Integrating systematic biological and proteomics strategies to explore the pharmacological mechanism of danshen yin modified on atherosclerosis

Abstract This research utilized the systematic biological and proteomics strategies to explore the regulatory mechanism of Danshen Yin Modified (DSYM) on atherosclerosis (AS) biological network. The traditional Chinese medicine database and HPLC was used to find the active compounds of DSYM, Pharmmapper database was used to predict potential targets, and OMIM database and GeneCards database were used to collect AS targets. String database was utilized to obtain the other protein of proteomics proteins and the protein‐protein interaction (PPI) data of DSYM targets, AS genes, proteomics proteins and other proteins. The Cytoscape 3.7.1 software was utilized to construct and analyse the network. The DAVID database is used to discover the biological processes and signalling pathways that these proteins aggregate. Finally, animal experiments and proteomics analysis were used to further verify the prediction results. The results showed that 140 active compounds, 405 DSYM targets and 590 AS genes were obtained, and 51 differentially expressed proteins were identified in the DSYM‐treated ApoE‐/‐ mouse AS model. A total of 4 major networks and a number of their derivative networks were constructed and analysed. The prediction results showed that DSYM can regulate AS‐related biological processes and signalling pathways. Animal experiments have also shown that DSYM has a therapeutic effect on ApoE‐/‐mouse AS model (P < .05). Therefore, this study proposed a new method based on systems biology, proteomics, and experimental pharmacology, and analysed the pharmacological mechanism of DSYM. DSYM may achieve therapeutic effects by regulating AS‐related signalling pathways and biological processes found in this research.

proteomics proteins and other proteins. The Cytoscape 3.7.1 software was utilized to construct and analyse the network. The DAVID database is used to discover the biological processes and signalling pathways that these proteins aggregate. Finally, animal experiments and proteomics analysis were used to further verify the prediction results. The results showed that 140 active compounds, 405 DSYM targets and 590 AS genes were obtained, and 51 differentially expressed proteins were identified in the DSYM-treated ApoE-/-mouse AS model. A total of 4 major networks and a number of their derivative networks were constructed and analysed. The prediction results showed that DSYM can regulate AS-related biological processes and signalling pathways. Animal experiments have also shown that DSYM has a therapeutic effect on ApoE-/-mouse AS model (P < .05). Therefore, this study proposed a new method based on systems biology, proteomics, and experimental pharmacology, and analysed the pharmacological mechanism of DSYM. DSYM may achieve therapeutic effects by regulating AS-related signalling pathways and biological processes found in this research.

| INTRODUC TI ON
Atherosclerosis (AS) is the most common disease in arteriosclerosis, mainly involving the elastic arteries and the more muscular fibres of the elastic fibres. The typical atherosclerotic lesion contains a large amount of 'ather-like' components of lipids and necrotic cells. 1 AS is a major disease that seriously endangers human health. The main clinical events of death are coronary heart disease and stroke.
Cardiovascular and cerebrovascular diseases are the leading causes of death worldwide. 1,2 In 2008, more than 17 million people worldwide died of cardiovascular and cerebrovascular diseases, of which 7.3 million died of coronary heart disease and 6.2 million died of stroke. It is estimated that by 2030, the number of deaths from cardiovascular and cerebrovascular diseases worldwide will reach 23.3 million. [1][2][3] Currently, it is believed that atherosclerotic disease is the result of a variety of complex factors interacting. 4 The current study found that it is mainly related to lipid metabolism disorders, endothelial damage, inflammatory response, wall shear stress and intestinal microflora imbalance. 5,6 The therapeutic drugs for AS include hypolipidemic drugs, antiplatelet drugs, dilated vascular drugs and treatment for coronary heart disease caused by ischaemia. [7][8][9] However, as these drugs require patients to take their medications for life, their side effects result in low patient compliance and reduced quality of life. 10,11 In addition, the treatment of AS-related cardiovascular and cerebrovascular diseases has entered a stage of diversified comprehensive treatment. Complementary and alternative medicine (CAM) has gradually entered mainstream medicine and has become a popular choice for patients. 12  Among them, Danshen Yin (DSY) is from the Shi Ge Fang Kuo, which has the effect of promoting blood circulation and relieving pain in Chinese medicine theory. As a classic prescription, it has been treating coronary heart disease and angina as its establishment. 13 Our previous clinical studies 13,14 and other reports 15 showed that DSY Modified (DSYM) can reduce the total myocardial ischaemic load in patients with unstable angina (low-risk group, intermediate-risk group) and has anti-ischaemic effects. Its specific mechanism may be related to lowering serum myocardial enzyme level, reducing myocardial infarct size, improving myocardial cell ultrastructure and inhibiting cardiomyocyte apoptosis. 16 Our previous work also showed that the mechanism of DSYM intervention in coronary heart disease may be related to autophagy and oxidative stress. 17,18 More importantly, previous researches have only studied single signal pathways or a few targets; and it is not easy to reveal the synergistic effect of 'multi-component-multi-target' herbs and the regulation effects of herbs on biological network of the disease from a holistic and comprehensive perspective. However, the mechanism of DSYM intervention in coronary heart disease has not been elaborated, especially its role in AS, the basic lesions of coronary heart disease. Our previous research successfully used systematic biological methods (such as network pharmacology and systematic pharmacology) to explore the mechanism of herbal formulae for treating diseases. [19][20][21][22][23] Hence, this study will explore the mechanism of DSYM regulating AS's biological network by an integrated systematic biological and proteomics strategies, and provide new ideas for drug development.
The research processes were shown in Figure S1.  29 The compounds with OB ≥ 30%, Caco-2> −0.4 and DL ≥ 0.18 were considered to be orally absorbable and pharmacologically active (namely, potential compounds). [19][20][21][22][23][24][25][26][27]29 Due to the limitation of predicting potential components based on pharmacokinetic parameters only, 30 in order to avoid omission of components, we searched a large number of literatures to supplement orally absorbable compounds with bioactive. Finally, after searching, a total of 34 components were added from the reference. 31-42

| DSYM's potential targets prediction and as genes collection
The molecular structures of DSYM's potential components were collected from the SciFinder (http://scifi nder.cas.org) and PubChem (https://pubch em.ncbi.nlm.nih.gov/), and were drawn by ChemBioDraw and saved as 'mol2' file format. Then, they were input into PharmMapper (http://lilab -ecust.cn/pharm mapper) to predict the potential targets of DSYM. 43 The UniProtKB (http:// www.unipr ot.org/) was utilized to collect the official symbols of the

K E Y W O R D S
ApoE-/-mouse, atherosclerosis, Danshen Yin Modified, proteomics, reverse transcription-PCR, systematic biology potential target's proteins with the species limited to 'Homo sapiens'. (see Table S1). Meanwhile, OMIM database (http://omim.org/) and Genecards (http://www.genec ards.org) were utilized to collect the AS-related disease genes and targets. 44,45 The AS-related genes and their relevance scores are shown in Table S2.

| Network Construction and Analysis Methods
The protein-protein interaction (PPI) data were obtained from the String database (http://strin g-db.org/). 46 The networks were constructed by Cytoscape 3.7.1 software (https://cytos cape. org/), a software to graphically display, analyse and edit the network. 47 Degree refers to the number of connections to the node.
Betweenness refers to the number of edges passing through the nodes. Degree and betweenness can determine the importance of the topology of the nodes in the network. The larger the value, the more important it is. 47 The networks were further analysed by the plug-in of Cytoscape, MCODE to found the clusters. 48 The definition and the methodology of acquisition of clusters were described in our previous work. [19][20][21][22][23]

| Gene ontology (GO) and pathway enrichment analysis
The genes and targets in clusters were input into DAVID (https:// david -d.ncifc rf.gov, ver. 6.8) to perform GO enrichment analysis. All of the genes and targets in the networks were also input into DAVID for Kyoto Encyclopedia of Genes and Genomes (KEGG) signalling pathway enrichment analysis. 48 P-value is Modified Fisher Exact P-Value, EASE score. 48 The smaller, the more enriched. after 10-20 minutes after boiling start). The decoctions were combined, filtered and concentrated to 1 g of original medicinal material/ mL. Finally, they were stored at 4°C.

| Experimental animal
Simvastatin + aspirin suspension: The tablets are dissolved in distilled water and concentrated to the solution containing simvastatin 0.2 mg/ mL and aspirin 1 mg/ mL.

| Animal models, grouping and drug administration
Twelve (12) C57BL/6L mice were set to blank group and fed with normal diet. Fifty (50) male ApoE (-/-) mice were fed a standard Western diet (containing 0.15% cholesterol and 21% fat wt/ wt). 49 After 12 weeks of modelling, they were randomly divided into model group, simvastatin + aspirin control group, DSYM low dose group, and DSYM high dose group. Among them, DSYM low dose group and DSYM high dose group contained 13 male mice per group, while the remaining groups contained 12 male mice per group.
After the successful modelling, the drug intervention was started. The dose was calculated according to the weight ratio of 60 kg human and 30 g mouse: the control group was administered with simvastatin at 2.52 mg/(kg·d) and aspirin at 12.60 mg/(kg·d); DSYM low dose group was administered with a solution containing 8.31 g/kg/d of crude drug; DSYM high dose group was administered with a solution containing 24.93 g/kg/d of crude drug. The blank group and the model group were given equal doses of ultrapure water per day according to the bodyweight ratio. The bodyweight was weighed once a week to adjust the dose, and the intervention was continued for 8 weeks.

| Specimen preparation method
After 0.5 h of the last administration, 2% sodium pentobarbital solution was prepared, and the mice were anaesthetized by intraperitoneal injection at a dose of 180 mg/kg. After the anaesthesia was effective, the abdominal cavity of the mouse was opened, and 1.5 mL of blood was taken through the abdominal aorta and allowed to stand for 30 minutes. The blood samples were centrifuged for 10 minutes at 4°C, 3 000 r/min within 1 hour and the supernatant was aspirated with a pipette, stored in a dry EP tube at −20°C for the detection of blood lipids, NO, ET, hs-CRP, VEGF, MMP-9, etc The mice were killed by cervical dislocation, and a small section of aortic specimen connected to the heart was quickly taken out about 1-2 cm under sterile conditions. Some specimens were fixed with 4% paraformaldehyde for HE staining, some were fixed by 2.5% glutaraldehyde for electron microscopy, and some were stored in liquid nitrogen for Western Blot, RT-PCR, etc

| Determination of Serum NO and ET
The NO is determined by the nitrate reductase method and is per- was performed according to the instructions of the ET kits. The radioactivity of the precipitate was measured on an automatic gamma counter, and the concentration of ET was calculated from the standard curve, and the result was automatically obtained by a computer.
The experiment was repeated three times, and the average value was taken.

| Determination of blood lipid
Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C) levels were measured by an automatic biochemical analyzer. It is responsible by the biochemical room of the First Affiliated Hospital of Hunan University of Chinese Medicine. The experiment was repeated three times, and the average value was taken.

| Determination of serum hs-CRP
The serum hs-CRP level was determined by chemiluminescence method. It is responsible by the biochemical immunization room of the First Affiliated Hospital of Hunan University of Chinese Medicine. The experiment was repeated three times, and the average value was taken.

| Determination of serum VEGF and MMP-9
The serum VEGF and MMP-9 levels were determined by doubleantibody sandwich enzyme-linked immunosorbent assay (ELISA) and performed in strict accordance with the instructions in the kit.
Allow the kit to warm to room temperature for at least 20 minutes before use. All reagents and samples are configured in advance.
The sample is provided with 3 duplicate holes. Blank wells, standard wells and sample wells were set; 100 μL of the sample dilution was added to the blank wells, and 100 μL of the standard or the sample was added to the remaining wells. The plate was coated with a cover and incubated at 37°C for 120 minutes; after the incubation is complete, the liquid is discarded. Then, 100 μL of biotin-labelled antibody working solution was added to each well and incubated at 37°C for 60 min; after the incubation, the liquid was discarded and the plate was washed. The horseradish peroxidase labelled avidin working solution (with biotinylated antibody working solution) 100ul was then added into each well and incubated at 37°C for 60 minutes; after the incubation, the liquid was discarded and the plate was washed. After that, the substrate solution was added into each well the colour was developed at 37°C in the dark for 30 minutes. Finally, the reaction was stopped by adding 50 ul of the stop solution to each well (the blue colour turned yellow at this time); and the optical density (OD value) of each well was measured sequentially at 450 nm using an enzyme-linked instrument (The reference wavelength is 630 nm).

| Morphological changes of aortic roots in mice observed by HE staining
The specimen was fixed with 4% paraformaldehyde, dehydrated with an upstream gradient, and paraffin embedded after hyalinized by xylene. The specimen was continuously cross-sectioned from the heart to the ascending aorta 5 μm up to the aortic root (100 μm above the aortic valve). 50 Approximately 20 slices were taken in succession, and one was taken every 5 sheets. Then, HE staining is performed.
Under the X100, X204 and X400 light microscope, it was observed that whether the intima of the vessel wall is bulged, whether there is accumulation of foam cells under the intima, whether the internal elastic membrane is intact, and photographed.

| Ultrastructural observation of aortic endothelial cells
One (1) mm 3 aortic root tissue in the mice of each group were taken out and fixed with 2.5% glutaraldehyde phosphate buffer for 2 hours or longer; Then, the tissue was rinsed with 0.1 mol/L phosphoric acid rinsing solution and fixed with 1% citrate fixative for 1 ~ 2 h. After dehydration, soaking, embedding, and curing, the specimen was cut into thin slices of 50-100 nm (70 nm) by LKB-III ultrathin microtome, and double-stained with 3% uranyl acetate and lead nitrate. Finally, FEI Tecnai G2 Spirit transmission electron microscope was used to observe and film.

| Tissue protein extraction
The frozen tissue was taken out from the liquid nitrogen, and lysis solution was added at a ratio of 500 ul of lysis solution per 0.1 mg of tissue. The tissue was broken up on a high-speed dispersing cutter on ice. Centrifuge at 12 000 rpm for 20 minutes in a refrigerated centrifuge and take the supernatant.

| Complete drying of the slide chip
Remove the slide chip and recombine at room temperature for 20-30 min. Open the package, uncover the seal and place the chip in a vacuum desiccator or dry at room temperature for 1-2 h.

| Chip operation
This process is carried out in strict accordance with the instructions in the QAM-CAA-4000 Antibody Protein Chip Kit: 100 μL of sample dilution was added to each well (final protein concentration of 500 μg/mL), and it was incubated for 30 minutes at room temperature on a shaker to block the quantitative antibody chip. After incubation, the buffer in each well was removed, 100 μl of standard and sample (diluted to 500 μg/mL) was added to the wells and incubated overnight at 4°C on the shaker. Then, 1.4 mL of the sample dilution was added to antibody mixture tubules, mixed well and the tubules were centrifuged; after that, 80 μL of detection antibody was added to each well and incubated for 2 hours on a shaker at room temperature. Then, 1.4 mL of the sample dilution was added to Cy3-streptavidin tubules, mixed well and the tubules were centrifuged; after that, 80 μL of Cy3-streptavidin was added to each well and incubated for 2 hours on a shaker in the dark. Finally, the Axon genePix were utilized to scan the signal using Cy3 or a green channel. The scanning parameters are as follows: PMT: 600, Wavelength: 532 nm, resolution: 10 μm. The data analysis was performed using QAM-CAA-4000 data analysis software.

| Validation of VEGF, MMP-9 and bFGF by RT-PCR
Aortic tissue samples were taken, and total RNA was extracted as described in the instructions of Trizol kit. After dilution with 2 μL of RNA, the RNA purity and concentration were determined by UV spectrophotometry. Using total RNA as a template, cDNA was synthesized according to the instructions of the reverse transcription kit, and the obtained DNA was amplified in a 25 μL system. The PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 minutes, 95°C for 10 seconds, 60°C for 1 minutes, and 40 cycles. The Ct value was read after the reaction was completed. The specificity of the PCR reaction of each sample was monitored by melting curve, and β-actin was used as an internal reference gene for data analysis. The primers were shown in Table 1. The experiment was repeated three times, and the average value was taken.

| DSYM sample preparation
According to the DSYM prescription, 66.28 g of dry herbal pieces was accurately weighed and placed in a 500 mL flat-bottomed flask.
The mixture was condensed and refluxed with 10 times and 8 times of water for 1 hour, filtered, and the filtrate was combined. The volume of filtrate was adjusted to 1000ml with distilled water, and the filtrate was filtered through a 0.45 μm PTEF microporous filter membrane, shaved with tin foil and placed in the refrigerator for later use.

| Standard sample preparation
According to the method in reference, 51 the tanshinone IIA, salvianolic acid B, tanshinol, rosmarinic acid, protocatechuic aldehyde were accurately weigh and distilled water were added to prepare a mixed solution per ml containing 160 μg of tanshinone IIA, 140 μg of salvianolic acid B, 16 μg of tanshinol, 10 μg of protocatechuic aldehyde and 23 μg of rosmarinic acid.

| Statistical analysis
All data were processed using SPSS 22.0 statistical software. Oneway analysis of variance was used for comparison of multiple groups, and the measurement data were expressed as mean ± standard deviation. The measurement data were measured by mean ± standard deviation. The test level P < .05 indicated that the difference was statistically significant.

| Atherosclerosis PPI network
Three thousand and seven hundred and twenty-nine (3729) ASrelated genes were required from GeneCards and OMIM database.

Primers
Forward primer Reverse primer Product length The PPI data of 590 genes whose relevance score ≥ 3 were obtained to construct the AS PPI network, and this network contains 531 nodes and 11 853 edges ( Figure 1A). These genes are arranged in descending order of the relevance score, the top 10 are as follows:

| Biological Processes of AS PPI Network
The AS PPI network was analysed by MCODE and returns 18 clusters (Table 2 and Figure S2). The genes in the clusters were input into DAVID for GO enrichment analysis.  (Table S3). The main biological processes in cluster 1 were used as an example shown in Figure 1B.

| DSYM fingerprint
After comparing with the retention time of the standard and the spectrum, the five chemical components contained in the DSYM were determined: tanshinone IIA, salvianolic acid B, tanshinol, rosmarinic acid and protocatechuic aldehyde ( Figure S3). and so on ( Figure S4).

| DSYM-AS PPI network
The DSYM-AS PPI network was constructed based on DSYM tar-

| Biological Processes of DSYM-AS PPI Network
The DSYM-AS PPI network was analysed by MCODE and returns 18 clusters (Table 3; Figure S5). The genes in the clusters were input into DAVID for GO enrichment analysis. ( Table S5). The main biological processes in cluster 1 were used as an example shown in Figure 2B.

| Pathway of DSYM-AS PPI network
After the pathway enrichment analysis, twenty-seven AS-related signalling pathways are obtained ( Figure 2C). The P values, fold enrichment and count of these signalling pathways were shown in Figure 2D. The details are described in Table S6.

| General observation
Before the experiment, there was no significant difference in the bodyweight between ApoE-/-mice and the same strain C57BL/6L mice (P > .05), which was comparable. During the modelling process, the colour, activity, and eating and drinking of the mice in each group were normal and there was no difference. At the 12th week and the 20th week, the bodyweight of each group was significantly increased (P < .01), and the body mass growth of ApoE-/-mice was more obvious than that of C57BL/6L mice (P < .01). At the end of the 20th week, the quality of the blank group and the model group still increased significantly (P < .01). Compared with the 12th weekend, the trend of body mass changes in the other experimental groups was not obvious. During the administration period after the model establishment, the mice in each group had less food intake than before, the hair colour was less lustrous, and the activity and drinking water were normal. In the low dose and high dose groups of DSYM, one mouse in each group died due to improper intragastric administration. Immediately after death, the thoracic and abdominal aorta was dissected, and pale yellow lipid streaks were observed, and then oxidized and melted soon. The number of mice that eventually entered the statistics was 12 in each group (see Table 4).

| Effect of DSYM on serum NO and ET levels in ApOE-/-mice
The serum NO level in the model group was significantly lower than that in the other group, and the ET content was significantly higher than that in the other group (P < .01). Drug   The DSYM high dose group was comparable to the simvastatin + aspirin control group in increasing NO (P > .05), but the DSYM group was superior to the western medicine group in reducing ET (P < .05). ( Figure 3A).

| Effect of DSYM on serum lipid in ApOE-/-mice
In the model group, the TC, TG, LDL-C increased significantly, HDL-C decreased significantly (P < .01), and the standard Western diet induced ApoE knockout mice to form severe hyperlipidemia. Compared with the model group, DSYM and simvastatin + aspirin can reduce TC and LDL-C and increase HDL-C (P < .05), but the lipid-lowering effect of the three groups is not statistically significant (P > .05). This indicates that the DSYM group is equivalent to the conventional western medicine treatment group. The increases of DSYM dose had no significant effect on its curative effect of lipid-lowering (P > .05).

| Effect of DSYM on serum hs-CRP in ApOE-/-mice
The hs-CRP level in the model group was significantly higher than that in the blank group (P < .01), which was also higher than the other groups (P < .05). The hs-CRP levels in the drug intervention F I G U R E 6 Results of Experimental Protein Network Analysis A, Experimental Protein Network (The larger the node size, the higher the degree of the node. The thicker the line, the greater the Edge Betweenness of the node.). B, Bubble chart of biological processes (X-axis is fold enrichment analysis). C, Pathway of Experimental Protein Network (Blue circle stands for proteins. Yellow circle stands for pathway. The larger the node size, the higher the degree of the node. The thicker the line, the greater the Edge Betweenness of the node.). D, Bubble chart of signalling pathway (X-axis is fold enrichment analysis) group decreased, and that in the DSYM high dose group and the simvastatin + aspirin group decreased. There was no significant difference between DSYM high dose group and the simvastatin + aspirin group (P > .05) ( Figure 3D).

| Effect of DSYM on serum VEGF and MMP-9 in ApOE-/-MIce
Serum VEGF and MMP-9 levels in the model group were significantly higher than those in the group (P < .01); drug intervention could down-regulate VEGF and MMP-9 levels (P < .05 or P < .01). In the reduction of VEGF and MMP-9, the efficacy of the DSYM high dose group was comparable to that of the simvastatin + aspirin group (P > .05), but better than the DSYM low dose group (P < .05). The efficacy of DSYM was dose dependent.

| Effect of DSYM on pathomorphology of aortic root in ApOE-/-mice
Blank group: the aortic wall was smooth, the thickness was uniform and no bulge, the endometrium, the media and the adventitia were not abnormal, and there was no AS lesion. Model group: the aortic wall is not smooth, the movement is not natural, the thickness is uneven, the intima is thickened, there is no protruding bulge and the lumen is narrowed, no obvious plaques are seen; Foam cell formation, smooth muscle cell proliferation, and inflammatory cell infiltration can be seen. The degree of change in the aorta of the remaining groups under HE staining was between that of the blank group and the model group ( Figure 4A).

| Effect of DSYM on ultrastructure of aortic vascular endothelial in ApOE-/-mice
In the model group, the endometrial endothelial cells of the aorta were detached and necrotic, the structure of the internal elastic membrane was irregular; the smooth muscle cells proliferated obviously, the mitochondria were extensively oedematous, vacuolated, the sputum was significantly reduced or disappeared, the nuclear structure was blurred, and the cytoplasm contains a large number of lipid droplets or even a string, suggesting a lipid-line stage, indicating successful modelling.
After drug intervention, endothelial cell injury was alleviated to varying degrees, indicating that DSYM, simvastatin + aspirin can improve the ultrastructure of aortic endothelial cells in ApoE-/-mice, and protect the injured endothelial cells; The DSYM high dose group was superior to the control group, while the control group was superior to the DSYM low dose group. (Figure 4B).

| Arrangement of 200 cytokines on membrane chips and expression profile of antibody chips
The arrangement and results of the 200 cytokines in the model group and the DSYM group on the membrane chip are shown in Figure 5.

| Antibody protein chip results data analysis
The results of the RayBio. Cytokine Antibody Arrays protein chip were analysed using the data of Normalization 2 Positive Control Normalization without Background. The signal value ratio method was used for chip analysis, and the difference between the signal value, Fold change and t test was selected. Select the signal value greater than 500, fold change greater than 2, less than 0.5 as the difference factor.
The analysis results of the two groups of sample chips showed that 51 models with significant differences were found in the model group/ DSYM group, see Table S7. The 4-1BB with the largest fold ratio has a low signal value in the model group, so it is rounded off. This study selected bFGF (whose signal value is maximum), Pro-MMP-9 (whose fold change ratio is maximum) and VEGF (which is closely related to vascular endothelial cell angiogenesis) for further study. (Table S7).

| Expression of VEGF, MMP-9 and bFGF mRNA in aortic lysates of each group
The expression of VEGF, MMP-9 and bFGF mRNA in C57BL/6L mice was weak, and the expression levels of VEGF, MMP-9 and bFGF mRNA in ApoE-/-mice were enhanced; and the expression of model group was significantly higher than that of other groups (P < .01).
The expression of MMP-9 mRNA between DSYM low dose group and high dose group was statistically significant (P < .05). There was  Figure S6).
To perform deep mining of proteomics data, we used systematic biological methods to further analyse proteomics data.

| Experimental Protein Network
The experimental proteins and their PPI network were utilized to construct the experimental protein network. This network contains 46 nodes and 350 edges ( Figure 6A).

| Biological Processes of Experimental Protein Network
The experimental protein network was analysed by MCODE and returns 2 clusters (Table 5 and Figure S7). The genes in the clusters were input into DAVID for GO enrichment analysis.
Cluster 1 is mainly involved in immune response (such as regulatory T cells, neutrophil chemotaxis), inflammatory response, angiogenesis and vascular endothelial barrier. Cluster 2 is associated with inflammatory responses, angiogenesis, cellular hypoxia and immune responses (Table S8). The main biological processes in cluster 1 were used as an example shown in Figure 6B.

| Biological processes of experimental protein-other proteins' PPI network
The experimental protein-other proteins' PPI Network was analysed by MCODE and returns 13 clusters (Table 6; Figure S8). The genes in the clusters were input into DAVID for GO enrichment analysis.
Cluster 1 is associated with immune reactions, inflammatory responses, angiogenesis, neutrophil chemotaxis, proliferation of monocyte macrophages and smooth muscle. Cluster 2 is associated with Proliferation and migration of smooth muscle, angiogenesis, and proliferation of endothelial cells and their signalling pathways. Cluster 3 is mainly related to inflammatory responses.
Cluster 6 is also involved in the inflammatory response. Cluster 7 is associated with Wnt signalling pathway. Cluster 8 is primarily involved in lipid metabolism. Cluster 5 and 13 do not return any human's biological processes. Cluster 4, 9, 10, 11, 12 failed to return AS-related biological processes (see Table S10). The main biological processes in cluster 1 were used as an example shown in Figure 7B.

| Pathway of experimental protein-other proteins' PPI network
After the pathway enrichment analysis, twenty-two AS-related signalling pathways are obtained ( Figure 7C). The P values, fold enrichment and count of these signalling pathways were shown in Figure 7D. The details are described in Table S11. The process of erosion or rupture of AS plaques involves a variety of inflammatory mechanisms including endothelial dysfunction, leucocyte migration, extracellular matrix degradation and platelet activation. 87 The main mechanism is: (1) Cytokines that regulate leucocyte activity in acute phase reactants, such as interleukin Platelets are also activated before significant ACS appears, which directly leads to the development of AS. [96][97][98] Our research showed that DSYM can interfere with these inflammatory molecules and their mediated biological processes.

| CON CLUS ION
This study proposed a new method based on systems biology, proteomics, and experimental pharmacology, and analysed the pharmacological mechanism of DSYM. DSYM may achieve therapeutic effects by regulating AS-related biological processes (such as coagulation pathway, inflammatory reaction, NO metabolism, vascular remodelling, lipid metabolism, neutrophil chemotaxis, and proliferation of macrophages and smooth muscle cells) and signalling pathways (such as PI3K-Akt, HIF-1, TNF, neurotrophin, adipocytokine signalling pathways, and complement and coagulation cascades).

ACK N OWLED G EM ENTS
This work is supported by the National Natural Science Foundation of China (No. 81774174).

CO N FLI C T O F I NTE R E S T
We declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in supplementary materials.