Study On The Treatment Of Atherosclerosis With Shanhuaxiaozhi Formula Based On Integrated Pharmacology

Background:Atherosclerosis(AS),a common vascular disease,is an important risk factor for the development of cardiovascular and cerebrovascular diseases.With the change of people’s lifestyles,the incidence of AS is increasing year by year and has become a serious threat to human health.SHXZF is based on the tea formula"Jiaweisanxianyin" in the "Integration of Medical Cases in Qing Palace",and has good clinical efficacy in reducing blood lipid level and improving the thickness of endothelial layer in AS patients.Our group has demonstrated that SHXZF can reduce total cholesterol and triglyceride levels in ApoE-/-mice induced by high-fat diet,improve lipid deposition and delay the progression of AS,but its mechanism of action is still unclear.In this study,the mechanism of action of SHXZF for the treatment of AS was explored based on an integrative pharmacological approach.Objective:To perform preliminary identification of the active chemical components of SHXZF using UPLC-ESI-QTOF-MSE technology based on an integrated pharmacological approach;to analyze the targets and molecular biological functions of the active chemical components of SHXZF that exert anti-AS effects using network pharmacology;to analyze the differentially expressed proteins of SHXZF acting on AS using proteomics technology;to integrate the results of network pharmacology and proteomics studies,to analyze the key core targets and key pathways of SHXZF for the treatment of AS,and to explore the potential mechanism of action of SHXZF for the treatment of AS.Methods:1.Identification of SHXZF components and network pharmacology study of anti-AS.The chemical composition database of the drugs contained in SHXZF was constructed based on the research literature and using TCMSP database and chemical specialty database;the constituent drugs of SHXZF were decocted proportionally and concentrated to aqueous decoction of 0.8 g/ml of raw drug,and the analysis was completed in an ultra-high liquid chromatography mass spectrometer;the results were compared with the constituent information in the chemical composition database,and the compounds were also identified based on the reference literature,Mass bank database provided fragment cleavage information to identify the compounds.The TCMSP database,Symmap database and TTD database were used to find the candidate targets of the above compounds and integrated and de-duplicated to obtain the gene targets regulated by SHXZF chemical components;the targets of AS in DisGeNET database and GeneCards database were searched,and the important related targets of AS were obtained by merging and taking the intersection;the jvenn online tool was used to compare the chemical components The key targets of SHXZF for AS were obtained by analyzing the candidate targets and the disease targets of AS using jvenn online tool;the chemical components corresponding to the above key targets were analyzed and the "drug-compound-target" network was constructed using Cytoscape 3.7.1 software;the key targets of SHXZF for AS were functionally enriched using DAVID database.The functional enrichment analysis of key targets of SHXZF for AS treatment was performed using DAVID database.2.Proteomics study of SHXZF for AS treatment.Twenty ApoE-/-mice were randomly divided into model group and Chinese medicine group,10 mice in each group were fed with high-fat diet.The mice in the model group were given saline gavage,and the herbal group was given SHXZF aqueous decoction by gavage,and the aortic arch of the mice was used for protein extraction after 8 weeks.Protein quantification was performed after passing the protein quantification test prompting proteolytic digestion and TMT labeling,and then the samples were analyzed by LC-MS/MS,and the obtained data were further analyzed by Proteome Discoverer software.The plausible proteins were screened according to the criteria of Score Sequest HT>0 and unique peptide≥1,and blank values were removed.Subsequently,the differential screening condition was set to Foldchange=1.5-fold and p-value<0.05,and the differentially expressed proteins were finally obtained.The screened differential proteins were analyzed by GO and KEGG Pathway using DAVID database.3.Combined network pharmacology and proteomics analysis.The SHXZF anti-AS targets predicted by network pharmacology and the differentially expressed proteins obtained by proteomics were intersected to obtain the co-expression targets of network pharmacology and proteomics,i.e.,the key core targets of SHXZF anti-AS;the compounds corresponding to the key core targets were screened,and the co-expression network of the compounds and the key core targets was constructed by using Cytoscape 3.7.1 software,and the co-expression network of SHXZF anti-AS was obtained by The network topology analysis was used to obtain the important compounds of SHXZF anti-AS;the expression level of related proteins was verified by Western blot method.Results:1.Identification of SHXZF components and network pharmacology study of anti-AS.A total of 14 chemical components of SHXZF were identified by mass spectrometry,and 833 candidate targets of 14 chemical components were obtained from the TCMSP database,the Symmap database,and the TTD database;1559 AS-related disease targets were obtained from the DisGeNET database and the Genecards database;363 candidate SHXZF targets for AS were obtained using the online tool jvenn.The network of "drug-compound-target" was constructed and the topology analysis was performed to determine that apigenin,kaempferol,naringenin,rutin,naringenin,1-Ocaffeoylquinic acid and hesperetin might be the key effective compounds of SHXZF for the treatment of AS based on the degree value.The molecular function of SHXZF against AS was mainly reflected in the binding activity with enzymes;the biological process mainly involved the regulation of the inflammatory response and smooth muscle cell proliferation;the cellular composition mainly involved the extracellular space,cytoplasm,outer plasma membrane and exosomes;the anti-AS effect of SHXZF could be through inflammatory pathways such as the TNF signaling pathway,TOLLlike receptor signaling pathway,HIF-1 signaling pathway,NF-KappaB signaling pathway,pathways affecting vascular endothelial cell function such as the cAMP signaling pathway,the VEGF signaling pathway and lipid metabolism-related pathways such as the PPAR signaling pathway.2.Proteomic study of SHXZF for AS treatment.The mouse aortic arch was selected for protein extraction,and three protein samples from each group were tested,and the protein concentration was verified to meet the experimental requirements.After the samples were detected by LC-MS/MS and library search,the qualitative and quantitative protein statistics were obtained,and 3991 proteins and 28388 peptides were finally identified under the screening conditions of protein FDR<0.01 and peptide FDR<0.01.The scoring criteria of Score Sequest HT>0 and unique peptide<1 and the elimination of blank values were used to obtain 3324 plausible proteins.Using Foldchange(FC)=1.5 and p-value<0.05 as screening conditions,167 differentially expressed proteins were obtained by comparing differentially expressed proteins between the model group and the TCM group,among which 129 proteins were up-regulated and 38 proteins were down-regulated.The results of GO functional enrichment analysis of differentially expressed proteins suggested that the related proteins were mainly involved in redox process,myocardial contraction process and the response process to zinc ions;they mainly contained mitochondria,mitochondrial inner membrane,peroxisomes and secretory granules;they also had molecular functions such as binding calcium ions,iron ions and heme;the results of KEGG pathway enrichment analysis showed that the related proteins were involved in lipid metabolism KEGG pathway enrichment analysis showed that related proteins are involved in lipid metabolism including steroid hormone biosynthesis,PPAR signaling pathway;carbohydrate metabolism related pathway including ascorbic acid and aldose metabolism pathway,fructose and mannose metabolism pathway;circulatory system including myocardial contraction pathway.3.Joint analysis of network pharmacology and proteomics.The net Venn diagram was drawn using the jvenn online tool,and 12 co-targets were obtained between 363 network pharmacology targets and 142 differentially expressed proteomic proteins,which were considered the key core targets of SHXZF against AS.Compounds corresponding to the 12 key core targets were extracted,the component-target network was constructed,and network topology analysis was performed,and the degree value was used as the basis of judgment,and combined with the results of the network pharmacology of the study,it suggested that rutin and hesperetin were the main compounds for SHXZF to exert its anti-AS effect;the KEGG pathway overlapped in the results of network pharmacology and proteomic analysis of student letters was the PPAR signaling pathway,which was considered SHXZF is a key pathway for the treatment of AS,and LPL and SCD expression is down-regulated in the PPAR signaling pathway among the 12 key core targets.Western Blot validation results showed that SHXZF can down-regulate LPL protein and SCD protein expression levels in aortic tissues of ApoE-/-mice.Conclusion1.Rutin and hesperetin,the important active compounds in SHXZF,may be the main active ingredients in the treatment of AS.2.SHXZF exerts its anti-AS effect through multiple targets and pathways,and its mechanism may be by inhibiting the expression of LPL and SCD genes in the PPAR signaling pathway,inhibiting the synthesis of fatty acids,and regulating the level of lipid metabolism in vivo,thus exerting its anti-AS therapeutic effect.