| Background Heart failure is a serious and terminal stage of many cardiovascular diseases,which is difficult to treatment,with poor prognosis and high mortality.Impaired myocardial energy metabolism is an important link throughout the development of heart failure,while excessive and persistent activation of the inflammatory response can promote cardiac enlargement and cardiac insufficiency,further aggravating the pathological process of heart failure.Traditional Chinese medicine have multi-component and multi-target properties and play a unique role in improving clinical symptoms of heart failure,but their development is limited by the fact that their potential therapeutic targets have not been fully investigated.Styrax(also known as Storax or Suhexiang)is an aromatic resin exuded from the trunk of Liquidambar orientalis Mill,a plant in the family Hamamelidae,which has the effect of “warming heart meridian”.The related prescriptions have been widely used in the treatment of coronary heart disease.However,its cardioprotective action mechanism is still unclear and lacks in-depth study under the guidance of a holistic view.Therefore,in this study,we constructed a rat model of heart failure through left anterior descending coronary artery(LAD)ligation,and studied the interventional effects,pharmacological substances and mechanism of Styrax in rats with heart failure,so as to provide a basis for the development and clinical application of Styrax.Objectives 1.To clarify the cardioprotective effect of Styrax based on pharmacodynamic experiments and screen the optimal dose;To clarify the metabolite profile of Styrax ameliorating metabolic disorders in heart failure rats based on metabonomics technology.2.Identification of the chemical components of the extracts of Styrax based on gas chromatography time-of-flight mass spectrometry(GC-TOF-MS)and ultra high performance liquid chromatography quadrupole exactive orbitrap mass spectrometry(UHPLC-QE-MS).3.To analyze the core targets and pathways of Styrax in the treatment of heart failure based on transcriptomics and network pharmacology;To screen the bioactive compounds of Styrax in the prevention and treatment of heart failure based on molecular docking technology.4.To investigate whether Styrax and its bioactive compounds exert cardioprotective effects by improving inflammatory factors and energy metabolism based on in vitro and in vivo experiments.Methods 1.Pharmacodynamic validation of Styrax against heart failure.Sixty rats were divided into the following six groups using the randomized number table method: shamoperated group(Sham),heart failure model group(Model),low-dose treatment group(ES-L,0.1 g/kg),medium-dose treatment group(ES-M,0.2 g/kg),high-dose treatment group(ES-H,0.4 g/kg),and trimetazidine treatment group(TMZ,6.25 mg/kg),and the corresponding dose was given to each group on the postoperative day consecutively by gavage once a day for 28 days.In this study,we used LAD ligation in rats to simulate a post-infarction heart failure model,echocardiography for evaluating cardiac function,light microscopy for observing myocardial histomorphological changes,transmission electron microscopy for observing myocardial mitochondrial morphology,TUNEL for detecting myocardial cell apoptosis,and biochemical indexes for detecting myocardial enzymes,energy metabolism and inflammatory factors to clarify the effect of Styrax on preventing and treating heart failure and to screen the optimal dose group.2.UHPLC-QE-MS metabolomics technique was applied to detect metabolites in plasma of Sham group,Model group and Styrax group(Styrax,0.4 g/kg).After data preprocessing,multivariate statistical analysis methods such as principal component analysis(PCA)and orthogonalpartial least square-discriminant analysis(OPLS-DA)were used.In addition,the variable importance prediction(VIP)values and the t-test results from the OPLS-DA model were combined to screen for potential biomarkers,then Metaboanalyst 5.0,a comprehensive metabolomics processing software,was applied to perform topological analysis to compare the signal transduction pathways and biochemical metabolic pathways involved in different metabolites among groups,and to find potential biomarkers related to the role of Styrax in the treatment of heart failure.3.Identification of the chemical components of the extracts of Styrax based on GC-TOFMS coupled with UHPLC-QE-MS.The preliminary qualitative analysis of the chemical constituents of Styrax was carried out by setting different ramp-up procedures,different solvent extracts and mobile phase ratios.Then,we confirmed the attribution of each major peak with the reference substance,secondary mass spectrometry information and databases,in order to provide a basis for the elucidation of the composition of Styrax.4.Integrated application of multi-omics techniques to identify the potential bioactive compounds of Styrax as well as the core targets for the treatment of heart failure.(i)Transcriptomics techniques were applied to obtain the expressed genes in Sham group,Model group and Styrax group,and then with P < 0.05,and | log2 Fold Change | > 1 is the standard for screening differentially expressed genes,finally combining network pharmacology to further explore analysis.(ii)The chemical composition of Styrax was collected by TCMSP database and mass spectrometry,and the potential medicinal substances of Styrax were screened based on the five principles of drug-like,i.e.,relative molecular mass ≤ 500,lipid-water partition coefficient ≤ 5,number of hydrogen bond donors ≤ 5,number of hydrogen bond acceptors ≤ 10 with rotational bond ≤ 10,and compound-target,target-disease,and compound-target-disease correlations were constructed.(iii)Integration analysis of transcriptomic differential genes with pharmacological targets of Styrax for the treatment of heart failure to screen out shared differential genes,and GO and KEGG enrichment analysis of these core crossover genes to obtain relevant biological functions and molecular pathways to explore the mechanism of action of Styrax.(iv)The core target proteins were selected as receptors and the active components of Styrax were targeted to screen the potential bioactive compounds of Styrax against heart failure based on molecular docking technology,which provided reference for the subsequent validation of efficacy and mechanism.5.Pharmacodynamic validation and mechanism investigation of Styrax and its bioactive compounds.(i)In vitro validation: oxygen-glucose deprivation-induced injury in H9c2 cardiomyocytes was used,with the control group(Control),the oxygen-glucose deprivation group(OGD),Cinnamyl cinnamate treatment group(CC,30 μM),Benzyl cinnamate treatment group(BC,30 μM)and the extract of Styrax treatment group(ES,10 μg/m L)to clarify the pharmacodynamic effects of Styrax and its bioactive compounds by CCK-8,Annexin V-FITC/PI double-stained flow cytometry,and JC-1 assay for mitochondrial membrane potential.(ii)In vivo validation: Thirty rats were divided into the following five groups using the random number table method: shamoperated group(Sham),heart failure model group(Model),Cinnamyl cinnamate treatment group(CC,120 mg/kg),Benzyl cinnamate treatment group(BC,120 mg/kg)and extract of Styrax treatment group(ES,0.4 g/kg).Echocardiography was performed to evaluate the cardiac function of rats,light microscopy was used to observe the histomorphological changes of myocardial tissue,transmission electron microscopy was performed to observe the morphology of the mitochondria,and biochemical assays of the myocardial enzymes,energy metabolism,and inflammatory factors was used to clarify the effects of Styrax and its bioactive compounds in treating heart failure.q RTPCR was also applied to validate the critical targets screened by omics(MMP9,PLG,ALB,PTGS2,Erb B4,NFKBIA),and the immunofluorescence technique was used to observe the fluorescence intensity of nuclear factor kappa-B(NF-κB),hypoxia-inducible factor-1alpha(HIF-1α),matrix metalloproteinases 9(MMP9),Erb-b2 receptor tyrosine kinase 4(Erb B4).Western blot technique to detect the protein of NF-κB,HIF-1α,MMP9,neuregulin 1(NRG-1),Erb B4,glucose transporter 4(GLUT4),hexokinase 2(HK2),pyruvate dehydrogenase kinase 4(PDK4)and citrate synthase(CS).Results 1.The results of pharmacodynamic study showed that all the treatment groups had different degrees of improvement on myocardial injury in rats with heart failure,especially the ES-H group had the best effect,which could improve cardiac function,myocardial histopathological damage,reduce the expression levels of NT-pro BNP and BNP,reduce the release of CK-MB,LDH and c Tn T,inhibit the levels of proinflammatory factors IL-1β,IL-6 and TNF-α,and increasing the level of IL-10;as well as maintaining myocardial energy supply,improving the functional structure of mitochondria,increasing the content of ATP in rat plasma and myocardial tissues,and reducing myocardial tissue apoptosis.2.The results of metabonomics studies showed that Styrax had an effect on improving the inflammatory microenvironment and energy metabolism,and that Styrax suppressed inflammation by reducing the levels of arachidonic acid-related metabolites(15-deoxy-12,14-PGJ2,leukotriene B5,6-trans-leukotriene B4)in rats.Also,it could increase the oxidative utilization of glucose by the myocardium,and improve myocardial energy metabolism by increasing the content of related metabolites citramalic acid and L-malic acid and decreasing succinic acid in the tricarboxylic acid(TCA)cycle.3.The results of mass spectrometry showed that the GC-TOF-MS method initially identified 25 volatile components in the extract of Styrax,accounting for 97.7% of the total measured components,with the main components being Cinnamyl cinnamate(29.46%),Cinnamyl alcohol(11.17%),Benzyl cinnamate(8.83%),Cinnamyl benzoate(6.69%),Caryophyllene(2.47%),Caryophyllene(2.47%),Cinnamaldehyde(2.02%),Styrene(0.77%),etc.;In addition,the UHPLC-QE-MS method was used to identify the other chemical components of Styrax.The results showed that 19 chemical components were identified in positive ion mode and 13 chemical components were identified in negative ion mode,mostly derivatives of cinnamic acid series.The above results serve as a reference for the screening of bioactive compounds for the prevention and treatment of heart failure in Styrax.4.The integration of transcriptomics and network pharmacology analysis yielded a total of 38 bioactive compounds candidates and 102 shared targets of Styrax.Based on protein-protein interaction(PPI)analysis,it was concluded that Styrax may exert therapeutic effects through the regulation of key targets such as MMP9,PLG,ALB,CRP,PTGS2,LCK,MAPK11,MPO,NFKBIA and Erb B4.The enrichment results of KEGG pathway showed that Styrax may exert cardioprotective effects through multiple pathways,including NF-κB pathway,TNF pathway,IL-17 pathway,Th17 pathway,lipid and atherosclerosis,and arachidonic acid pathway.In addition,based on the screening results of molecular docking,the bioactive compounds of Styrax(Cinnamyl cinnamate and Benzyl cinnamate)were found to have the highest docking activity with 10 key targets,suggesting that these two bioactive compounds may play an important role in the prevention and treatment of heart failure.5.The cardioprotective effects of Styrax through anti-inflammation and improvement of energy metabolism were confirmed.In a model of OGD-induced cardiomyocyte injury,both Styrax and its bioactive compounds were found to enhance H9c2 cardiomyocyte activity,reduce apoptosis and stabilize mitochondrial membrane potential,but the pharmacological effects of both bioactive compounds were not as strong as those of the Styrax extract group.In the LAD-induced heart failure model in rats,Styrax and its bioactive compounds(Cinnamyl cinnamate and Benzyl cinnamate),were found to improve echocardiography and myocardial histopathological damage,reduce the expression of heart failure markers NT-pro BNP and BNP.The BC and ES groups reduced the release of inflammatory factors IL-1β,IL-6,TNF-α and PGE2 in rats with heart failure,while the CC and ES groups increased ATP production,NAD+/NADH ratio and CS activity,thus improving energy remodeling and exerting myocardial protective effects.Then,the q RT-PCR results suggested that Styrax and itsbioactive compounds could regulate the m RNA levels of the key targets such as MMP9,PLG,ALB,PTGS2,NFKBIA,and Erb B4.Immunofluorescence and western blotting experiments showed that Styrax could inhibit the expression levels of NF-κB,HIF-1α and MMP9,activate NRG-1 and Erb B4,and upregulate the expression of GLUT4,HK2 and CS proteins in myocardial tissues,which represents that Styrax may exert cardioprotective effects by inhibiting the expression of NF-κB/HIF-1α/MMP9 and activating NRG-1/Erb B4 pathway,and this result further elaborates that the molecular mechanism of Styrax in the treatment of heart failure may be achieved through the inflammatory microenvironment and energy metabolic remodeling.Conclusions In this study,we investigated the pharmacological effects and molecular mechanisms of Styrax in rats with heart failure by combined omics techniques,molecular biology techniques and the animal cell model,and dissected the effects of Styrax on improving the inflammatory microenvironment and energy metabolic remodeling in heart failure at the holistic-cellular-molecular level.In addition,we also clarified the potential pathological targets,bioactive compounds and key pathways of the effect of Styrax against heart failure,and revealed the potential molecular docking pattern of the bioactive compounds(Cinnamyl cinnamate and Benzyl cinnamate)and the core targets in Styrax,suggesting that the cardioprotective effect of Styrax is exerted through “warming heart meridian”.Finally,the NF-κB/HIF-1α/MMP9 and NRG-1/Erb B4 signaling pathways were explored to verify the anti-inflammatory and energy metabolism-improving effects of Styrax and its bioactive compounds,which provided a new research idea to clarify the pharmacodynamic substances and their potential targets of action in traditional Chinese medicine,and also provides an exploration example to interpret the scientific connotation of clinical efficacy of traditional Chinese medicine by using modern technology. |
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