Study On Mechanism Of Fuzi Water-soluble Alkaloids And Renshen Total Ginsenosides In The Treatment Of Acute Heart Failure Based On Metabolomics And Network Pharmacology

Objective:The aim of this research was to study the therapeutic effect of Fuzi water-soluble alkaloids（FWA）and Renshen total ginsenosides（RTG）,the main active components of Fuzi-Renshen herb pairs,on acute heart failure,and to elucidate the mechanism of their compatibility through metabolomics and network pharmacology.Methods:1.The acute heart failure rat model was established by injecting propafenone hydrochloride injection（16.5 mg/kg）into the femoral vein.All rats were randomly divided into 5 groups:blank control group（BCG）,model group（MG）,Fuzi water-soluble alkaloids group（FZG）,Renshen total ginsenosides group（RSG）and compatibility group（FZRSG）.Among them,BCG rats were given distilled water,and MG,FZG,RSG,and FZRSG rats were respectively given distilled water,FWA（0.48g/kg）,RTG（0.6 g/kg）,and FWA（0.48 g/kg）-RTG（0.6 g/kg）mixture after modeling.Hemodynamic indexes(±LV dp/dt_max,HR),electrocardiogram index（QRS wave time）and heart failure related serum biochemical factors（TNF-α,Ang-Ⅱ,Nt-pro BNP,ANP）of the rats were detected using physiological recorder and ELISA kits after administrating FWA,RTG alone or compatibility for 60 min.2.The LC-MS technology based on UPLC-LTQ Orbitrap XL MS/MS was used to study the metabolomics of the serum of rats in each group after 1h administration.The changes of the whole level of endogenous small molecule metabolites in the serum were detected.The differential metabolites of drug efficacy in rats with acute heart failure were searched by multivariate statistical analysis.After that,qualitative and quantitative analysis of the differential metabolites was carried out.The KEGG pathway analysis was used to find metabolic pathways related to the differential metabolites,and the possible mechanism of FWA-RTG combination in the treatment of acute heart failure was revealed from the metabolic level of the body.3.The chemical components in FWA and RTG were collected based on literature retrieval and online databases,and the potential active components were screened by ADME properties.Then the targets of active components and acute heart failure were predicted and screened by online databases.The potential therapeutic targets of FWA-RTG for acute heart failure were selected by integrating drug targets and disease targets.The network of"active components-potential targets"was constructed,and the network topology analysis,PPI,GO enrichment analysis and KEGG pathway analysis were conducted to explore the main components,key targets and signal pathways of FWA-RTG compatibility therapy for acute heart failure.Using the molecular docking technology based on Autodock software,the important active components and key target proteins involved in the signaling pathway obtained from the network pharmacology research were respectively docked.The interactions between the components and the targets were studied by computer simulation.The specific mechanism of FWA-RTG compatibility in the treatment of acute heart failure was further verified.4.Integrating the results of the above studies,we found that the combination of FWA-RTG in the treatment of acute heart failure may closely related to NOS3,β2-AR,Ca MKⅡδand other targets in calcium signaling pathway.Therefore,the influences of FWA-RTG on NOS3 and NO in serum and NO in myocardial tissue before and after administration were tested with the kits.The m RNA and protein expressions ofβ2-AR,SERCA2a,NOS3,Ca MKⅡδ,PI3k and Akt were detected by molecular biological methods,including RT-q PCR and western blotting,so as to study the pathway mechanism of the FWA-RTG compatibility on acute heart failure from the molecular level.Results:1.When the+LV dp/dt_max of rats decreased to less than 50%of the normal value,and maintained for more than 5 min,and there was no upward trend,it indicated that the acute heart failure model was successful.The hemodynamic changes within 60min after administration were monitored.After modeling,+LV dp/dt_max and HR were significantly decreased,and-LV dp/dt_max was significantly increased.After administration,the hemodynamic indexes of rats were restored to a certain extent,that was to say,+LV dp/dt_max,HR increased,and-LV dp/dt_max decreased.The recovery speed and degree of FZRSG were faster and higher than that of single administration groups.The+LV dp/dt_max of FZRSG was significantly higher than that of FZG at 10min,and FZG,RSG at 30 min after administration,and was significantly higher than that of MG at 60 min after administration.The-LV dp/dt_max of FZRSG at 5 and 60min after administration were significantly lower than that in MG rats.HR of RSG rats was significantly different from that of MG at 5 min after administration,HR of FZRSG rats was significantly different from that of MG at 5,20,30 and 60 min after administration.The increase of HR of FZRSG was higher than that of single administration groups,and there was a significant difference between FZG and FZRSG at 30 min after administration.After modeling,the QRS wave became wide and deformed,the amplitude of Q wave was deepened,and the QRS duration was significantly prolonged.After 60 min of administration,the QRS duration of the electrocardiogram of rats in each group had different degrees of recovery,and the QRS duration of FZRSG was significantly shorter than that of MG.The levels of TNF-α,ANP,Ang-Ⅱand NT-pro BNP in MG rats were significantly higher than those in BCG rats.After administration,except for ANP of FZG,other indexes in each group decreased.The contents of TNF-αand NT-pro BNP in FZRSG were significantly less than those in MG,and the contents of NT-pro BNP in FZG and RSG were significantly less than those in MG.Compared with the single administration groups,the NT-pro BNP content of FZRSG was significantly less,and the TNF-αcontent of FZRSG was significantly less than that of RSG.2.Metabonomics study showed that there were six differential metabolites associated with FWA-RTG in the treatment of acute heart failure in rats:L-pipecolic acid,L-arginine,uric acid,N-benzoylglycine,sphingosine-1-phosphate and phosphatidylinositol lyso 16:0.The serum levels of L-pipecolic acid,L-arginine,uric acid and N-benzoylglycine in MG rats were significantly lower than those in BCG rats,while the levels of sphingosine-1-phosphate and phosphatidylinositol hemolysin16:0 were significantly higher than those in BCG rats.After administration,the levels of metabolites recovered to different degrees.The contents of L-pipecolic acid,L-arginine,uric acid,N-benzoylglycine in FZRSG were increased,and the contents of sphingosine-1-phosphate and phosphatidylinositol hemolysin 16:0 of FZRSG were significantly less than those of MG.The L-arginine level of FZRSG was significantly higher than that of FZG and RSG;the N-benzoylglycine level of FZRSG was significantly higher than that of RSG;the phosphatidylinositol lyso 16:0 of FZRSG was significantly lower than that of RSG.The metabolic pathways involved in these differential metabolites included lysine degradation,aminoacyl-t RNA biosynthesis,arginine and proline metabolism,purine metabolism,D-arginine and D-ornidine metabolism,sphingolipid metabolism,phenylalanine metabolism.3.35 potential active components in FWA-RTG and 99 key target proteins in the combination of FWA-RTG for the treatment of acute heart failure were obtained by network pharmacology.The material basis of the FWA-RTG combination in the treatment of heart failure might be higenamine,ginsenoside-Rh3,songorine,beiwutinine,3-deoxyaconine.IL-6,VEGFA,TNF,PTGS2 and NOS3 may be the key targets of FWA-RTG in the treatment of acute heart failure.Calcium signaling pathway,c GMP-PKG signaling pathway,c AMP signaling pathway,PI3k-Akt signaling pathway were the signal pathways associated with the potential targets.Higenamine,ginsenoside-Rh3,songorine,beiwutinine,3-deoxyaconine were selected for molecular docking with the proteins of calcium signaling pathway.The results showed that there was a certain trend of molecular binding.Songorine,higenamine and ginsenoside-Rh3 had a strong binding ability with key target proteins in calcium signaling pathway,such as AA2BR,TNNC1,AA2AR.Among all the target proteins,ADRB1（β1-AR）and ADRB2（β2-AR）were well combined with the components,indicatingβ-adrenergic receptor might be an important target of FWA-RTG compatibility.4.The content of NO in myocardial tissue of MG was significantly less than that of BCG,while the level of NO in myocardial tissue of FZRSG was significantly higher than that of MG.The serum NO content of MG was significantly more than that of BCG,and the serum NO contents of FZG,RSG and FZRSG were significantly less than MG.The serum NOS3 content of MG was significantly higher than that of BCG.After administration,serum NOS3 content in rats of FZG,RSG and FZRSG decreased,and the level of NOS3 in FZRSG was significantly less than FZG and MG.Theβ2-AR,SERCA2a,NOS3,Ca MKⅡδm RNA expression levels of myocardial tissue of MG rats significantly decreased compared with BCG.The above m RNA levels in the administration groups were increased to different degrees compared with that of MG.Ca MKⅡδm RNA expression of FZG rats significantly raised.Theβ2-AR,SERCA2a,NOS3 and Ca MKⅡδm RNA expression of FZRSG rats also increased significantly,compared with MG.The m RNA levels ofβ2-AR,NOS3 and Ca MKⅡδin FZRSG were significantly higher than those in FZG and RSG,and the m RNA expression of SERCA2a in FZRSG was also significantly higher than that in FZG.Compared with BCG rats,the proteins expression ofβ2-AR,NOS3,p-NOS3,p-Akt,Ca MKⅡδand SERCA2a in MG rats were significantly decreased,and the ratio of p-NOS3/NOS3 was also significantly decreased.After administration,the protein expressions ofβ2-AR,Ca MKⅡδ,NOS3 and p-NOS3 in FZG,RSG and FZRSG rats were significantly increased,the p-NOS3/NOS3 ratio of RSG and FZRSG were significantly increased,and the p-Akt expression of FZRSG was significantly increased.The results showed that the p-NOS3/NOS3 ratio and SERCA2a expression level of FZRSG were significantly higher than those of FZG and RSG.In addition,the Ca MKⅡδlevel of FZRSG was also significantly higher than that of FZG.Conclusion:1.FWA-RTG compatibility had a good therapeutic effect on acute heart failure induced by propafenone hydrochloride in rats.It improved the hemodynamics and electrocardiogram abnormalities of rats with acute heart failure,reduced the levels of cytokines related to heart failure.The effect of compatibility was better than that of single administration.2.The compatibility of FWA-RTG might treat acute heart failure by regulating the disturbed metabolic pathways including arginine and proline metabolism,and the signaling pathways including calcium signaling pathway.Furthermore,the activation ofβ2-AR,Akt/NOS3/NO signal pathway,Ca MKⅡδ/SERCA2a signal pathway,and thereby promoting myocardial tissue NO generation,regulating myocardial intracellular calcium ions might be the important mechanisms of FWA-RTG compatibility to treat acute heart failure.