Analysis method of active ingredients and mechanism of cold-asthma Zupao granules for treating asthma

Abstract

The invention belongs to the field of traditional Chinese medicine research and development, and relates to a method for analyzing active ingredients and mechanisms of cold-asthma Zupa granules for treating asthma, which comprises the following steps: determining blood entering ingredients of the cold-asthma Zupa granules for treating asthma; searching known active ingredients, combining the known active ingredients with the blood entering ingredients, and removing weight to obtain potential active ingredients; predicting a potential active ingredient action target spot, taking an intersection target spot of the potential active ingredient action target spot and a known asthma related target spot as a potential target spot, and performing GO enrichment analysis and KEGG signal path analysis; constructing a protein-protein interaction network to obtain a core target spot; analyzing enriched pathways by utilizing the potential active components, the potential targets and the signal pathways to construct a network diagram of medicine-component-disease-target-pathway so as to obtain key active components; and determining possible action targets in combination with the core targets and literature reports, and verifying to obtain actual action targets. The method provides reliable data support for the drug effect, material basis and action mechanism of the cold-asthma Zupa granules for treating asthma.

Description

Technical Field

[0001] The present invention belongs to the field of research and development of traditional Chinese medicine, and specifically relates to an analytical method for studying the active ingredients and mechanisms of Hanchuanzupa granules in treating asthma based on UPLC-MS / MS, network pharmacology and in vitro and in vivo experiments. Background Art

[0002] Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. Respiratory symptoms generally include wheezing, shortness of breath, chest tightness and cough. Airway inflammation is the main pathological basis of asthma. Various inflammatory mediators are involved in the inflammatory process, aggravating the damage of respiratory tract and alveolar tissue. Various inflammatory mediators can stimulate excessive proliferation of airway smooth muscle cells, causing airway remodeling, which can cause reduced lung ventilation function. Although inhaled corticosteroids are widely used to control asthma, this method cannot effectively improve the airway remodeling process. Traditional Chinese medicine has the characteristics of comprehensive treatment with all-round and multi-channel in the treatment of asthma, and has the advantages of reliable efficacy and few side effects.

[0003] Hanchuanzupa granules are a commonly used local medicine, widely used in Xinjiang Uygur Autonomous Region, China. It is one of the three Chinese patent medicines that can be used during asthma attacks as specified in the Guidelines for Clinical Use of Chinese Patent Medicines: Respiratory Diseases. It is composed of 9 Chinese medicines, including fennel, hyssop, maidenhair fern, celery seeds, licorice extract, fenugreek, rue, rose, and nettle seeds. The prescription is mainly composed of dry heat drugs, which have the effects of expelling cold and relieving cough, warming the lungs and relieving asthma. Modern pharmacology has confirmed that it has the effects of relieving cough, relieving asthma, and protecting the lungs. However, its material basis and mechanism of action for the treatment of asthma are still unclear, so exploring its mechanism and material basis is of great significance for the screening and development of anti-asthma drugs. Summary of the invention

[0004] In view of this, the present invention provides a method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules in treating asthma. The method identifies the chemical components in the serum containing Hanchuanzupa granules through UPLC-MS / MS, and explores the active ingredients and mechanisms of action of Hanchuanzupa granules in treating asthma through network pharmacology, animal experiments and cell experiments.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides a method for analyzing the active ingredients and mechanism of Hanchuanzupa granules in treating asthma, comprising the following steps: A mouse asthma model was established and Hanchuanzupa granules were administered intragastrically. The chemical components of Hanchuanzupa granules and the components in mouse serum were identified and compared to obtain the blood-entering components of Hanchuanzupa granules. Find known active ingredients of Hanchuanzupa granules for treating asthma, combine them with the blood-entering components, remove duplicates, and obtain potential active ingredients of Hanchuanzupa granules for treating asthma; Predict the targets of potential active ingredients of Hanchuanzupa granules for the treatment of asthma, take the intersection targets of the targets and known asthma-related targets as potential targets of Hanchuanzupa granules for the treatment of asthma, and perform GO enrichment and KEGG signaling pathway analysis on the potential targets; Using the potential targets to construct a protein interaction network, and performing visualization and network topology analysis to obtain core targets; Using the potential active ingredients, the potential targets and the top 20 pathways enriched in the signal pathway, a drug-ingredient-disease-target-pathway network diagram is constructed and a network topology analysis is performed to obtain key active ingredients; The possible targets of Hanchuanzupa Granules for treating asthma were determined by combining the core targets and literature reports, and then verified to obtain the actual targets of Hanchuanzupa Granules for treating asthma.

[0006] Furthermore, OVA was used to stimulate Balb / c mice to establish an asthma model, and low, medium and high doses of Hanchuanzupa granules were administered, and mouse plasma and left lung tissues were collected to detect the levels of IL-4, IL-5, IL-13 and IgE in the serum of each group of mice. The anti-inflammatory effect of Hanchuanzupa granules was evaluated by microscopic observation and HE staining of lung pathological sections, and the anti-inflammatory effect of Hanchuanzupa granules was determined, and the components in mouse serum were identified.

[0007] Furthermore, the chemical components in the Hanchuanzupa granule powder and the serum of mice in the drug administration group and the model group were identified by UPLC-MS / MS, and the detection conditions of the UPLC-MS / MS were as follows: The sheath gas flow rate was 30Arb, the auxiliary gas flow rate was 10Arb, the ion transfer tube temperature was 350Arb, the evaporator temperature was 350°C, the full millisecond resolution was 60000, the MS / MS resolution was 15000, the collision energy was 16 / 38 / 42, the ionization mode was electrospray ionization, and the spray voltage was 5.5KV (positive) or -4KV (negative); Chromatographic conditions: mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, the gradient was as follows: 0-3.5 min, 95-85% A; 3.5-6 min, 85-70% A; 6-6.5 min, 70-70% A; 6.5-12 min, 70-30% A; 12-12.5 min, 30-30% A; 12.5-18 min, 30-0% A; 18-25 min, 0-0% A; 25-26 min, 0-95% A; 26-30 min, 95-95% A; the flow rate was 0.4 mL / min, and the injection volume was 5 μL.

[0008] Furthermore, the known active ingredients of Hanchuanzupa Granules were searched through TCMSP, PubMed, and China National Knowledge Infrastructure. Combined with the blood-entering components of Hanchuanzupa Granules, they were sorted and removed to obtain the potential active ingredients of Hanchuanzupa Granules.

[0009] Furthermore, predicting the target of the potential active ingredients of Hanchuanzupa granules for treating asthma is to import the potential active ingredients into the Swiss Target Prediction database to find the target gene corresponding to each ingredient; Known asthma-related targets were obtained by the following method: the asthma keyword Asthma was input into the GeneCards, TTD, OMIM, and DrugBank databases to obtain the target genes corresponding to the disease; The method for obtaining potential targets includes: taking the intersection of the target gene corresponding to the potential active ingredient and the asthma-related target, which is the potential target of the active ingredient of Hanchuanzupa Granules for treating asthma.

[0010] Furthermore, the specific process of constructing the protein interaction network is as follows: the obtained potential targets are input into the STRING database, the species is selected as Homo sapiens, the protein interaction information is obtained, and it is imported into the Cytoscape software for visualization analysis.

[0011] Furthermore, the top 100 core targets in the protein interaction network were imported into Cytoscape software for cluster analysis to further verify the importance of the core targets; the cluster analysis was to import the top 100 targets in the protein interaction network information into Cytoscape software and use the MOCDE plug-in for cluster analysis.

[0012] Furthermore, the specific process of the GO enrichment analysis and KEGG signal pathway analysis is: importing the potential targets into the DAVID database, selecting "Homo species" in the DAVID database, and further analyzing the relevant results of Hanchuanzupa granules in the treatment of asthma. The relevant results of the analysis include signal pathways, biological processes involved in potential targets, cellular components acted on, and molecular functions of the potential targets.

[0013] Furthermore, the specific process of constructing the drug-ingredient-disease-target-pathway network diagram is as follows: constructing the relationship between the potential active ingredients and potential targets, and the top 20 pathways enriched by signal pathway analysis, and importing them into the Cytoscape software to obtain the constructed drug-ingredient-disease-target-pathway network diagram and perform visual analysis, and further screening based on the aforementioned protein interaction network diagram to obtain key potential targets.

[0014] Furthermore, based on the results of the above-mentioned protein interaction network visualization analysis, the active ingredients with the highest degree values ​​are ranked and selected. The active ingredients with the highest degree values ​​can also be selected in combination with literature and experience.

[0015] Furthermore, possible action targets were determined based on the core targets and relevant literature, and then the possible action targets were selected and verified using the cell inflammation model to obtain the actual action targets of Hanchuanzupa Granules for the treatment of asthma. The verification process is as follows: BEAS-2B cells were stimulated with LPS to construct a cell inflammation model, and the cells were treated with Hanchuanzupa Granules in low, medium, and high doses; the levels of inflammatory factors were detected to evaluate the anti-inflammatory effect of Hanchuanzupa Granules.

[0016] The present invention also provides an analysis device for a traditional Chinese medicine action mechanism model based on network pharmacology, the device comprising at least a processor and a storage device, the processor being used to read a program in the storage device to execute any of the analysis methods described above.

[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: (1) The present invention uses UPLC-MS / MS to identify the chemical components of Hanchuanzupa Granules and their blood-entering components, combines the in vivo and in vitro chemical component detection information of Hanchuanzupa Granules, analyzes the active components of Hanchuanzupa Granules, and combines the components retrieved from TCMSP, PubMed and the active components reported in the literature related to Hanchuanzupa Granules in CNKI, sorts and removes duplicates, predicts the targets of these active components and asthma-related targets, searches for intersection targets, and constructs protein interaction networks, drug-component-target-disease-pathway networks, KEGG and GO networks. Through enrichment analysis and other steps, the core genes and core components of Hanchuanzupa granules in the treatment of asthma were predicted, and the Balb / c mouse asthma model and BEAS-2B cell inflammation model were constructed. The therapeutic effect of Hanchuanzupa granules on the most important pathological feature of airway inflammation was evaluated by qRT-PCR. At the same time, according to the prediction results of network pharmacology, the mRNA expression levels and protein expression levels of related core targets were verified, and the actual targets of Hanchuanzupa granules in the treatment of asthma were explored. Reliable data support was provided for the efficacy, material basis and mechanism of action of Hanchuanzupa granules in the treatment of asthma, which provided a certain scientific basis for the rational utilization and research of ethnic medicine resources, and had certain significance for the development of Xinjiang characteristic ethnic medicine.

[0018] (2) The method provided by the present invention makes up for the deficiency of the traditional "single drug single target" research model that it is difficult to clarify the material basis and mechanism of action of traditional Chinese medicine. Through network pharmacology, network construction, core target screening, key active ingredient screening, functional enrichment analysis and other technical means are used to explore the mechanism of action and material basis of efficacy of traditional Chinese medicine with "multi-target-multi-component" characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a bar graph of the levels of inflammatory factors in the serum of each group of mice detected by ELISA; Figure 2 The following are the pathological sections of the lungs of mice in each group; Figure 3 This is the total ion current diagram of Hanchuanzupa granules in negative ion mode; Figure 4 This is the total ion current diagram of the negative ion mode of the serum of mice in the drug administration group; Figure 5 This is the total ion current diagram of the negative ion mode of serum of mice in the model group; Figure 6 This is a Venn diagram of the targets of the active ingredients of Hanchuan Zupa Granules and the intersection targets of asthma; Figure 7 This is the PPI diagram of potential targets of Hanchuan Zupa Granules; A is the PPI network diagram, and B is the PPI diagram of the top 100 targets in terms of degree value; Figure 8 The figure is a cluster analysis diagram of the top 100 potential targets; A~E are the top 6 subnetworks obtained by MOCDE cluster analysis; Fig. 9 It is a KEGG and GO enrichment analysis diagram; A is the GO enriched BP item, that is, the biological process; B is the CC item, that is, the cellular component; C is MF, that is, the molecular function; D is the visualization diagram of the top 20 pathways of KEGG enrichment analysis; Fig.10 This is a drug-ingredient-disease-target-pathway network diagram; orange is the disease asthma, green triangles are pathways, blue is the target, pink is the ingredient, and green squares are the drug Hanchuanzu Pa'e Granules; Fig.11 This is a bar graph showing the inhibitory effects of low, medium and high doses of Hanchuanzupa granules on the expression of inflammatory factors in the BEAS-2B cell inflammation model; Fig.12 This is a bar graph obtained by qRT-PCR detection of the effects of low, medium and high doses of Hanchuanzupa granules on the mRNA expression of three key target proteins in the BEAS-2B cell inflammation model; Fig.13 This is a Western Blot diagram detecting the effects of low, medium and high doses of Hanchuanzupa granules on the expression levels of three key target proteins in the BEAS-2B cell inflammation model. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0022] Throughout the specification, unless otherwise specified, the terms used herein should be understood as the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0023] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0024] The effects of the present application will be described in detail below in conjunction with the examples and experimental data. Unless otherwise specified, the technical means used in the examples include protein extraction, Western Blot, RNA extraction, qRT-PCR, and RNA ethanol precipitation recovery, which are conventional means well known to those skilled in the art.

[0025] The main idea of ​​the analysis method of the active ingredients and mechanisms of Hanchuanzupa granules for the treatment of asthma based on UPLC-MS / MS, network pharmacology and in vitro and in vivo experiments provided by the present invention is: First, find out the potential active ingredients in Hanchuan Zupa Granules; Second, to find out the potential target of Hanchuan Zupa Granule in treating asthma; Third, potential targets were imported into the STRING database to construct a PPI network, and visualized and analyzed using the Cytoscape software to further analyze and obtain core targets; Fourth, cluster analysis was performed on the top 100 potential targets to further demonstrate the importance of core targets; Fifth, potential targets were imported into the DAVID database for KEGG enrichment analysis and GO enrichment analysis; First, construct a drug-ingredient-disease-target-pathway network diagram using Cytoscape software for potential targets, potential active ingredients, and the top 20 pathways from enrichment analysis, and further screen key active ingredients; Second, combine the obtained core targets and literature reports to determine the possible targets of Hanchuanzupa Granules in the treatment of asthma; construct a BEAS-2B cell inflammation model, evaluate the anti-airway inflammation effect of Hanchuanzupa Granules by qRT-PCR, and verify the possible targets by qRT-PCR and Western Blot, so as to obtain the actual targets of Hanchuanzupa Granules in the treatment of asthma, that is, the mechanism of action.

[0026] Example 1 Analytical method for the active components and mechanisms of Hanchuanzupa granules against asthma based on UPLC-MS / MS, network pharmacology and in vitro and in vivo experiments 1. Therapeutic effect of Hanchuanzupa granules on OVA-induced asthma mice 1. Construction of Balb / c mouse asthma model and drug administration Balb / c mice were randomly divided into 6 groups (n=10): control group, OVA group, OVA + dexamethasone (2 mg / kg), OVA + Hanchuanzupa granules group (1.8g / kg, 3.6g / kg and 7.2 g / kg) treatment group. On days 1 and 14, each mouse was injected with 200μL of OVA sensitization solution. On day 21, 20μL of OVA provocation solution was inhaled intranasally, and Hanchuanzupa granules (7.2g / kg, 3.6g / kg and 1.8 g / kg) were administered by gavage from day 28 to day 35, and dexamethasone (2 mg / kg) was injected intraperitoneally. The mice in the control group were only injected intraperitoneally with 0.9% saline. The mice were anesthetized with 20% urethane (0.01 mL / g) 3 hours before the last treatment and then treated with 20 μL of OVA challenge solution by intranasal inhalation. The eyeballs were removed and plasma was collected in anticoagulant tubes containing EDTAK2. The mice were killed and the left lung tissue was collected.

[0027] 2. Plasma preparation The collected plasma from each group of mice was centrifuged at 3500 rpm for 10 min, and the supernatant was taken to obtain serum.

[0028] 3. ELISA method was used to detect the levels of inflammatory factors in the serum of each group of mice Take out the strips from the aluminum foil bag equilibrated at room temperature for 60 min; set up blank wells, standard wells and sample wells, add 50 μL of standards of different concentrations to each standard well, and do not add to the blank wells; add 10 μL of the sample to be tested to the sample well, and add 40 μL of universal sample diluent; add 100 μL of enzyme-labeled detection antibody to each well except the blank well, standard well and sample well, seal the plate with sealing film, and incubate in a 37 ℃ constant temperature box for 60 min; discard the liquid, pat dry on absorbent paper, add 350 μL of washing solution to each well, let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat this 5 times; add 50 μL of substrate A and B to each well; incubate in a 37 ℃ constant temperature box for 20 min; add 50 μL of stop solution to each well, and detect the absorbance value at 450 nm on the microplate reader.

[0029] The results are as follows Figure 1 As shown, compared with the control group, OVA can significantly increase the levels of inflammatory factors IL-4, IL-5, IL-15 and IgE; compared with the model group, the administration of Hanchuanzupa granules significantly reduced the levels of IL-4, IL-5, IL-15 and IgE.

[0030] 4. HE staining The left lung lobe of the mouse was collected to prevent scratching, immersed in sterile PBS to clean the blood, and then placed in 10% formaldehyde solution. After fixation and dehydration, it was embedded in paraffin, cut into 3μm sections, and stained with H&E. After the sections were made, the alveolar structure and inflammatory cell infiltration were observed under a microscope.

[0031] The results are as follows Figure 2 As shown in the figure, the alveolar septa of the mice in the control group were normal, the alveolar wall structure was intact, there was no inflammatory exudate between the alveoli and in the bronchi, the bronchial morphology was normal and the endothelium was smooth, there was no obvious hyperplasia of epithelial cells, no obvious inflammatory cell infiltration around the bronchi, and no obvious scattered lymphocytes. The alveolar septa of the mice in the model group were thickened, the alveolar wall surface was rough, the inflammatory secretions between the alveoli increased, the bronchial wall was thickened and the endothelium was rough, the inside of the bronchi was infiltrated by inflammatory cells and there was obvious inflammatory cell infiltration around, and the lymphocytes were scattered significantly, especially with eosinophil infiltration and edema under the mucosa, and mucus plugs formed in the bronchi. Compared with the model group, the alveolar septa of the group administered with Hanchuanzupa granules were clear and gradually returned to normal, the alveolar wall was intact, the thickness of the alveolar wall was significantly improved compared with the model group, and the inflammatory secretions in the bronchi and between the alveoli were reduced, especially in the high-dose group administered with Hanchuanzupa granules.

[0032] 2. Identification of the components of Hanchuanzupa granules by UPLC-MS / MS 1. Preparation of Hanchuanzupa Granule Samples Weigh 100 mg of Hanchuanzupa granules, add 500 μL of extract (volume ratio of methanol to water = 4:1, internal standard concentration is 10 μg / mL); vortex for 30 seconds, homogenize at 45 Hz for 4 minutes, and ultrasonicate in an ice-water bath for 1 hour; after standing at -40°C for 1 hour, centrifuge the sample at 4°C, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 minutes; remove the supernatant and filter it through a 0.22 μm filter membrane into a sample injection bottle for testing.

[0033] 3. Serum sample preparation Take 300 μL of the plasma sample collected above, add 30 μL of hydrochloric acid (2 mol / L); vortex for 1 min, let stand at 4°C for 15 min; repeat vortexing and let stand four times, add 1.2 mL of acetonitrile; vortex for 5 min, centrifuge at 12000 rpm for 5 min, take 1350 μL of supernatant and blow dry with nitrogen; add 112.5 μL of 80% methanol (internal standard concentration is 10 μg / mL) for re-dissolution, vortex for 5 min, centrifuge at 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 5 min; take 100 μL of supernatant into the injection bottle for detection.

[0034] 3. UPLC-MS / MS detection conditions Mass spectrometer: Orbitrap Exploris 120 mass spectrometer from Thermo Fisher Scientific, USA; Mass spectrometry conditions: sheath gas flow rate of 30Arb, auxiliary gas flow rate of 10Arb, ion transfer tube temperature of 350Arb, evaporator temperature of 350°C, full millisecond resolution of 60000, MS / MS resolution of 15000, collision energy of 16 / 38 / 42, ionization mode of electrospray ionization, spray voltage of 5.5KV (positive) or -4KV (negative); Chromatographic column: UPLC BEH C18 column (1.7 μm * 2.1 * 100 mm) from Waters, USA Chromatographic conditions: mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, and the gradient was as follows: 0~3.5 min, 95~85% A; 3.5~6 min, 85~70% A; 6 6.5 min, 70~70% A; 6.5~12 min, 70~30% A; 12~12.5 min, 30~30% A; 12.5~18min, 30~0%A; 18~25min, 0~0%A; 25~26min, 0~95%A; 26~30min, 95~95%A; the flow rate was 0.4 mL / min, and the injection volume was 5 μL.

[0035] 4. UPLC-MS / MS spectrum acquisition The chemical composition spectrum of Hanchuanzupa granules was collected based on ChromaTOF for BT software, and peak extraction, peak alignment, peak matching and peak intensity correction were performed to obtain a csv format file containing the compound retention time and peak area information.

[0036] The chemical components of Hanchuanzupa granules and the components in mouse serum were identified and compared to obtain the blood-entering components of Hanchuanzupa granules. A total of 42 blood-entering components of Hanchuanzupa granules were analyzed and identified, as shown in Table 1 and Figures 3-5 .

[0037] Table 1 Components of Hanchuanzupa Granules entering the blood 3. Potential Targets of Hanchuanzupa Granules in Treating Asthma Predict active ingredient targets and collect asthma targets Combined with the aforementioned 42 blood-entering components of Hanchuanzupa Granules, TCMSP was used to search for the active ingredients of traditional Chinese medicine contained in Hanchuanzupa Granules, and the active ingredients reported in the literature related to Hanchuanzupa Granules in PubMed and CNKI were retrieved to supplement them. The results were sorted and deduplicated to obtain the potential active ingredients of Hanchuanzupa Granules for the treatment of asthma. The "Canonical SMILES" information of each active ingredient was obtained in the Pubchem database, and then screened in SwissADME. The screening conditions were: "GI absorption: High" and Druglikeness: 2 or more "Yes" (including 2) in the first 5 columns. A total of 249 active compounds that met the conditions were obtained, and then the "Canonical SMILES" of each component was entered in the SwissTarget Prediction database to predict the target of the potential active ingredients of Hanchuanzupa Granules for the treatment of asthma.

[0038] Using "asthma" as the keyword, relevant target information was retrieved through GeneCards, TTD, OMIM, and DrugBank databases. The data targets were sorted in Excel tables and merged with the above-mentioned predicted targets to obtain asthma-related targets.

[0039] The results are as follows Figure 6As shown, 2644 targets related to 249 active ingredients were retrieved from the SwissTarget Prediction database, and 1123 asthma-related targets were obtained from the GeneCards, TTD, OMIM, and DrugBank databases. The intersection was taken as potential targets for Hanchuanzupa Granules in the treatment of asthma, with a total of 420 targets.

[0040] 4. Construction of protein interaction network The intersection of known related targets of potential active ingredients and asthma-related targets is the potential target of Hanchuanzupa Granules in treating asthma.

[0041] Potential targets were imported into the STRING database, and Organism: Homosapiens was searched under Multiple Proteins to obtain the target protein interaction network and tsv. data. The data were imported into Cytoscape 3.9.1 and visualized using the CentiScape2.2 plug-in. The final results are shown in the figure. Figure 7 As shown in Table 2 , the core targets include IL 6, TP53, STAT3, TNF, IL1B, EGFR, AKT1, SRC, CTNNB1, HSP90AA1, TLR4, HIF1A, ESR1, NFKB1, CXCL8, BCL2, PTGS2, JUN, HSP90AB1, and MMP9.

[0042] By reviewing the literature, STAT3 (signal transducer and activator of transcription 3) has been shown to promote inflammatory response; AKT1 is a key component of the PI3K-AKt pathway, and abnormal activation of the PI3K-AKt pathway can enhance the expression and secretion of proinflammatory cytokines; NFKB1 is a key protein in the NF-κB signaling pathway, and plays an important role in the expression regulation of inflammatory cytokines, adhesion factors, and immune response. Combined with the above network pharmacology prediction results, it is believed that STAT3, AKT1, and NFKB1 may be the targets of Hanchuanzupa granules in inhibiting airway inflammation and anti-asthma, and subsequent verification will be carried out.

[0043] Table 2. Topological parameters of core target genes in the intersection genes of Hanchuanzupa granules and asthma 5. Cluster Analysis Based on the results of the above protein interaction network visualization analysis, the top 100 core targets were ranked according to their degree values, and cluster analysis was performed using the MCODE plug-in in Cytoscape 3.9.1 software. The results are shown in Figure 8 .

[0044] Depend on Figure 8It can be seen that the 20 core targets including STAT3, AKT1, and NFKB1 all fall into the first two sub-networks, and the MOCDE Scores of the two sub-networks are both greater than 10, which further illustrates that STAT3, AKT1, and NFKB1 may be the core targets of Hanchuanzupa Granules in the treatment of asthma.

[0045] VI. KEGG pathway enrichment analysis and GO enrichment analysis The DAVID database was used to perform GO enrichment and KEGG pathway enrichment analysis on the potential targets obtained above. "Homo species" was selected on the DAVID database; the biological process (BP), cellular component (CC), molecular function (MF) and signaling pathway of Hanchuanzupa granules in the treatment of asthma were further analyzed; and the results were visualized using the microbial information platform.

[0046] The results are as follows Fig. 9 As shown: The potential targets of Hanchuanzupa granules in treating asthma were analyzed through KEGG pathway analysis, and the top 20 pathways were visualized, including tumor signaling pathways (Pathways in cancer), neuroactive ligand-receptor interaction (Neuroactiveligand-receptor interaction), lipid and atherosclerosis (Lipid and atherosclerosis), fluid shear stress and atherosclerosis (Fluid shear stress and atherosclerosis), etc.

[0047] GO enrichment analysis showed that potential targets may be involved in biological processes such as inflammatory response, positive regulation of cytoplasmic calcium ion concentration, positive regulation of MAPK cascade, and negative regulation of cell apoptosis. In terms of cellular components, they mainly act on the plasma membrane, the overall composition of the plasma membrane, membrane rafts, etc. The main molecular functions are: protein serine / threonine / tyrosine kinase activity, protein tyrosine kinase, enzyme binding, etc.

[0048] 7. Constructing a drug-ingredient-disease-target-pathway network The potential active ingredients, potential targets and the top 20 KEGG-enriched pathways of Hanchuanzupa granules were used to construct a drug-ingredient-disease-target-pathway network using Cytoscape 3.9.1 software and perform visual analysis to obtain the key active ingredients.

[0049] According to the visual analysis results, the top active ingredients in terms of moderate values ​​are: Schizandrin A, Anhydroicaritin, 2-benzylidenehexanal, Cicaprost, and Coulterone. Fig.10 The structural formula of Coulterone is as follows:

[0050] 8. Construction of BEAS-2B cell inflammation model and evaluation of the anti-asthma effect of Hanchuanzupa granules A cellular inflammation model was constructed, and Hanchuanzupa granules were administered to evaluate the anti-airway inflammation effect of Hanchuanzupa granules. Combined with the core targets obtained above and literature reports, the cellular inflammation model was used to further verify the possible targets of Hanchuanzupa granules in the treatment of asthma, and finally the actual targets of Hanchuanzupa granules in the treatment of asthma were obtained.

[0051] 1. Cytotoxicity The cells were inoculated in a 96-well plate, with 100 μL per well, and divided into a blank group, a control group, a group receiving Hanchuanzupa granules at different concentrations, a group receiving LPS at different concentrations, and a group receiving dexamethasone at different concentrations. After culturing in a cell culture incubator for 24 h, the cells were dosed according to the grouping and cultured in the incubator for 12 h. The culture medium was taken out and the supernatant was discarded. 100 μL of complete culture medium mixed with 10% CCK-8 reagent was added to each well and incubated in the incubator for 2 h. The absorbance was detected at 450 nm on a microplate reader, and the cell viability of each group was calculated. According to the results, the optimal concentration was selected for subsequent experiments.

[0052] The results are as follows: LPS showed cytotoxicity at 10 μg / mL; Hanchuanzupa granules showed cytotoxicity at 384 mg / L; dexamethasone showed cytotoxicity at 2000 nmol / L.

[0053] 2. qRT-PCR detection of mRNA expression levels of inflammatory factors RNA extraction: The cells were inoculated in 6-well plates at a density of 5×105 wells, and 2 mL of culture medium was added to each well. The cells were cultured in an incubator for 24 hours, and then taken out. The control group, model group, dexamethasone group, and low-, medium-, and high-dose groups of Hanchuanzupa granules were given drugs according to the control group, model group, dexamethasone group, and low-, medium-, and high-dose groups of Hanchuanzupa granules. The cells were incubated in an incubator for 3 hours, and then taken out. LPS was added to the remaining groups except the control group to make the final concentration 1μg / mL, and then cultured in an incubator for 24 hours. The 6-well plate after the treatment was taken out, the supernatant was discarded, and the cells were rinsed twice with 1 mL PBS. 1 mL RNAisoPlus was added to each well to fully lyse the cells, and the liquid was transferred to a 1.5 mL EP tube. 0.2 mL of chloroform was added and shaken to mix. The plates were allowed to stand at room temperature for 5 minutes, and then centrifuged at 4 ℃ 12000rpm for 15 minutes. The colorless liquid on the upper layer of the EP tube was aspirated and transferred to a new EP tube. 0.5 mL of pre-cooled isopropanol was added, the plates were mixed by inversion, and then placed on ice for 10 minutes. Centrifuged at 4 ℃ 10000rpm for 10 minutes. Discard the supernatant, add 1 mL of 75% ethanol, wash by inverting, centrifuge at 7500 rpm for 5 min at 4 °C, and discard the supernatant. Dry the RNA until it is transparent, add 50 μL of DEPC water, test the RNA concentration and purity, and store at -80 °C for later use.

[0054] cDNA preparation: Calculate the total RNA volume added to each well based on the measured RNA concentration. Melt the components in the PrimeScriptTMRT reverse transcription kit on ice and add each component in sequence according to the instructions. The reverse transcription reaction system is 20 μL. The reverse transcription program is set as: 37 ℃ 15 min, 85 ℃ 5 sec, 4 ℃ ∞.

[0055] qRT-PCR: All components of TB Green Premix Ex Taq TM kit, cDNA and primers were melted on ice, and the reaction system was 20 μL, and the detection was performed on the machine. The CT value obtained was analyzed by the 2-ΔΔct method to calculate the relative expression of the target gene.

[0056] After LPS modeling, the expression levels of IL-6, IL-8, and TNF-α in BEAS-2B cells were significantly increased compared with the control group; compared with the model group, Hanchuanzupa granules could significantly reduce the expression levels of IL-6, IL-8, and TNF-α in BEAS-2B cells, and the anti-inflammatory effect was more significant. Fig.11 .

[0057] 9. qRT-PCR detection of STAT3, AKT1, and NFKB1 mRNA expression RNA extraction: Cells were seeded in 6-well plates at a density of 5 × 10 52 mL of culture medium was added to each well, and the cells were cultured in the incubator for 24 h. The cells were taken out and administered to the control group, model group, dexamethasone group, and low-, medium-, and high-dose groups of Hanchuanzupa granules. The cells were incubated in the incubator for 3 h, and the cells were taken out. LPS was added to the remaining groups except the control group to make the final concentration 1 μg / mL, and the cells were cultured in the incubator for 24 h. The 6-well plate at the end of the treatment was taken out, the supernatant was discarded, and the cells were rinsed twice with 1 mL PBS. 1 mL RNAiso Plus was added to each well to fully lyse the cells, and the liquid was transferred to a 1.5 mL EP tube. 0.2 mL of chloroform was added and shaken to mix. The cells were allowed to stand at room temperature for 5 min and centrifuged at 4 ℃ 12000 rpm for 15 min. The colorless liquid on the upper layer of the EP tube was aspirated and transferred to a new EP tube. 0.5 mL of pre-cooled isopropanol was added, the mixture was mixed by inversion, and the cells were placed on ice for 10 min. The cells were centrifuged at 4 ℃ 10000 rpm for 10 min. Discard the supernatant, add 1 mL 75% ethanol, wash by inverting, centrifuge at 7500 rpm for 5 min at 4 °C, and discard the supernatant. Dry the RNA until it is transparent, add 50 μL DEPC water, test the RNA concentration and purity, and store at -80 °C for later use.

[0058] cDNA preparation: Calculate the total RNA volume added to each well based on the measured RNA concentration. Melt the components in the PrimeScriptTMRT reverse transcription kit on ice and add each component in sequence according to the instructions. The reverse transcription reaction system is 20 μL. The reverse transcription program is set as: 37 ℃ 15 min, 85 ℃ 5 sec, 4 ℃ ∞.

[0059] qRT-PCR: All components of TB Green Premix Ex Taq TM kit, cDNA and primers were melted on ice, and the reaction system was 20 μL, and the detection was performed on the machine. The CT value obtained was analyzed by the 2-ΔΔct method to calculate the relative expression of the target gene.

[0060] Compared with the control group, the mRNA expressions of STAT3, AKT1, and NFKB1 in the LPS model group were significantly increased; compared with the model group, the expression levels of STAT3, AKT1, and NFKB1 in the Hanchuanzupa granule group were significantly decreased, suggesting that Hanchuanzupa granule may exert anti-asthma effects through the three core targets of STAT3, AKT1, and NFKB1. Fig.12 .

[0061] 10. Western Blot Detection of Protein Expression of STAT3, AKT1, and NFKB1 Protein extraction: Take out the 6-well plate, discard the supernatant, wash the cells in each well twice with pre-cooled PBS, add 100 μL of cell lysis buffer to each well (1 mL RIPA lysis buffer: 100 mM PMSF: phosphatase inhibitor = 100:1:1), lyse on ice for 30 minutes, transfer the cell lysate to a 1.5 ml centrifuge tube with a cell scraper, and centrifuge at 4°C, 10,000 x g for 10 minutes. Divide the centrifuged supernatant into clean 1.5 ml centrifuge tubes.

[0062] BCA protein quantification: Take part of the supernatant and place it in a new centrifuge tube for BCA concentration determination. Dilute the protein 20 times, mix well and place on ice. Prepare standard samples with different concentration gradients and add 100μL of sample diluent. After mixing, transfer 100μL to the next tube, and so on. Take 40μL of each standard sample in a new centrifuge tube. Prepare BCA working solution of 20μg / ml. Dilute the protein to 80μg / ml with PBS, dilute and mix with 4× loading buffer. Boil in a 100-degree water bath for 10 minutes, centrifuge at 10000 for 10 minutes, take the supernatant, slowly cool down, and store at -20℃.

[0063] Western Blot: The proteins in each sample were separated by 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with 5% blocking solution at room temperature for 1 hour and incubated with the primary antibody at 4°C overnight. After washing with TBST, the membrane was incubated with the secondary antibody for 30 minutes at room temperature. The ECL chemiluminescence method was used for exposure and development in a Tanon 5200 Multi fully automatic chemiluminescence imager. Image J software was used to calculate the gray value of each band and the relative expression of the target protein in the sample.

[0064] Compared with the control group, the protein expressions of STAT3, AKT1 and NFKB1 in the model group were significantly increased; compared with the model group, the protein expressions of STAT3, AKT1 and NFKB1 in the Hanchuanzupa granule group were significantly decreased, indicating that Hanchuanzupa granule may exert anti-asthma effects through the three core targets of STAT3, AKT1 and NFKB1. Fig.13 .

[0065] In summary, the present invention clarifies that STAT3, AKT1, and NFKB1 may be the key targets of Hanchuanzupa granules in the treatment of asthma, schisandrin, dehydrated icariin, α-methylcinnamaldehyde, cicaprost, and coulterone may be the key active ingredients of Hanchuanzupa granules, and Hanchuanzupa granules may exert a therapeutic effect on asthma through signal pathways such as tumor signaling pathways, neuroactive ligand-receptor interactions, lipids and atherosclerosis, fluid shear stress and atherosclerosis.

[0066] The present invention adopts UPLC-MS / MS to detect the chemical components of Hanchuanzupa granules, detects the blood components from the plasma of mice in the model group and the Hanchuanzupa granules administration group, integrates TCMSP and components in the literature, conducts network pharmacology research, and verifies through qRT-PCR and Western Blot, so as to obtain the material basis and mechanism of action of Hanchuanzupa granules in treating asthma.

[0067] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules in treating asthma, characterized in that: The following steps are involved: A mouse asthma model was established and Hanchuanzupa granules were administered intragastrically. The chemical components of Hanchuanzupa granules and the components in mouse serum were identified and compared to obtain the blood-entering components of Hanchuanzupa granules. Find known active ingredients of Hanchuanzupa granules for treating asthma, combine them with the blood-entering components, remove duplicates, and obtain potential active ingredients of Hanchuanzupa granules for treating asthma; Predict the targets of potential active ingredients of Hanchuanzupa granules for treating asthma, take the intersection targets of the targets and known asthma-related targets as potential targets of Hanchuanzupa granules for treating asthma, and perform GO enrichment analysis and KEGG signaling pathway analysis on the potential targets; Using the potential targets to construct a protein interaction network, and perform visualization and network topology analysis to obtain core targets; Using the potential active ingredients, the potential targets and the top 20 pathways enriched by signal pathway analysis, a drug-ingredient-disease-target-pathway network diagram is constructed to obtain key active ingredients; The possible targets of Hanchuanzupa Granules for treating asthma were determined by combining the core targets and literature reports, and then verified to obtain the actual targets of Hanchuanzupa Granules for treating asthma.

2. The method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules for treating asthma according to claim 1, characterized in that: The chemical components in Hanchuanzupa granules and mouse serum were identified by UPLC-MS / MS, and the detection conditions of the UPLC-MS / MS were as follows: The sheath gas flow rate was 30Arb, the auxiliary gas flow rate was 10Arb, the ion transfer tube temperature was 350Arb, the evaporator temperature was 350°C, the full millisecond resolution was 60000, the MS / MS resolution was 15000, the collision energy was 16 / 38 / 42, the ionization mode was electrospray ionization, and the spray voltage was 5.5KV or -4KV; Chromatographic conditions: mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, and the gradient was as follows: 0-3.5 min, 95-85% A; 3.5-6 min, 85-70% A; 6-6.5 min, 70-70% A; 6.5~12 min, 70~30% A; 12~12.5 min, 30~30% A; 12.5~18min, 30~0%A; 18~25min, 0~0%A; 25~26min, 0~95%A; 26~30min, 95~95%A; the flow rate was 0.4 mL / min, and the injection volume was 5 μL.

3. The method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules for treating asthma according to claim 1, characterized in that: Through TCMSP, PubMed, and CNKI, we searched for the known active ingredients of Hanchuanzupa Granules for the treatment of asthma. Combined with the blood-entering components of Hanchuanzupa Granules, we sorted and removed duplicates to obtain the potential active ingredients of Hanchuanzupa Granules.

4. The method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules for treating asthma according to claim 1, characterized in that: The target of the potential active ingredients of Hanchuanzupa granules for treating asthma is predicted by importing the potential active ingredients into the Swiss Target Prediction database to find the target gene corresponding to each ingredient; Known asthma-related targets were found from GeneCards, TTD, OMIM, and DrugBank databases; The intersection of the target genes corresponding to the potential active ingredients and the asthma-related targets is the potential target of the active ingredients of Hanchuanzupa Granules for treating asthma.

5. The method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules for treating asthma according to claim 1, characterized in that: The specific process of constructing the protein interaction network is as follows: the potential targets obtained are input into the STRING database, Homo sapiens is selected as the species, the protein interaction information is obtained, and it is imported into the Cytoscape software for visualization analysis.

6. The method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules for treating asthma according to claim 1, characterized in that: The top 100 core targets in the protein interaction network were clustered to further verify the importance of the core targets.

7. The method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules for treating asthma according to claim 1, characterized in that: The specific process of the GO enrichment analysis and KEGG signaling pathway analysis is: importing the potential targets into the DAVID database, selecting "Homo species" in the DAVID database, and further analyzing the relevant results of Hanchuanzupa granules in the treatment of asthma. The relevant results of the analysis include signaling pathways, involved biological processes, affected cellular components and molecular functions of the potential targets.

8. The method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules for treating asthma according to claim 1, characterized in that: The specific process of constructing the drug-ingredient-disease-target-pathway network diagram is as follows: constructing the relationship between the potential active ingredients and the potential targets, and the top 20 pathways enriched by signal pathway analysis, and importing them into the Cytoscape software to obtain the constructed drug-ingredient-disease-target-pathway network diagram and perform visual analysis.

9. The method for analyzing the active ingredients and mechanisms of Hanchuanzupa granules for treating asthma according to claim 1, characterized in that: The key active ingredients of Hanchuanzupa Granules for the treatment of asthma are schisandra chinensis, dehydrated icariin, α-methylcinnamaldehyde, cicaprost and coulterone; The targets of Hanchuanzupa granules in treating asthma are STAT3, AKT1 and NFKB1; The signaling pathways of Hanchuanzupa Granules in treating asthma are tumor signaling pathway, neuroactive ligand-receptor interaction, lipid and atherosclerosis pathway, or fluid shear stress and atherosclerosis pathway.

10. An analysis device for a Chinese medicine action mechanism model based on network pharmacology, characterized in that: The analysis device at least includes a processor and a storage device, and the processor is used to read the program in the storage device to execute the analysis method according to any one of claims 1 to 9.

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