Use of jinkui shenqi pills in preparation of drug to prevent or treat neural tube defects

Abstract

To provide a novel use of Jinkui Shenqi pills (JSP) in the preparation of a drug to prevent or treat neural tube defects (NTD).SOLUTION: Provided is a use of a pharmaceutical composition and a formulation thereof in the preparation of a drug for preventing or treating neural tube malformation, the raw herbal ingredients of the pharmaceutical composition including 20 to 28 pts.wt. of Dried root of Rehmannia glutinosa, 4 to 8 pts.wt. of Cornus officinalis fruit, 4 to 8 pts.wt. of Dioscorea Rhizome, 4 to 8 pts.wt. of Alisma Tuber, 15 to 20 pts.wt. of Poria Sclerotium, 4 to 8 pts.wt. of Moutan Bark, 4 to 8 pts.wt.of Achyranthes Root, and 0.5 to 1.5 pts.wt.of Processed Aconite Root, preferably, the pharmaceutical composition further including 4 to 8 pts.wt. of Cinnamon Rhizome and / or 4 to 8 pts.wt. of Plantago Seed.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention belongs to the technical field of drug use and pharmacological research, and particularly relates to a novel use of Jingui Jinqi Wan in preparing a drug for preventing or treating neural tube defects. [Background technology]

[0002] Neural tube defects (NTDs) are congenital nervous system defects that are influenced by genetic and environmental factors. They are caused by incomplete closure of the neural tube during fetal development and are the most common congenital anomalies occurring in the central nervous system (CNS). They usually include anencephaly, spina bifida, and encephalocele. Research has shown that neural tube defects are an important cause of neural tube defects, and various clinical symptoms are seen due to incomplete closure at different positions of the neural axis. Most patients with symptomatic diseases suffer from incontinence and learning disabilities and remain wheelchair bound for life. Currently, the incidence of NTDs can be reduced to some extent by taking folic acid, but the mechanism of NTD development is not completely clear, and folic acid cannot prevent or treat all NTDs. Existing studies have shown that the development of NTDs is closely related to multiple signaling pathways, including the canonical Wnt / β-catenin pathway, the non-canonical Wnt / planar cell polarity (PCP) signaling pathway, changes in bone morphology (bone morphogenetic protein (BMP)), and retinoic acid (RA), and is also associated with the PI3K / Akt pathway.

[0003] In the theory of traditional Chinese medicine, innate essence is also called yuan essence. In addition to the innate essence scattered in the limbs and bones, other innate essences are concentrated in the kidneys, which is the manifestation of the function of "kidneys have the function of storage" and "kidneys store essence". In "Suwen: Strange Diseases," it is written that "the kidneys store essence, essence fills the bones to produce bone marrow, bone marrow gathers to become the brain, bone marrow fills the brain, and when essence is gone, the brain disappears." From this description, we can see that the kidneys are the source of bone marrow production, and when the bone marrow is sufficient, the brain is healthy. When parents' kidney essence is deficient, the fetus will have a lack of innate essence, which will affect the growth and development of the fetus and cause neural tube defects, which is basically consistent with the mechanism of NTD development.

[0004] Jingui shenqi pill (JSP: Jingui shenqi pill) was first published in the Jingui Yaolue: Blood Paralysis Deficiency Labor Disease Edition, and is also called Eight Flavors Jingui Pill. The formula contains a total of eight kinds of herbal medicines, including Rehmannia Root, Cornus officinalis, Chinese Yao, Zeaxanthemum Root, Poria Cocos, and Danpi, which have the effect of nourishing kidney yin, and Cinnamon Bark and Aconite Root, which have the effect of nourishing yang. This formula is the first to "request yang from yin", and can exert the effect of strengthening both yin and yang. It is a famous formula from ancient times to strengthen the kidneys, support yang, and help essence become qi. JSP has been widely studied in osteoporosis, diabetes, anti-aging, etc., but no experimental reports on NTDs have been seen yet. Therefore, the field is expected to develop more novel uses of drugs, which is of positive significance for the development of traditional high-quality formulas. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, the technical problem to be solved by the present invention is to provide a novel use of Jingui Jinki Wan (JSP) in preparing a medicament for preventing or treating neural tube defects.

[0006] The second technical problem to be solved by the present invention is to provide a pharmaceutical preparation for preventing and treating neural tube defects. [Means for solving the problem]

[0007] To solve the above technical problems, the present invention discloses the use of Jingui Jinqi Wan (JSP) in preparing a medicament for preventing or treating neural tube defects (NTDs).

[0008] The present invention also discloses the use of Jingui Jinki Wan (JSP) in the preparation of a medicament for preventing or treating neural tube defects caused by NTDs.

[0009] The present invention also discloses the use of Jingui Jinki Wan (JSP) in the preparation of a PI3K / Akt pathway inhibitor.

[0010] The present invention also relates to a use of a pharmaceutical composition and its formulation in preparing a medicament for preventing or treating neural tube defects (NTD), the pharmaceutical composition comprising raw herbal ingredients including 20-28 parts by weight of Rehmannia Root, 4-8 parts by weight of Cornus Root, 4-8 parts by weight of Chinese Yam, 4-8 parts by weight of Zelkova Root, 15-20 parts by weight of Poria Root, 4-8 parts by weight of Danpi Root, 4-8 parts by weight of Cinnamon Bark, and 0.5-1.5 parts by weight of Aconite Root; Preferably, the pharmaceutical composition further comprises 4-8 parts by weight of osmanthus and / or 4-8 parts by weight of psyllium sap. The pharmaceutical composition and its formulation use are disclosed.

[0011] Preferably, the raw herbal medicines of the pharmaceutical composition include 24 parts by weight of Rehmannia Root, 6 parts by weight of Cornus Officinalis Root, 6 parts by weight of Chinese Yam, 6 parts by weight of Zelkova Root, 17.3 parts by weight of Poria Cocos Root, 6 parts by weight of Danpi Root, 6 parts by weight of Cinnamon Bark, and 1 part by weight of Aconite Root; Preferably, the pharmaceutical composition further comprises 6 parts by weight of calfskin and / or 6 parts by weight of psyllium.

[0012] The present invention also relates to a use of a pharmaceutical composition and its formulation in preparing a medicament for preventing or treating neural tube defects caused by NTD, the pharmaceutical composition comprising 20-28 parts by weight of Dried Rehmannia Root, 4-8 parts by weight of Cornus Root, 4-8 parts by weight of Chinese Yam, 4-8 parts by weight of Zelkova Root, 15-20 parts by weight of Poria Root, 4-8 parts by weight of Danpi Root, 4-8 parts by weight of Cinnamon Bark, and 0.5-1.5 parts by weight of Aconite Root; Preferably, the pharmaceutical composition further comprises 4-8 parts by weight of osmanthus and / or 4-8 parts by weight of psyllium sap. The pharmaceutical composition and its formulation use are disclosed.

[0013] Preferably, the raw herbal medicines of the pharmaceutical composition include 24 parts by weight of Rehmannia Root, 6 parts by weight of Cornus Officinalis Root, 6 parts by weight of Chinese Yam, 6 parts by weight of Zelkova Root, 17.3 parts by weight of Poria Cocos Root, 6 parts by weight of Danpi Root, 6 parts by weight of Cinnamon Bark, and 1 part by weight of Aconite Root; Preferably, the pharmaceutical composition further comprises 6 parts by weight of calfskin and / or 6 parts by weight of psyllium.

[0014] The present invention also relates to a pharmaceutical composition and its preparation for use in preparing a PI3K / Akt pathway inhibitor, the pharmaceutical composition comprising 20-28 weight parts of Dried Rehmannia Root, 4-8 weight parts of Cornus Root, 4-8 weight parts of Chinese Yam, 4-8 weight parts of Zelkova Root, 15-20 weight parts of Poria Root, 4-8 weight parts of Danpi, 4-8 weight parts of Cinnamon Bark, and 0.5-1.5 weight parts of Aconite Root; Preferably, the pharmaceutical composition further comprises 4-8 parts by weight of osmanthus and / or 4-8 parts by weight of psyllium sap. The pharmaceutical composition and its formulation use are disclosed.

[0015] Preferably, the content is 24 parts by weight of Ryujihuang, 6 parts by weight of Japanese yam, 6 parts by weight of wild herbs, 6 parts by weight of Zetan, 17.3 parts by weight of Bouling, 6 parts by weight of Danpi, 6 parts by weight of Guizhi, and 1 part by weight of Fuzi. Preferably, the pharmaceutical composition further comprises 6 parts by weight of calfskin and / or 6 parts by weight of psyllium.

[0016] The present invention also discloses the use of the composition in the preparation of a medicament for preventing or treating neural tube defects (NTD), wherein the main active ingredients of the composition include Quercetin, Wogonin, Sitosterol, Kaempferol, and Stigmasterol.

[0017] The present invention also discloses the use of the composition in the preparation of a medicament for preventing or treating neural tube defects caused by NTDs, wherein the main active ingredients of the composition include Quercetin, Wogonin, Sitosterol, Kaempferol, and Stigmasterol.

[0018] The present invention also discloses the use of the composition in the preparation of a PI3K / Akt pathway inhibitor, wherein the main active ingredients of the composition include Quercetin, Wogonin, Sitosterol, Kaempferol, and Stigmasterol. Effect of the Invention

[0019] This invention uses network pharmacology analysis methods to develop and verify a novel use of Jingui Jinqi Wan (JSP) to prevent or treat NTDs, analyzes the active ingredients, action targets and signaling pathways of JSP to prevent and inhibit NTDs, conducts preliminary experimental verification of the main targets and pathways of JSP to prevent and inhibit NTDs, and provides a theoretical basis for the clinical application of JSP in preventing and inhibiting NTDs.

[0020] In the present invention, the targets and active ingredients of JSP that prevent and suppress NTDs were analyzed through network pharmacology, and 26 major active ingredients, including Quercetin, Wogonin, Beta-Sitosterol, Kaempferol, and Stigmasterol, and 64 major targets, including IL-10, IL-6, and Bcl-2, were obtained. KEGG pathway analysis revealed that JSP can act by controlling signaling pathways such as the PI3K / Akt pathway and Sonic hedgehog factor (shh), and molecular docking showed that JSP can effectively bind to proteins related to the PI3K / Akt pathway. This is consistent with the network pharmacological results presented in previous studies that the occurrence of NTDs is related to the PI3K / Akt pathway, and further verified the action and effectiveness of JSP in preventing and suppressing NTDs.

[0021] Through animal experiments, the present invention further demonstrates that JSP can promote the activation of the PI3K / Akt pathway, and the mechanism may be related to downstream regulated biological processes such as cell proliferation and apoptosis, inflammation, and reactive oxygen metabolism. Akt is the core protein of the PI3K / Akt pathway, and when phosphorylated, it acts on the Bcl-2 protein family, among which Bcl-2 can play a role in inhibiting cell apoptosis. Bax protein is also one of the Bcl-2 protein family, and when cells undergo apoptosis, Bax can promote the release of cytochrome C and accelerate apoptosis, and the higher the value of Bcl-2 / Bax, the lower the degree of apoptosis. The close relationship between the PI3K / Akt pathway and apoptosis is consistent with the mechanism of apoptosis in neural tube epithelial cells during the development and progression of NTD. The animal experimental results of the present invention combined with the literature further demonstrated that the main active components in JSP, namely quercetin, wogonin, sitosterol, kaempferol, and stigmasterol, have the potential to inhibit the occurrence of apoptosis, further confirming the important role of the PI3K / Akt pathway in the prevention and suppression of NTDs by JSP.

[0022] This invention explores the mechanism of action of JSP in the prevention and inhibition of NTDs based on network pharmacology combined with in vivo experiments. Through network pharmacology, we preliminarily investigated the possible action targets and pathways of JSP in the treatment of NTDs, and combined with the verification of animal experiments, we demonstrated that JSP can reduce the incidence of NTDs in mouse fetuses, reduce the apoptosis of neural tube epithelial cells, and increase the expression of PI3K / Akt signaling pathway-related proteins. As mentioned above, the role of JSP in the prevention and inhibition of NTDs is likely to be achieved by activating the PI3K / Akt pathway to reduce the occurrence of cell apoptosis, which provides a theoretical basis for the deeper application of JSP and fills the gap in the prior art that there is no research and application of JSP to NTDs. [Brief description of the drawings]

[0023] In order to facilitate a clearer understanding of the present invention, the present invention will be described in more detail below based on specific embodiments of the present invention with reference to the drawings.

[0024] [Figure 1] Schematic diagram of drug-active ingredient-disease-target network, where square nodes represent action targets, oval nodes represent active ingredients, circular nodes represent drug names, and each edge represents an interaction relationship between the target and the active ingredient. [Diagram 2] FIG. 1 is a schematic of a Venn diagram of JSP-NTD targets. [Diagram 3] Schematic diagram of the common target PPI network of JSP-NTD and screening of core targets, where the size and color change of the node represent the magnitude of the degree value, and the larger the node and the darker the color, the higher the corresponding degree value and the more edges connected to it, which indicates its importance. [Figure 4] FIG. 1 is a schematic diagram of the “active ingredient-target-disease” network. [Diagram 5] Results of GO functional enrichment analysis of core targets for NTD prevention and suppression by WYP. [Figure 6] KEGG pathway analysis (first 20) of core targets for NTD prevention and suppression by WYP. The vertical axis represents the most enriched pathways from top to bottom, and the horizontal axis represents the ratio of the number of genes enriched in the target pathway in the gene list to the total number of genes in the gene list. The larger the dot, the more targets are enriched in the pathway. [Figure 7] This figure shows the binding energy of docking between the main active ingredient and the core target. The darker the color, the lower the binding energy, and the lighter the color, the higher the binding energy. When the binding energy is less than 0, it indicates that there is binding activity between the molecules. When the binding energy is less than -5.0 kcal·mol-1, it indicates that the binding activity between the molecules is strong. The lower the energy, the stronger the binding ability. [Figure 8]Schematic diagram of docking of main active ingredients with core targets, where (A) quercetin-Akt1, (B) wogonin-IL6, (C) β-sitosterol-PIK3CG, (D) kaempferol-Akt2, and (E) stigmasterol-IL10. The dark molecule in the center is the drug, the lighter shades are the core protein, and the short dotted bar and the darker parts of the adjacent small molecules are amino acid residues through which hydrogen bonds connect the drug and the core protein. [Figure 9] Stereomicroscope images and HE staining images of normal mouse fetuses and NTD mouse fetuses, where (A) is fetal morphology of the normal group under a stereomicroscope (8x), (B) is HE staining of a cross section of the neck of the normal group (5x), (C) is HE staining of a cross section of the neck of the normal group (10x), (D) is an NTD embryo under a stereomicroscope (8x), (E) is HE staining of a cross section of the neck of the model group (5x), and (F) is HE staining of a cross section of the neck of the model group (10x). [Figure 10] The effect of each concentration of JSP on the incidence of NTD in mouse fetuses in each group, △ indicates △P<0.05, △△P<0.01 compared with the normal group, * indicates *P<0.05, **P<0.01 compared with the model group, and # indicates #P<0.05 compared with the folic acid group. [Figure 11] The expression results of PI3K / Akt pathway proteins in each group, △ indicates △P<0.05, △△P<0.01 compared with the normal group, * indicates *P<0.05, **P<0.01 compared with the model group, and # indicates #P<0.05 compared with the folic acid group. [Figure 12] The expression results of apoptosis-related proteins in each group, *P<0.05, **P<0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In the following examples of the present invention, network pharmacology methods are used to examine the action and pharmacological studies of JSP. EXAMPLES

[0026] Example 1 Obtaining a pharmaceutical active ingredient In this example, the traditional Chinese medicine systems pharmacology database AND analysis platform (TCMSP) was used to search for all active ingredients in JSP prescriptions, including Zea mays, Cornus officinalis, Chinese yam, Caucasus oleracea, Peony bark, Cinnamon bark, Aconite fructus, Poria cocos, Rehmannia rhizome, and Plantago sativa.The biologically active ingredients of WYP were obtained by screening under the limiting conditions of oral bioavailability (OB)≧30% and drug-likeness (DL)≧0.18.

[0027] In this embodiment, the reported active ingredients of 10 kinds of Chinese herbal medicines in JSP were searched through the TCMSP database under the screening conditions of OB≧30% and DL≧0.18, and as a result, 27 were obtained from Zeaxanthemum Root, 132 from Cornus officinalis, 44 from Dahurian, 67 from Chou-an-chi, 33 from Peony Bark, 187 from Aconite Root, 19 from Poria Cocos, 20 from Rehmannia Root, 41 from Rehmannia Root, and 37 from Plantago Root. After removing duplicate molecules and those without corresponding targets, a total of 125 kinds of active ingredients were obtained. The specific ingredients are shown in Table 1 below.

[0028] [Table 1] JPEG2025015461000003.jpg244164 JPEG2025015461000004.jpg71164

[0029] Example 2 Screening and prediction of potential action targets of WYP active ingredients For the JSP biologically active ingredients obtained in Example 1, relevant potential targets were found through the TCMSP platform, and then the search function of UniProKBt in the UniPort database was used to obtain the human genes corresponding to the target proteins of each active ingredient, the species was limited to human, and all target gene names were amended to official gene symbols. If there was no target in the TCMSP database, its CAS / InChI number was confirmed and it was complemented by combining it with the Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ) database and the SwissTargetPrediction (http: / / www.swisstargetprediction.ch / ) analysis platform.

[0030] In this example, a total of 172 working targets were obtained after the above operations and removal of duplicate targets, the details of which are shown in Table 2 below.

[0031] [Table 2] JPEG2025015461000006.jpg85164

[0032] Example 3 Obtaining NTD disease targets The Gencards (http: / / www.genecards.org / ) database was searched using the keyword "Neural Tube Defects" and the median value of "Relevancescore" >2 was used as the screening criteria to collect relevant targets. Then, it was complemented with the OMIM (https: / / omim.org / ) and TTD (http: / / db.idrblab.net / ttd / ) databases.

[0033] In this example, combined with the 290 targets supplemented by the TTD database, a total of 1585 targets were screened, of which 180 targets are listed in Table 3 below.

[0034] [Table 3] TIFF2025015461000008.tif60164

[0035] Example 4: Construction of a “drug-active ingredient-target” network In this example, the above active ingredients obtained by screening were used to sequentially import the obtained disease genes into cytoscape software to construct a “drug-active ingredient-disease-target” graph as shown in Figure 1. It was found that the network had a total of 237 nodes and 527 edges, which more intuitively displayed the interrelationships between multiple ingredients and multiple targets.

[0036] Example 5: Screening for common targets between drugs and diseases and constructing protein interaction networks In this example, we first found the intersection between JSP targets and NTD targets through online Venn (http: / / www.bioinformatics.com.cn), obtained 64 common targets of JSP-NTD, and created a Venn diagram as shown in Figure 2 .

[0037] Next, the above common action targets of JSP-NTD were imported into the STRING (https: / / string-db.org) database, protein-protein interaction (PPI) was performed, and the obtained results were imported into Cytoscape software for visualization analysis to construct a PPI network for screening core targets. As can be seen from the results shown in Figure 3, a network graph consisting of 64 nodes and 897 edges was obtained.

[0038] Through the above "drug-active ingredient-disease-target" network, the common part genes of the drug and disease target obtained by the Venn diagram were obtained, and the "active ingredient-target-disease" network was created by Cytoscape software as shown in Figure 4. Core components were extracted based on the common part genes, and the frequency values ​​were calculated through the Network Analyzer plug-in. The main medicinal ingredients were analyzed and screened, and the small molecule ligands were docked to the molecules. It was revealed that a total of 26 core components and 64 common part genes were obtained, and the frequency values ​​were calculated to rank the JSP active ingredients, and the top five active ingredients were quercetin, wogonin, sitosterol, kaempferol, and stigmasterol, which were selected as the main active ingredients for NTD treatment by JSP and used for molecular docking.

[0039] Example 6 GO function and KEGG pathway enrichment analysis In this example, the Metascape platform was used to perform GO and KEGG analysis by importing the core targets into the Metascape database. “Homosapiens” was selected to perform Gene Ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis for the core targets of JSP NTD prevention and suppression.

[0040] In this example, the GO analysis results were summarized as follows: there were 1117 biological processes (BP), 45 cellular components (CC), and 95 molecular functions (MF), as shown in detail in Figure 5. GO function enrichment was most represented by BP entries, which mainly involved biological processes such as the action of cytokines, the control of oxidative stress, and the control of apoptosis processes. CC mainly involved RNA polymerase II transcription regulatory complex, cytosol, cytoplasm, etc. MF mainly involved RNA polymerase II transcription factor binding, transcription coregulator binding, adenosine triphosphate assembly, steroid binding, etc. This indicated that the treatment of NTDs by JSP may be the result of multiple actions.

[0041] In this example, 158 pathways were enriched through KEGG pathway analysis (P<0.05). The top 10 results are shown in Figure 6. These mainly include pathways in cancer, Hepatitis B, phosphatidylinositol-3-kinase / protein kinase B (PI3K / Akt) signaling pathway, Hepatitis C, and other signaling pathways. The most relevant pathway was the PI3K / Akt pathway, which is related to various biological processes, and therefore was chosen as the validation pathway for subsequent animal experiments.

[0042] Example 7 Validation of molecular docking In this embodiment, the intersection of the active ingredient-disease-target network is obtained, and the core ingredient is extracted based on the intersection gene, and the core target is obtained by protein interaction network analysis, and finally determined by combining its pathway analysis and literature research. As shown in Figure 7, through Autodock software, the core active ingredient is selected as a docking ligand to dock with the core target and the main pathway protein, and the binding energy represents the affinity and conformational stability of the active ingredient and the protein.

[0043] In this example, it is necessary to select a protein structure in the PDB (https: / / www.rcsb.org) database that has a close publication date, high resolution, and complete structure, and use PyMOL software to remove water and small molecules, then add hydrogen through autodock software, export it as a pdbqt file, and set it as a ligand. The file of the drug active ingredient was downloaded from TCMSP, and some ingredients need to be downloaded from Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ) to obtain sdf files, convert them to mol2 files using openbabel software, add hydrogen in Autodock software, detect torsion bonds, select torsion bonds, set them as ligands, and export them as pdbqt files. The docking box was set, exported as a gpf file, run Autogrid, set docking parameters (50 docking times) and calculation methods, exported as a dpf file, and run Autodock. The results were examined, and the results with the highest binding energy were exported as pdbqt files and visualized using PyMOL software.

[0044] In this example, structural matching analysis was performed on some of the results using PyMoL software, and as shown in detail in Figure 8, the structures tend to be stabilized by the formation of interactions such as hydrogen bonds between the components and the target.

[0045] Example 8 Animal experiment verification Animals: healthy adult C57BL / 6 mice, male to female ratio 2:1, weighing (23±2) g, provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., Animal Certificate No. SCXK(Kyoto)2021-0006. The rearing environment was well ventilated, with free access to water and food, lighting according to the circadian rhythm, room temperature (22±2) °C, and humidity 45%-55%.

[0046] Drugs and Reagents: JSP (consisting of Zeaxanthin, Cornus officinalis, Chinese yam, Cauliflower, Peony bark, Cinnamon bark, Aconite Root, Poria Cocos, Rehmannia Root, and Plantago Root) is manufactured by the Tongrentang Pharmaceutical Factory of Beijing Tongrentang Co., Ltd. (Lot No. 2035248). After reviewing a large number of literature, it was found that the high, medium, and low doses of JSP administered intragastrically to mice were 0.7, 1.4, and 2.8 g / kg (doses were converted based on body surface area, equivalent to 0.5, 1, and 2 times the clinically recommended daily dose for adults, respectively). All-trans-retinoic acid (atRA) was purchased from Sigma, USA. HE staining kit (lot number 20201016), BSA (lot number 9048-46-8), and 1× TBST (lot number T1086) were purchased from Solarbio. One-step PAGE gel rapid preparation kit (10%) was purchased from YaMei Biopharmaceutical Technology Co., Ltd. (Lot No. PG212). 1× Electrophoretic Transfer Buffer was purchased from Solarbio (Lot No. D1061). Electrophoresis solution of 1× Tris-glycine electrophoresis buffer was purchased from Solarbio (Lot No. T1072). Ultrasensitive ECL Chemiluminescence Instant Substrate (Lot No. AR1197) and BCA Protein Concentration Assay Kit (Lot No. AR0197) were purchased from Boster Biological Technology. PVDF membrane was purchased from Millipore (Lot No. IPVH00010). Goat anti-rabbit (lot no. ZJ2020-R) and β-actin antibody (lot no. AP0060) were purchased from Bioword. Akt (lot no. 4685S), p-Akt (lot no. 4060S), PI3K (lot no. 4249S), p-PI3K (lot no. 4228S), Bcl-2 (#3498), Bax (#14796), and cleaved caspase-3 (#9664) were purchased from Cell Signaling.

[0047] Equipment: BS224S type 4-digit electronic balance (Sartorius, Doyle); Gel imaging analyzer (Azure Biosystems, USA); SMZ-168 stereo microscope (MOTIC CHINA GROUP CO., LTD.); vertical electrophoresis tank (Bio-Rad, USA); Membrane transfer apparatus (Bio-Rad, USA).

[0048] Establishment of a mouse fetal neural tube malformation model C57BL / 6 male and female mice were placed in the same cage at 8 pm in a ratio of 1:2 and then placed in separate cages at 8 am the next day to check for vaginal plugs, and mice with vaginal plugs were considered pregnant. 8 am on the day the vaginal plug was found was defined as 0.5 days of embryo (Embryo, E). AtRA powder was dissolved in olive oil and treated by intraperitoneal injection at a dose of 50 mg / kg into E7.5d pregnant mice. A preliminary study by the research team found that when E7.5d pregnant mice were treated with ATRA, the mouse fetuses began to develop neural tube malformations from E9.5d, and the occurrence of neural tube malformations continued until E11.5d (when neural tube development was complete). Therefore, in this experiment, E11.5d mouse fetuses were selected for the study. E11.5d pregnant mice were dissected, mouse fetuses were removed, and the morphology of the neural tube of the mouse fetuses was observed under an optical microscope with reference to the normal group to confirm the occurrence of neural tube malformations.

[0049] Grouping: After the mice were adaptively reared for one week, the mice in which vaginal plugs were found were divided into a normal group, a model group, a JSP low dose (0.7 g / kg)-atRA co-treatment group, a JSP medium dose (1.4 g / kg)-atRA co-treatment group, a JSP high dose (2.8 g / kg)-atRA co-treatment group, and a folic acid-atRA co-treatment group (see Table 4 below). There were five pregnant mice in each group, and their fetuses were used for the study.

[0050] [Table 4]

[0051] Dosage: Pregnant mice were randomly assigned to groups and marked as gestational (E) day 0.5 (E0.5d) at 8 am on the day when the vaginal plug was found. On E7.5d, the pregnant mice in the experimental groups were intraperitoneally injected with atRA (7.5 mg / kg / d), except for the normal control group, which was intraperitoneally injected with the same amount of olive oil. From E0.5d to E11.5d, the JSP and FA groups continued to be treated with the drug intragastrically once a day, whereas the normal and atRA groups received the same amount of saline. On E11.5d, the pregnant mice were anesthetized and sacrificed by cervical dislocation, and the embryos were isolated by Caesarean section.

[0052] Sample preparation and detection of indicators On E11.5d, pregnant mice from each group were sacrificed by cervical dislocation and their limbs were fixed to the operating table in a supine position. The skin and muscles were cut at the midline of the abdomen to expose the abdominal cavity, the uterus was exposed, the mesometrium was cut, the uterus was removed, washed with PBS, and transferred to a glass petri dish filled with PBS. The placenta and fetal membrane tissue were carefully peeled off under a dissecting microscope, and the embryos were removed. The occurrence of neural tube malformations was confirmed under a light microscope and expressed as the incidence of neural tube malformations (number of mouse fetuses with neural tube malformations per pregnant mouse / total number of mouse fetuses per pregnant mouse × 100%).

[0053] Hematoxylin and eosin (HE) staining The frozen sections prepared above were taken, stained in hematoxylin reagent for 3 minutes at room temperature, drained, washed with a small amount of water for 5 minutes, quickly blued, differentiated in 0.5% hydrochloric acid alcohol for 15 s, rinsed with running water for 2 minutes, stained with eosin stain for 10 minutes, and then quickly washed with a small amount of running water for 3 minutes to remove the stain. They were dehydrated in gradient alcohol (70% alcohol for 30 s, 70% alcohol for 30 s, 80% alcohol for 30 s, 95% alcohol for 30 s, 100% alcohol for 1 minute, 100% alcohol for 2 minutes), and cleared in xylene solution (alcohol: xylene for 1 minute, 100% xylene for 2 minutes, 100% xylene for 5 minutes). The sections were air-dried and then sealed with neutral gum.

[0054] Western Blot Experiment The mouse fetal tissue was taken out of the -80°C refrigerator, weighed, and the mouse fetal tissue was placed in a grinder, and tissue dissolution solution was added (10 μL of dissolution solution containing 1 μL of phosphatase inhibitor and 1 μL of 1 mM protease inhibitor PMSF was added per 1 mg of tissue sample), and thoroughly ground on ice until no obvious tissue fragments were found, and then completely dissolved for 30 min. The obtained tissue suspension was transferred to a pre-cooled EP tube, centrifuged at 12,000 rpm for 20 min in a 4°C centrifuge, and the supernatant was collected and individually packaged and stored in a -80°C refrigerator, and the obtained supernatant is the mouse fetal tissue protein. Western blot technique was used to detect the expression of core proteins of the PI3K / Akt signaling pathway (Akt, p-Akt, PI3K, p-PI3K) and downstream apoptosis-related proteins of the pathway (Bcl-2, Bax, cleaved caspase-3) in the tissues of mouse fetuses from each group.

[0055] statistical methods Data were analyzed using SPSS 17.0 statistical software, and one-way analysis of variance (one-wayANO-VA) was used to compare the means between groups. P<0.05 indicated that the differences were statistically significant.

[0056] Example 9: Animal experiment verification results Morphological and pathological observations of neural tube closure in atRA-induced NTD mouse fetuses Spina bifida and anencephaly are the most typical malformations of NTD. The results shown in Figure 9 show that the appearance and development of mouse fetuses were observed under a stereomicroscope. In normal mouse fetuses, structures such as the midbrain, hindbrain, telencephalon, diencephalon, telencephalon, and fourth ventricle are well developed, with a smooth appearance and good development. In NTD mouse fetuses, the midbrain, diencephalon, telencephalon, hindbrain, and telencephalon were damaged, and neural tissue was exposed. HE staining was used to observe the pathological changes in the coronal sections of normal and NTD mouse fetuses. The HE staining results showed that the neural tube of normal mouse fetuses had a regular lumen, good closure, clear structures, and orderly arranged cells. Meanwhile, in NTD mouse fetuses, the neural tube showed an incomplete closure.

[0057] Effect of JSP on the incidence of NTDs in mouse fetuses of each group In this example, the incidence of NTD in mouse fetuses in each group was statistically analyzed through stereomicroscopy, as shown in Figure 10. Compared with the normal group, the incidence of NTD in mouse fetuses in the atRA group was significantly improved (P<0.01); compared with the atRA group, the JSP low dose group, JSP medium dose group, JSP high dose group and FA group could all reduce the malformation rate of mouse fetuses (P<0.01); and the atRA+2.8g / kg / d group had a better effect than the atRA+FA group (P<0.01).

[0058] JSP can promote expression of the PI3K / Akt pathway In this example, the expression levels of PI3K / Akt pathway Akt, p-Akt, PI3K, and p-PI3K pathway proteins by JSP were detected by Western blot technique, and the results are shown in Figure 11. It was found that, compared with the normal group, the expression levels of both p-PI3K and p-Akt were significantly decreased in the atRA group (P<0.01), compared with the atRA group, the expression of p-PI3K was improved in the JSP and FA treatment groups (P<0.01), the expression of p-Akt was significantly increased in JSP (P<0.01), the expression level of p-Akt was increased in FA (P<0.05), and compared with the FA group, the expression of both p-PI3K and p-Akt was increased in the JSP group (P<0.05).

[0059] JSP can reduce the expression of apoptotic proteins By Western blot technique, the expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins by JSP were detected, and the results are shown in Figure 12. In the atRA group, the expression level of Bcl-2 was significantly decreased (P<0.01), and the expression levels of both Bax and cleaved caspase-3 were significantly increased (P<0.01), and the Bcl-2 / Bax ratio was decreased (P<0.01). Compared with the atRA group, in the JSP group, the expression of Bcl-2 was increased (P<0.01), and both Bax and cleaved caspase-3 were significantly decreased (P<0.01), and the Bcl-2 / Bax ratio was increased (P<0.01). Compared with the FA group, in the JSP group, the expression of Bcl-2 was increased (P<0.05), the expression of both Bax and cleaved caspase-3 was decreased (P<0.05), and the Bcl-2 / Bax ratio was increased (P<0.05).

[0060] As described above, in this invention, based on network pharmacology combined with in vivo experiments, we explored the mechanism of action of JSP in preventing and inhibiting neural tube defects (NTD) in mice, collected the active ingredients of JSP and the targets of JSP for treating NTD through databases, constructed a protein-protein interaction (PPI) network, and performed gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. The molecular docking of the main action targets and the main active ingredients was verified through AutoDock software. 125 active ingredients of JSP were screened, and 172 targets and 64 core targets of JSP for treating NTD were obtained. KEGG pathway enrichment analysis showed that the phosphatidylinositol-3-kinase (PI3K) / Akt signaling pathway was one of the important mechanisms of JSP's anti-NTD. Molecular docking showed that the main active components of JSP had relatively stable binding activity with Akt1, Akt2, PIK3CG, PI3GCA and other core action targets.

[0061] As described above, in order to verify the present invention through animal experiments and establish a mouse fetal neural tube malformation model, all-trans-retinoic acid (atRA) was intraperitoneally injected into pregnant mice, and low, medium, and high doses of JSP (0.7, 1.4, 2.8 g / kg) and folic acid (37 μg / kg) were administered for prevention and suppression, and the pregnant mice were dissected on E11.5d to remove the mouse fetuses. The number of malformations in the mouse fetuses was counted, the malformation rate of each group was analyzed, and pathological changes in the brain tissue were examined using hematoxylin-eosin (HE) staining. Western blot was used to detect the expression of protein kinase B (Akt), phosphorylated Akt (p-Akt), phosphatidylinositol 3-kinase (PI3K), and phosphorylated phosphatidylinositol-3-kinase (p-PI3K) proteins in whole mouse fetuses.The results of the animal experiment showed that the incidence of NTD in mouse fetuses was significantly increased in the atRA group compared with the normal group (P<0.01), and compared with the atRA group, the JSP low dose group, JSP medium dose group, JSP high dose group and FA group could all reduce the deformity rate of mouse fetuses (P<0.01), and the atRA+2.8mg / kg JSP group had a better effect than the atRA+FA group (P<0.05). HE staining showed that the neural tube in normal mouse fetuses had a regular lumen, good closure, distinct structures, and well-arranged cells, whereas the neural tube in NTD mouse fetuses showed no closure.As shown by the results of Western blot detection, compared with the normal group, the expression levels of both p-PI3K and p-Akt were significantly decreased in the atRA group (P<0.01), the expression of p-PI3K was increased in the JSP and FA treatment groups (P<0.01), the expression of p-Akt was significantly increased in the JSP group (P<0.01), the expression of p-Akt was increased in the FA group (P<0.05), and compared with the FA group, the expression of both p-PI3K and p-Akt was increased in the JSP group (P<0.05). In the atRA group, the expression level of Bcl-2 was significantly decreased (P<0.01), and the expression levels of both Bax and cleaved caspase-3 were significantly increased (P<0.01), and the Bcl-2 / Bax ratio was decreased (P<0.01). Compared with the atRA group, the expression of Bcl-2 was increased (P<0.01), both Bax and cleaved caspase-3 were significantly decreased (P<0.01), and the Bcl-2 / Bax ratio was increased (P<0.01) in the JSP group.Compared with the FA group, the expression of Bcl-2 was increased (P<0.05), both Bax and cleaved caspase-3 were decreased (P<0.05), and the Bcl-2 / Bax ratio was increased (P<0.05) in the JSP group.

[0062] As described above, the above examples of the present invention not only verify the novel use and effect of JSP in a drug for preventing or treating neural tube defects, but also verify the mechanism by which JSP can prevent or suppress neural tube defects by inhibiting cell apoptosis through its action to activate the PI3K / Akt signaling pathway, providing theoretical support for the further development and application of JSP.

[0063] Obviously, the above examples are merely examples for clarifying the embodiments, and are not intended to limit the embodiments. Those skilled in the art may make various other changes or modifications based on the above description. It is not necessary and impossible to comprehensively cover all the embodiments here. Any obvious changes or modifications derived therefrom shall still fall within the scope of protection of the present invention.

Claims

1. 23. Use of Jingui Jinqi Wan in preparing a medicament for preventing or treating neural tube defects.

2. 23. Use of Jingui Jinqi Wan in the preparation of a medicament for preventing or treating neural tube defects caused by NTD.

3. Use of Jingui Jinqi Wan in the preparation of PI3K / Akt pathway inhibitors.

4. Use of a pharmaceutical composition and its formulation in the preparation of a medicament for preventing or treating neural tube defects, comprising: The composition of the raw herbal medicines of the pharmaceutical composition includes 20-28 parts by weight of Rehmannia Root, 4-8 parts by weight of Cornus Officinalis Root, 4-8 parts by weight of Chinese Yam, 4-8 parts by weight of Zelkova Root, 15-20 parts by weight of Poria Cocos Root, 4-8 parts by weight of Danpi Root, 4-8 parts by weight of Cinnamon Bark, and 0.5-1.5 parts by weight of Aconite Root; Preferably, the pharmaceutical composition further comprises 4-8 parts by weight of osmanthus and / or 4-8 parts by weight of psyllium sap.

5. Use of a drug composition and its formulation in the preparation of a medicament for preventing or treating a neural tube defect caused by an NTD, comprising: The composition of the raw herbal medicines of the pharmaceutical composition includes 20-28 parts by weight of Rehmannia Root, 4-8 parts by weight of Cornus Officinalis Root, 4-8 parts by weight of Chinese Yam, 4-8 parts by weight of Zelkova Root, 15-20 parts by weight of Poria Cocos Root, 4-8 parts by weight of Danpi Root, 4-8 parts by weight of Cinnamon Bark, and 0.5-1.5 parts by weight of Aconite Root; Preferably, the pharmaceutical composition further comprises 4-8 parts by weight of osmanthus and / or 4-8 parts by weight of psyllium sap.

6. Use of a pharmaceutical composition and its formulation in the preparation of a PI3K / Akt pathway inhibitor, comprising: The composition of the raw herbal medicines of the pharmaceutical composition includes 20-28 parts by weight of Rehmannia Root, 4-8 parts by weight of Cornus Officinalis Root, 4-8 parts by weight of Chinese Yam, 4-8 parts by weight of Zelkova Root, 15-20 parts by weight of Poria Cocos Root, 4-8 parts by weight of Danpi Root, 4-8 parts by weight of Cinnamon Bark, and 0.5-1.5 parts by weight of Aconite Root; Preferably, the pharmaceutical composition further comprises 4-8 parts by weight of osmanthus and / or 4-8 parts by weight of psyllium sap.

7. 1. Use of a composition in the preparation of a medicament for preventing or treating a neural tube defect, comprising:

2. Use of a composition, characterized in that the main active ingredients of said composition include quercetin, wogonin, sitosterol, kaempferol, and stigmasterol.

8. Use of a composition in the preparation of a medicament for preventing or treating a neural tube defect caused by an NTD, comprising:

2. Use of a composition, characterized in that the main active ingredients of said composition include quercetin, wogonin, sitosterol, kaempferol, and stigmasterol.

9. Use of a composition in the preparation of a PI3K / Akt pathway inhibitor, comprising:

2. Use of a composition, characterized in that the main active ingredients of said composition include quercetin, wogonin, sitosterol, kaempferol, and stigmasterol.

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