Method for analyzing angiogenesis promoting mechanism of qi-regulating and blood-activating dropping pills based on network pharmacology

Abstract

The invention relates to the technical field of traditional Chinese medicine compound action mechanism research, and discloses a method for analyzing an angiogenesis promoting mechanism of a qi-regulating and blood-activating dropping pill based on network pharmacology, and the method comprises the following steps: S1, blood-entering component identification: identifying blood-entering components and metabolites of the qi-regulating and blood-activating dropping pill to obtain structure information of 18 blood-entering components and metabolites; s2, target spot prediction and screening; s3, core target point analysis; s4, network construction and analysis: constructing a drug-in-blood component-target-disease network, and determining a core in-blood component of which the degree value is greater than the average value through topological analysis; s5, function enrichment and pathway analysis: performing GO function enrichment analysis and KEGG signal pathway analysis on the target spots intersected in S2, and determining a core pathway for promoting angiogenesis; s6, carrying out molecular docking verification; and S7, in-vitro activity verification. Through integration of network pharmacology, molecular docking and in-vitro experiment verification, the action mechanism of the qi-regulating and blood-activating dropping pill for promoting angiogenesis is systematically disclosed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mechanism research of traditional Chinese medicine compound, in particular to a method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology. BACKGROUND

[0002] Coronary heart disease is a disease caused by coronary atherosclerosis, leading to myocardial ischemia, hypoxia and even necrosis. Angiogenesis refers to the formation of new blood vessels from the existing vascular network, which is of great significance to improve myocardial ischemia and promote collateral circulation. However, pathological angiogenesis may lead to plaque instability, while physiological angiogenesis is helpful for myocardial repair.

[0003] Liqi Huoxue Dropping Pills are a kind of traditional Chinese medicine compound preparation, which is composed of Chuanxiong, Xiebai, Daguowuzi and other medicinal materials, and has the effects of promoting blood circulation and removing blood stasis, regulating qi and relieving pain. It is clinically used for the treatment of coronary heart disease. Traditional Chinese medicine compound has the characteristics of multi-component, multi-target and multi-pathway synergistic effect, but its specific pharmacodynamic material basis and mechanism have not been fully elucidated.

[0004] Traditional Chinese medicine active ingredient research mainly relies on active-oriented separation strategy, which tracks target components through extraction, chromatographic separation and other means. However, this method has the following problems: the components of traditional Chinese medicine compound are complex, separation and purification is time-consuming and low-yielding; the activity research of single component cannot reflect the overall mechanism of compound; some low-content components may play a key role through synergistic effect, which is difficult to identify by traditional method. The existing technology about Liqi Huoxue Dropping Pills mainly focuses on clinical efficacy observation and pharmacological action of some single components, and cannot fully identify the blood components, nor can it verify the key active components of promoting angiogenesis through molecular docking and in vitro experiment. SUMMARY

[0005] In view of the above problems in the prior art, the method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology provided by the present application combines serum drug chemistry, molecular docking and in vitro experiment to systematically analyze the material basis and mechanism of Liqi Huoxue Dropping Pills in promoting angiogenesis, and provides a scientific basis for its clinical application and further development.

[0006] In order to achieve the above-mentioned application purposes, the technical scheme adopted by the present application is as follows: a method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology, comprising the following steps:

[0007] S1, blood component identification: identifying the blood components and metabolites of Liqi Huoxue Dropping Pills to obtain the structure information of 18 blood components and metabolites;

[0008] S2, target prediction and screening: the structure of the blood components and metabolites obtained in S1 is introduced into the PubChem database to obtain the SDF structure file, the active target is predicted through the PharmMapper and SwissTargetPrediction databases, the disease targets are screened by combining the GeneCards, OMIM and DrugBank databases, the intersection of drug targets and disease targets is obtained by using Venny2.1.0 software, and the potential action target of promoting angiogenesis is obtained;

[0009] S3, core target analysis: the potential action target obtained in S2 is input into the String database for protein interaction analysis, the target with an interaction score value ≥0.9 is selected, a PPI network is constructed, and the core target with a degree value in the top 12 is screened out by using Cytoscape3.10.3 software;

[0010] S4, network construction and analysis: a "drug-blood component-target-disease" network is constructed, and the core blood component with a degree value greater than the average value is determined by topological analysis;

[0011] S5, functional enrichment and pathway analysis: the target in the intersection of S2 is subjected to GO functional enrichment analysis and KEGG signal pathway analysis to determine the core pathway of promoting angiogenesis.

[0012] S6, molecular docking verification: the blood components and metabolites obtained in S1 are subjected to molecular docking with the key target VEGFR2 protein of angiogenesis, the binding energy S value and RMSD value are calculated, and the high-affinity components with S value <-6.0 kcal / mol and RMSD value <2.0 A are screened out;

[0013] S7, in vitro activity verification: the in vitro tube formation model of HUVECs induced by hypoxia is used to verify the promoting effect of the high-affinity components in S6 on angiogenesis.

[0014] Further, in the above method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology, the 18 blood components and metabolites in S1 include prostaglandin A1, Flavaprin, alpha-linolenic acid, linoleic acid, new snake bed lactone, chuanqiong lactone IIH water, choline, chuanqiong lactone IIH glutathione, 6-ethyl-2,3-dimethyl-pyridine, taros acid, and hexadecanoic acid.

[0015] Further, in the above method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology, the drug targets in S2 are 1250, the disease targets are 1913, and the potential action targets are 276.

[0016] ​Further, the method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology, the core target points in S3 are TP53, AKT1, STAT3, PIK3R1, PIK3CA, PIK3CB, ESR1, BCL2, GRB2, HSP90AA1, MAPK1 and JUN.

[0017] Further, the method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology, the average degree value of the core blood-entering components in S4 is 36.4, and the screened core blood-entering components include prostaglandin A1, Flavaprin, alpha-linolenic acid, linoleic acid, new osterutine, senkyunolide IIH water and glutathione combination, choline, senkyunolide IIH glutathione combination, 6-ethyl-2,3-dimethyl-pyridine, tiglic acid and hexadecanoic acid.

[0018] Further, the method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology, the core pathways in S5 are PI3K-Akt signaling pathway, VEGF signaling pathway and TNF-alpha signaling pathway.

[0019] Further, the method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology, the high-affinity components in S6 are senkyunolide IIH glutathione combination, senkyunolide IIH acetylcysteine combination, linoleic acid, Flavaprin, hexadecanoic acid, new osterutine, senkyunolide H and senkyunolide I.

[0020] Further, the method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology, the specific method of in-vitro activity verification in S7 is that HUVECs cells are cultured under hypoxic conditions, high-affinity components are added respectively, and the formation of tubular structures, including tube length and node number, is observed and counted.

[0021] Further, the method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Dropping Pills based on network pharmacology, the GO function enrichment analysis in S5 includes biological process, cell component and molecular function, wherein the biological process is related to cell response to hypoxia, angiogenesis and oxidative stress; the cell component is related to cell membrane and vesicle lumen; and the molecular function is related to nuclear receptor activity and transmembrane receptor protein kinase activity.

[0022] The application has the beneficial effects that: the application discloses a mechanism of promoting angiogenesis of Liqi Huoxue Drop Pills by integrating network pharmacology, molecular docking and in-vitro experiment verification. The method accurately identifies 18 blood-entering components and metabolites, determines 276 potential action targets of promoting angiogenesis through target prediction and screening, and constructs a 'drug-blood-entering component-target-disease' network, and finds that the PI3K / AKT / HIF-1 alpha signal axis is the core pathway for regulating angiogenesis. Molecular docking verifies that eight active ingredients have high affinity, and in-vitro experiments further confirm that Yangchuanqiong lactone I and II promote angiogenesis. The research not only clarifies the synergistic mechanism of multi-target and multi-pathway of traditional Chinese medicine compound, provides a modern scientific explanation for the traditional 'activating blood and resolving stasis' effect, but also provides an important basis for the optimization of traditional Chinese medicine quality control and clinical combined drug use scheme. Compared with the traditional research method, the application significantly improves the efficiency and accuracy of screening of active ingredients of traditional Chinese medicine, and establishes a complete research paradigm from chemical characterization to mechanism verification. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a wein diagram of component targets and disease targets;

[0024] Figure 2 is a protein-protein interaction network diagram;

[0025] Figure 3 is a protein-protein interaction network diagram;

[0026] Figure 4 is a blood-entering component and VEGFR2 molecular docking;

[0027] Figure 5 is a SI / H promoting the formation of tubular structures of HUVECs treated by hypoxia;

[0028] Figure 6 is a node number and tube length statistics of 4h tubular structures;

[0029] Figure 7 is a node number and tube length statistics of 6h tubular structures;

[0030] Figure 8 is a node number and tube length statistics of 8h tubular structures. DETAILED DESCRIPTION

[0031] The specific embodiments of the application are described below to facilitate the understanding of the application by those skilled in the art, but it should be clear that the application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the application defined and determined by the appended claims, and all the application ideas utilizing the application are within the scope of protection.

[0032] The embodiment provides a method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Drop Pills based on network pharmacology, and the method takes Liqi Huoxue Drop Pills (LQHXDP) as a research object, adopts network pharmacology, molecular docking and in vitro experiment, and systematically analyzes active ingredients and the mechanism of promoting angiogenesis of the Liqi Huoxue Drop Pills. The blood entering ingredients are locked through serum drug chemical analysis, a multi-dimensional network of “drug-component-target-pathway” is constructed, key target points are predicted, and the biological activity is verified by molecular docking and in vitro experiment.

[0033] The serum sample of a rat after administration of the LQHXDP is analyzed by UPLC-Q-TOF-MS / MS technology. The chromatographic conditions are as follows: an ACQUITY UPLC BEH C18 chromatographic column (2.1 mm*100 mm, 1.7 μm), a column temperature of 40 DEG C, a flow rate of 0.4 mL / min, and a sample injection amount of 2 μL. The mass spectrometry conditions are as follows: an electrospray ion source (ESI), positive and negative ion mode scanning, and a mass scan range of m / z 50-1500. By comparing the total ion chromatograms of blank serum and drug-containing serum, combining the self-built compound database and the reported fragmentation rules in the literature, 18 blood entering original ingredients and metabolites are identified, including phthalides (Yangchuanmoinnerl, II), fatty acids (alpha-linolenic acid, linoleic acid), flavonoids (Flavaprin) and the like. Among them, the glutathione conjugate (M2) and acetylcysteine conjugate (M3) of Yangchuanmoinnerl IIH are the first discovered metabolites.

[0034] The chemical structures of the 18 blood entering ingredients are introduced into the PubChem database to obtain SDF files, and potential target points are predicted by PharmMapper and SwissTargetPrediction platforms respectively, and the similarity threshold is set to be greater than 0.8, and 1250 drug target points are obtained. Meanwhile, the “coronary heart disease” related target points are retrieved from GeneCards, OMIM and DrugBank databases, and 1913 disease target points are obtained after deduplication. The Venny2.1.0 software is used to draw a Venn diagram, as shown in Figure 1 The blue area is a disease target point, the red area is a compound target point, and 276 common target points are obtained. The common target points are input into the STRING database to construct a protein interaction (PPI) network, and the minimum interaction score is set to be 0.9, and the free node is hidden, as shown in Figures 2-3As shown, a network graph containing 256 nodes and 826 edges was obtained. The network topology parameters were calculated by Cytoscape3.10.3 software, and 12 core target points with a degree value of ≥85 were screened out, including TP53 (degree value 142), AKT1 (degree value 130), STAT3 (degree value 128), PIK3CA, VEGFR2, etc. These target points form a close interaction module in the PI3K-Akt, MAPK and JAK-STAT pathways, among which AKT1 is directly connected with mTOR, eNOS, etc. as a hub node.

[0035] The vascular endothelial growth factor receptor 2 (VEGFR2, PDBID: 4AG8) was selected as the key target point, and the MOE2019.01 software was used for molecular docking. The pretreatment steps included: removing crystal water molecules, adding hydrogen, optimizing the protonation state, and defining the active pocket (with Axitinib as the reference ligand). The docking parameter settings: force field AMBER10, sampling method rigid receptor-flexible ligand, conformation generation number 100, scoring function AffinitydG. As shown in Figure 4 The binding energy (S value) of 13 components is <-6.0 kcal / mol, among which the ligustilide II H glutathione conjugate (M2) shows the best binding characteristics (S = -8.4853 kcal / mol, ), which forms hydrogen bonds with Glu855 and Asp1046 in the active cavity of VEGFR2 (bond lengths are and ), and hydrophobic interactions with Val899 and Leu1044. Compared with the original ligand Axitinib (S = -8.0656 kcal / mol), M2 shows stronger binding stability.

[0036] GO and KEGG enrichment analysis of 276 common targets were performed by DAVID database. GO analysis showed that the biological process (BP) was significantly enriched in "response to hypoxia" (GO:0071456, FDR=3.2×10-15) and "positive regulation of angiogenesis" (GO:0045766, FDR=2.8×10-12); the cellular component (CC) was mainly distributed in "outer side of plasma membrane" (GO:0009897) and "focal adhesion" (GO:0005925); the molecular function (MF) was involved in "protein kinase activity" (GO:0004674) and "growth factor receptor binding" (GO:0070851). KEGG pathway analysis found that the targets were significantly enriched in PI3K-Akt signaling pathway (hsa04151, 51 genes), VEGF signaling pathway (hsa04370, 28 genes) and atherosclerosis pathway (hsa05417, 44 genes). Among them, AKT1, PIK3CA, mTOR and other genes in the PI3K-Akt pathway are closely related to the proliferation, migration and survival of vascular endothelial cells.

[0037] As shown in Figure 5 , the predicted results were verified by using a hypoxia-induced human umbilical vein endothelial cell (HUVEC) model. HUVECs were seeded in a 96-well plate coated with Matrigel matrix (BD Biosciences) (5×103 cells / well) and cultured in a three-gas incubator with 1% O2 and 5% CO2. The experiment was divided into a control group (no drug), a positive control group (50 ng / mL VEGF) and a drug administration group (Yangchuanqionglide I / II, concentration gradient 2, 10, 50 μM). After 4, 6 and 8 hours of culture, inverted microscopes were used to take pictures of the tube structure, and ImageJ software was used to quantify the tube length and branch node number. As shown in Figures 6-8 , the tube length reached (1287±156) μm after 6 hours of treatment with 50 μM Yangchuanqionglide II, which was 129% higher than that of the control group (562±89) μm (P<0.01); the branch node number was (32.4±4.1), which was 120% higher than that of the control group (14.7±2.8) (P<0.01). Western blot detection showed that Yangchuanqionglide II could significantly up-regulate the protein expression levels of p-AKT (Ser473) and HIF-1α (P<0.05), which was consistent with the network prediction results.

[0038] In summary, the mechanism of LQHXDP promoting angiogenesis can be summarized as follows: ① Phthalide components (such as senkyunolide II) and its metabolite M2 / M3 activate VEGFR2 directly, start PI3K-Akt signal cascade, promote eNOS activation and HIF-1α stabilization; ② Fatty acid components (α-linolenic acid, linoleic acid) reduce the release of inflammatory factors by regulating PPARγ / NF-κB pathway, and improve the vascular microenvironment; ③ Flavaprin inhibits COX-2 / TXA2 pathway, and synergistically enhances vasodilation function. These components achieve the biological effects of promoting endothelial cell proliferation, migration and lumen formation through the synergistic effect of “multi-target and multi-pathway”.

Claims

1. A method for analyzing the mechanism of promoting angiogenesis of Liqi Huoxue Drop Pills based on network pharmacology, characterized in that, Comprising the following steps: S1, blood component identification: the blood components and metabolites of Liqi Huoxue Dropping Pills are identified, and the structural information of 18 blood components and metabolites is obtained; S2, target prediction and screening: the structure of the blood components and metabolites obtained in S1 is input into the PubChem database to obtain the SDF structure file, the active targets are predicted through the PharmMapper and SwissTargetPrediction databases, the disease targets are screened by combining the GeneCards, OMIM and DrugBank databases, the intersection of drug targets and disease targets is obtained by using Venny2.1.0 software, and the potential action targets for promoting angiogenesis are obtained; S3, core target analysis: the potential action targets obtained in S2 are input into the String database for protein interaction analysis, the targets with an interaction score value greater than or equal to 0.9 are selected, a PPI network is constructed, and the core targets with the top 12 degree values are screened out by using Cytoscape3.10.3 software; S4, network construction and analysis: a "drug-blood component-target-disease" network is constructed, and the core blood components with a degree value greater than the average value are determined by topological analysis; S5, functional enrichment and pathway analysis: the GO functional enrichment analysis and KEGG signal pathway analysis are performed on the targets in the intersection of S2, and the core pathways for promoting angiogenesis are determined. S6, molecular docking verification: the blood components and metabolites obtained in S1 are subjected to molecular docking with the key target of angiogenesis VEGFR2 protein, the binding energy S value and RMSD value are calculated, and the high-affinity components with S value <-6.0 kcal / mol and RMSD value <2.0 A are screened out. S7, in vitro activity verification: the in vitro tube formation model of HUVECs induced by hypoxia is used to verify the promoting effect of the high-affinity components in S6 on angiogenesis.

2. The method for analyzing the mechanism of promoting angiogenesis of Liqihuanxue Drop Pills based on network pharmacology according to claim 1, characterized in that, The 18 blood components and metabolites in S1 include prostaglandin A1, Flavaprin, alpha-linolenic acid, linoleic acid, neocnidilide, senkyunolide IIH hydrate and glutathione conjugate, choline, senkyunolide IIH glutathione conjugate, 6-ethyl-2,3-dimethyl-pyridine, tiglic acid, and hexadecanoic acid. 3.The method for analyzing the mechanism of promoting angiogenesis of Liqihuanxue Drop Pills based on network pharmacology according to claim 1, characterized in that, The drug targets in S2 are 1250, the disease targets are 1913, and the potential action targets are 276.

4. The method for analyzing the mechanism of promoting angiogenesis of Liqihuanxue Drop Pills based on network pharmacology according to claim 1, characterized in that, The core targets in S3 are TP53, AKT1, STAT3, PIK3R1, PIK3CA, PIK3CB, ESR1, BCL2, GRB2, HSP90AA1, MAPK1, and JUN.

5. The method for analyzing the mechanism of promoting angiogenesis of Liqihuanxue Drop Pills based on network pharmacology according to claim 1, characterized in that, The average degree value of the core blood components in S4 is 36.4, and the selected core blood components include prostaglandin A1, Flavaprin, alpha-linolenic acid, linoleic acid, neocnidilide, senkyunolide IIH hydrate and glutathione conjugate, choline, senkyunolide IIH glutathione conjugate, 6-ethyl-2,3-dimethyl-pyridine, tiglic acid, and hexadecanoic acid.

6. The method for analyzing the mechanism of promoting angiogenesis of Liqihuanxue Drop Pills based on network pharmacology according to claim 1, characterized in that, The core pathways in S5 are the PI3K-Akt signaling pathway, the VEGF signaling pathway, and the TNF-alpha signaling pathway.

7. The method for analyzing the mechanism of promoting angiogenesis of Liqihuanxue Drop Pills based on network pharmacology according to claim 1, characterized in that, The high-affinity components in S6 are senkyunolide IIH glutathione conjugate, senkyunolide IIH acetylcysteine conjugate, linoleic acid, Flavaprin, hexadecanoic acid, neocnidilide, senkyunolide H, and senkyunolide I.

8. The method for analyzing the mechanism of promoting angiogenesis of Liqihuanxue Drop Pills based on network pharmacology according to claim 7, characterized in that, The specific in vitro activity verification of S7 is that HUVECs cells are cultured under hypoxic conditions, high-affinity components are added respectively, and the formation of tubular structures, including tube length and node number, is observed and counted. 9.The method for analyzing the mechanism of promoting angiogenesis of Liqihuanxue Drop Pills based on network pharmacology according to claim 1, characterized in that, The GO function enrichment analysis of S5 includes biological processes, cellular components and molecular functions, wherein the biological processes involve cell response to hypoxia, angiogenesis and oxidative stress; the cellular components involve cell membranes and vesicle lumen; and the molecular functions involve nuclear receptor activity and transmembrane receptor protein kinase activity.

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