CN118903430A - Application of PI3K/Akt in preparing medicament for preventing, relieving or treating blood deficiency - Google Patents

Abstract

The present invention provides the use of PI3K/Akt in the preparation of a drug for treating blood deficiency. The present invention discovers a new function of the PI3K/Akt pathway, that is, activating the PI3K/Akt pathway can increase the expression of hematopoietic factors, inhibit kidney inflammatory factors, and increase the expression of Bcl-2. Based on the role of PI3K/Akt in preventing, alleviating and/or treating blood deficiency, it can be used to prepare a drug for preventing, alleviating and/or treating blood deficiency.

Description

Application of PI3K/Akt in the preparation of drugs for preventing, alleviating or treating blood deficiency

Technical Field

The invention belongs to the field of gene function and application, and particularly relates to an application of PI3K/Akt in preparing a drug for preventing, alleviating and/or treating blood deficiency.

Background Art

According to traditional Chinese medicine, the pathogenesis of blood deficiency syndrome is due to excessive blood loss and insufficient hematopoiesis, and clinical manifestations include pale face, dizziness, pale lips, general weakness and mental weakness. Due to the damage of the body's hematopoietic function, it often leads to pancytopenia, visceral dysfunction, decreased immunity, and even bone marrow suppression in severe cases. Modern medicine believes that anemia patients are often accompanied by symptoms of blood deficiency, which are common in renal anemia, iron deficiency anemia, aplastic anemia and anemia after radiotherapy and chemotherapy. Due to factors such as the aging population, dietary habits, drug treatment and the fast-paced modern lifestyle, the prevalence of anemia has increased year by year and tends to be younger. For this reason, the study of blood deficiency syndrome has attracted more and more attention and has become a hot spot in current drug research and development.

The PI3K/Akt signaling pathway consists of two parts: 1) phosphatidylinositol 3-kinase (PI3K) and 2) its downstream molecule, serine/threonine protein kinase B (PKB/Akt). PI3K consists of two subunits, the catalytic subunit pll0 and the regulatory subunit p85, and has the activity of serine/threonine kinase and phosphatidylinositol kinase. PI3K binds to the activated receptor tyrosine kinase RTK of the intracellular segment of the cytokine receptor and is recruited to the plasma membrane, making its catalytic subunit close to the phosphatidylinositol in the inner leaflet of the plasma membrane. Akt is a downstream substrate of phosphatidylinositol 3-kinase (PI3K), has a high homology with PKA and PKC, is composed of 480 amino acid residues, and has three subtypes of protein structure: Akt1, Akt2 and Akt3, located on human chromosomes 14q32, 19ql3 and lq44, respectively. Akt has two phosphorylation-regulated active sites (Ser473 and Thr308), which are located in the catalytic domain (PH domain) and the carboxyl-terminal regulatory domain, respectively. Akt can only be fully activated after phosphorylation of these sites. The PH domain is the phosphatidylinositol binding region, and its mutation or deletion can lead to reduced or lost Akt activity.

The PI3K/Akt signaling pathway is involved in a variety of physiological processes and is an important signaling pathway for many diseases, especially tumor development. It can regulate cell survival, metastasis and metabolism, and play a role in angiogenesis and recruitment of inflammatory factors. The present invention studies blood deficiency syndrome with 16SrRNA sequencing and metagenomic sequencing as the focus, reveals the relationship between the PI3K/Akt signaling pathway and the pathogenesis of blood deficiency syndrome, and provides a new idea for the design and screening of blood deficiency syndrome drugs.

Summary of the invention

In view of the problems existing in the prior art, the purpose of the present invention is to determine the relationship between the PI3K/Akt signaling pathway and blood deficiency syndrome, and to provide an application of the PI3K/Akt signaling pathway in the preparation of a drug for preventing, alleviating and/or treating blood deficiency syndrome.

The purpose of the present invention is achieved through the following technical solutions:

The first aspect of the present invention provides the use of PI3K/Akt in the preparation of a drug for treating blood deficiency syndrome.

In a second aspect, the present invention provides a method for screening drugs for treating blood deficiency using the PI3K/Akt signaling pathway, comprising the following steps: (1) preparing an animal model of blood deficiency; (2) pathological detection; (3) detection of proteins related to the kidney PI3K/Akt signaling pathway; (4) screening of active compounds; (5) docking of active compounds and target molecules; and (6) Western blot detection of the effects of active compounds on key signaling pathway proteins in rat kidneys.

In a third aspect, the present invention provides a pharmaceutical preparation comprising a PI3K/Akt pathway protein and a pharmaceutically acceptable carrier or excipient.

Preferably, the pharmaceutical preparation includes a solid preparation, a liquid dosage form, or a gel preparation.

Preferably, the pharmaceutical preparation is an oral preparation or an injection.

Preferably, the oral dosage form is selected from the group consisting of tablets, capsules, powders, granules, mixtures, pills and oral solutions.

In a fourth aspect, the present invention provides a drug for treating blood deficiency obtained by screening the PI3K/Akt signaling pathway, the drug comprising the following active ingredients: at least one of astragaloside IV, stilbene glycoside, paeoniflorin, and wedeloyl lactone, or a combination thereof.

The fifth aspect of the present invention provides a drug for treating blood deficiency syndrome obtained by screening PI3K/Akt, wherein the drug is Shengxuebao mixture.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The present invention discovered a new function of the PI3K/Akt pathway, namely, activating the PI3K/Akt pathway can increase the expression of hematopoietic factors, inhibit kidney inflammatory factors, and increase the expression of Bcl-2.

(2) Based on the role of PI3K/Akt in preventing, alleviating and/or treating blood deficiency syndrome, it can be used to prepare drugs for preventing, alleviating and/or treating blood deficiency syndrome.

(3) Based on the role of PI3K/Akt in the prevention, alleviation and/or treatment of blood deficiency syndrome, drugs for the treatment of blood deficiency syndrome were screened by using the PI3K/Akt signaling pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a PPI network diagram of shared targets (A) Shared target network diagram of Shengxuebao mixture in the treatment of BDS (B) Top 20 key targets;

Figure 2 is a network diagram of Shengxuebao mixture-ingredients-targets-diseases;

Figure 3 is a GO functional enrichment analysis diagram;

Figure 4 is a KEGG pathway analysis diagram;

Figure 5 shows the 3D and 2D images of the molecular docking results;

Figure 6 shows the HE staining results of kidney tissues of rats in each group, and the black arrows indicate glomeruli;

Figure 7 shows the expression of EPO and TNF-α in the serum of rats in each group;

Figure 8 shows the effect of Shengxuebao mixture on IL-6 and IL-1β in the serum of BDS rats;

Figure 9 shows the effect of Shengxuebao mixture on key signaling pathway proteins in rat kidney.

DETAILED DESCRIPTION

The inventive concept of the present invention will be illustrated below with reference to specific embodiments.

1. Materials

1.1 Database and software

Function database Website Compound and target disease target protein name conversion TCMSPTCMIDGenecardsUniprot https://old.tcmsp-e.com/tcmsp.php https://bidd.group/TCMID/https://www.genecards.org/https://www.uniprot.org/ Common target analysis Protein interaction network analysis Venny2.1.0STRING https://bioinfogp.cnb.csic.es/tools/venny/ https://cn.string-db.org/ GO and KEGG analysis DAVID6.8 https://david.ncifcrf.gov/ Draw a bubble chart ImageGP http://www.ehbio.com/ImageGP/index.php/home/in dex/upsetview.html Network construction and analysis Molecular simulation and docking Cytoscape3.7.2DiscoveryStudio2020 https://cytoscape.org/ Installation Package

1.2 Experimental instruments

instrument factory model ELISA reader Rayto RT-6100 Shaker Servicebio TSY-B Electrophoresis Apparatus Shanghai Tianneng Technology Co., Ltd. G0002 Vortex mixer USA/Scilogex MX-S Chemiluminescence imaging system Nikon Japan Nikon DS-U3 Scanner EPSON V370 Grayscale analysis software AlphaInnitech alphaEaseFC Desktop high speed refrigerated centrifuge DRAGONLAB D3024R Chemiluminescence analyzer CLINX 6300 Paper Cutter Deli No.8014 Ice Maker SIMAG SPR80

1.3 Experimental reagents and consumables

Reagents factory Part Number EPO Antibody Proteintech Biotechnology Co., Ltd. 66975-1-Ig EPOR Antibody SABSignalwayAntibody #38497-1 PI3K Antibodies Affinity Biotechnology Centre Ltd AF6241 P-PI3K Antibody Affinity Biotechnology Centre Ltd AF3241 AKT Antibody Proteintech Biotechnology Co., Ltd. 60203-2-Ig P-AKT Antibody Affinity Biotechnology Centre Ltd 66444-1-Ig TNF-α Antibody Abcam Ab205587 Bcl-2 Antibody Abcam ab194583 β-actin Abcam Ab8226 Rat EPO ELISA Kit Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. HO51 Rat IL-6 ELISA Kit Shenzhen Xinbosheng Biological Co., Ltd. ERC003 Rat Il-1β ELISA Kit Shenzhen Xinbosheng Biological Co., Ltd. ERC007 RIPA Lysis Buffer Beyotime P0013C 5× Protein Loading Buffer MCMBiotech WB2001 Protein molecular weight markers Proteintech PL00001 Twain 20 Solarbio TB220 ECL Proteintech PK1003 Goat anti-rabbit Abcam Ab150077 Goat anti-mouse Abcam Ab6789 Electrophoresis fluid Servicebio G2018-1L Transfer solution Servicebio G2019-1L Blood-producing mixture Tsinghua Deren Xi'an Xingfu Pharmaceutical Co., Ltd. 220823 Acetylphenylhydrazine Sinopharm Chemical Reagent Co., Ltd. A100226 Cyclophosphamide Jiangsu Hengrui Medicine Co., Ltd. 20221129 Compound donkey-hide gelatin paste Shandong Dong'e Ejiao Co., Ltd. 2211047 4% paraformaldehyde Servicebio G1101-500ML Anhydrous ethanol Sinopharm Chemical Reagent Co., Ltd. 100092693 Sodium Pentobarbital Merck, USA 57-33-0 0.9% Sodium Chloride Shandong Chenxin Pharmaceutical Co., Ltd. E23021322

2. Methods

2.1 Network Pharmacology

Network pharmacology and molecular docking technology play an important role in the field of traditional Chinese medicine compound. It can visualize the analysis of traditional Chinese medicine compound, find the main components, key targets and pathways from the database, explore the mechanism of traditional Chinese medicine compound in an integrated and coherent manner, and finally verify it in vitro and in vivo experiments. The authenticity and scientificity of computer simulation are guaranteed.

2.1.1 Collection and screening of active ingredients of Shengxuebao mixture

TCMSP and TCMID databases were selected as websites for searching information on Chinese herbal medicine components and structures. Based on OB (oral bioavailability) and DL (drug similarity), OB ≥ 30% and DL ≥ 0.18 were determined as the key ingredient screening conditions. Shengxuebao mixture contains 7 Chinese herbs. "Prepared Polygonum multiflorum", "Astragalus", "White Peony", "Eclipta prostrata", "Ligustrum lucidum", "Mulberry", and "Ciphala cibotii" were set as keywords to collect chemical components on the TCMSP and TCMID platforms.

2.1.2 Active ingredient target acquisition

In order to standardize the names of protein targets, the protein targets corresponding to the active ingredients screened from the TCMSP database were entered into the Uniprot database at one time, the species was limited to "Homosapiens", and the format was converted to "Genesymbol" to obtain the protein target information corresponding to the ingredient.

2.1.3 Disease target collection

A search was conducted in the GeneCards database using the keyword "Blood deficiency syndrome" and the species selected was "Homo Sapiens". The collected targets were saved in an EXCEL table.

2.1.4 Acquisition of drug-disease common targets

After deleting duplicates from all ingredient targets in the Shengxuebao mixture, enter the Venny website, with ingredient targets as List1 and disease targets as List2. The intersection of the two is the common target of the drug and the disease.

2.1.5 Construction of protein-protein interaction network

The common targets of drugs and diseases were analyzed through the STRING database, and "HomoSapiens" was selected with a confidence level of 0.900 (Highestconfidence>0.9) to draw a PPI network diagram. The network visualization software Cytoscape was then used for data analysis and processing, and NetworkAnalyzer was selected to analyze the network topology characteristic values such as degree centrality (Degree) and combined score (combinedscore).

2.1.6 Network construction, GO enrichment and KEGG pathway analysis

To explore the biological process, molecular function, cellular components and related pathways of Shengxuebao mixture in the treatment of BDS. The enriched results were obtained through the DAVID database, and the results ranked in the top 20 by Count (number of genes) were selected, and the bubble chart was drawn using the ImageGP online tool. Finally, the "drug-ingredient-disease-target" network diagram was constructed in the Cytoscape software and analyzed with "NetworkAnalyzer".

2.1.7 Molecular simulation docking

The molecular structures of active compounds and protein structures of overlapping targets were downloaded from the compound database and PD website, respectively. Virtual docking of compounds and targets was performed using the DS20 program and visualized using PyMOL software. The specific analysis is as follows: First, the ligands of active compounds in Shengxuebao mixture were collected (MOL2 format) and the ligands were processed. Second, the water molecules in the original ligands and protein receptors were removed, saved in PDB format, and then converted to PDBQT format. Finally, molecular docking was performed: AutoGrid was used to set the lattice size and parameters. After the docking was completed, the binding energy was used as a reference to screen out molecules with stronger binding ability in Shengxuebao mixture. The binding energy ≤-5.0kJ/mol was used as a standard to evaluate the good binding ability of molecules with targets.

2.2 Animal model preparation and treatment

Male Sprague Dawley rats were purchased from the Experimental Animal Center of the Air Force Medical University. The standard license number for experimental animals is IACUC-20231256. The body weight is (250±20) g. The temperature and humidity during the feeding process are 22±2℃ and 50-70%, respectively. The rats are then allowed to adapt to the environment and fed for seven days before the next experiment. All animals and operations comply with the relevant regulations on experimental animal ethics and are licensed and approved by the Air Force Medical University Experimental Animal Ethics Review. The clinical medication of Shengxuebao mixture stipulates that the dosage is 15ml/time, three times a day. According to the body surface area of rats and humans, the dosage of rats is 5.25ml/kg. The medium-dose group of Shengxuebao mixture is set at 2.62ml/kg, and the low-dose group is set at 1.31ml/kg. The experimental animals were divided into six groups according to the random number table method: blank group (Control), model group (Model group), Fufang ejiao Jiang (FEJ) group (6ml/kg), high dose (Shengxuebao mixture high dose, Shengxuebao mixture-H) 5.24ml/kg, middle dose (Shengxuebao mixture middle dose, Shengxuebao mixture-M) 2.62ml/kg and low dose (Shengxuebao mixture low dose, Shengxuebao mixture-L) 1.31ml/kg. The BDS model was established by subcutaneous injection of APH and intraperitoneal injection of CTX. The specific method was as follows: on the basis of the blank control group, the Model group, Shengxuebao mixture group and FEJ group were subcutaneously injected with APH 20 mg·kg-1 on the first day, subcutaneously injected with APH 40 mg·kg-1 on the fourth day, and intraperitoneally injected with CTX 25 mg·kg-1 2h later. The model can be replicated by continuous injection for 4 days. The blank control group was injected with an equal volume of normal saline. The mice were gavaged from the first day and the drug was administered for a total of 10 days.

2.2.1 Hematoxylin-eosin pathology test

Kidney tissue was fixed with 4% paraformaldehyde, then trimmed, dehydrated, embedded, sliced, stained, and sealed. Tissue sections were observed in detail at different magnifications to detect basic pathological changes in the sections, such as congestion, necrosis, and inflammatory changes. The sections were analyzed under a light microscope and photographed with an objective lens.

(1) Embedding and sectioning: The fixed kidney slices were transferred to a dehydration box and dehydrated with 75%, 85%, 90%, 95% and 100% alcohol at different time sequences; the tissues were then immersed in xylene solution, the wax-impregnated tissues were placed in an embedding machine and cut into parallel straight lines using a small paraffin cutter.

(2) Hematoxylin-eosin staining: After hematoxylin staining for 30 minutes, rinse with running water for 1 minute, then decolorize with 1% hydrochloric acid-alcohol for 2 seconds, restain with ammonia water for 1 minute, and finally stain with eosin for 20 to 40 seconds.

(3) Dehydration and sealing: Dehydrate the slices in different concentrations of ethanol, then air dry and seal with neutral gum. Avoid bubbles during the entire operation.

(4) Photographing and analyzing results: Observe and collect images using a microscope for result analysis.

2.2.2 ELISA method to detect the level of hematopoietic factors in serum

According to the instructions of the kit, enzyme-linked immunosorbent assay (ELISA) was used to detect EPO and TNF-u, IL-6 and IL-1β in rat serum.

2.2.3 Western blot detection of proteins related to the renal PI3K/Akt signaling pathway

1) Protein extraction: Weigh 20 mg of kidney tissue on an analytical balance, place in a 1.5 mL centrifuge tube and place on ice, prepare RIPA lysis buffer and protein phosphatase inhibitor in a ratio of 100:1, add 300 uL of the prepared lysis buffer to each sample for grinding, place on ice for lysis for 30 min, then centrifuge at 12000 rpm for 30 min in a 4° centrifuge, collect the supernatant in a new EP tube for later use.

2) Protein quantification: Prepare the BCA quantification kit and configure the standard and AB working solution according to its instructions. First add the prepared concentration gradient standard to the 96-well plate, then add 2uL of sample, and then immediately add 200uL of working solution, incubate at 37° for 30min, measure the absorbance at 576nm and draw a standard curve. After calculating the protein concentration, add 5X protein loading buffer to each group and shake and mix, boil the protein for 10min, and place it in a -20° refrigerator after it returns to room temperature.

3) Glue preparation: After cleaning and draining the device used for gel preparation, add double distilled water to check for leaks for 10 minutes. At the same time, prepare the separation gel and concentrated gel. First add the separation gel and let it solidify, then add the concentrated gel 40 minutes later. Immediately insert the comb vertically, and wait for 30 minutes for solidification before adding the sample.

4) Electrophoresis and transfer: Prepare samples and protein markers, add samples and perform electrophoresis under the parameters of S1 (80V, 30min) and S2 (120V, 1.5h). After electrophoresis, soak the PVDF membrane in methanol for 30s, introduce transfer solution and transfer device, lay layers into a sandwich structure, and transfer under the conditions of (100V, 1h).

5) Sealing and primary antibody incubation: Prepare 5% skim milk. After the membrane is transferred, take out the PVDF membrane in the clip and put it into skim milk. Slowly shake it on a shaker for 1 hour and then seal it. After sealing, wash it with PBST three times, 10 minutes each time, and incubate it with the prepared primary antibody dilutions EPO (1:1000), EPOR (1:1000), PI3K (1:500), p-PI3K (1:500), Akt (1:800), p-AKT (1:800), TNF-α (1:700), Bcl-2 (1:1000). After sealing, wash it with PBST three times, 10 minutes each time, and then incubate it with the prepared primary antibody dilutions at 4° for 8 hours.

6) Incubation with secondary antibody: Place the primary antibody at room temperature for 30 minutes, rinse with PBST three times, 10 minutes each time, incubate with the corresponding type of antibody at room temperature for 1 hour, and then rinse with PBST three times, 10 minutes each time.

7) Luminescence and grayscale analysis: Prepare AB luminescent liquid with a ratio of A:B=1:1, apply the luminescent liquid on the surface of the strip, and detect it through an imaging system and computer software ImageLab. After the detection, use ImageJ software for grayscale analysis.

2.3 Statistical analysis

All data were expressed as mean ± standard deviation (X ± SD) and analyzed using statistical software Graphpad Prism 8.0. One-way ANOVA was used to analyze the significance of the differences among the groups. P < 0.05 indicated that the results were statistically significant, and P < 0.01 indicated that the differences were significant.

3. Results

3.1 Network pharmacology results

3.1.1 Screening of active ingredients in Shengxuebao mixture

The components after searching and screening in the TCMSP database are shown in Table 1. A total of 62 components were collected after screening.

Table 3-1 Chemical components screened from Shengxuebao mixture

MolID MoleculeName Oral bioavailability OB (%) Drug-like DL MOL010300 dIDP(6Z,10E,14E,18E)-2,6,10,15,19,23- 41.08 0.57 MOL002372 hexamethyltetracosa-2,6,10,14,18,22-hexaene 33.55 0.42 MOL006209 Cyanin 47.42 0.76 MOL000737 Morin 46.23 0.27 MOL002773 beta-carotene 37.18 0.58 MOL000098 Quercetin 46.43 0.28 MOL000358 beta-sitosterol 36.91 0.75 MOL000422 Kaempferol 41.88 0.24 MOL004576 Taxifolin 57.84 0.27 MOL005146 LucidumosideD 48.87 0.71 MOL005147 LucidumosideD_qt 54.41 0.47 MOL005169 (20S)-24-ene-3β,20-diol-3-acetate 40.23 0.82 MOL005190 Eriodictyol 71.79 0.24 MOL005195 syringaresinoldiglucoside_qt 83.12 0.80 MOL005209 Lucidusculine 30.11 0.75 MOL005211 Olitoriside 65.45 0.23 MOL005212 Olitoriside_qt 103.23 0.78 MOL000006 Luteolin 36.16 0.25 MOL000098 Quercetin 46.43 0.28 MOL001790 Linarin 39.84 0.71 MOL001689 Acacetin 34.97 0.24 MOL002975 Butin 69.94 0.21 MOL003378 1,3,8,9-tetrahydroxybenzofurano[3,2-c]chromen-6-one 33.94 0.43 MOL003389 3'-O-Methylorobol 57.41 0.27 MOL003398 Pratensein 39.06 0.28 MOL003402 Demethylwedelolactone 72.13 0.43 MOL003404 Wedelolactone 49.6 0.48 MOL000006 Luteolin 36.16 0.25 MOL000098 Quercetin11alpha,12alpha-epoxy-3beta-23- 46.43 0.28 MOL001910 dihydroxy-30-norolean-20-en-28,12beta-olide 64.77 0.38 MOL001918 Paeoniflorgenone(3S,5R,8R,9R,10S,14S)-3,17-dihydroxy-4,4,8,10,14- 87.59 0.37 MOL001919 pentamethyl-2,3,5,6,7,9-hexahydro-1H-cyclopenta[a]phenanthrene-15,16-dione 43.56 0.53 MOL001921 Lactiflorin 49.12 0.80 MOL001924 Paeoniflorin 53.87 0.79 MOL001925 paeoniflorin_qt 68.18 0.40 MOL001928 albiflorin_qt 66.64 0.33 MOL001930 benzoylpaeoniflorin 31.27 0.75 MOL000211 Mairin 55.38 0.78 MOL000358 beta-sitosterol 36.91 0.75 MOL000359 Sitosterol 36.91 0.75 MOL000422 Kaempferol 41.88 0.24 MOL000492 (+)-catechin 54.83 0.24 MOL000211 Mairin 55.38 0.78 MOL000239 Jaranol 50.83 0.29 MOL000296 Hederagenin(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl- 36.91 0.75 MOL000033 17-[(2R,5S)-5-propan-2-yloctan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H- cyclopenta[a]phenanthren-3-ol 36.23 0.78 MOL000354 isorhamnetin 49.6 0.31 MOL000371 3,9-di-O-methylnissolin 53.74 0.48 MOL000374 5'-hydroxyiso-muronulatol-2',5'-di-O-glucoside 41.72 0.69 MOL000378 7-O-methylisomucronulatol 74.69 0.30 MOL000379 9,10-dimethoxypterocarpan-3-O-β-D-glucoside 36.74 0.92 MOL000380 dihydro-6H-benzofurano[3,2-c]chromen-3-ol 64.26 0.42 MOL000387 Bifendate 31.1 0.67 MOL000392 formononetin 69.67 0.21 MOL000398 isoflavanone 109.99 0.3 MOL000417 Calycosin 47.75 0.24 MOL000422 kaempferol 41.88 0.24 MOL000433 FA 68.96 0.71 MOL000098 Quercetin 46.43 0.28 MOL000438 (3R)-3-(2-hydroxy-3,4-dimethoxyphenyl)chroman-7-ol 67.67 0.26 MOL000439 isomucronulatol-7,2'-di-O-glucosiole 49.28 0.62 MOL000442 1,7-Dihydroxy-3,9-dimethoxypterocarpene 39.05 0.48

3.1.2 Obtaining common targets

The targets corresponding to the active ingredients of Shengxuebao mixture were deduplicated and imported into the Uniprot database to convert gene names, resulting in a total of 324 targets. 615 anemia disease targets with a score greater than 15 were screened out using the Genecards database, and 66 potential targets of Shengxuebao mixture for the treatment of BDS were obtained by taking the intersection in Venny.

3.1.3 Results of PPI network construction of Shengxuebao mixture regulating BDS

The common targets of drug components and diseases that were screened were imported into the STRING database, and a network model of protein-protein interaction and PPI enrichment was established. The degree centrality and comprehensive score of the network topology feature values were analyzed using NetworkAnalyzer, and the results showed that the main targets were TNF, IL-6, AKT1, etc., as shown in Figure 1.

3.1.4 Network diagram of common targets of Shengxuebao mixture, compound and BDS

The common targets of Shengxuebao mixture and seven medicinal materials-compound components-BDS diseases were summarized into a table and imported into Cytoscape3.7.2 software to construct a drug-compound-disease common target network diagram, see Figure 2.

3.1.5 Analysis of enrichment of BDS predicted targets regulated by Shengxuebao mixture

The DAVID online analysis tool was used to perform GO enrichment analysis on the common target proteins of Shengxuebao mixture components-BDS, including biological processes, molecular functions and cellular components. The results showed that the main biological processes of BDS regulated by Shengxuebao mixture included negative regulation of RNA polymerase II promoter transcription, apoptosis process, drug response, immune response, etc., as shown in Figure 3. The DAVID online analysis tool was used to perform KEGG enrichment analysis on Shengxuebao mixture. The results showed that the mechanism of Shengxuebao mixture regulating BDS was mainly related to signaling pathways such as PI3K/Akt and HIF-1. The KEGG bubble diagram of the top 20 pathways was drawn by sorting by P <0.01 value, as shown in Figure 4.

3.1.6 Docking of active compounds and important target molecules

Nine compounds with high ranking in the "active compound-target-pathway" network, including astragaloside, 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-Glucoside, paeoniflorin, demethylwedelolactone, specnuezhenide, and isoflavanone, were selected for molecular simulation docking with the main target genes IL-6, TNF-α, and AKT1. The lower the binding energy, the more stable the conformation. Binding energy <-7.0 kcal/mol indicates that the molecule has a strong binding ability and a certain stability with the target. Ki (inhibition rate constant) is less than 10 μmol/L, and the molecules have good inhibitory activity against the target. The docking results in Table 2-2 show that PI3K has a good binding score with paeoniflorin (-7.2), AKT1 has a good binding score with demethylwedeholactone (-7.3), IL6, TNF-α, and IL-1β have a good binding score with diphenylethylene glycoside (-5.2), isoflavone (-5.8), and astragaloside IV (-6.1), respectively. The results show that the 9 active ingredients have good binding activity and stability with the 5 core targets, showing good affinity. The subsequent visualization and analysis of compound-protein binding is shown in Figure 5.

Table 3-2 Molecular docking results

Target Stilbene Glycol Paeoniflorin Astragaloside IV Ligustrum lucidum Morin Wedelolide Dogwood fern Isoflavones Chrysophanol PI3K -6.5 -7.2 -8.4 -8.1 -8.2 -6.5 -8 -7.4 -8.6 AKT1 -9.2 -9.2 -10.2 -7.9 -9.9 -7.3 -8.7 -4.4 -9.7 IL-6 -5.2 -5.3 -4.7 -4.2 -4.4 -4.9 -3.8 -5.1 -5.5 TNF-α -8.7 -10.9 -7.6 -8.9 -1 -7.5 -8.4 -5.8 -10.1 IL-1β -4.7 -5.5 -6.1 -5.1 -5 -5.2 -8.1 -4.7 -5.1

3.2 Effect of Shengxuebao mixture on renal pathological damage in BDS rats

HE staining was used to observe the pathological state of the kidney tissue of BDS rats treated with Shengxuebao mixture. The results are shown in Figure 6. Compared with the C group, the glomeruli of the rats in the M group were reduced, a small amount of cell necrosis appeared in the kidney tissue, the number of RBCs in the renal interstitial capillaries decreased, and the glomerular capsule cavity and renal tubular lumen were dilated. Compared with the rats in the M group, the dilation of the glomerular capsule cavity and renal tubular lumen in the Shengxuebao mixture group and the FEJ group was significantly reduced, the density of necrotic cells decreased, and the RBC content in the interstitial capillaries increased. The staining showed that Shengxuebao mixture can restore the kidney damage caused by BDS rats.

3.3 Effect of Shengxuebao mixture on EPO and TNF-α levels in serum of BDS rats

Since blood deficiency syndrome can cause different degrees of damage to the kidney and bone marrow, the kidney's ability to synthesize EPO is reduced, and the secretion of hematopoietic factors is disordered, resulting in a decrease in the body's hematopoietic ability. In mammals, EPO produced by the kidney is essential for the production of RBC. After adulthood, the kidney becomes the main site of EPO production, and TNF-α, as an important inflammatory factor and negative hematopoietic factor, is essential for hematopoiesis. As shown in Figure 7, EPO and TNF-α in serum were detected by ELISA. Compared with group C, the EPO level in group M was significantly reduced ( P < 0.05), and the expression of TNF-α was significantly increased ( P < 0.05). Both the Shengxuebao mixture group and the FEJ group could upregulate EPO ( P < 0.05) and downregulate TNF-α ( P < 0.05), indicating that Shengxuebao mixture can promote the secretion and synthesis of renal EPO, reduce renal inflammatory response, and have a good improvement on renal function.

3.4 Effect of Shengxuebao mixture on positive regulatory factors of hematopoiesis in BDS rats: IL-6 and IL-1β

The connection between hematopoiesis and bone marrow hematopoiesis was described earlier. Maintaining the balance of hematopoietic factors plays an important role in restoring the body's hematopoietic function. IL-6 and IL-1β are important positive regulatory factors for hematopoiesis. By detecting these two indicators, it can be indirectly reflected whether the treatment of BDS rats with Shengxuebao mixture is related to the regulation of hematopoietic factors in the body. IL-6 is a cytokine secreted by bone marrow stromal cells, which has multiple functions such as regulating immune response, acute phase response and hematopoiesis. IL-1β is a subtype of IL-1, which can promote the proliferation of hematopoietic stem cells (HSC) and induce other cells to produce hematopoietic factors to promote hematopoiesis. As shown in Figure 8, the ELISA method was used to detect IL-6 and IL-1β in the serum of rats in each group. It was found that compared with group C, IL-6 and IL-1β in group M were significantly downregulated ( P < 0.05), and IL-6 and IL-1β were upregulated in each group treated with Shengxuebao mixture and the FEJ group ( P < 0.05). This result suggests that Shengxuebao mixture can improve the level of hematopoietic factors in BDS rats, thereby regulating the hematopoietic function in the body and playing a blood-tonifying role.

3.5 Effect of Shengxuebao mixture on PI3K/Akt signaling pathway proteins in BDS rats

According to the previous network pharmacology analysis results, the PI3K/Akt signaling pathway was also predicted as one of the pathways for treating BDS, among which TNF-α, AKT1, etc. were predicted as important targets for treating BDS. In order to verify the prediction, the expression of important targets and related targets on the PI3K/Akt signaling pathway by Shengxuebao mixture was studied. EPO, as an important hematopoietic factor in the body, can stimulate hematopoiesis with EPOR to treat anemia. At the same time, EPO also has non-hematopoietic functions such as anti-inflammation and anti-apoptosis. Bcl-2 is an important anti-apoptotic gene. The Western blot results are shown in Figure 9. Compared with group C, EPO and EPOR in group M decreased ( P < 0.05), the phosphorylation levels of p-PI3K and p-Akt decreased ( P < 0.05), the expression of Bcl-2 decreased ( P < 0.05), and the expression of TNF-α increased ( P < 0.05). After treatment with Shengxuebao mixture and FEJ, the expression of EPO and EPOR proteins was significantly increased, the phosphorylation level of PI3K/Akt was increased, TNF-α was inhibited, and Bcl-2 expression was promoted. According to the above results, EPO, phosphorylated PI3K, phosphorylated AKT, and Bcl-2 were significantly increased in the Shengxuebao mixture group and the FEJ group, indicating that each therapeutic drug not only restored the expression of renal EPO, but also activated the PI3K/AKT pathway, increased the Bcl-2 content, and played an anti-apoptotic and anti-inflammatory role in renal tissue. In short, the improvement of blood deficiency symptoms by Shengxuebao mixture is related to the activation of PI3K/Akt pathway by EPO, the upregulation of Bcl-2 level, and the anti-apoptotic and anti-inflammatory effects on renal tissue.

4. Discussion

Traditional Chinese medicine research has the characteristics of multiple targets, multiple routes, and multiple pathways, but it is also a double-edged sword. It has unique advantages for traditional Chinese medicine, but it also brings difficulties to the development and research of traditional Chinese medicine. The difficulty of its research lies in the complex mechanism of interaction between different components of traditional Chinese medicine and between different components and the body, which restricts research and development. However, with the rise of network pharmacology, it provides new options for traditional Chinese medicine research, which is more in line with the characteristics of multi-target and multi-pathway of traditional Chinese medicine compound. Its integrity and comprehensiveness further promote the development of traditional Chinese medicine. Therefore, this chapter is based on the network pharmacology method to screen out the key components, targets and pathways that meet the conditions, so as to clarify the protective effect of Shengxuebao mixture on BDS and provide evidence support for later experimental verification.

Through network pharmacology, it was found that the main active ingredients in Shengxuebao mixture, astragaloside, 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-Glucoside, paeoniflorin, demethylwedelolactone, specnuezhenide, isoflavanone, chrysophanol, morin and Woodwardic acid, have many targets and play the main pharmacological effects. Astragaloside and isoflavones are the main components of Astragalus. Astragaloside has a significant effect on anemia after chemotherapy. Stilbene glycoside and chrysophanol are the main components of Polygonum multiflorum. Stilbene glycoside can increase the number of WBC after chemotherapy and improve bone marrow hematopoietic function. Chrysophanol can fight against atherosclerosis, inhibit the nuclear factor kappa B (NF-κB) signaling pathway, reduce the level of inflammatory factors and play an anti-inflammatory role. Ligustrum lucidum glycoside is the main component of Ligustrum lucidum. Studies have reported that it can treat bone marrow suppression caused by CTX and regulate the expression of hematopoietic factors in the body. Morin is a natural bioflavonoid widely found in different species of Moraceae. Some experiments have shown that it can improve antioxidant levels, reverse cell apoptosis, autophagy, etc. by regulating the PI3K/Akt/mTOR signaling pathway, proving that morin can produce chemical protection against acute neurotoxicity induced by the chemotherapy drug ifosfamide by intervening in inflammation and apoptosis markers, such as NF-κB and B-cell lymphoma-2 (Bcl-2). Paeoniflorin, a water-soluble monoterpene glycoside, is the main bioactive component of white peony root. Studies have shown that peoniflorin has a wide range of in vitro and in vivo pharmacological effects, and can significantly increase the number of peripheral blood WBC, CFU-GM, CFU-E and CFU-M in radiation-induced BDS mice. Dog spine fern is an effective ingredient in dog spine, which can not only enhance the liver and kidney function of the body, but also play a role in postpartum hemostasis, anti-inflammatory and antiviral effects. Based on this, the present invention confirms that the above components are the main components of Shengxuebao mixture.

According to the kidney theory of traditional Chinese medicine, blood production is closely related to kidney function. The kidney stores essence and produces blood, and the erythropoietin secreted by the kidney stimulates bone marrow hematopoiesis, thereby maintaining normal hematopoiesis of the body. Mature EPO is produced in renal interstitial fibroblasts. EPO acts on the bone marrow hematopoietic system, promotes the differentiation and maturation of primitive RBCs, and accelerates RBC production. 90% of EPO in the body originates from the kidneys and has multiple functions, especially protecting renal function. The hematopoietic ability of EPO is to stimulate hematopoiesis through EPOR and treat anemia [128]. Studies have reported that EPO deficiency limits the survival of animals. The number of RBCs and HCT of mice with EPO knockout genes are significantly reduced, and renal function is impaired. EPO and EPOR are important regulators of red blood cell differentiation, proliferation, and cell survival. The interaction between them triggers the activation of multiple signaling pathways, including PI3K/Akt, JAK/STAT, and MAPK. Studies have shown that the use of Angelica polysaccharides to treat renal anemia can restore the production of EPO and the expression of EPORmRNA, activate EPOR downstream of the PI3K/Akt signaling pathway, thereby inducing its target genes Bcl-xL and Fam132b, and increasing the Bcl-2/Bax ratio in the bone marrow mononuclear cells of rats with chronic kidney disease (CKD); at the same time, the PI3K/Akt pathway can play a role in blood, metabolism, and various tumor diseases. The PI3K/Akt signaling pathway transmits intracellular signal cascades and participates in the regulation of multiple cellular processes, such as cell proliferation, differentiation, and apoptosis. It can be seen that the study of renal EPO secretion and the renal protective effect of the PI3K/Akt pathway is of great significance.

Therefore, the present invention firstly observed the basic functions of rat kidneys through renal pathology. According to previous network pharmacology analysis, the PI3K/Akt pathway was predicted to be one of the ways to treat BDS. In order to verify the prediction, the expression of important targets and related targets on the PI3K/Akt signaling pathway by Shengxuebao mixture was studied. The results showed that the renal index of the rats in group M was significantly increased ( P < 0.05), and the kidneys also showed pathological conditions, resulting in different degrees of kidney damage in the rats in group M. After treatment with the drug Shengxuebao mixture, it was shown that the renal pathological conditions could be improved and the renal function could be protected. The results of Western blot and ELISA experiments showed that the rats in group M had a resistance to the synthesis and secretion of EPO ( P < 0.05). After treatment with Shengxuebao mixture and FEJ group, the levels of EPO and EPOR could be increased ( P < 0.05), indicating that Shengxuebao mixture could improve the EPO production of rats in group M and restore the symptoms of blood deficiency. At the same time, hematopoietic function is inseparable from the regulation of hematopoietic factors in the body. IL-6 is a cytokine secreted by bone marrow stromal cells, which has multiple functions such as regulating immune response, acute phase response and hematopoiesis. The study showed that Siwu Decoction can significantly improve the anemia state by regulating the expression of serum hematopoietic cytokine IL-6 to exert hematopoietic function [133]. IL-1β is a subtype of IL-1, which can promote the proliferation of hematopoietic stem cells (HSC) and induce other cells to produce hematopoietic factors to promote hematopoiesis. Related studies have confirmed that FEJ can positively regulate the body's hematopoiesis by promoting the expression of related hematopoietic growth factor IL-1β in anemic mice. Secondly, the expression of TNF-α in the kidneys of rats in the M group increased, and the level of Bcl-2 decreased ( P <0.05). After treatment with the Shengxuebao mixture group and the FEJ group, the TNF-α level was reduced and the expression of Bcl-2 was increased ( P <0.05). Shengxuebao mixture can improve the state of rats with blood deficiency syndrome, and the mechanism may be related to promoting EPO/PI3K/Akt-mediated erythropoiesis in the kidneys and reducing renal inflammatory response.

Based on mechanism research, it was found that Shengxuebao mixture can induce the expression of EPO and EPOR, activate the PI3K/Akt signaling pathway, inhibit the expression of TNF-α, promote the anti-apoptosis effect of the kidney, maintain the kidney structure, and improve the symptoms of blood deficiency, thereby playing a role in protecting renal tissue.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (8)

1. Application of PI3K/Akt in preparing medicine for preventing, relieving and/or treating blood deficiency.
2. 2. A method for screening a medicament for treating blood deficiency by using a PI3K/Akt signal pathway, which is characterized by comprising the following steps: (1) preparation of an animal model of blood deficiency; (2) pathology detection; (3) detection of kidney PI3K/Akt signal channel related proteins; (4) screening of active compounds; (5) docking the active compound with a target molecule; (6) Western blot detects the effect of active compounds on rat kidney key signal pathway proteins.
3. 3. A pharmaceutical formulation comprising a PI3K/Akt pathway protein, and a pharmaceutically acceptable carrier or excipient.
4. 4. A pharmaceutical formulation according to claim 3, wherein the pharmaceutical formulation comprises a solid formulation, a liquid formulation, or a gel formulation.
5. 5. A pharmaceutical formulation according to claim 3, wherein the pharmaceutical formulation is an oral formulation or an injection.
6. 6. The pharmaceutical formulation of claim 5, wherein the oral dosage form is selected from the group consisting of: tablets, capsules, powders, granules, mixtures, pills and oral liquids.
7. 7. A medicament for treating blood deficiency obtained by screening a PI3K/Akt signal pathway, the medicament comprising the following active ingredients: at least one of astragaloside IV, stilbene glucoside, paeoniflorin and demethylwedelolactone or a combination thereof.
8. 8. The medicament of claim 7, wherein the medicament is a blood born mixture.