| ObjectiveAtherosclerosis is the main cause of acute cardio-cerebrovascular events,and "stagnation of heat" is an important etiology and pathogenesis of atherosclerosis.Previous studies have found that Yang Xin Shu Mai Granule(YXSMG)has certain advantages in the treatment of stable coronary heart disease.whether it can inhibit the progression of atherosclerotic plaques and stabilize vulnerable plaques remains to be further explored.The purpose of this study is to explore the mechanism of YXSMG on atherosclerosis through mass spectrometry analysis,network pharmacology,in vivo and in vitro experimental studies,and to explore the relationship between YXSMG and TLR9/MyD88/NF-κB pathway and macrophage polarization,in order to reveal the mechanism of YXSMG in stabilizing atherosclerotic vulnerable plaque from material basis,biological network,whole animal and cellular molecular level.To explain the modern scientific connotation of Professor Fang Zhuyuan’s academic thoughts on atherosclerosis "pulse arthralgia" and "blood stasis and heat knot".Methods1.HPLC-MS/MSThe active components of YXSMG were determined by high performance liquid chromatography-mass spectrometry/mass spectrometry(HPLC-MS/MS).The data collected by high resolution liquid quality were sorted out through the preliminary data,then searched and compared in the database,and then screened by mzCloud Best Match≥ 70 according to the matching degree of mzCloud database.Finally,the information of effective components and related values were obtained to reveal the material basis of the therapeutic effect of YXSMG.2.Network pharmacology(1)Collection and prediction of effective active components of YXSMG:the active components of YXSMG were searched by SwissTargetPrediction and PubChem database platform respectively,and the potential drug component target information was obtained.(2)Collection and screening of atherosclerosis-related genes:disease-related targets were searched through TTD,DisGeNET,DrugBank,GeneCards and OMIM databases with the key words of "atherosclerosis".According to the screening conditions of different databases,the screening results were summarized,and all the relevant gene target information of atherosclerosis was obtained.(3)Target prediction,protein interaction network construction and core target screening:firstly,the common target proteins of disease and drug components were obtained and uploaded to STRING platform to establish drug target protein-disease target protein interaction network(PPI).Then the core targets were screened by Betweenness Centralities,Closeness Centralities and Degree Centralities.(4)GO and KEGG bioenrichment analysis:the selected PPI network targets were imported into the OmicShare cloud platform database,and the results of GO and KEGG enrichment analysis were obtained by database visualization analysis.(5)Construction of "disease-component-target-pathway" network map:after 20 KEGG pathways involved in atherosclerosis were obtained,the active components and core targets of YXSMG were arranged in excel file format together with the active components and core targets of YXSMG,which were uploaded to Cytoscape3.7.0 software to get the multidimensional network diagram of "disease-component-target-pathway".(6)Docking analysis of core components and core target molecules:select appropriate ligand molecular structure,save and name it lig.pdbqt,receptor protein molecule save and name rep.pdbqt,set 10 docking output models,input docking function,docking mode is rigid docking,get docking results log.txt and lig out.pdbqt files,open lig out.pdbqt and rep.pdbqt files simultaneously with PyMOL,and export 10 docking results.Choose the best binding conformation of affinity.3.In vivo experiment(1)Effect of YXSMG on atherosclerotic plaque in ApoE-/-mice:ApoE-/-mice were randomly divided into Model group(Model),YXSMG-L group(YXSMG-L,5.2g/kg/d)and YXSMG-H group(YXSMG-H,11.7g/kg/d).C57BL/6J was used as blank control group(C57).The model was established for 8 weeks and intervened for 4 weeks.Body weight,food intake and blood lipids were measured.The pathological changes of aortic sinus plaques in each group were observed by oil red staining,Hype staining and Sirius red staining.(2)Effect of YXSMG on inflammatory expression of atherosclerotic plaque in ApoE-/mice:ELISA detected the expression of serum inflammatory factors(IL-6,IL-1β,TNF-α and MCP-1),qPCR detected the expression level of IL-6,IL-1β and TNF-α mRNA in aorta,IF and WB detected the expression level of IL-6 and IL-1β inflammation related protein in aorta of each group.(3)Effect of YXSMG on TLR9/MyD88/NF-κB pathway in atherosclerotic plaque of ApoE-/-mice:the expression of TLR9 mRNA in aorta of each group was detected by qPCR,and the expression of TLR9,MyD88,p-IκB and p-p65 protein in aorta of mice in each group was detected by WB and IF.(4)Effect of YXSMG on M1/M2 polarization of macrophages in atherosclerotic plaques of ApoE-/-mice:the phenotypic protein expression levels of CD86 and CD 163 in aortic sinus macrophages of mice in each group were detected by IF.4.In vitro experiment(1)The effect of YXSMG on the activity of RAW264.7 macrophages and the activation effect of ODN1826 on RAW264.7 macrophages TLR9:the concentration and time of ODN1826 model were screened by CCK-8 and WB,the protective effect of YXSMG on inflammatory injury of RAW264.7 macrophages activated by ODN1826 was observed,and two effective concentrations of YXSMG(YXSMG-L,YXSMG-H)were selected.WB and qPCR were used to detect the expression of TLR9 in RAW264.7 macrophages activated by OND1826.(2)Effect of YXSMG on TLR9/MyD88/NF-κB pathway in RAW264.7 macrophages activated by ODN1826:the expression of TLR9/MyD88/NF-κB pathway protein in RAW264.7 macrophages activated by OND1826 was detected by IF and WB.(3)Effect of YXSMG on M1/M2 polarization of RAW264.7 macrophages activated by ODN1826:ELISA,qPCR and IF were used to detect the expression level of M1/M2-related phenotypic molecules in RAW264.7 macrophages activated by OND1826.(4)Inhibition of TLR9 on TLR9/MyD88/NF-κB pathway and M1/M2 polarization of RAW264.7 macrophages and intervention of YXSMG:concentration and time of ODN2088 screening by CCK-8 and WB,WB and IF detection of ODN2088 intervention and YXSMG on the expression of proteins related to TLR9/MyD88/NF-κB pathway in ODN1826-activated RAW264.7 macrophages.IF and flow cytometry were used to detect the expression of M1/M2related phenotypic molecules in ODN 1826-activated RAW264.7 macrophages treated with ODN2088 and YXSMG.Results1.HPLC-MS/MSThe active components of YXSMG were determined by HPLC-MS/MS,and five active components of catechin,formononetin,tanshinone ⅡA,cryptotanshinone and glycitein were obtained,which provided a certain basis for the standard of effective components of YXSMG and provided reference for drug research and follow-up experiments.2.Network pharmacology22 key targets were screened by network pharmacology,including EGFR,IL-6,JUN,RELA,TNF,TLR9 and so on.The results of GO enrichment analysis showed that the biological process,cellular composition and molecular function of YXSMG in intervention of atherosclerosis were mainly related to metabolism,lipid,oxidative stress and intracellular signal transduction,suggesting that YXSMG may regulate lipid metabolism by mediating intracellular signal transduction and thus interfere with the biological,cellular and molecular mechanisms related to atherosclerosis.From the results of KEGG pathway enrichment analysis,it can be seen that the intervention of YXSMG on atherosclerosis mainly involves fluid shear force,inflammation,toll-like receptor pathway,cell polarization and so on,suggesting that YXSMG may mediate toll-like receptor to regulate macrophage polarization to inhibit inflammation and improve the formation and development of atherosclerotic plaque.Through the verification of computer virtual simulation docking,it is found that the core compound has good binding ability to the core target protein,which can preliminarily reveal the "multicomponent-multitargetmultipathway" interaction network between YXSMG and atherosclerosis.3.In vivo experiment(1)YXSMG can reduce the body weight of ApoE-/-mice fed with high fat,reduce the levels of serum TC,TG and LDL-C blood lipids,increase the level of serum HDL-C,and improve the size of atherosclerotic plaque and inflammatory infiltration.(2)YXSMG can reduce the levels of serum IL-1β,IL-6,MCP-1 and TNF-α in ApoE-/-mice fed with high fat diet,decrease the levels of IL-1β,TNF-α and IL-6 mRNA in aorta,and decrease the expression of IL-1β and IL-6 protein in aorta.(3)YXSMG can reduce the protein expression of TLR9,MyD88,p-IκB and p-p65 in aorta.The inhibitory effect of YXSMG on TLR9/MyD88/NF-κB signal pathway was verified in vivo.(4)YXSMG can reduce the expression level of CD86 in aortic sinus and increase the expression level of CD 163.4.In vitro experiment(1)YXSMG can reduce the expression of TLR9 mRNA and the expression of IL-6 and IL1β in RAW264.7 macrophages activated by ODN1826.(2)YXSMG could inhibit the expression of TLR9,MyD88 and p-IκB in RAW264.7 macrophages activated by ODN1826,and decrease the expression level of p65 in nucleus after intervention with YXSMG,which further confirmed the inhibitory effect of YXSMG on TLR9/MyD88/NF-κB signal pathway.(3)YXSMG can reduce the expression of TNF-α,IL-6 and IL-1β mRNA in RAW264.7 macrophages activated by ODN1826,and increase the expression of Arg1 and IL-10 mRNA.It is further confirmed that YXSMG can inhibit the expression of inflammatory molecules in M1 macrophages and promote the expression of related molecules in M2 macrophages.(4)ODN2088 inhibited the expression of TLR9/MyD88/NF-κB pathway-related proteins in ODN1826-activated RAW264.7 macrophages,and ODN2088 and YXSMG had a synergistic effect.(5)YXSMG and ODN2088 could decrease the expression of CD86 and increase the expression of CD 163 in RAW264.7 macrophages activated by ODN1826.ConclusionYXSMG can reduce the level of blood lipid and improve the size of atherosclerotic plaque and inflammatory infiltration in ApoE-/-mice fed with high fat.It is concluded that YXSMG can improve the mechanism of atherosclerotic plaque by inhibiting TLR9/MyD88/NF-κB pathway reprogramming macrophage M1/M2 polarization and reducing arterial inflammation. |
PDF Full Text Request