Li-Li Hou, Hai-Xin Liu, Qing-Shan Li?

1. Institute of Pharmaceutical & Food Engineering,Shanxi University of Chinese Medicine,Jinzhong 030600,China,Shanxi University of Chinese Medicine,Jinzhong 030600,China

2. Shanxi Key Laboratory of Innovative Drug for the Treatment of Serious Diseases Basing on the Chronic Inflammation,Shanxi University of Chinese Medicine,Jinzhong 030600,China

Keywords:Network pharmacology Promoting blood circulation and removing blood stasis Angiogenesis

ABSTRACT Objective: Reveal the material basis and mechanism of angiogenesis effect of Danshenchuanxiong herb-partners. Methods: The effective components of Danshen and chuanxiong were searched in the TCM System Pharmacology Database (TCMSP), and the drug targets were searched in the Swiss Target Prediction database; in the Disease-Gene Database(DisGeNET) ) Search for angiogenesis-promoting and inhibiting angiogenesis targets; use the protein interaction database (STRING) database and Cytoscape software to analyze the network core targets; use the DAVID database to enrich the drug-disease target intersection for GO and KEGG; RT-qPCR experiments verified the effect of luteolin on MMP9 gene expression; in vitro tube formation experiments analyzed the effect of luteolin on endothelial cell neovascularization. Results: After analysis, it is concluded that Danshen has 10 active ingredients and 50 angiogenesis targets; Chuanxiong has 2 active ingredients and 4 angiogenesis targets. The intersection of drug-taking targets yielded 9 targets, including AR,PARP1, MMP9, MMP2, MMP12, AKR1B10, ABCC1, CDK6, and STAT3.After analyzing the active ingredient-target network graph of Danshen/Chuanxiong, it was concluded that luteolin is the key chemical ingredient. After protein interaction (PPI) analysis, the results showed that MMP9 is the core target. The KEGG pathway is mainly enriched in 5 signaling pathways, the most important pathway is cancer-related pathways. There are 22 main biological processes of GO, which mainly involve collagen catabolism, protein extracellular matrix, metal endopeptidase activity, etc. The preliminary analysis of tubule formation experiment shows that luteolin inhibits angiogenesis and down-regulates the expression of MMP9 gene. Conclusion: Luteolin, the key chemical component of the Danshen-Ligusticum chuanxiong drug pair,inhibits angiogenesis by regulating the MMP9 target, and develops the reverse regulation of angiogenesis for the traditional Chinese medicine for promoting blood circulation and removing blood stasis on the basis of the known treatment of ischemic diseases. Provide a new perspective and theoretical basis for the use of Danshen and Chuanxiong medicine in clinical cancer patients. Reveal the material basis and mechanism of angiogenesis effect of Danshenchuanxiong herb-partners.

1. Introduction

Angiogenesis is a process of neovascularization, which can promote embryonic development and regulate some key biological processes. Abnormal angiogenesis is also the basis of various pathological processes, including tumor metastasis and atherosclerotic plaque formation [1]. Many kinds of traditional Chinese medicine can regulate angiogenesis, such as drugs for promoting blood circulation and removing blood stasis. Some researchers have found that drugs for promoting blood circulation and removing blood stasis have a two-way regulatory effect on angiogenesis [2-5]. In clinic, the drugs for promoting blood circulation and removing blood stasis are commonly used in combination with Salvia miltiorrhiza and Ligusticum chuanxiong. The study found that it can promote angiogenesis in rats with myocardial ischemia[6,7]. At the same time, it can inhibit angiogenesis in tumor model[8,9]. Clinically, inhibition of angiogenesis can be used for tumor treatment, while promoting angiogenesis can be used for ischemic diseases. However, the effect of Salvia miltiorrhiza and Ligusticum chuanxiong on angiogenesis, the key effective components and regulatory mechanism are not clear. Network pharmacology is a new method to study the action and mechanism of drugs based on biology, computer, pharmacology and other subjects, which opens a new way for the study of the effect and mechanism of traditional Chinese medicine and compatible prescriptions on diseases. In this study, the method of network pharmacology combined with experimental verification was used to study the effect and regulation mechanism of Salvia miltiorrhiza and Ligusticum chuanxiong on angiogenesis, which has a certain guiding effect on the clinical use of drugs for promoting blood circulation and removing blood stasis.

2. Experimental materials and methods

2.1 Experimental materials

2.1.1 Experimental cell lineHuman umbilical vein endothelial cell EA.hy926, was purchased from the Cell Resource Center of Shanghai Academy of Life Sciences, Chinese Academy of Sciences.

2.1.2 Experimental reagent

Luteolin (Dalian Meilun Biotechnology Co., Ltd., item No.:MB2172-1); Phosphate buffer (PhD de Biotechnology Co., Ltd.,Ref.: PYG14040); DMEM High Sugar medium (Gibco, No.:12800017); Green-streptomycin (Hycolne, Ref.: J180034); 0.05%trypsin solution (including EDTA) (Biotechnology Co., Ltd., Ref.:PYG0014); Trizol (Takara, No.: 9108). Reverse transcription kit(Takara, item number: AJ92003A); PCR amplification kit (Takara,item number: 638314); matrigel base glue (BD, item number:356231)

2.1.3 Experimental instrumentsFluorescence quantitative PCR (App Bio, QuantStudio3); high content (MD, ImageXpress Micro4.0)

2.2 Experimental methods

2.2.1 Information collection of Salvia miltiorrhiza and Rhizoma Chuanxiong

The active components of Salvia miltiorrhiza and Ligusticum chuanxiong, two kinds of traditional Chinese medicine for promoting blood circulation and removing blood stasis, were searched in the traditional Chinese medicine system pharmacology database (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, TCMSP) (http://lsp.nwu.edu.cn/tcmsp.php) platform [11,12]. Because there are many ingredients in traditional Chinese medicine, and the structure of each component is also different, it is necessary to screen the effective components of traditional Chinese medicine according to (DL) and bioavailability(OB). OB is the bioavailability of drugs and an important index of active components in traditional Chinese medicine in the treatment of diseases; DL stands for drug-like properties and is an index to predict the properties of effective active compounds. Based on the conditions of bioavailability OB ≥ 30% and DL ≥ 0.18, the effective data of Salvia miltiorrhiza and Ligusticum chuanxiong were screened. The targets of Salvia miltiorrhiza and Ligusticum chuanxiong were predicted by Swiss Target Prediction website,and the effective targets of Salvia miltiorrhiza and Ligusticum chuanxiong were screened under the condition that Probability ≥0.6[14,15].

2.2.2 Target analysis of angiogenesis

The targets related to angiogenesis were searched with "Inhibit angiogenesis" and "Promote angiogenesis" in the disease-gene database (DisGeNET) (http://www.disgenet.org/) [16,17], and the targets related to angiogenesis were obtained after merging.

2.2.3 Drug-disease core target analysisThe targets of the active components of Salvia miltiorrhiza and Ligusticum chuanxiong were merged and the repetitive values were deleted, and the intersection targets of drugs and diseases were obtained by intersecting the obtained component targets with the targets of angiogenesis, and the Wayne diagram was drawn. The nine intersecting targets were analyzed by STRING database [18]and Cytoscape3.7.2 software, and the corresponding PPI network diagram was obtained.

2.2.4 GO and KEGG enrichment analysisNine drug-disease intersection targets were introduced into DAVID database [19,20]. Under the screening condition of P ≤ 0.05, the final effective data of KEGG pathway and GO biological pathway were obtained.

2.2.5 Salvia miltiorrhiza-Ligusticum chuanxiong-active ingredient-angiogenesis target network

The effective components and targets of Salvia miltiorrhiza and Ligusticum chuanxiong and the targets of angiogenesis were imported into Cytoscape software according to a certain format to build a network diagram.

2.2.6 Molecular docking

Collect protein conformations in the protein bank database (PDB,https://www.rcsb.org/). The screening conditions are set as follows:(1) the protein structure is obtained by X-crystal diffraction; (2) the crystal resolution of the protein is less than 2.8); (3) the preferred selection of the protein structure reported in the molecular docking literature; (4) the creature comes from Homo sapiens. Then,SYBYL-X 2.0 is used for molecular docking calculation.

2.2.7 Cell culture

EA.Hy926 cells were cultured in DMEM complete medium(containing 10% fetal bovine serum and 1% double antibody).The cell culture conditions were 37 ℃, constant temperature and humidity, 5% CO2.

2.2.8 RT-qPCR experimentIn quantitative RT-qPCR analysis, 0.1% FBS medium was added in the blank control group, and luteolin (10-6mol/L) diluted with 0.1% FBS medium was added to the cell culture medium of the intervention group, each well was 100 μ L system, after 24 hours of treatment. Use Trizol reagent to extract total RNA from the cell, as described by the manufacturer. The extracted RNA was reverse transcribed into cDNA by reverse transcription kit. CDNA was amplified by amplification kit. The system of RT-qPCR is as follows: pre-denaturation at 95 ℃ for 15 minutes, denaturation at 95 ℃ for 10 s, annealing at 60 ℃ for 20 s, extension at 72 ℃ for 30 s and 40 cycles. The sequence of primers used for RT-qPCR is as follows: MMP-9 primer sequence: sense chain: 5'-AGTCCACCCTTGTGCTCTTCCC-3', internal reference: 5'-TCTGCCACCCGAGTGTAACCAT-3'; GAPDH primer sequence: sense chain:5'-AGCGAGCATCCCCCAAAGTT-3', internal reference: 5'-GGGCACGAAGGCTCATCATT-3'.

2.2.9 Angiogenesis analysis experimentIn this experiment, 10 generations of cells were selected for Tube Formation Assay experiment in vitro. After resuscitation, the cells were passaged at least twice and the experiment was carried out when the cell vitality was exuberant. The resuscitation cells were routinely inoculated in a 25 cm2 culture bottle, and when the cells were fused to 80% ～ 90%, they were subcultured in a 75 cm2 culture bottle. The cells were adhered to the wall at least twice and observed under the microscope. When the morphology and vitality of the cells were good and fused to 80%-90%, the cells were synchronized and starved for 24 hours in a serum-free medium.The BD matrix glue was thawed to liquid state at least 24 h at 4 ℃in advance, and the gun head of 1 ml aseptic liquid separator, 96-hole blackboard and off-board machine bracket were pre-cooled in advance in the refrigerator at 4℃. During the operation of the Tube Formation Assay experiment (all operations in this experiment need to be aseptic), take out the ice box and use the ultraviolet ice box to beat a box of ice and put it in the super clean table, put the 96-hole blackboard on the ice box, and quickly take it out of the 4℃refrigerator and insert it into the ice box when using BD base glue.Using the liquid separator to insert the gun head precooled at 4℃,the matrix glue was quickly added to the middle position at the bottom of each hole (50 μL per hole, all operations in this experiment need to avoid the generation of bubbles in the hole). After the glue was laid, the 96-hole blackboard was moderately shaken to cover the whole hole bottom, and the matrix glue was placed flat in the refrigerator at 4℃ for 15 min. After setting, remove the blackboard and place it on the bracket of the precooled separator (the 96-hole blackboard of the other bracket needs to be leveled before centrifugation, and 55 μL ddH2O is added to the corresponding hole)centrifuge for 2500 rpm,15 min. After centrifugation, the blackboard with bubble removal and BD base glue was placed in a 37℃, 5%CO2 incubator to balance 30 min～60 min. The cells were collected by trypsin digestion, and the cells were counted and inoculated in the 96-well plate at the density of 1.5 × 104 cells per well. The blank control group added 0.1% FBS medium, the intervention group added luteolin (10-6mol/L) diluted with 0.1% FBS medium, and the positive control group added VEGF (50 ng/mL), 100 μ L per well.After all the operation, the 96-hole blackboard was incubated for 6 hours and 16 hours in a 5% CO2 incubator at 37℃ (note that the 96-hole blackboard should not be shaken in the first hour after it was placed in the incubator). After the 96-hole blackboard was incubated in the incubator for about 8 hours, the vascular lumen formed by the cells in the hole could be observed under the microscope. After the lumen was successfully observed, the cytoplasm was stained with Calcein AM (1 μmol/L) and washed for 3 times in 37℃, 5%CO2 incubator for 3 times. Under the high connotation analysis instrument, 9 middle visual fields and 10 times objective lenses were selected to take pictures, and the number of vascular mesh structures in each hole was calculated.

2.2.10 Data processing methodThe experimental data of each group were counted by GraphPad Prism 5, and the significance was tested by analysis of variance.

3. Result

3.1 Active components and targets of Salvia miltiorrhiza and Ligusticum chuanxiong

There are 3714 components of Salvia miltiorrhiza and 602 components of Ligusticum chuanxiong in traditional Chinese medicine system pharmacology database TCMSP. After screening with OB ≥ 30% DL ≥ 0.18, it is concluded that Salvia miltiorrhiza has 10 active ingredients and Chuanxiong has 2 active ingredients.The component targets were predicted by Swiss Target Prediction website. After removing the repetition value, it was concluded that there were 50 targets in Salvia miltiorrhiza and 4 targets in Ligusticum chuanxiong.

3.2 Targets for bidirectional regulation of angiogenesis

The targets of angiogenesis were searched in DisGeNET database,and 526 targets for promoting and inhibiting angiogenesis were obtained.

3.3 Effect of Salvia miltiorrhiza / Ligusticum chuanxiong on effective components-target-signal pathway-angiogenesis in bidirectional regulation of angiogenesis

Based on the data of the active components of Salvia miltiorrhiza and Ligusticum chuanxiong and the related target data of angiogenesis, 9 intersection targets of disease and drugs were obtained. It is concluded that KEGG pathway is mainly enriched in 5 signal pathways (Table 2). KEGG pathway is the target enrichment pathway of Salvia miltiorrhiza and Ligusticum chuanxiong, which represents the signal pathway of drug regulation related diseases[11]. The map of Salvia miltiorrhiza/Ligusticum chuanxiong-active ingredient-target-signal pathway-angiogenesis network was made by Cytoscape3.7.2 software (Figure 1). The nodes in the figure represent the active components of Salvia miltiorrhiza and Ligusticum chuanxiong and the active components of the two drugs, as well as the intersection targets of diseases and two drugs, while the edges represent the relationship between Salvia miltiorrhiza-Ligusticum chuanxiong and its active components, active components and intersection targets, disease and core pathways, and core pathways and intersection targets. It can be seen from the network diagram that the five active components of Salvia miltiorrhiza and Ligusticum chuanxiong can regulate 9 targets, and these 9 targets are enriched in 5 KEGG pathways, that is, each target gene protein is enriched in different signal pathways, and each active component also regulates different target gene proteins, reflecting the synergistic and bidirectional regulation of angiogenesis by multi-components, multitargets and multi-pathways of Salvia miltiorrhiza and Ligusticum.

Figure 1 Danshen/chuanxiong-active ingredient-target-signal pathwayangiogenesis network diagram

3.4 Effective components and targets of Radix Salviae Miltiorrhizae / Ligusticum chuanxiong in bidirectional regulation of angiogenesis

Through the intersection of Salvia miltiorrhiza / Ligusticum chuanxiong drug pair targets and bidirectional angiogenesis regulation targets, there are 9 disease and drug intersection targets(Figure 2). They are AR, PARP1, MMP9, MMP2, MMP12,AKR1B10, ABCC1, CDK6 and STAT3, which are potential targets for Salvia miltiorrhiza-Ligusticum chuanxiong in the treatment of angiogenesis-related diseases. Nine targets were found in the data of Salvia miltiorrhiza and Ligusticum chuanxiong, and the corresponding active components of the targets were obtained (Table 1). The interaction between active components and target proteins was analyzed by Cytoscape (Figure 3). As shown in figure 2, a single compound can regulate multiple targets to trigger biological effects,which may play a vital role in regulating angiogenesis. The results show that luteolin has the highest Degree value (Degree=7) and can regulate PARP1, MMP9, MMP2, MMP12, AKR1B10, ABCC1,CDK6 and other targets, so luteolin as a key active component has become the object of further study.

Figure 2 The intersection of Danshen/chuanxiong active ingredient targets and angiogenesis targets

Figure 3 Danshen/chuanxiong active ingredient-target network diagram (the greater the Degree value, the greater the node)

Table 1 Drug-disease intersection targets and components

3.5 MMP9 is the core target of Salvia miltiorrhiza /Ligusticum chuanxiong on bidirectional regulation of angiogenesis

Through the active ingredient-target network map of Salvia miltiorrhiza / Ligusticum chuanxiong, luteolin was found to be the key effective component affecting angiogenesis in the drug pair of Salvia miltiorrhiza / Ligusticum chuanxiong. Therefore, the target of luteolin on angiogenesis was imported into string website to obtain protein-protein interaction and analyzed by Cytoscape3.7.2 software to obtain protein-protein interaction network map (Figure 4). Figure 4 shows 7 interaction nodes (PARP1, MMP9, MMP2, MMP12,CDK6, ABCC1, AKR1B10) and 12 edges of the key targets of Salvia miltiorrhiza and Ligusticum chuanxiong in the treatment of angiogenesis-related diseases. The nodes in the figure represent drug-disease intersection target proteins, while the edges represent the cross-linking relationship between intersection target proteins. In addition, it is shown in figure 3 that MMP9 has the highest Degree value (Degree=10), so MMP9 may be the core target of Salvia miltiorrhiza-Ligusticum chuanxiong on angiogenesis.

Figure 4 Protein-protein interaction (PPI) network. Each node represents a related target gene, and a larger degree of protein is described by a larger node and a darker color.

3.6 main biological processes and core pathways of Salvia miltiorrhiza / Ligusticum chuanxiong on bidirectional regulation of angiogenesis

Core pathway analysis showed that the main pathways of Salvia miltiorrhiza / Ligusticum chuanxiong drugs on bidirectional regulation of angiogenesis were enriched in 5 signal pathways(Table 2), KEGG pathway is the target enrichment pathway of Salvia miltiorrhiza / Ligusticum chuanxiong drugs, representing the signal pathways of drug regulation-related diseases [11]. The results showed that the main pathways affected by Salvia miltiorrhiza and Ligusticum chuanxiong were tumor-related pathways and MicroRNAs signaling pathways in tumors. Among them, the pathway with the lowest P value is the tumor-related pathway.Moreover, all the five pathways contained the target MMP9, so the results of protein-protein interaction analysis confirmed that MMP9 was the core target of Salvia miltiorrhiza / Ligusticum chuanxiong on bidirectional regulation of angiogenesis. GO analysis showed that there were 13 pathways of molecular function, 1 pathway of biological process and 8 pathways of cellular components. The results showed that (Figure 6), GO functional enrichment is mainly related to collagen catabolism, protein extracellular matrix, metal endopeptidase activity and so on, which are closely related to the core target MMP9.

Table 2 KEGG pathway analysis

Figure 5 Bubble diagram of KEGG pathway

Figure 6 GO biological pathway diagram

3.7 Molecular docking simulation

Based on the active component-target network map of Salvia miltiorrhiza / Ligusticum chuanxiong, PPI network and KEGG signal pathway analysis, we selected MMP9 core target gene for molecular docking with luteolin. In figure 7, luteolin is observed to enter the active pocket of the protein (Figure 7). Luteolin small molecules mainly form three hydrogen bonds with TYR64, ARG116 and GLU117 on MMP9. The binding score is 4.1652, which meets the conditions for further verification.

Figure 7 Analysis of target-compound docking simulation.

3.8 effect of luteolin on tubule formation in vitro

In order to verify the effect of luteolin on tubule formation in vitro,the experimental method of angiogenesis analysis was used in this study. Results as shown in figure 8A, compared with the blank group,luteolin inhibited the angiogenesis of EA.hy926 cells in vitro and promoted the angiogenesis of EA.hy926 cells in vitro after VEGF treatment. After the statistics of the number of circular structures, the results of single factor analysis of variance showed that compared with the blank group, the number of annular structures of EA.hy926 cells treated with luteolin decreased significantly (P < 0.01), while that of EA.hy926 cells treated with VEGF increased significantly (P< 0.0l) (Figure 8B). The results showed that luteolin could inhibit the angiogenesis of endothelial cells in EA.hy926 cells.

3.9 Effect of luteolin on mRNA expression of MMP9 and VEGF-A

In order to further analyze the angiogenic effect of luteolin and the transcriptional level of the target, RT-qPCR experiment was used to verify whether luteolin, the core active component, had an effect on the expression of core target MMP9. At the same time, VEGFAn is an important target of angiogenesis [21], so it is important to verify whether luteolin has an effect on its expression. The results of paired t-test showed that the expression of MMP9 in EA.Hy926 cells treated with luteolin was significantly lower than that in the blank group (P < 0.01) (Figure 9A), and the expression of VEGF-An in EA.hy926 cells treated with luteolin was also significantly decreased(P < 0.05) (Figure 9B). Luteolin could inhibit the transcription of MMP9 and VEGF-A, and the expression of MMP9 and VEGFAn in the treatment group was lower than that in the blank group,indicating that luteolin, the main component of Salvia miltiorrhiza /Chuanxiong pair, inversely regulates angiogenesis through MMP9,which is consistent with the results of angiogenesis analysis.

Figure 9 RT-qPCR detection of the effect of luteolin on the mRNA expression of MMP9 and VEGF-A. Figure A shows that luteolin inhibits the expression of MMP9 (P<0.01); Figure B shows that luteolin inhibits the expression of VEGF-A (P<0.05).

4. Discussion

Angiogenesis is a process of new blood vessels formed by preexisting blood vessels, a process that participates in physiological conditions (such as development and wound healing), and many pathological conditions (including cancer, infectious arthritis and psoriasis) [22]. It includes several steps characterized by unique endothelial cell function, such as proliferation, migration, lumen formation, differentiation and maturation [23]. Each step involves a variety of growth factors, receptors, and molecules that affect the diversity of signal transduction pathways that affect the pathogenicity of angiogenesis in different diseases [24].

Salvia miltiorrhiza is a kind of traditional Chinese medicine in China, which has the functions of promoting blood circulation and removing blood stasis, cooling blood and clearing heart, nourishing blood and calming nerves. The crude extract of Salvia miltiorrhiza and Salvianolic acid B (a component of Salvia miltiorrhiza)enhance angiogenesis in rat endothelial cell lines by up-regulating the expression of VEGF and VEGF-R2 genes. Cryptotanshinone can reduce the protein expression of matrix metalloproteinase-2(MMP-2) and MMP-9 in vitro. It may also inhibit tumor cellinduced endothelial cell angiogenesis and rat aortic ring angiogenesis in vitro by inhibiting angiogenesis-related factors [27]. Chuanxiong Xin San Wen Tong, with the function of promoting blood circulation and removing blood stasis, warming meridians [28]. Total phenols of Ligusticum chuanxiong can improve cardiac function by promoting angiogenesis of ischemic myocardium [29]. Salvia miltiorrhiza has the best effect on atherosclerosis. It is reported that its main active components tetramethylpyrazine and paeoniflorin can reduce atherosclerosis. The combination of tetramethylpyrazine and paeoniflorin inhibits ox-LDL-induced HUVEC angiogenesis by inhibiting VEGF / VEGFR2 and Jagged1 / Notch1 signaling pathways, which may contribute to the stability of atherosclerotic plaques [30]. However, the mechanism of the combination of Salvia miltiorrhiza and Ligusticum chuanxiong on angiogenesis is still unclear.

In this paper, the active components, action targets and related pathways of the combination of Salvia miltiorrhiza and Ligusticum chuanxiong on angiogenesis were discussed by using the method of network pharmacology. There were 10 active components in Salvia miltiorrhiza, 2 active components in Ligusticum chuanxiong, 50 targets in Salvia miltiorrhiza and 4 targets in Ligusticum chuanxiong.Luteolin is the most important active ingredient. Luteolin is a polyphenolic flavonoid compound, which has a variety of biological activities such as anti-inflammation, antioxidation, anti-tumor and so on. Some studies have shown that luteolin has an inhibitory effect on angiogenesis, thus achieving the effect of anti-cancer [33].

There are 7 nodes and 24 sides in the protein interaction network of Salvia miltiorrhiza and Ligusticum chuanxiong, in which MMP9 gene plays an important role in the network, which may be an important target for the combination of Salvia miltiorrhiza and Ligusticum chuanxiong to affect angiogenesis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases that can degrade extracellular matrix. MMP9 is an important member of the MMP family and plays an important role in angiogenesis and cell migration. MMP9 was first found to be up-regulated in inflammation and some cancers, and its role in tumor invasion and metastasis is closely related to its promotion of angiogenesis [34].

Through the KEGG enrichment analysis of the predictive targets of Salvia miltiorrhiza and Ligusticum chuanxiong on angiogenesis,it can be found that the combination of Salvia miltiorrhiza and Ligusticum chuanxiong can regulate the cancer pathway by affecting angiogenesis. In 1971, Folkman proposed a whole new way of thinking about cancer in the New England Journal of Medicine.Through the application of effective strategies to inhibit the key participants driving tumor angiogenesis, so as to make the tumor"starve to death" [35]. Since then, Gullino proved in 1976 that cells in precancerous tissue acquire the ability of angiogenesis in the process of carcinogenesis, and put forward anti-angiogenesis as a strategy to prevent cancer. As a revolutionary method for the treatment of cancer, it has become one of the most exciting areas of scientific exploration today [36].

To sum up, this study studied the mechanism of the combination of Salvia miltiorrhiza and Ligusticum chuanxiong on angiogenesis through the method of network pharmacology combined with experimental verification. It was found that the combination of Salvia miltiorrhiza and Ligusticum chuanxiong could achieve the anticancer effect by affecting angiogenesis, and its mechanism may be related to MMP9, MMP2 and other targets. Based on the analysis of the effective components, target proteins and action pathways of the two drugs, it is concluded that Salvia miltiorrhiza-Ligusticum chuanxiong plays a role in angiogenesis-related diseases through multiple targets, multiple pathways and multiple components.

Author contribution

Hou Lili: network Pharmacology, Molecular docking and PCR experiment.Liu Haixin: experimental part of Angiogenesis Analysis and guiding experiment.

Li Qingshan: the guidance of exper