Study On The Mechanism Of Shenfu Qiangxin Pill In The Treatment Of Heart Failure Based On Network Pharmacology And Metabolomics

Heart failure(HF),a complex clinical syndrome,is associated with increased mortality and frequent hospitalizations.Although optimal medical managements have been effective in reducing all-cause mortality and readmission rate of HF,a considerable proportion of patients remains at high risk of cardiovascular events.Therefore,a broader range of therapeutic agents than is currently available is essential to reduce the residual risks of cardiovascular diseases.The "multi-component,multi-target" nature of traditional Chinese medicine(TCM)and good safety profile enable TCM as effective and safe agents for prophylaxis and treatment of complex diseases,such as HF.Shenfu Qiangxin Pill(SFQX)is a representative TCM in the treatment of HF,however the research on its mechanism is relatively single at present,so further study is needed to investigate the mechanism of SFQX in the treatment of HF.Network pharmacology is based on the multi-level network to holistically predict drug targets and pathways of action.It is a key technology for discovering drug-disease linkages.Metabolomics,the terminal of systems biology,can examine the changes in the overall endogenous metabolome of organisms under the effects of TCM.These two methods are similar to the concept of the " holistic view" of Chinese Medicine.Therefore,the mechanisms of the SFQX in the treatment of HF can be elucidated at a greater depth by combining the results from network pharmacology and metabolomics.However,network pharmacology only predicts the underlying mechanism and metabolomics is still immature,so the potential mechanism will be verified through molecular biology technology(experiments in vivo and in vitro)and intensive study will be conducted at the cellular level.Part I Pharmacodynamic study of SFQX on rats with HF after myocardial infarctionObjective:To evaluate the efficacy of SFQX on rats with HF after myocardial infarction.Methods:We ligated the left anterior descending coronary artery for 2 weeks to construct HF after myocardial infarction rat models.According to the left ventricular ejection fraction(LVEF)and random number table,rats were divided into model group,positive drug group(perindopril tert-butylamine),SFQX high-dose group,SFQX medium-dose and SFQX low-dose group,sham operation group.Rats were orally administered for 4 weeks.Cardiac function of rats in each group was assessed by echocardiography,the levels of atrial natriuretic peptide(ANP)and B-type natriuretic peptide(BNP)in serum were detected by ELISA,the heart histology and morphology was observed by hematoxylin-eosin(HE)staining and Masson stainingResults:After treatment for 4 weeks,LVEF and left ventricular fractional shortening(LVFS)of rats in SFQX high-dose group and positive drug group increased(P<0.05)compared with model group.Meanwhile,levels of ANP and BNP in serum were decreased(P<0.05).HE staining results showed that the structure of myocardial fibers in the model group was disordered,and the muscle fibers became thick and wavy.Compared with model group,the arrangement of myocardial fibers was clearer,the gaps of myocardial cell bundles and inflammatory cell infiltration were reduced in the positive drug group and the high-dose SFQX group.The Masson staining results showed the collagen fibers in the positive drug group and SFQX groups were significantly reduced(P<0.05)Conclusion:SFQX can improve cardiac function in rats with HF(increasing LVEF and LVFS),reduce the levels of HF biomarkers(ANP and BNP)and delay ventricular remodeling.Part II Integrating network pharmacology and metabolomics to predict the mechanism of SFQX on HFObjective:Combinating network pharmacology and metabolomics analysis to investigate the primary pathways and metabolites of SFQX on HF.Afterwards,integrating key targets and related pathways and performing targeted metabolomics quantification to explore its underlying mechanisms1 Network pharmacologyMethods:All candidate bioactive ingredients and target prediction of SFQX were obtained from BATMAN-TCM database.The targets related to HF were obtained from the DisGeNet,OMIM,and TTD databases.To explore the potential relationships among the overlapping targets,we used search tool for the retrieval of interacting genes(STRING)to evaluate protein-protein interaction(PPI)information and constructed a PPI network Afterwards,The nodes with higher degree of interaction were selected as hub genes.The Cytoscape 3.8.0 software was used to visualize the "Traditional Chinese medicine-Component-Target candidate-Disease" networks.Finally,the overlapping targets were classified in line with gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)pathways using the database for annotation,visualization and integrated discovery(DAVID)for annotation to explore the possible mechanism of SFQX in the treatment of HF.Results:A total of 215 active ingredients and 497 potential targets for SFQX in the treatment of HF have been identified.Furthermore,the pathways enriched by these targets were mainly related to MAPK signaling pathway,PI3K-Akt signaling pathway,Sphingolipid signaling pathway,Apoptosis,AMPK signaling pathway,etc.2 Non-targeted metabolomics study of SFQX on rats with HFMethods:The SFQX high-dose group with the best effect in the pharmacodynamics was selected as the SFQX group and the rats were divided into sham operation group,model group,and SFQX group.In this study,serum metabolomics profiling with liquid chromatography-mass spectrometry(LC-MS)and multivariate data method was used to discover the potential regulation mechanisms of SFQX on HF rats.Afterward,the crucial targets in the shared pathways of metabolomics and network pharmacology were predictedResults:The results showed that SFQX could exert excellent effects on HF probably through regulating sphingolipid metabolism,pyrimidine metabolism,amino acyl-tRN A biosynthesis,nitrogen metabolism,glutathione metabolism,cysteine and methionine metabolism,arginine and proline metabolism,and other metabolic pathways,which were reflected by the altered levels of proline,cytosine nucleoside,5-methylcytidine,pipecolic acid,cytosine and phytosphingosine,etc.Through the integration of network pharmacology and metabolomics,the main targets of SFQX in the treatment of HF were AKT1,MAPK1/3(ERK1/2)and TP53 which were closely related to autophagy and apoptosis3 Study on amino acid targeted metabolomics of SFQX on rats with HFMethods:Integrating the results of network pharmacology and metabolomics,we found that most of the overlapping metabolic pathways were related to amino acid metabolism.To further screen the alterations in amino acid metabolome regulated by SFQX in the treatment of HF,we performed targeted metabolomics analysis using LC-MS to investigate the amino acid metabolism in the serum of model rats,sham-operated rats and SFQX ratsResults:Serum concentrations of histidine,methionine,phenylalanine,tyrosine,citrulline,hypotaurine,ornithine,tryptophan,kynurenine were significantly higher in HF group than in sham operation group.A total of 4 potential biomarkers including phenylalanine,citrulline,tryptophan and kynurenine were screened out,showing a significant change after the treatment of SFQXConclusion:Network pharmacology combined with metabolomics exhibited a powerful mean to investigate the mechanism of SFQX.The present study reveals the possible mechanisms of SFQX for HF are the regulation of cardiomyocyte autophagy and apoptosis.Part III Molecular Biology Study of SFQX in the treatment of HFPreliminary prediction results indicated that SFQX may regulate the autophagy and apoptosis of cardiomyocytes in HF.The functional relationship between autophagy and apoptosis is complex in cardiomyocytes.It is unclear whether SFQX affects the crosstalk between autophagy and apoptosis.The possible forms of interaction and the pathway need to be further investigated.Therefore,classic molecules closely related to autophagy and apoptosis were selected to study mechanisms of SFQX for HF in vivo and in vitro experiments.Furthermore,experiments about whether SFQX affected the crosstalk of autophagy and apoptosis were constructed.Finally,we explore its putative pathway.1 Effects of SFQX on autophagy and apoptosis of cardiac myocytes in rats with HFObjective:To clarify that SFQX can treat HF by regulating autophagy and apoptosis.Methods:The rat model of HF after myocardial infarction was established by ligating the anterior descending branch of the left coronary artery.Rats were orally administered for 4 weeks.Cardiomyocyte apoptosis of rats in each group was evaluated by TUNEL and immunofluorescence.The protein expression of Bcl-2,Bax and Cleaved-caspase3,which were closely related to apoptosis,and LC3-II,Beclin 1 and SQSTM1/p62 which were closely related to autophagy,was detected by western blot(WB).Results:Compared with the model group,the green fluorescent spots marked by TUNEL staining in positive drug group and the SFQX groups were significantly reduced,and the apoptosis index was significantly decreased(P<0.01).Compared with the model group,the Bax fluorescence expression of the positive drug group and the SFQX groups was reduced,and the Bcl-2 fluorescence expression was increased.The WB detections showed that the expression of Cleaved-caspase3 in the positive drug group and the SFQX groups significantly decreased compared with the model group(P<0.01).Compared with the model group,the expression of Bax in the positive drug group and the high-dose SFQX group was significantly decreased(P<0.01),and Bax/Bcl-2 was decreased(P<0.05).Compared with the model group,the expression of Bcl-2 in the positive drug group and the SFQX with high and medium dose groups was increased(P<0.05).Compared with the model group,the expression of LC3-II in the positive drug group and the high-dose SFQX group was increased(P<0.05).The expression of Beclin 1 in the positive drug group and the SFQX groups was increased(P<0.05).There was no statistically significant difference in the expression of SQSTM1 between the groups(P>0.05).Conclusion:SFQX can inhibit the apoptosis of myocardial cells in rats with HF after myocardial infarction by reducing the expression of Bax and Cleaved-caspase3,and increasing the expression of Bcl-2.It can also increase the expression of LC3-II and Beclin 1 to promote autophagy of cardiomyocytes.2 Study of SFQX in regulating the crosstalk between autophagy and apoptosis in H9C2 induced by oxygen-glucose deprivationBackground:Previous studies have shown that SFQX are related to MAPK signaling pathways(JNK,ERK1/2,p38).MST1 is the upstream of the MAPK signaling pathway,and JNK is the switch of the MAPK signaling pathway.Both MST1 and JNK can be involved in autophagy and apoptosis,and the MST1/JNK signaling pathway is crucial in HF.Objective:To study whether SFQX can regulate the crosstalk between autophagy and apoptosis,and whether is related to MST1/JNK pathway.Methods:H9C2 was subjected to oxygen-glucose deprivation(OGD)to construct a cardiomyocyte injury model.CCK-8 method and lactate dehydrogenase method were used to screen the optimal intervention time of OGD and the optimal intervention concentration of SFQX.Cardiomyocyte apoptosis was detected by flow cytometry.The expression of Caspase-3,Beclin 1 were detected by WB.Rapamycin and 3-methyladenine(3-MA)were used to further investigate the crosstalk between autophagy and apoptosis in H9C2.In addition,expression levels of MST1,JNK and p-JNK were detected by WB to investigate whether the mechanism of SFQX regulating autophagy and apoptosis of H9C2 was related to MST1/JNK pathway.Results:The results showed SFQX could reduce the apoptosis rate of cardiomyocytes induced by OGD(P<0.05),and WB testing showed that SFQX could reduce the expression of apoptosis-related protein Caspase-3(P<0.05),up-regulate the expression of Beclin 1(P<0.05)to inhibit cardiomyocyte apoptosis and promote cardiomyocyte autophagy.3-MA attenuated the SFQX-promoted autophagy and antiapoptotic effects,whereas rapamycin had no additional effects compared with SFQX alone.Meanwhile,The results indicated that SFQX can inhibit the expression of MST1(P<0.05),the key protein of the interaction between autophagy and apoptosis,reduce JNK phosphorylation(P<0.01)Conclusion:These results suggest that autophagy is a protective mechanism in H9C2 subjected to OGD injury and that SFQX may inhibit OGD-induced apoptosis to promote autophagy via MST1/JNK pathway.