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. 2021 May 25;6(22):14677–14691. doi: 10.1021/acsomega.1c01847

Study on the Mechanism of the Danggui–Chuanxiong Herb Pair on Treating Thrombus through Network Pharmacology and Zebrafish Models

Mengqi Zhang †,‡, Peihai Li †,‡, Shanshan Zhang †,‡, Xuanming Zhang †,‡, Lizhen Wang †,‡, Yun Zhang †, Xiaobin Li †,§,*, Kechun Liu †,*
PMCID: PMC8190889  PMID: 34124490

Abstract

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Danggui–Chuanxiong (DC) is a commonly used nourishing and activating blood medicine pair in many gynecological prescriptions and modern Chinese medicine. However, its activating blood mechanism has not been clearly elucidated. Our research aimed at investigating the activating blood mechanisms of DC using network pharmacology and zebrafish experiments. Network pharmacology was used to excavate the potential targets and mechanisms of DC in treating thrombus. The antithrombotic, anti-inflammatory, antioxidant, and vasculogenesis activities of DC and the main components of DC, ferulic acid (DC2), ligustilide (DC7), and levistilide A (DC17), were evaluated by zebrafish models in vivo. A total of 24 compounds were selected as the active ingredients with favorable pharmacological parameters for this herb pair. A total of 89 targets and 18 pathways related to the thrombus process were gathered for active compounds. The genes, TNF, CXCR4, IL2, ESR1, FGF2, HIF1A, CXCL8, AR, FOS, MMP2, MMP9, STAT3, and RHOA, might be the main targets for this herb pair to exert cardiovascular activity from the analysis of protein–protein interaction and KEGG pathway results, which were mainly related to inflammation, vasculogenesis, immunity, hormones, and so forth. The zebrafish experiment results showed that DC had antithrombotic, anti-inflammatory, antioxidant, and vasculogenesis activities. The main compounds had different effects of zebrafish activities. Especially, the antithrombotic activity of the DC17H group, anti-inflammatory activities of DCH and DC2H groups, antioxidant activities of DCM, DCH, DC2, DC7, and DC17 groups, and vasculogenesis activities of DCM, DCH, and DC2 groups were stronger than those of the positive group. The integrated method coupled zebrafish models with network pharmacology provided the insights into the mechanisms of DC in treating thrombus.

1. Introduction

Thrombotic diseases are a group of diseases that can affect tissues and organs throughout the body and seriously endanger human health and life. After thrombosis, blood flow is blocked, or the downstream blood flow is interrupted by the embolism, which will cause ischemia and necrosis of tissues and organs and further induce various serious cardiovascular and cerebrovascular diseases.1

Traditional Chinese medicine (TCM) is an effective method to prevent and treat thrombosis due to its synergistic effect and few side effects.2 Danggui (the radix of Angelica sinensi (Oliv.) Diels) and Chuanxiong (the radix of Ligusticum chuanxiong Hort.) were both first recorded in the traditional Chinese medicine classics of Shennong Bencao Jing (200–300 A.D., Han Dynasty). Danggui and Chuanxiong have basically the same effect. They are blood-activating drugs and are usually used for the treatment of cardiovascular diseases. Danggui still has a certain effect of nourishing blood in the application.3 Paired use of these two drugs can enhance the effects of promoting blood circulation, removing blood stasis, and nourishing blood.4 High-dose extracts of the Danggui–Chuanxiong herb pair (DC, 80 mg/kg) had an antagonistic effect on the formation of thrombus in experimental rats.5 In addition, DC showed certain anti-platelet aggregation and elimination of blood stasis effects.6,7

Network pharmacology has become a novel and effective method to observe the effects of drugs at the organ and organism levels and to explore the pharmacological mechanism of TCMs. Network pharmacology based on database information of genes, proteins, diseases, and drugs systematically observes the intervention and influence of drugs on the disease, revealing the synergy features of multimolecule drugs.8,9 The mechanisms of the herb pair Danggui–Honghua for treating blood stasis syndrome were predicted by the network pharmacology approach.10 The effect of Shexiang Baoxin Pill on regulating disease by gene interactions was uncovered by network pharmacology.11

Zebrafish is a small tropical freshwater fish that can be used as a vertebrate model for human disease research.12 Compared with other common in vivo models such as mice, zebrafish has many advantages,13 such as low cost, short life cycle, easy experimental operation, low demand weight of test compounds, and high-throughput screening.14 The zebrafish model has a thrombosis process similar to that of mammals.15 Using zebrafish as a model to study the effects of drugs on thrombosis has certain advantages.

In this study, we used network pharmacology and zebrafish models to analyze the mechanism of DC in the treatment of thrombus (Figure 1). It might provide a research foundation for better application of DC in treating thrombus.

Figure 1.

Figure 1

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Conceptual framework of this study.

2. Results and Discussion

2.1. Active Ingredients

The constituents in Danggui and Chuanxiong were gathered from TcmSP and manually replenished through extensive text mining methods. The major components of Danggui and Chuanxiong are both phthalides and organic acids.16,17 Moreover, Chuanxiong is also rich in alkaloids.17 A total of 288 (mutual 27) constituents were gathered from Danggui (125) and Chuanxiong (190) (Table S1).

Two key ADME (absorption, distribution, metabolism, and excretion) parameters, oral bioavailability (OB) and druglikeness (DL), were used to screen the active compounds in DC. In addition, some compounds with high content and biological activity were also selected, even though they did not meet the two criteria. The ultrahigh-performance liquid chromatography Q-Exactive HF mass spectrometry (UHPLC-Q-Exactive HF/MS) method was used to confirm that the selected active compound was included in the DC extract (Figure S1). A total of 24 compounds were selected as the active ingredients for this herb pair (Table 1).

Table 1. Active Compounds and Their ADME Parameters of DCa.

2.1.

2.1.

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a

AS stands for A. sinensis (Oliv.) Diels; LC stands for L. chuanxiong Hort.

The main active ingredients were organic acids (DC1–DC6) and phthalides (DC7–DC18). DC2 is the quality-control component of Danggui and Chuanxiong in the Chinese Pharmacopoeia.18 It can significantly improve blood flow, inhibit platelet aggregation, and reduce blood lipids to prevent thrombosis.19 DC17 is the quality-control component of Chuanxiong in the Chinese Pharmacopoeia.18 DC4, the ester of ferulic acid, is abundant in Danggui and also has vasodilating and antioxidant effects.20,21 DC1 has antioxidant, anti-inflammatory, and anti-cancer effects.22 Moreover, DC1 and its ester, DC3, are reported to have antioxidant and cardio-protective properties.23 Although phthalides have low DL values, they have significant effects on the cardiovascular and cerebrovascular systems.17 DC7 and DC9 possess anti-platelet aggregation and anti-thrombosis effects and regulate heart function.24,25 DC12 and DC13 could induce heme oxygenase-1 expression and inhibit smooth muscle cell proliferation.26,27 DC16 could alleviate cardiac fibrosis in aging rats after myocardial infarction.28 DC18 can be used as a pharmaceutical composition for treating or preventing cardiovascular diseases.29 DC20 is considered to be the effective component of Chuanxiong with anti-platelet aggregation, vasodilation, and anti-portal hypertension effects.30

2.2. Target Proteins of DC

A total of 89 targets related to the thrombus process for active compounds were gathered from similarity ensemble approach (SEA) and STITCH databases and filtrated with the therapeutic target database (TTD), comparative toxicogenomics database (CTD) and existing literature reports (Table 2). ABCB1, ABCC4, and ABCG2 (ABC transporters) in the heart are related to the outflow of therapeutic and cardiotoxic agents from the heart. They also affect the conduction characteristics of the heart KATP potassium channels (SUR proteins) or contribute to the lysosomal function.31 ESR1 and ESR2 in the vascular system are relevant to the functional recovery of injured blood vessels.32 Carbonic anhydrase (CA) expression in the ventricle may be along with ventricular hypertrophy/failure. Elevated CA can be used as a biomarker for early detection of cardiac hypertrophy and heart failure. CA inhibition can improve the function of the heart.33

Table 2. Target Information of DC.

no. target Uniprot ID gene name
T1 multidrug resistance protein 1 P13568 ABCB1
T2 multidrug resistance-associated protein 4 O15439 ABCC4
T3 ATP-binding cassette sub-family G member 2 Q9UNQ0 ABCG2
T4 arachidonate 5-lipoxygenase P09917 ALOX5
T5 5-lipoxygenase-activating protein P20292 ALOX5AP
T6 DNA-(apurinic or apyrimidinic site) lyase P27695 APEX1
T7 androgen receptor P10275 AR
T8 CA 1 P00915 CA1
T9 CA 12 O43570 CA12
T10 CA 13 Q8N1Q1 CA13
T11 CA 14 Q9ULX7 CA14
T12 CA 2 P00918 CA2
T13 CA 3 P07451 CA3
T14 CA 6 P23280 CA6
T15 CA 7 P43166 CA7
T16 CA 9 Q16790 CA9
T17 T-cell surface glycoprotein CD4 P01730 CD4
T18 CDGSH iron–sulfur domain-containing protein 1 Q9NZ45 CISD1
T19 C-X-C chemokine receptor type 4 P61073 CXCR4
T20 steroid 17-α-hydroxylase/17,20 lyase P05093 CYP17A1
T21 cytochrome P450 19A1 P11511 CYP19A1
T22 cytochrome P450 1A1 P00185 CYP1A1
T23 cytochrome P450 1B1 Q16678 CYP1B1
T24 1,25-dihydroxyvitamin d(3)24-hydroxylase, mitochondrial Q07973 CYP24A1
T25 7-dehydrocholesterol reductase Q9UBM7 DHCR7
T26 dihydrofolate reductase P9WNX1 DHFR
T27 dynamin-1 Q05193 DNM1
T28 estrogen receptor P03372 ESR1
T29 estrogen receptor β Q92731 ESR2
T30 proteinase-activated receptor 1 P25116 F2R
T31 fatty acid amide hydrolase 1 O00519 FAAH
T32 fatty acid-binding protein, heart P05413 FABP3
T33 fibroblast growth factor 1 P05230 FGF1
T34 fibroblast growth factor 2 P09038 FGF2
T35 vascular endothelial growth factor receptor 1 P17948 FLT1
T36 glutamate carboxypeptidase 2 Q04609 FOLH1
T37 folate receptor α P15328 FOLR1
T38 proto-oncogene c-Fos P01100 FOS
T39 glucose-6-phosphate 1-dehydrogenase P11413 G6PD
T40 vitamin D-binding protein P02774 GC
T41 lactoylglutathione lyase Q04760 GLO1
T42 G-protein-coupled bile acid receptor 1 Q8TDU6 GPBAR1
T43 metabotropic glutamate receptor 2 Q14416 GRM2
T44 glutathione reductase, mitochondrial P00390 GSR
T45 hypoxia-inducible factor 1-α Q16665 HIF1A
T46 3-hydroxy-3-methylglutaryl-coenzyme A reductase P04035 HMGCR
T47 insulin-like growth factor-binding protein 1 P08833 IGFBP1
T48 insulin-like growth factor-binding protein 2 P18065 IGFBP2
T49 insulin-like growth factor-binding protein 4 P22692 IGFBP4
T50 interleukin-2 P60568 IL2
T51 interleukin-8 P10145 IL8
T52 klotho Q9UEF7 KL
T53 microtubule-associated protein tau P10636 MAPT
T54 72 kDa type IV collagenase P08253 MMP2
T55 matrix metalloproteinase-9 P14780 MMP9
T56 methionine synthase P13009 MTR
T57 NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 4-like 2 Q9NRX3 NDUFA4L2
T58 acyl carrier protein, mitochondrial O14561 NDUFAB1
T59 nuclear factor erythroid 2-related factor 2 Q14494 NFE2L2
T60 nuclear factor NF-kappa-B p105 subunit P19838 NFKB1
T61 NPC1-like intracellular cholesterol transporter 1 Q71UR5 NPC1L1
T62 puromycin-sensitive aminopeptidase P55786 NPEPPS
T63 oxysterols receptor LXR-α Q13133 NR1H3
T64 bile acid receptor Q96RI1 NR1H4
T65 programmed cell death protein 4 Q53EL6 PDCD4
T66 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase γ-1 P19174 PLCG1
T67 PTPsigma-(brain) Q13332 PTPsigma
T68 dihydropteridine reductase P09417 QDPR
T69 transforming protein RhoA P61586 RHOA
T70 corticosteroid-binding globulin P08185 SERPINA6
T71 sex hormone-binding globulin P04278 SHBG
T72 NAD-dependent protein deacylase sirtuin-5, mitochondrial Q9NXA8 SIRT5
T73 probable global transcription activator SNF2L2 P51531 SMARCA2
T74 serine palmitoyltransferase 2 O15270 SPTLC2
T75 3-oxo-5-α-steroid 4-dehydrogenase 2 P31213 SRD5A2
T76 somatostatin P61278 SST
T77 signal transducer and activator of transcription 3 P40763 STAT3
T78 tumor necrosis factor P01375 TNF
T79 transient receptor potential cation channel subfamily A member 1 O75762 TRPA1
T80 short transient receptor potential channel 3 Q8NET8 TRPC3
T81 short transient receptor potential channel 6 Q9H1D0 TRPC6
T82 transthyretin P02766 TTR
T83 tubulin β-1 chain Q9H4B7 TUBB1
T84 tubulin β-3 chain Q13509 TUBB3
T85 thymidylate synthase P0A884 TYMS
T86 tyrosinase P14679 TYR
T87 urotensin-2 receptor Q9UKP6 UTS2R
T88 transitional endoplasmic reticulum ATPase P55072 VCP
T89 vitamin D3 receptor P11473 VDR
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2.3. Network Construction and Mechanism Analysis

2.3.1. Component–Target Network

The component–target (C–T) network was constructed using Cytoscape 3.6.1 software to visualize the corresponding relationship between compounds and targets. A total of 130 associations were generated between 24 active ingredients and 89 targets for DC (Figure 2). The average degree of the potential target per active compound from Danggui was 6.65 and that from Chuanxiong was 4.25. The compounds with high interconnections illustrated the multirelationships between compounds and targets in the C–T network. DC1 (degree = 23), DC21 (degree = 18), DC2 (degree = 13), and DC4 (degree = 13) showed higher relevance degrees, which may play important roles in treating thrombus for DC. This is consistent with the fact that phenolic acid compounds are the active ingredients of DC in the literature.34 As shown in the C–T network (Figure 2), the efficacy of this herb pair was not only concentrated on modulating the crucial targets involved in the thrombosis (F2R, TUBB1, HIF1A, MMP2, and MMP9) but also, more essentially, focused on the regulation of the other proteins mediating inflammation and vascular systems (IL2, TNF, ESR1, ESR2, MAPT, FGF1, and FGF2).

Figure 2.

Figure 2

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C–T network of DC. The squares are active compounds, and the ellipses represent targets. The red squares are active compounds from Danggui, the yellow ones represent those from Chuanxiong, and the orange ones are those shared. The chemical names of DC1–DC24 can be found in Table 1. The light-green ellipses are targets hit by the compounds of Danggui, the green ones represent those by Chuanxiong, and the blue ones are those shared.

2.3.2. Target–Pathway Network

The selected target protein information was imported into the database for annotation, visualization, and integrated discovery (DAVID) to get the pathways of the target enrichment. The target–pathway (T–P) network was constructed using Cytoscape 3.6.1 software to visualize the corresponding relationship between targets and pathways (Figure 3). The main pathways in the T–P network included the metabolic pathways (degree = 19), pathways in cancer (degree = 13), proteoglycans in cancer (degree = 10), nitrogen metabolism (degree = 9), microRNAs in cancer (degree = 9), and T cell receptor signaling pathway (degree = 7). In addition to these highly relevant pathways, there were several pathways about hormones, including the steroid hormone biosynthesis, prolactin signaling pathway, ovarian steroidogenesis, estrogen signaling pathway, and steroid biosynthesis. The major targets were NFKB1 (degree = 7), PLCG1 (degree = 7), RHOA (degree = 6), STAT3 (degree = 6), MMP9 (degree = 5), FOS (degree = 5), ESR1 (degree = 4), MMP2 (degree = 4), TNF (degree = 4), and CYP17A1 (degree = 4).

Figure 3.

Figure 3

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T–P network of DC. The ellipses are targets, and the arrows represent pathways of which the size is proportional to its degree.

The formation of thrombus is one of the important mechanisms that cause cardiovascular disease (CVD). In various cardiovascular diseases, antithrombotic therapy has played a positive role in reducing the disability and mortality.35,36 The T–P network was constructed; the leading pathway was metabolic pathways. Hypertension, thrombosis, and other diseases are defined as metabolic syndrome and cause endothelial dysfunction, which in turn accelerates atherosclerosis.37 In addition, CVD is a major vascular complication of metabolic disease hyperglycemia. The link between hyperglycemia and the CVD focuses on the role of mediators and pathways, containing oxidative stress, glucose metabolic pathway, and nonoxidative glucose pathways.38 Nitrogen metabolism is another important pathway with a high degree. Nitric oxide (NO) and nitrite are the intermediate metabolites in nitrogen metabolism. Nitrite can be converted into NO under pathological conditions and therefore represents a physiologically relevant NO capacity in blood or tissue.39 NO produced physiologically has a useful function in the cardiovascular system, including controlling the vascular tone, leukocyte adhesion, and platelet aggregation.40,41 In addition, NO also has anti-inflammatory and antioxidant effects.40,42 The T cell receptor signaling pathway is the vital regulated pathway because it controls the oxygen diffusion to the central part of the heart.43

2.3.3. Protein–Protein Interaction Network

In order to further explore the importance of the selected targets, the protein–protein interaction (PPI) of target proteins selected above was built from the STRING platform (https://string-db.org/),44 and the PPI network was obtained through Cytoscape 3.6.1 software. There were 85 nodes and 346 edges in the PPI network, and the more the number of the edge, the stronger the interaction of the protein (Figure 4). ESR1, CXCL8, TNF, AR, FOS, FGF2, CXCR4, MMP2, IL2, HIF1A, MMP9, and STAT3 showed stronger interactions with other factors, which maybe the main targets for DC in treating thrombus. These targets are mainly regulating factors related to inflammation, vasculogenesis, immunity, hormones, and so forth.45−47

Figure 4.

Figure 4

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PPI network of intersection targets.

2.4. Zebrafish Activities of DC In Vivo

From the abovementioned results, the main genes analyzed from the PPI network and KEGG pathway were mainly related to inflammation, vasculogenesis, immunity, hormones, and so forth. The pharmacological experiments on zebrafish models included anti-thrombotic, anti-inflammatory, antioxidant, and vasculogenesis activity experiments. Danggui and Chuanxiong both contain DC2, DC7, and other components. Among them, DC2 is the main component of Danggui and Chuanxiong. It is also an index component for the quality control of both Danggui and Chuanxiong in the 2020 Chinese Pharmacopoeia.18 DC17 is the quality-control component of Chuanxiong in the Chinese Pharmacopoeia.18 Therefore, the zebrafish activities of DC2, DC7, and DC17 components were also evaluated.

2.4.1. Antithrombotic Activity

First, the activating blood effect of DC was proven using a zebrafish thrombosis model. Zebrafish larvae have a heart → dorsal aorta and dorsal longitudinal anastomotic → caudal vein → heart blood circulation system. A thrombus formed in the zebrafish caudal vein results in the reduction of heart red blood cells (RBCs).48 Therefore, the staining intensity of the heart RBCs was used to evaluate the degree of thrombus formation. The model of arachidonic acid (AA)-induced zebrafish thrombosis was used to evaluate the activating blood effects of DC, DC2, DC7, and DC17 in vivo. In the model control group, the stained RBC intensity declined compared with that in the normal control group (circled in the red line, P < 0.01) (Figure 5). The intensity of heart RBCs in DC, DC2, and DC17 groups increased compared with that in the model control group. The activities in DC and DC17 groups were concentration-dependent. Especially, the RBC intensity of the DC17H group was more than that of the positive control group. These results suggested that DC and DC17 could prevent the thrombus formation induced by AA. DC17 may be the main pharmacodynamic substance for DC to exert antithrombotic activity.

Figure 5.

Figure 5

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(A) Antithrombotic activity of DC, DC2, DC7, and DC17. (B) Representative pictures of DC, DC2, DC7, and DC17 groups. ##P < 0.01, compared with the normal control group. **P < 0.01; *P < 0.05, compared with the model control group. DCL, DCM, and DCH, administrated with DC of 20, 40, and 80 μg/mL; DC2L, DC2M, and DC2H, treated with DC2 of 1, 5, and 10 μg/mL; DC7L, DC7M, and DC7H, given DC7 of 1, 5, and 10 μg/mL; and DC17L, DC17M, and DC17H, administrated with DC17 of 1, 5, and 10 μg/mL.

2.4.2. Anti-inflammatory Activity

Inflammatory cytokines can stimulate and regulate the coagulation system; the two interact and are closely related to thrombosis.49 CuSO4 as an inorganic salt can induce neuromast damage in the zebrafish lateral line system and can easily cause the characteristics of inflammation, namely, the infiltration of inflammatory cells.50 In the present study, the anti-inflammatory activities of DC, DC2, DC7, and DC17 were assessed in the zebrafish inflammation model induced by CuSO4. The results showed that the inflammatory cells of zebrafish migrated significantly after CuSO4 treatment, namely, green fluorescent cells above the red horizontal line (Figure 6). All test groups had varying degrees of anti-inflammatory effects. Especially, the effects of DCH and DC2H groups (P < 0.01) were stronger than those of the positive control group. These results indicated that DC and DC2 had a much stronger effect of preventing CuSO4-induced inflammation. DC2 may be the main pharmacodynamic substance for DC to exert the anti-inflammatory activity.

Figure 6.

Figure 6

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(A) Anti-inflammatory activity of DC, DC2, DC7, and DC17. (B) Representative pictures of DC, DC2, DC7, and DC17 groups. ##P < 0.01, compared with the normal control group. **P < 0.01; *P < 0.05, compared with the model control group. DCL, DCM, and DCH, administrated with DC of 20, 40, and 80 μg/mL; DC2L, DC2M, and DC2H, treated with DC2 of 1, 5, and 10 μg/mL; DC7L, DC7M, and DC7H, given DC7 of 1, 5, and 10 μg/mL; and DC17L, DC17M, and DC17H, administrated with DC17 of 1, 5, and 10 μg/mL.

2.4.3. Antioxidant Activity

In the vascular system, oxidative stress helps in regulating vasoconstriction, platelet aggregation, angiogenesis, and other physiological reactions.51 Many studies have shown that oxidative stress is involved in the pathological process of thrombotic diseases, such as arterial atherosclerosis, cerebral apoplexy, cardiac muscle infarction, and pulmonary embolism.52,53 For evaluating antioxidant activities of DC, DC2, DC7, and DC17, metronidazole (MTZ) was used as the modeling drug because it can result in reactive oxygen species overproduction, skin cell apoptosis, and skin fluorescent spot (FS) reduction.54 The FS amount of the model control group was significantly less than that of the normal control group (Figure 7). The amounts of FS increased slightly when treated with DC, DC2, DC7, and DC17. These results suggested that DC, DC2, DC7, and DC17 could alleviate the oxidative stress response induced by MTZ.

Figure 7.

Figure 7

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(A) Antioxidant activity of DC, DC2, DC7, and DC17. (B) Representative pictures of DC, DC2, DC7, and DC17 groups. ##P < 0.01, compared with the normal control group. *P < 0.05, compared with the model control group. DCL, DCM, and DCH, administrated with DC of 20, 40, and 80 μg/mL; DC2L, DC2M, and DC2H, treated with DC2 of 1, 5, and 10 μg/mL; DC7L, DC7M, and DC7H, given DC7 of 1, 5, and 10 μg/mL; and DC17L, DC17M, and DC17H, administrated with DC17 of 1, 5, and 10 μg/mL.

2.4.4. Vasculogenesis Activity

Vascular damage, reduced blood flow, and increased clotting are the main causes of thrombosis.55 Vatalanib (PTK787) is an inhibitor of angiogenesis.56 In the vasculogenesis activity experiment, the intersegmental vessel (ISV) number of the model control group lessened obviously after PTK787 treatment, revealing a serious vascular injury (Figure 8). The ISV number of DC and DC2 treatment groups increased compared with that of the model control group. Especially, the ISV numbers of DCH, DC2L, DC2M, and DC2H groups were more than those of the positive control group (P < 0.05). The activities in DC and DC2 groups were concentration-dependent. The result indicated that DC and DC2 had preventive effects against PTK787-induced vascular injury. However, the vasculogenesis activity of the DC7 groups declined as the concentration increased. DC17 groups even had the effect of inhibiting angiogenesis. DC7 and DC17 may not be the main pharmacodynamic substances for DC to exert vasculogenesis activity.

Figure 8.

Figure 8

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(A) Vasculogenesis activity of DC, DC2, DC7, and DC17. (B) Representative pictures of DC, DC2, DC7, and DC17 groups. ##P < 0.01, compared with the normal control group. **P < 0.01; *P < 0.05, compared with the model control group. DCL, DCM, and DCH, administrated with DC of 20, 40, and 80 μg/mL; DC2L, DC2M, and DC2H, treated with DC2 of 1, 5, and 10 μg/mL; DC7L, DC7M, and DC7H, given DC7 of 1, 5, and 10 μg/mL; and DC17L, DC17M, and DC17H, administrated with DC17 of 1, 5, and 10 μg/mL.

The main genes of the DC herb pair analyzed from the network pharmacology were mainly related to inflammation, vasculogenesis, immunity, hormones, and so on. DC displayed anti-inflammatory, antioxidant, and vasculogenesis activities in zebrafish experiments. Additionally, the main compounds of DC, DC2, DC7, and DC17 showed different effects of anti-inflammatory, antioxidant, and vasculogenesis activities. The anti-thrombotic effect of DC should be through the combined effects of different phytochemical components acting on different targets. The zebrafish experiments verified the results of network pharmacology in vivo, and the combination of these two experiments conducted a preliminary exploration of the anti-thrombotic mechanism of DC. Systematic studies are necessary to explore the basis and mechanism of DC responsible for the pharmacological activities, which will be conducted in our further study.

3. Conclusions

Danggui and Chuanxiong are blood-activating drugs and usually used for treating cardiovascular diseases. Danggui still has a certain effect of nourishing blood. The effects of promoting blood circulation, removing blood stasis, and nourishing blood can be enhanced with paired use of these two drugs. In this study, we used network pharmacology and zebrafish models to analyze the mechanism of DC in the treatment of thrombus. In the results of network pharmacology, 24 active ingredients and 89 related targets were obtained for DC treatment of thrombus. By combining the PPI and KEGG pathway analysis, the main targets for the herb were mainly related to inflammation, vasculogenesis, immunity, hormones, and so forth. In addition, the zebrafish experiment results showed that DC had antithrombotic, anti-inflammatory, antioxidant, and vasculogenesis activities. The main compounds of DC, DC2, DC7, and DC17 had different effects on zebrafish activities. The antithrombotic mechanisms of DC may be through its phytochemical components acting on different targets of anti-inflammatory, antioxidation, vasculogenesis, and so forth. These results offer a support for the medicinal uses of DC.

4. Materials and Methods

4.1. Network Pharmacology Analysis

4.1.1. Chemical Ingredient Database Building and Active Ingredient Screening

The construction of Danggui (the radix of A. sinensis (Oliv.) Diels) and Chuanxiong (the radix of L. chuanxiong Hort) chemical composition database is mainly based on the TcmSP (http://tcmspw.com/tcmsp.php),57 Chinese Academy of Sciences database (www.organchem.csdb.cn), Scifinder (https://www.cas.org/products/scifinder), and China Knowledge Network databases. The compounds were saved using the Drug Bank server (https://www.drugbank.ca/) or Chemdraw software in the mol2 format and SMILES format for later analysis. Meanwhile, important pharmacological parameters of the compound were obtained from TcmSP, including the molecular weight, OB, DL, number of donor atoms for H-bonds (nHDon), and number of acceptor atoms for H-bonds (nHAcc).

The chemical components with physical and chemical parameters OB > 30% and DL ≥ 0.2 were selected as active ingredients. Additionally, chemical constituents with cardiovascular pharmacological activity found in previous literature investigations were also selected as active ingredients for subsequent analysis.

4.1.2. Target Fishing

To predict potential targets of active compounds in DC, compounds in the SMILES format were imported into the databases of SEA (http://sea.bkslab.org/) and STITCH (http://stitch.embl.de/).58,59 The potential targets were filtrated using the TTD (http://db.idrblab.net/ttd/) and CTD (http://ctdbase.org/) (screening species was “Homo sapiens”), combining with the existing literature reports to obtain targets related to the CVD process. The target proteins selected above were used to build a PPI network model on the STRING (https://string-db.org/) platform.44

4.1.3. KEGG Pathway Analysis

The selected target protein information was imported into the DAVID (http://david.abcc.ncifcrf.gov) to get the pathways of the target enrichment.60 OFFICIAL GENE SYMBOL and Homo sapiens were selected as the background. A wide range of pathways were excluded using P < 0.05 as the screening condition.

4.1.4. Network Construction

The data of the correlation between the active ingredient and selected target and the selected target and pathway obtained by screening were integrated in the excel tables. Then, C–T and T–P networks of the data were constructed using Cytoscape 3.6.1 software. The PPI of target proteins selected was built from the STRING (https://string-db.org/) platform, and the PPI network was obtained through Cytoscape 3.6.1 software.

4.2. Chemicals and Regents

MTZ, o-dianisidine, tricaine, Vc, and AA were obtained from Solarbio (Beijing, China); aspirin, copper sulfate, and ibuprofen were from Bioleaf Biotech Co., Ltd. (Shanghai, China); PTK787 was from Abcam (Cambridge, UK); Danhong injection was from Heze Buchang Pharmaceutical Co., Ltd. (Shandong, China); ferulic acid, cis-ligustilide, and levistolid A were from Shanghai Jianglai Biochemical Co., Ltd. (Shanghai, China); and dimethyl sulfoxide (DMSO) was supplied by Shanghai Shenggong Co., Ltd. (Shanghai, China). The other chemicals and reagents used in this study were of analytical grade.

4.3. Medicinal Materials and Extraction

Danggui (voucher specimen no. 2020042001) and Chuanxiong (voucher specimen no. 2020042002) medicinal materials were bought from Jinan Jianlian Chinese Medicine Shop and stored at Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan, China. These two materials were ground into crude powder, mixed in proportion (1:1), and then extracted with distilled water (1:8, w/v) twice under reflux for 2 h. The filtrate was merged and concentrated at 40 °C and then freeze-dried to obtain the extract of DC.

4.4. Animals and Treatment

The zebrafish of AB strains, Tg: Lyz-EGFP, Tg: (krt4:NTR-hKikGR)cy17 (supplied by Chung-Der Hsiao from Chung Yuan Christian University), and Tg: fli1-EGFP were used for antithrombotic, anti-inflammatory, antioxidant, and vasculogenesis activity experiments, respectively. The zebrafish were kept under a 14 h light/10 h dark cycle at 28.0 ± 0.5 °C. Healthy larvae were selected and then maintained in a light incubator at 28.0 ± 0.5 °C for the activity experiments. The experiments were conducted according to the standard ethical guidelines. The procedures were approved by the Ethics Committee of the Biology Institute of Shandong Academy of Science (no. SWS20200601).

4.4.1. Antithrombotic Activity Experiment

The intensity of RBCs of the heart in the antithrombotic activity experiment was used to assess the activating blood activity capability of DC. A total of 15 groups were contained in the antithrombotic experiment, including the normal control group; positive control group; model control group; and low-, medium-, and high-dosage of DC, DC2, DC7, and DC17 groups (DCL, DCM, and DCH; DC2L, DC2M, and DC2H; DC7L, DC7M, and DC7H; and DC17L, DC17M, and DC17H, respectively). AB strain zebrafish larvae of 3 dpf were randomly assigned to 24-well microplates (10 per well). The normal control group was given DMSO (0.1% v/v), and the positive control group was given aspirin (30 μg/mL). The larvae in the DC groups were administrated with three concentrations of DC of 20, 40, and 80 μg/mL. The larvae in the DC2, DC7, and DC17 groups were treated with three concentrations of DC2, DC7, and DC17 of 1, 5, and 10 μg/mL, respectively. After incubation for 6 h in the light incubator, all groups except the normal control group were treated with AA (80 μM). Incubating for another hour, the zebrafish larvae were stained with 1.0 mg/mL o-dianisidine for 10 min. The venous thrombosis of the zebrafish larvae in each group was observed and photographed using an inverted fluorescence microscope (Olympus IX53, Japan). Image Pro Plus 5.1 processing software was used to quantitatively analyze the erythrocytes in the heart.

4.4.2. Anti-inflammatory Activity Experiment

The divided groups and the dosages of DC were the same as those in the antithrombotic activity experiment. Tg (Lyz:EGFP) strain zebrafish larvae of 3 dpf were randomly divided into 24-well microplates (10 per well). The normal control group was given DMSO (0.1% v/v), and the positive control group was given ibuprofen (20 μM). The larvae in the DC groups were treated with three concentrations of DC of 20, 40, and 80 μg/mL. The larvae in the DC2, DC7, and DC17 groups were given three concentrations of DC2, DC7, and DC17 of 1, 5, and 10 μg/mL, respectively. After incubation for 6 h in the light incubator, all groups except the normal control group were administrated with CuSO4 (20 μM). After incubating for another hour, the zebrafish larvae were anesthetized with tricaine (0.16%, w/v) and photographed using the inverted fluorescence microscope. The number of inflammatory cells that migrated above the caudal notochord was counted.

4.4.3. Antioxidant Activity Assay

The divided groups and dosages of DC were both the same as those in the antithrombotic activity experiment. The zebrafish larvae of 1 dpf of Tg (krt4:NTR-hKikGR)cy17 were used and randomly divided into 24-well microplates (10 per well). The normal control group was treated with DMSO (0.1% v/v), the model control group was treated with MTZ (5 mM), and the positive control group was treated with MTZ (5 mM) and Vc (0.2 mM). The larvae in the DC groups were administrated with MTZ (5 mM) and three concentrations of DC of 20, 40, and 80 μg/mL. The larvae in the DC2, DC7, and DC17 groups were treated with MTZ (5 mM) and three concentrations of DC2, DC7, and DC17 of 1, 5, and 10 μg/mL, respectively. After incubation for 24 h, we observed the skin FSs using an inverted fluorescence microscope and counted their number using Image Pro Plus 5.1.

4.4.4. Vasculogenesis Activity Experiment

In the vasculogenesis activity experiment, the divided groups were the same as those in the antithrombotic activity experiment. The zebrafish larvae of 24 hpf of Tg (fli1:EGFP) strain were used and randomly divided into 24-well microplates (10 per well). The normal control group was treated with DMSO (0.1% v/v), the model control group was treated with PTK787 (0.225 μg/mL), and the positive control group was treated with PTK787 (0.225 μg/mL) and Danhong injection (9 μL/mL). The larvae in the DC groups were given PTK787 (0.225 μg/mL) and three concentrations of DC of 20, 40, and 80 μg/mL. The larvae in the DC2, DC7, and DC17 groups were administrated with PTK787 (0.225 μg/mL) and three concentrations of DC2, DC7, and DC17 of 1, 5, and 10 μg/mL, respectively. After incubation for 24 h in the light incubator, the zebrafish larvae were imaged using the fluorescence microscope. The number of ISV was counted to assess the angiogenic activity.

Acknowledgments

This work was funded by China Postdoctoral Science Foundation (2020M672100 and 2019M662418), the Shandong Provincial Natural Science Foundation (ZR2020QD103), the Foundation of State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of Technology, Shandong Academy of Sciences (ZZ20190406), the Major Project of Bioengineering Technology Innovation Center of Shandong Province (2019JSWGCCXZX003, 2018JGX114), the Major Innovation Projects of the Pilot Project for the Integration of Science, Education and Industry, Qilu University of Technology, Shandong Academy of Sciences (2020KJC-ZD08), and the Postdoctoral Innovation Program of Shandong Province (202002047).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c01847.

  • Detailed information of all compounds in DC; total ion chromatograms of the active compounds of DC by ultrahigh-performance liquid chromatography Q-Exactive HF mass spectrometry (UHPLC-Q-Exactive HF/MS) in negative and positive ion modes; and UHPLC-Q-Exactive HF/MS analysis of DC (PDF)

Author Contributions

M.Z. and P.L. contributed equally to this work. K.L., X.L., P.L., and M.Z. established research ideas and designed methods. M.Z., S.Z., and X.Z. conducted experimental operations; P.L. and L.W. collected data; P.L. and Y.Z. analyzed the data; and M.Z, K.L., and X.L. led the manuscript writing. All authors reviewed the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao1c01847_si_001.pdf (209.1KB, pdf)

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