like changes were found.The pathological morphological structure of KXYX group was significantly improved after the intervention of Kangxian Yixin Decoction.2.Based on proteomic analysis,there were 159 differentially expressed proteins between model group and control group,of which 42 proteins were up-regulated and 117 down-regulated.(unique peptide ? 1line fold Change >1.2or Fold Change < 0.83(p < 0.05).Compared with model group,51 differential proteins were obtained in KXYX group,of which 34 were up-regulated and 17 were down-regulated.(unique peptide ? 1 and Fold Change < 0.83(p < 0.05).The differential proteins such as carbonic anhydrase4(Ca4),mitochondrial mitotic factor(Mff)and mitochondrial sulfur dioxygenase(Ethe1)were significantly decreased in DCM.Through bioinformatics analysis,it was found that the cellular localization of differential proteins among the three groups was mainly concentrated in mitochondria and cytoplasm.The main biological processes of differential proteins in model group compared with control mainly include energy metabolism,calcium reaction,redox,muscle structure development,etc.,and the main molecular functions include cytoskeleton protein binding,A-TPase regulatory activity,protein complex binding,redox activity and so on.Compared with model,the main biological processes of differential proteins in KXYX include sulfur compound metabolism,cellular amide metabolism,Golgi vesicle transport mediated by endoplasmic reticulum,and cellular amino acid metabolism;the main MF includes catalytic activity,coenzyme binding,collagen binding,NAD binding,actin binding,oxidoreductase activity and so on.By KEGG enrichment pathway analysis,it was found that the main enrichment pathways of model than control included dilated cardiomyopathy pathway(disease pathway),myocardial contraction pathway,metabolic pathway,thermogenic pathway,protein processing in endoplasmic reticulum and so on.The main enrichment pathways of KXYX than model include protein processing in endoplasmic reticulum,myocardial contraction,amino acid biosynthesis,thermogenic pathway and so on.3.Through metabonomic analysis,there were 64 up-regulated and 49down-regulated metabolites between model group and control group(p < 0.05).Compared with model group,there were 140 differential metabolites in);KXYX group,of which 112 were up-regulated and 28 down-regulated(p <0.05).Through analysis,it was found that 27 differential metabolites such as adipic acid,linoleic acid and L-carnitine decreased or increased significantly in model group.Among the callback metabolites,by ROC curve analysis,it was found that 6 kinds of differential metabolites were involved in model group and AUC > 0.9 in control group,and 10 kinds of differential metabolites were involved after intervention of Kangxian Yixin Decoction.The pathway analysis of differential metabolites showed that model group was more involved in amino sugar and nucleotide sugar metabolism,sulfur metabolism,TCA cycle,glycolysis and glucose regeneration than control group.KXYX group to model group found that Kangxian Yixin Decoction mainly through regulating amino acid synthesis and metabolism,purine metabolism,sulfur metabolism,phenylalanine metabolism,TCA cycle,linoleic acid metabolism and other pathways play a therapeutic effect on DCM.4.Through the combined analysis of proteomics and metabonomics,it was found that there were 263 pathways involved in differential metabolites and differential proteins in model group compared with control.They are mainly dilated cardiomyopathy(disease pathway),purine metabolism,sulfur metabolism,carbon metabolism,glycolysis / gluconeogenesis,thermogenesis,oxidative phosphorylation,myocardial contraction,calcium signal pathway,PI3K-Akt signal pathway,Apelin signal pathway,c AMP signal pathway,aldosterone synthesis and secretion,actin cytoskeleton regulation,c GMP-PKG signal pathway,endoplasmic reticulum protein processing,MAPK signal pathway and so on.There are 77 pathways involved in differential proteins or metabolites compared with model in KXYX group,including purine metabolism,sulfur metabolism,TCA cycle,ABC transporter,vitamin digestion and absorption,amino acid synthesis,protein digestion and absorption,thermogenic pathway,myocardial contractile function,endoplasmic reticulum protein processing,c AMP signaling and so on.Based on the comprehensive analysis of differential proteins and differential metabolites,Kangxian Yixin Decoction plays an important role in regulating the pathway of sulfur metabolism.5.91 potential targets of Kangxian Yixin Decoction in the treatment of DCM were screened by network pharmacology.The targets of each component of Kangxian Yixin Decoction showed 43 kinds of monarch drugs(47%),71 kinds of subject drugs(77%),41 kinds of adjuvants(45%)and 12 kinds of drugs(13%).1011 kinds of biological processes,133 kinds of molecular functions,84 kinds of cell composition,147 KEGG-related pathways and 120 kinds of Reactome pathways were obtained by analyzing Kangxian Yixin Decoction by String database.Through Metascape,the functions closely related to egg white function were classified into 20 subsets,involving 722 pathways,including blood circulation,nuclear receptor transcription pathway,muscle system related process,active oxygen metabolism process and so on.There are 357 ways of action of Astragalus membranaceus in Kangxian Yixin prescription,which are exactly the same as the total prescription,and 18 functional subsets compared with the total prescription,which occupies the most important position in Kangxian Yixin prescription.A total of 489 ways of action of subject drugs are exactly the same as the total prescription,which strengthens and complements the action mechanism of Junyao.6.The expression of differential proteins Ca4,Ethe1,Mff,myosin light chain 4(Myl4)and GTP binding protein(Sar1b)was verified by Western Blot.The results showed that the expression of Ca4,Ethe1,Mff and Sar1 b in model group was significantly lower than that in control group,and the differences of Ca4,Ethe1 and Mff had statistical meaning(P < 0.05,P < 0.01).After treatment with Kangxian Yixin Decoction,the protein expression of Ca4,Ethe1,Mff and Sar1 b increased,and the difference of Mff was statistically significant.Compared with control,the amount of Myl4 protein in model group increased significantly(P < 0.01),and Myl4 decreased significantly after intervention with Kangxian Yixin Decoction(P < 0.05).These differential proteins may be the targets of Kangxian Yixin Decoction in the treatment of DCM,which are mainly related to oxygen transport and reversible hydration of CO2,mitochondrial division and fusion,mitochondrial oxidative phosphorylation,endoplasmic reticulum protein processing and myocardial contraction.Conclusions3.1.The cardiac function of furazolidone-induced DCM rat model was significantly decreased,the morphological structure of myocardial tissue was damaged,the degree of myocardial fibrosis was significantly increased,and the ultrastructure of myocardial mitochondria was seriously damaged.Kangxian Yixin Decoction intervention can significantly improve cardiac function,alleviate pathological morphological and structural changes,improve myocardial fibrosis and reduce mitochondrial injury in DCM rats.4.2.Proteomic techniques were used to analyze and identify the differential proteins related to the development of DCM and the prognosis of Kangxian Yixin Formula.Bioinformatics analysis showed that the cellular localization of differential proteins was concentrated in mitochondria and cells.It is revealed that the intervention mechanism of Kangxian Yixin Decoction may be the regulation of energy metabolism,sulfur metabolism pathway,protein processing in endoplasmic reticulum,myocardial contractile function,amino acid biosynthesis and so on.5.3.Metabonomics research technology was used to analyze and identify the differential metabolites involved in the development of DCM and the intervention of Kangxian Yixin Formula.Bioinformatics analysis revealed that the intervention mechanism of Kangxian Yixin Decoction may be the regulation of TCA cycle,amino acid synthesis and metabolism,sulfur metabolism,purine metabolism,linoleic acid metabolism and so on.6.4.51 differential proteins were identified and screened from the myocardial tissue of DCM rats,and 140 metabolites were related to the intervention mechanism of Kangxian Yixin Decoction.Through the joint analysis of differential metabolites and proteins,it was revealed that the mechanism of KXYXYF involved in energy metabolism,purine metabolism,amino acid biosynthesis,sulfur metabolism,ABC transport of egg white,vitamin digestion and absorption,thermogenic pathway,myocardial contraction,endoplasmic reticulum protein processing and other pathways.5.Through the study of network pharmacology,it is found that Kangxian Yixin Decoction plays a role in the treatment of DCM by regulating blood circulation,nuclear receptor transcription pathway,muscle system related processes,active oxygen metabolism,regulating ion transport,regulating hormone levels,regulating apoptosis signal pathway and so on.In the action mechanism of Kangxian Yixin Decoction in the treatment of DCM,Junyao plays a major role,and the subject medicine enhances and complements the role of Junyao.7.6.The expression of Ca4,Ethe1,Mff,Myl4 and Sar1 b was verified by Western Blot.These differential proteins may be the targets of Kangxian Yixin Decoction,which are mainly related to oxygen transport and reversible hydration of CO2,mitochondrial division and fusion,mitochondrial oxidative phosphorylation,endoplasmic reticulum protein processing and myocardial contractile function.Through comprehensive analysis,the potential targets of Kangxian Yixin Decoction are mainly concentrated in mitochondria and cytoplasm.its regulation of mitochondrial function is related to mitochondrial fusion and division,mitochondrial biosynthesis,mitochondrial fatty acid oxidation,mitochondrial oxidative phosphorylation and the maintenance of ion homeostasis.Kangxian Yixin Decoction can treat DCM by regulating energy metabolism,mitochondrial function,sulfur metabolism,endoplasmic reticulum protein processing,myocardial contractile function and so on.