Method for treating leucopenia by analyzing effective components in Qiguo Liangbai capsules based on network pharmacology and application of method

Abstract

The invention discloses a method for treating leucopenia by analyzing active ingredients of a Qigui Shengbai capsule based on network pharmacology and application of the method. According to the method, main effective components of the Qiguo Liangbai capsule are identified firstly, potential targets and mechanisms of the Qiguo Liangbai capsule are further predicted, and a component-target-signal channel network diagram of the Qiguo Liangbai capsule is constructed by combining network pharmacological analysis of transcriptome and metabonomics; and molecular biology is utilized to verify the component-target-signal path network diagram, and a theoretical basis is further provided for clinical application of the Qigui Liangbai capsule to treatment of leucopenia.

Description

Technical Field

[0001] The present invention relates to the field of bioinformatics, and in particular to a method for treating leukopenia by analyzing the effective ingredients of Qijiao Shengbai capsule based on network pharmacology and application thereof. Background Art

[0002] The World Health Organization states that the incidence of cancer is on the rise and is the leading cause of death. Chemotherapy is widely used in the treatment of cancer, but it can cause many adverse reactions, including leukopenia, that is, the number of peripheral white blood cells is less than 4.0×109 / L. Leukopenia is a common adverse reaction associated with abnormal bone marrow hematopoiesis, especially during cancer-related radiotherapy. At present, there are many drugs used to treat leukopenia, including traditional Chinese medicine, Western medicine, and biological hormones. For example, granulocyte colony stimulating factor (G-CSF) drugs and biological hormones can cause side effects, significantly increase treatment costs and cause adverse reactions. Leukopenia belongs to the category of "deficiency and fatigue" in traditional Chinese medicine. The causes of its occurrence are mostly due to insufficient congenital endowment, postnatal malnutrition, long-term illness, internal injuries, and the use of chemotherapy drugs that damage the body's positive energy. Clinically, it is mostly manifested as symptoms such as insufficient qi and yin, and deficiency of both qi and blood. Traditional Chinese medicine has become an alternative choice for people because of its small side effects, low toxicity and high efficiency. It has been widely used in the treatment of leukopenia in clinical practice in my country.

[0003] Qijiao Shengbai Capsule (hereinafter referred to as QJSB) is a large variety of Miao medicine. It is a capsule preparation refined by scientific research based on the precious prescriptions of Miao medicine. It is mainly composed of donkey-hide gelatin, sophora flavescens, jujube, astragalus, angelica, blood ginseng, and epimedium. It is mainly suitable for leukopenia in clinical practice. The "Textbook of Chinese Medicine Hematology" describes the treatment of "leukopenia and agranulocytosis (asthenia disease)" in detail, and Qijiao Shengbai Capsule is listed as the first choice of modern Chinese patent medicine in clinical treatment. At the same time, in the "Interpretation of Chinese Medicine Diagnosis and Treatment Program and Pathway", Qijiao Shengbai Capsule is also recommended as a Chinese patent medicine for the syndrome differentiation and treatment of "leukopenia" and "leukopenia after chemotherapy". Compared with synthetic drugs, Chinese medicine has the characteristics of complex ingredients, multiple targets, and multiple pathways. It induces a series of biotransformation processes in the patient's body to enhance the immune system as a whole, thereby achieving the purpose of treating diseases. However, due to the diversity and complexity of Chinese medicine ingredients, the research on the effective material basis of Chinese medicine is challenging. Therefore, systems pharmacology techniques such as network pharmacology, transcriptomics, and metabolomics are widely used to analyze the active ingredients and mechanisms of action of traditional Chinese medicine.

[0004] Network pharmacology uses modern information technology to mine and analyze drug data and related disease data, and then summarizes and analyzes the relevant information to construct a component-target-pathway network of drugs acting on diseases. This process is complete and systematic, which coincides with the traditional Chinese medicine's "holistic view" and "differentiation and treatment" viewpoints. Therefore, it is widely used to study the efficacy and functional mechanism of Chinese medicine. In recent years, the application of integrated systems biology and network pharmacology is a promising method to describe the effects and mechanisms of Chinese medicine prescriptions. However, the mechanism of Qijiao Shengbai Capsule in the treatment of leukopenia is still incomplete, and it is still necessary to explore the effective ingredients and mechanisms of Qijiao Shengbai Capsule in the treatment of leukopenia. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes a method for treating leukopenia by analyzing the effective ingredients of Qijiao Shengbai Capsule based on network pharmacology and its application.

[0006] The present invention analyzes and identifies the chemical components of Qijiao Shengbai Capsule by ultra-high performance liquid chromatography-mass spectrometry technology, and compares and analyzes the blank plasma after oral administration of Qijiao Shengbai Capsule to determine the effective components in the blood. Then, based on the active components in the body, network pharmacology is performed to predict the potential targets of Qijiao Shengbai Capsule for treating leukopenia, and gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis are performed to construct a component-target-pathway network. A leukopenia mouse model induced by cyclophosphamide is used, and Qijiao Shengbai Capsule is given for treatment. The number of peripheral leukocytes and spleen immune response are analyzed by serum biochemical detection, histopathological analysis and flow cytometry. The differentially expressed genes between the control group and the model group, and between the model group and the medium-dose group of Qijiao Shengbai Capsule are analyzed by transcriptomics method. The differentially expressed metabolites between the control group and the model group, and between the model group and the medium-dose group of Qijiao Shengbai Capsule are analyzed by metabolomics analysis method. Common targets and pathways are obtained by integrating transcriptomics, metabolomics, network pharmacology and other methods, and are identified and verified by in vivo experiments.

[0007] The present invention provides a method for treating leukopenia based on network pharmacology analysis of the effective ingredients of Qijiao Shengbai Capsule, the method comprising the steps of:

[0008] (1) Using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry technology to detect the Qijiao Shengbai capsule sample, and obtain the effective ingredients in the Qijiao Shengbai capsule;

[0009] (2) using the Swiss Target Prediction platform to perform target prediction on the active ingredient obtained in step (1) to obtain the target of the active ingredient;

[0010] (3) using the David database to perform gene ontology function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis on the target obtained in step (2) to obtain the biological process, molecular function, cellular component and related signaling pathway of the target;

[0011] (4) Cyclophosphamide was used to construct a leukopenia mouse model to verify the effectiveness of Qijiao Shengbai Capsule on leukopenia;

[0012] (5) Transcriptomics was used to analyze the differentially expressed genes between the control group and the model group, and between the model group and the QJSB group;

[0013] (6) Metabolomics was used to analyze the differentially expressed metabolites between the control group and the model group, and between the model group and the QJSB group;

[0014] (7) By integrating the common targets and signal pathways obtained in the above steps (1)-(6), a component-target-signal pathway network diagram of Qijiao Shengbai Capsule was obtained.

[0015] Furthermore, the Qijiao Shengbai Capsule sample in step (1) is a Qijiao Shengbai Capsule powder sample, a mouse plasma sample after oral administration of Qijiao Shengbai Capsule, and a mouse plasma sample not orally administered with Qijiao Shengbai Capsule.

[0016] Furthermore, the active ingredients in step (1) are Nicotinamide, Sophoridine, Matrine, dibutyl phthalate, Calycosin-7-0-glc, Palmiticacid, Azelaic acid, 5alpha-Hydroxysophocarpine, Linoleicacid, Citricacid, n-methylcytisine, Steariicacid, oleic acid, Coumarin, methyl palmitate, and Daidzein.

[0017] Furthermore, in steps (4)-(5), the control group was injected intraperitoneally with 0.9% saline for 3 consecutive days, and the model group was injected with 100 mg / kg cyclophosphamide for 3 consecutive days; the QJSB group was injected with 100 mg / kg cyclophosphamide for 3 consecutive days and then gavage with Qijiao Shengbai Capsule solution began on the 4th day.

[0018] Furthermore, the common targets in step (7) are Bcl-2, Bax, Caspase-3, and Cytochrome c.

[0019] Furthermore, the signaling pathway in step (7) is a PI3K-AKT signaling pathway.

[0020] The invention also provides an application of using the method to improve the therapeutic effect of treating leukopenia.

[0021] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0022] The present invention explores the mechanism of action of Qijiao Shengbai Capsule on leukopenia by combining network pharmacology, transcriptomics and metabolomics. A total of 95 chemical components were identified from Qijiao Shengbai Capsule, and a total of 16 active ingredients were identified in mouse plasma. Qijiao Shengbai Capsule can significantly improve the pathological damage of spleen tissue in leukopenic mice. Compared with the model group mice, the Qijiao Shengbai Capsule group can significantly increase the weight, white blood cell count, and bone marrow nucleated cell count of mice, and can also increase CD4 + and CD8 + The percentage of lymphocytes was significantly increased by 1.54%, which significantly promoted lymphocyte proliferation. Integrated network pharmacology, transcriptomics and metabolomics analysis showed that Bcl-2, Bax, Caspase-3 and Cytochrome c were the main common targets, mainly involved in the PI3K-AKT signaling pathway. The activation of Bcl-2 in the spleen tissue of mice in the model group was significantly reduced, but increased in the treatment of Qijiao Shengbai capsule. The PI3K-AKT signaling pathway was verified, and it was found that the expression level of Bcl-2 was reduced in mice in the model group, but increased after treatment with Qijiao Shengbai capsule.

[0023] In summary, Qijiao Shengbai Capsule can effectively reduce the negative effects of cyclophosphamide on spleen damage and peripheral blood count in mice. It can also regulate immune function, promote cell proliferation, and increase the number of white blood cells, which is most likely achieved through the PI3K-AKT signaling pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work. Figure 1 This is the "ingredient-target-signaling pathway" diagram of the Qijiao Shengbai Capsule of the present invention; wherein Figure 1 A represents the potential 182 target for the treatment of leukopenia by the active ingredients of QJSB; Figure 1 B represents the protein-protein interaction results of 182 potential targets of QJSB for the treatment of leukopenia; Figure 1 C represents the KEGG pathway results of the active ingredients of QJSB in treating leukopenia; Figure 1D represents the GO enrichment analysis results of the effective ingredients of QJSB for the treatment of leukopenia;

[0025] Figure 2 It is a network diagram of "ingredients-targets-signaling pathways" of Qijiao Shengbai Capsule of the present invention;

[0026] Figure 3 This is a graph showing the effect of the Qijiao Shengbai capsule of the present invention on the expression of apoptotic proteins in spleen tissue of patients with leukopenia;

[0027] Figure 4 The results of serum analysis and lymphocyte analysis of the animal model induced by cyclophosphamide by Qijiao Shengbai Capsule of the present invention; A represents the effect of Qijiao Shengbai Capsule on peripheral leukocytes of mice induced by cyclophosphamide-induced leukopenia; B represents that after 2 weeks of treatment, Qijiao Shengbai Capsule increases peripheral leukocytes; C\D represents the thymus index in the spleen of mice detected by flow cytometry by Qijiao Shengbai Capsule; E represents the thymus index in the spleen of mice detected by flow cytometry by Qijiao Shengbai Capsule;

[0028] Figure 5 This is a histopathological diagram showing the effect of the Qijiao Shengbai capsule of the present invention on the spleen tissue of mice. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0030] The present invention firstly uses ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-ESI-Q-Exactive Plus Orbitrap-MS) to detect and preliminarily identify the chemical components of Qijiao Shengbai Capsule (hereinafter referred to as QJSB) and the components that migrate into the blood circulation. Then, the potential targets and mechanisms of QJSB in treating leukopenia are further studied with the help of network pharmacology. Second, the effectiveness of QJSB on leukopenia is verified by pharmacodynamic experiments, suggesting that QJSB has the effect of improving the body's immunity. Third, transcriptomics reveals the DEGs and related signaling pathways of leukopenic mice treated with QJSB at the gene level. Metabolomics analyzes the metabolites, targets and pathways of QJSB in treating leukopenic mice. Finally, by integrating network pharmacology, transcriptomics and metabolomics analysis, the key targets and signaling pathways of QJSB in treating leukopenic mice are obtained, and western blot analysis is performed for mechanism verification. Therefore, the present invention explores the mechanism of QJSB intervention in leukopenia on the basis of clinical medication and traditional therapeutic effects, and further provides a theoretical basis for studying the clinical application of QJSB in treating leukopenia mice.

[0031] The Qijiao Shengbai capsules (Guizhou, China) used in the present invention were purchased from Guizhou Hanfang Pharmaceutical Co., Ltd., batch number Z2522012;

[0032] Cyclophosphamide was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd., batch number: HJ20160467;

[0033] Antibodies used for Western blot: anti-Bcl-2 (68103-1-lg), anti-Bax (660267-1-lg), anti-Caspase3 (66470-2-lg), and anti-Cytochrome C (66264-1-lg) antibodies were purchased from Proteintech;

[0034] Tandem mas tag (TMT) kit was purchased from Thermo 88Fisher Scientific; RPMI-1640 (HyClone, Thermo Scientific Co, Waltham, MA, USA), red blood cell (RBC) lysis buffer was purchased from Jimei Biotechnology (Beijing, China);

[0035] Phosphate buffered saline (PBS, USA), anti-CD3e (Percp), anti-CD4 ((PE)), and anti-CD8a (FITC) were purchased from Merck. Except for UPLC-grade reagents (formic acid, 89 acetonitrile, and methanol), the rest of the reagents and 90 chemicals were analytical;

[0036] All SPF male ICR mice, 6–8 weeks old and weighing 18–22 g, were purchased from the Animal Center of Guizhou Medical University (Beijing, China, approval number: SCXK:2016-0006). The mice were placed in an animal room (temperature 24 ± 2 °C, relative humidity 60 ± 5%) with a 12 h light / dark cycle. Before the experiment, the rats were given drinking water and then fed with standard laboratory food to acclimate to the environment for one week. All experimental projects were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals and have been approved by the Ethics Committee of Guizhou Medical University. The mice were housed in a standard laboratory with a 12 / 12 h light / dark cycle, a temperature of 25 ± 1 °C, and a humidity of 60 ± 5%, with free access to water and food.

[0037] Example 1

[0038] A method for treating leukopenia by analyzing the active ingredients of Qijiaoshengbai capsules based on network pharmacology.

[0039] 1. Experimental Methods

[0040] 1. Identification of the active ingredients of Qijiaoshengbai Capsules

[0041] 100 mg of the powder in Qijiao Shengbai capsules was added with 1000 μL of 80% methanol solution and grinding beads, ground for 5 min, vortexed for 10 min, centrifuged at 12000 r / min for 10 min at 4°C, and the supernatant was filtered and tested by UHPLC-ESI-Q-Exactive Plus Orbitrap MS.

[0042] To preliminarily detect the potential active ingredients of QJSB extract, the extract was separated by Thermo Vanquish Horizon ultra-high performance liquid chromatography equipped with a Hypersil Gold C18 column (2.1×100 mm2, 1.9 μm). The optimized gradient consisted of phase A (0.1% formic acid in water) and phase B (0.1% formic acid in acetonitrile) as follows: injection volume, flow rate and column temperature were 2 μL, 0.3 mL / min, and 40°C, respectively. The mass spectrometry was acquired using an ESI-Q-xactive Plus Orbitrap system, and the samples were detected in negative and positive ion modes. The mass spectrometry parameters were as follows: 3.5 / 2.5 Kv (+ / -) spray voltage; 350°C evaporator temperature; 320°C capillary temperature; fullms-ddms general method; 100-1500 m / z scan range; 70000 resolution (MS1); 17500 MS / MS resolution; 20, 40 and 60 normalized collision energies (NCE). The raw data were imported into Compound Discoverer 3.2, and the relative molecular mass deviation was set to less than 5 ppm. The analysis results were based on the accurate relative molecular mass and primary and secondary mass spectrometry fragmentation information in Xcalibur software, and combined with standard spectra and fragmentation information in m / z Cloud, m / z Vault, Masslist, MoNA and ChemSpider databases and literature. The advanced identification algorithm mzLogic was used to increase the credibility of the results, and Mass Frontier 7.0 software was used for fragment-assisted inference and verification to identify the active ingredients of QJSB.

[0043] SPF ICR male mice, 5-7 weeks old, weighing 18-22g, were kept in the same environment as 2.1. The QJSB-administered group was given 4 times the dose (human dose) of QJSB by gavage for 3 days, once a day, and the blank group was given an equal amount of normal saline. Blood was collected from the eye sockets at 6 time points, 0.5h, 1h, 1.5h, 2h, 3h, and 4h after the last administration. The blood was placed in a heparinized anticoagulant tube and mixed, and then centrifuged at 3500rpm for 10min at 4°C. The upper plasma was taken and stored at -80°C for later use.

[0044] Take 1 ml of plasma from the blank group and 1 ml of plasma from the QJSB-treated group, add 2 ml of methanol and 2 ml of acetonitrile, vortex mix, ultrasonicate in an ice bath for 10 min, centrifuge at 12000 rpm at 4°C for 10 min, take the supernatant, blow dry with N2, redissolve in 1 ml of methanol, and blow dry again with N2, redissolve the residue with 200 ul of methanol, vortex for 2 min, ultrasonicate for 10 min, centrifuge at 15000 rpm at 4°C for 10 min, take the supernatant, and use UHPLC-ESI-Q-Exactive Plus Orbitrap MS for sampling and detection to identify the components of QJSB that migrate into the blood circulation.

[0045] 2. Construction of QJSB component-target-pathway network

[0046] The molecular structure of the active components identified in mouse plasma was drawn with ChemDraw software and exported in SMILES format. Swiss TargetPrediction was then used to predict the targets of the components, where the species was limited to "human", and the target names were standardized using the UniProtKB search function in the UniProt database. Targets related to the pathogenesis of leukopenia were collected from OMIM, GeneCards, and DrugBank databases. Duplicate targets were deleted. Overlapping targets between components and diseases were obtained by constructing a Venn diagram.

[0047] Active components and overlapping targets were imported into Cytoscape for analysis of their interactions. Topological parameters, including “degree”, “closeness centrality” and “betweenness centrality”, were calculated using NetworkAnalyzer. Nodes with degree ≥ 10 were selected as the main active components. Protein-protein interactions (PPIs) were explored using the STRING database, and the main targets were selected based on the criterion that the topological parameters of potential targets exceeded the average value of the PPIs network.

[0048] David database was used for gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to elucidate the classification and function of these targets and their roles in signal transduction pathways. GO functions include biological process (BP), molecular function (MF), and cellular component (CC). Active components, potential targets, and the top 10 pathways obtained by KEGG pathway analysis were used to construct a component-target-pathway network.

[0049] 3. Animal Experiments

[0050] Experimental study on cyclophosphamide-induced leukopenia mouse model

[0051] (1) Serum biochemical analysis

[0052] Sixty SPF ICR mice were fed adaptively for one week, and 60 mice with temperature fluctuation less than 0.5℃ were selected to start the experiment. Then the 60 mice were randomly divided into 5 groups: control group, cyclophosphamide (CTX) group, leukojun (20mg / kg / d) group, QJSB (0.5g / kg / d, 1g / kg / d, 2g / kg / d, for 14 consecutive days). The leukopenia model was prepared by intraperitoneal injection of CTX (100mg / kg, for 3 consecutive days) as the model group; the normal group mice were intraperitoneally injected with an equal amount of 0.9% saline for 3 consecutive days; the QJSB group and the model group were modeled in the same way, and QJSB solution was gavaged on the 4th day after modeling, while the normal group and the model group mice were given the same solvent (distilled water) according to the administration method and dosage of the QJSB group. After the modeling was completed (i.e., the 4th day of the experiment), 50μL of blood from the medial canthus of the orbits of 6 mice in each group was randomly collected, and the leukocyte classification and counting were performed using the American IDXX blood cell analyzer. On the 18th day of the experiment, blood was collected from the ophthalmic venous plexus and the mice were killed. The plasma was centrifuged at 13000rpm for 10min at 4℃ and stored at -80℃ for metabolomics determination. Mouse bone marrow cells were collected for research; spleens of 3 mice in the control group, model group, and QJSB group (1g / kg / d) were randomly selected, rinsed with cold phosphate buffered saline (PBS), and stored in 10% paraformaldehyde. Histopathological examination was performed after hematoxylin-eosin staining. Spleen samples were taken from the control group, model group, and QJSB (1g / kg / d) for lymphocyte isolation and identification and transcriptomics research.

[0053] (2) Histopathological analysis

[0054] Mouse spleen tissue was taken, washed with cold phosphate buffered saline (PBS), fixed with 4% paraformaldehyde, embedded in paraffin, and sliced ​​at 4-5 μm to observe the pathological changes of spleen tissue. These spleen tissue sections were then stained with hematoxylin and eosin (H&E). The pathological changes of spleen tissue were evaluated by optical microscopy.

[0055] Mice were killed by cervical dislocation and immediately immersed in 75% alcohol for 5 min. Unilateral femur was removed under sterile conditions, and the muscles and connective tissue on the femur were removed as much as possible. 1 ml of RPMI1640 culture medium was drawn with a syringe, and the bone marrow was flushed from the bone marrow cavity into a 1.5 ml sterile EP tube. This was repeated 3-5 times to ensure that the bone marrow was completely flushed out. A 200 ul pipette was then used to repeatedly blow and beat to make a single cell suspension for counting bone marrow nucleated cells.

[0056] (3) CD4 + 、CD8 + T lymphocyte percentage analysis

[0057] Fresh spleens (8 in each group) were placed in RPMI-1640 containing 10% bovine serum, ground with a syringe core on a 200-mesh nylon mesh, and filtered to make a crude spleen cell suspension. After centrifugation (1600rpm, 10min, 4°C), cells were collected from the interphase and placed in 2.0ml red blood cell lysis buffer to lyse red blood cells. After treatment for 10min, 4.0ml RPMI1640 was added to the spleen cell suspension to terminate lysis, and then centrifuged at 1600rpm for 10min and washed twice with RPMI-1640. The suspension was centrifuged at 1600rpm for 10min, and the cells in each group were adjusted to 1×10 7 Cells / ml. Flow cytometry was used for lymphocyte phenotyping. Splenocytes were incubated with anti-CD3E (PERCP), anti-CD4 (PE), and anti-CD8A (FITC) monoclonal antibodies in the dark for 30 min and then washed with 1.5 ml PBS. After centrifugation (1600 rpm, 10 min, 4 ° C), cells were collected and washed twice with PBS, then added to 500 μL PBS and analyzed using a Facscalibur II flow cytometer (BD Biosciencies, USA).

[0058] (4) Western blot analysis

[0059] Total protein was extracted from spleen tissue according to the instructions of RIPA lysis buffer (Kangwei Century Biotechnology, China). Protein concentration was determined using a BCA protein assay kit (Kangwei Century, China). Proteins were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 1.5 h at room temperature, the membranes were incubated with anti-Bcl-2 (1:2000), anti-BAX (1:5000), anti-CASPASE3 (1:1000), anti-CYTOCHROMEC (1:5000), and anti-GAPDH (1:200) at 4 °C overnight. After washing three times with TBST, the membranes were incubated with HRP mouse secondary antibody (1:200) at room temperature for 1 h. Finally, protein signals were detected using an enhanced chemiluminescence (ECL) detection kit (Proteintech), photographed with the aid of a gel imager, and analyzed using Image J software.

[0060] (5) Metabolomics analysis

[0061] Endogenous metabolites in spleen tissue were extracted with organic reagents. 100 mg spleen tissue samples were extracted with 0.5 mL of pre-cooled methanol / acetonitrile aqueous solution (2:2:1, v / v / v), homogenized, and vortexed and sonicated at low temperature for 15 minutes. To precipitate proteins, the mixture was placed at -20 ° C overnight, then centrifuged at 15000 rpm for 15 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and then injected into a sample bottle for metabolomics analysis.

[0062] Similarly, the UHPLC-HPI-Q-Exactive Plus Orbitrap-MS was equipped with a ZORBAX Eclipse Plus C18 column (2.1 × 100 mm2, 1.8 μm; Agilent Technologies (USA) for metabolomics analysis. The optimal gradient of water (0.1% formic acid, A) and acetonitrile (0.1% formic acid, B) was 0-2.5 min, 2-2% B; 2.5-5 min, 2-40% B; 5-12 min, 40-100% B; 12-16 min, 100-100% B; 16-16.1 min, 100-2% B; 16.1-19 min, 2-2% B, with a flow rate of 0.3 mL / min. The ion spray voltage was 3.5 / 2.8 kV (+ / -), and the scan range was 100-1500 m / z. The auxiliary gas heater and capillary temperatures were 350°C and 320°C, respectively. Dynamic exclusion was 3 seconds, and the S-lens RF level was 50. Compound Discoverer 3.2 software was used for preprocessing of mass spectrometric data, including peak identification, peak matching, and retention time correction. SIMCA-P OPLS-DA and PCA analysis were performed using the software Umetrics 14.1 (Umetrics, AB, Sweden). In addition, we used the KEGG (https: / / www.genome.jp / kegg / ) and HMDB databases ( http: / / www.hmdb.ca / ) Endogenous metabolites were identified by comparing primary and secondary mass spectrometry fragmentation information; we used an in-house metabolite fragmentation library to verify their composition by MS2 spectra. MetabolAnalyst 5.0 was used to analyze the changes in plasma metabolites after QJSB treatment. Metabolic pathways with effect values ​​greater than 0.1 were considered significant.

[0063] (6) Transcriptomic analysis

[0064] The spleens of 9 mice (n=3) in the control group, model group and QJSB group were selected for transcriptomic analysis. Total RNA was isolated from spleen tissue and purified using Trizol (Beyotime, Shanghai, China) reagent according to the manufacturer's instructions. The concentration and purity of the extracted RNA were detected by Nanodrop 2000 (Nanodrop, Wilmington, DE, United States). At the same time, RNA integrity was analyzed by agarose gel electrophoresis, initial quantification was performed using Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA), and RNA integrity was detected by Qsep400 bioanalyzer. The total RNA content ≥1 μg and the concentration ≥35 ng were required for the establishment of a single database. The sequencing library was sequenced using Illumina Truseq TM RNA library preparation kit (Illumina, Nebraska, USA) was constructed according to the manufacturer's protocol. Mouse spleen tissue passed the quality check and was subjected to RNA sequencing using the Illumina high-throughput sequencing platform.

[0065] The data were uploaded to the Maiwei Cloud Online Platform (https: / / cloud.metware.cn / ). Differential expression analysis was performed using DESeq2 software. Whether there were significant differentially expressed genes (DEGs) between groups was determined based on p-value ≤ 0.05 and |log2FoldChange| ≥ 0.585. In addition, we performed functional enrichment analysis, including GO (Gene Ontology) and KEGG, to determine which DEGs were significantly enriched in GO terms and metabolic pathways compared with the whole transcriptome background, with p-value ≤ 0.05. GO functional enrichment and KEGG pathway analysis were performed using metscape (https: / / metascape.org). Finally, the HMDB database (https: / / hmdb.ca / ) was used for correlation analysis of metabolomics and transcriptomics.

[0066] (7) Data analysis

[0067] All data are expressed as mean ± standard deviation (SD) and statistically analyzed by GraphPadPrism 8.0.2 (San Diego, CA, USA). One-way analysis of variance (ANOVA) was used to analyze differences between groups, p < 0.05 was considered statistically significant, and p < 0.01 was considered statistically significant.

[0068] 2. Experimental Results

[0069] 1. The main active ingredients of Qijiao Shengbai Capsule for the treatment of leukopenia

[0070] The present invention analyzed the in vivo and in vitro material basis of QJSB by UHPLC-ESI-Q-Exactive Plus Orbitrap MS, identified 16 prototype compounds from the serum of mice after administration, and revealed the potential active substances of the formula. The results are shown in Table 1:

[0071] Table 1 Specific names of the main active ingredients in Qijiao Shengbai Capsules for the treatment of leukocytopenia

[0072]

[0073]

[0074] 2. QJSB “component-target-signaling pathway” network diagram

[0075] By importing the above 16 main active ingredients into Swiss Target Prediction, a total of 670 component targets were identified. After removing duplicate values, a total of 2303 leukopenia-related targets were obtained from OMIM, GeneCards, and DrugBank databases. Figure 1 As shown in A, the above two groups of targets were introduced into the jvenn online tool to identify intersections. Therefore, 182 potential targets of QJSB for the treatment of leukopenia were obtained. The 182 overlapping targets were uploaded to the STRING database, and the Cytoscape3.7.1 software platform was used to obtain the PPI ( Figure 1 B). The size and color of the nodes reflect the importance of the degree. The average degree, closeness centrality, and betweenness centrality of the component nodes were 34.1105, 0.5135, and 0.0056, respectively. 50 targets with scores greater than the average were identified. The top 7 targets (degree>80) were AKT1, EGFR, IL6, STAT3, BCL-2, CASP3, and JUN, which may be the main targets of QJSB for the treatment of leukopenia.

[0076] like Figure 1 As shown in D, in GO enrichment analysis, responses to protein phosphorylation, negative regulation of apoptotic processes, and positive regulation of cell proliferation had high correlations in biological processes. Among cellular components, targets mainly involved plasma membrane, cell surface, and mitochondria. Molecular function results showed strong correlations with ATP binding, protein kinase activity, and protein serine / threonine / tyrosine kinase activity in QJSB treatment of leukopenia.

[0077] A total of 158 enrichment results with p-value < 0.05 were obtained in the KEGG pathway enrichment analysis. The top ranked pathways based on their p-values ​​were visualized using the Cytoscape 3.7.1 software platform. Figure 1 As shown in Figure C, QJSB may play a role in the treatment of leukopenia through the PI3K / AKT signaling pathway, Foxo signaling pathway and other pathways. The PI3K / AKT signaling pathway involves 40 potential targets and is one of the important pathways for controlling cell division, autophagy, survival and differentiation, and may be a key pathway. The component-target-signaling pathway network shows that multiple components are associated with multiple targets, and different targets also correspond to multiple mechanistic pathways. Cytoscape3.7.1 was used to construct the blood component-target-signaling pathway diagram, as shown in Figure 3. Figure 2 As shown, the interaction network shows that all components have 5 or more therapeutic targets, nearly 50% of the targets are regulated by more than 2 components, and all enriched pathways have more than 10 therapeutic targets.

[0078] 3. Animal experiment verification results

[0079] The in vitro and in vivo material basis of QJSB was analyzed by UHPLC-ESI-Q-Exactive Plus Orbitrap MS, and 16 prototype compounds were identified from the serum of mice after administration, which revealed the potential active substances of the formula to a certain extent (Table 1). Among them, it was found that Matrine restored sepsis-induced STRT1 downregulation and deacetylation of NF-kB p65 subunit and p53, thereby inactivating the NF-kB pathway and inhibiting the p53-induced apoptosis pathway in sepsis lungs. At the same time, sophoridine has a protective effect on brain cells in pMCAO rats, and its mechanism may be related to upregulating Bcl-2 and downregulating Bax and Caspase-3 protein expression levels ( Figure 3 ), thereby inhibiting cell apoptosis.

[0080] The experimental results of cyclophosphamide (CTX)-induced leukopenia mouse model, such as Figure 4 and Figure 5 It was shown that QJSB can significantly increase the number of peripheral leukocytes in CTX-induced mice. After taking QJSB, the spleen index of the model group mice was significantly improved, indicating that QJSB may be involved in regulating hematopoiesis and immune function to alleviate CTX-induced leukopenia. In further research of the present invention, it was found that the effect of QJSB medium-dose group in increasing the number of peripheral leukocytes and improving spleen index was better than that of high-dose QJSB (2g / kg / d) and low-dose QJSB (0.5g / kg / d), indicating that QJSB is not dose-dependent. Therefore, the present invention selected the control group, model group and QJSB group for pathological tissue analysis, CD4 +、CD8 + T lymphocyte analysis and multi-omics studies.

[0081] Transcriptomics studies have shown that the regulated differentially expressed genes are mainly involved in the PI3K-AKT signaling pathway, the MAPK signaling pathway, and focal adhesion kinase (FAK). In order to study how QJSB achieves the above-mentioned pharmacodynamic effects through in vivo molecular mechanisms, the present invention integrates system pharmacology analysis methods including network pharmacology, transcriptomics, and metabolomics, and identifies common targets, namely, the gene FOS associated with breast cancer, the transcription factor AP-1 family member JUN, and the hepatocyte growth factor (HGF); further suggesting that QJSB may be involved in regulating hematopoiesis and immune function, affecting cell proliferation and apoptosis, improving CTX-induced leukopenia, and increasing the number of white blood cells. In the present invention, the results show that after administration of QJSB, the differentially expressed genes of JUN, HGF, and Fos are upregulated, thus suggesting that QJSB may improve leukopenia by regulating the genes on these pathways.

[0082] In addition, this study found that after QJSB administration, spleen index and spleen injury were significantly improved and lymphocyte CD4 + Studies on animal models have shown that persistent lymphocytopenia is caused by apoptosis, and inhibiting T cell apoptosis can improve the survival rate of animals. It is reported that the potential mechanism of T cell reduction and dysfunction is related to mitochondria-mediated apoptosis, in which Caspase-3 and Caspase-9 are key enzymes. Therefore, we speculate that QJSB may promote T lymphocyte proliferation and inhibit CD4 + Cell apoptosis, thereby regulating the expression of mitochondrial pathway-related proteins, thereby protecting the spleen from damage and improving leukopenia. Mitochondrial pathway apoptosis is usually activated by cell stress or damage, and is regulated by the interaction of Bcl-2 family and Caspase family members in mitochondria. Under normal circumstances, Bax binds to Bcl-2-related proteins to avoid cytotoxicity caused by its high level. When cells are stimulated by toxic drugs, Bax located on the outer membrane of mitochondria can form a release channel, enhance the permeability of the mitochondrial membrane, and thus release apoptosis-related factors from mitochondria. In the present invention, Bax expression in CTX treatment increases and Bcl-2 expression decreases, while the results of the QJSB treatment group are the opposite. The results show that CTX, as a stimulating factor, promotes the formation of Bax and its conversion from a bound state to a free state. QJSB promotes the expression of Bcl-2 protein, thereby strengthening the inhibitory effect on Bax.

[0083] The imbalance between Bax and Bcl-2 will lead to the release of Cytochrome-C (hereinafter referred to as Cyt-C). The released Cyt-C binds to Apaf-1 and triggers conformational changes, thereby recruiting Caspase-9 to the apoptotic body complex and forming a dimeric complex structure with Caspase-9. The allosteric activation of Caspase-9 in the lysosome leads to proteolysis and activation of the downstream actuator Caspase-3, which cuts hundreds of substrates, some of which are complex cell apoptosis. In the present invention, the expression of Cyt-C in the CTX treatment group increased significantly, while the expression of Cyt-C in the QJSB group decreased. Therefore, QJSB can significantly reduce the release of Cyt-C, thereby protecting the integrity of mitochondria. The expression of Caspase-3 protein was further measured, and the results showed that the QJSB group effectively reduced the expression of Caspase-3, which suggests that QJSB can effectively regulate the expression of proteins related to the mitochondrial pathway, thereby inhibiting CD4 + Apoptosis of T lymphocytes, increase the number of T lymphocytes, and improve leukopenia.

[0084] In summary, the present invention established a CTX-induced leukopenia mouse model. On this basis, network pharmacology, transcriptomics and metabolomics analysis were comprehensively used to screen out key targets and signaling pathways. The effects of QJSB on spleen and peripheral serum metabolites and their mechanisms were studied through in vivo experiments. The results showed that QJSB can effectively regulate the expression of mitochondrial pathway-related proteins, thereby inhibiting CD4 + Apoptosis of T lymphocytes, increase the number of T lymphocytes, and improve leukopenia.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for treating leukopenia based on network pharmacology analysis of the active ingredients of Qijiao Shengbai Capsule, characterized in that: The method comprises the steps of: (1) Using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry technology to detect the Qijiao Shengbai capsule sample, and obtain the effective ingredients in the Qijiao Shengbai capsule; (2) using the Swiss Target Prediction platform to perform target prediction on the active ingredient obtained in step (1) to obtain the target of the active ingredient; (3) using the David database to perform gene ontology functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis on the target obtained in step (2) to obtain the biological process, molecular function, cellular component and related signaling pathway of the target; (4) Cyclophosphamide was used to construct a leukopenia mouse model to verify the effectiveness of Qijiao Shengbai Capsule on leukopenia; (5) Transcriptomics was used to analyze the differentially expressed genes between the control group and the model group, and between the model group and the QJSB group; (6) Metabolomics was used to analyze the differentially expressed metabolites between the control group and the model group, and between the model group and the QJSB group; (7) By integrating the common targets and signal pathways obtained in the above steps (1)-(6), a component-target-signal pathway network diagram of Qijiao Shengbai Capsule was obtained.

2. The method according to claim 1, characterized in that The Qijiao Shengbai Capsule sample in step (1) is a Qijiao Shengbai Capsule powder sample, a mouse plasma sample after oral administration of Qijiao Shengbai Capsule, and a mouse plasma sample without oral administration of Qijiao Shengbai Capsule.

3. The method according to claim 1, characterized in that: The active ingredients in step (1) are Nicotinamide, Sophoridine, Matrine, dibutyl phthalate, Calycosin-7-0-glc, Palmiticacid, Azelaic acid, 5alpha-Hydroxysophocarpine, Linoleicacid, Citric acid, n-methylcytisine, Steariicacid, oleic acid, Coumarin, methyl palmitate, and Daidzein.

4. The method according to claim 1, characterized in that In steps (4)-(5), the control group was intraperitoneally injected with 0.9% saline for 3 consecutive days, and the model group was injected with 100 mg / kg cyclophosphamide for 3 consecutive days. The QJSB group was injected with 100 mg / kg cyclophosphamide for 3 consecutive days and then gavaged with Qijiao Shengbai Capsule solution on the 4th day.

5. The method according to claim 1, characterized in that The common targets in step (7) are Bcl-2, Bax, Caspase-3, and Cytochrome-C.

6. The method according to claim 1, characterized in that The signaling pathway in step (7) is the PI3K-AKT signaling pathway.

7. Use of the method according to any one of claims 1 to 6 to improve the therapeutic effect of treating leukopenia.

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