Use of Jingui Jinqi Wan in preparing drugs for preventing or treating 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 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 of the central nervous system (CNS). These include anencephaly, spina bifida, and encephalocele. Research has shown that neural tube defects are an important cause of neural tube defects, with various clinical symptoms resulting from incomplete closure at different locations along the neural axis. Most patients with symptomatic disease suffer from incontinence, learning disabilities, and lifelong wheelchair use. While folic acid supplementation can currently reduce the incidence of NTDs to some extent, the underlying mechanisms of NTD development remain unclear, 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, bone morphogenetic protein (BMP) and retinoic acid (RA) signaling pathway, and is also associated with the PI3K / Akt pathway.

[0003] In traditional Chinese medicine, innate essence, also known as yuan essence, is present in the kidneys. In addition to the innate essence scattered throughout the limbs and bones, other innate essences are concentrated in the kidneys. This is a manifestation of the kidneys' storage function and their ability to store essence. The Su Wen: Treatise on Strange Diseases states, "The kidneys store essence, which fills the bones and produces bone marrow. The bone marrow then gathers to form the brain, and the marrow fills the brain with it. When essence is depleted, the brain disappears." This statement indicates that the kidneys are the source of bone marrow production, and that sufficient bone marrow contributes to a healthy brain. Deficiency of kidney essence in both parents can result in a lack of innate essence in the fetus, which can affect fetal growth and development and cause neural tube defects, which are fundamentally consistent with the pathogenesis of NTDs.

[0004] Jingui Shenqi Pill (JSP), also known as Eight Flavors Jingui Shenqi Pill, was first published in the Jingui Essentials: Blood Paralysis, Deficiency, and Labor Diseases. This formula contains eight herbs, including Rehmannia glutinosa, Cornus officinalis, Daphnia serrata, Zelkova, Poria cocos, and Danpi root, which nourish kidney yin, and Cinnamon bark and Aconite root, which nourish yang. This formula, originally designed to "require yin to seek yang," has the effect of strengthening both yin and yang. It has been a timeless formula for strengthening the kidneys, supporting yang, and facilitating the transformation of essence into qi. While JSP has been widely studied for osteoporosis, diabetes, and anti-aging, no experimental studies have been reported for its use in NTDs. Therefore, the field is expected to develop more novel uses for the drug, which will have positive implications for the development of high-quality traditional 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 Jinqi 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 drug 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 Jinqi 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 Jinqi Wan (JSP) in the preparation of a PI3K / Akt pathway inhibitor.

[0010] The present invention also relates to a pharmaceutical composition and its formulation use in preparing a medicament for preventing or treating neural tube defects (NTD), wherein the raw herbal ingredients of the pharmaceutical composition include 20-28 parts by weight of Rehmannia Root, 4-8 parts by weight of Cornus Root, 4-8 parts by weight of Daphnia Root, 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 to 8 parts by weight of osmanthus and / or 4 to 8 parts by weight of psyllium sap. The present invention discloses a pharmaceutical composition and its formulation use.

[0011] Preferably, the raw herbal ingredients of the pharmaceutical composition include 24 parts by weight of Rehmannia Root, 6 parts by weight of Cornus Root, 6 parts by weight of Daphnia Root, 6 parts by weight of Zelkova Root, 17.3 parts by weight of Poria Cocos, 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 osmanthus and / or 6 parts by weight of psyllium.

[0012] The present invention also relates to a pharmaceutical composition and its formulation for use in preparing a medicament for preventing or treating neural tube defects caused by NTDs, wherein the raw herbal ingredients of the pharmaceutical composition include 20-28 parts by weight of Rehmannia Root, 4-8 parts by weight of Cornus Root, 4-8 parts by weight of Daphnia Root, 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 to 8 parts by weight of osmanthus and / or 4 to 8 parts by weight of psyllium sap. The present invention discloses a pharmaceutical composition and its formulation use.

[0013] Preferably, the raw herbal ingredients of the pharmaceutical composition include 24 parts by weight of Rehmannia Root, 6 parts by weight of Cornus Root, 6 parts by weight of Daphnia Root, 6 parts by weight of Zelkova Root, 17.3 parts by weight of Poria Cocos, 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 osmanthus and / or 6 parts by weight of psyllium.

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

[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 osmanthus and / or 6 parts by weight of psyllium.

[0016] The present invention also discloses the use of a composition in the preparation of a medicament for preventing or treating neural tube defects (NTDs), wherein the main active ingredients of the composition include quercetin, wogonin, sitosterol (Beta-Sitosterol), kaempferol, and stigmasterol.

[0017] The present invention also discloses the use of a 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 (beta-sitosterol), kaempferol, and stigmasterol.

[0018] The present invention also discloses the use of a composition in the preparation of a PI3K / Akt pathway inhibitor, wherein the main active ingredients of the composition include quercetin, wogonin, sitosterol (Beta-Sitosterol), kaempferol, and stigmasterol. [Effects 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 for preventing and inhibiting NTDs, and conducts preliminary experimental verification of the main targets and pathways of JSP for preventing and inhibiting NTDs, providing a theoretical basis for the clinical application of JSP in preventing and inhibiting NTDs.

[0020] In this study, we used network pharmacology to analyze the targets and active components of JSP, which prevent and suppress NTDs. We identified 26 major active components, including quercetin, wogonin, beta-sitosterol, kaempferol, and stigmasterol, and 64 major targets, including IL-10, IL-6, and Bcl-2. KEGG pathway analysis revealed that JSP acts by regulating signaling pathways such as the PI3K / Akt pathway and Sonic hedgehog (shh) pathway. Furthermore, molecular docking showed that JSP can effectively bind to proteins related to the PI3K / Akt pathway. This is consistent with previous network pharmacological findings that NTD development is related to the PI3K / Akt pathway, further validating the role and efficacy of JSP in preventing and suppressing NTDs.

[0021] Through animal experiments, the present invention further demonstrates that JSP can promote activation of the PI3K / Akt pathway, and that this mechanism may be related to downstream biological processes such as cell proliferation and apoptosis, inflammation, and reactive oxygen metabolism. Akt is a core protein of the PI3K / Akt pathway. Upon phosphorylation, it acts on the Bcl-2 protein family, among which Bcl-2 plays a role in inhibiting cell apoptosis. Bax, also a member of the Bcl-2 protein family, promotes the release of cytochrome C during apoptosis, accelerating apoptosis. The higher the Bcl-2 / Bax level, 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 results of our animal experiments, combined with the literature, further demonstrated that the major 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 study explores the mechanism of action of JSP in the prevention and control of NTDs using network pharmacology combined with in vivo experiments. Through network pharmacology, we preliminarily investigated the possible targets and pathways of action of JSP in the treatment of NTDs. Combined with animal experiments, we demonstrated that JSP can reduce the incidence of NTDs in mouse fetuses, decrease apoptosis in neural tube epithelial cells, and increase the expression of proteins associated with the PI3K / Akt signaling pathway. Thus, JSP's role in the prevention and control of NTDs is likely achieved by activating the PI3K / Akt pathway to reduce cell apoptosis. This provides a theoretical basis for further application of JSP and fills the gap in the prior art in the lack of research and application of JSP in NTDs. [Brief explanation of the drawings]

[0023] In order to make the contents of the present invention clearer and easier to understand, 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 the 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 the interaction relationship between the target and the active ingredient. [Figure 2] FIG. 1 is a schematic representation of a Venn diagram of JSP-NTD targets. [Figure 3] Schematic diagram of the JSP-NTD common target PPI network and core target screening. Here, the size and color of the nodes represent the magnitude of their degree values. The larger and darker the node, the higher the corresponding degree value and the more edges connected to it, indicating its importance. [Figure 4] FIG. 1 is a schematic diagram of the “active ingredient-target-disease” network. [Figure 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 included in the gene list. The larger the dot, the more targets are enriched in the pathway. [Figure 7] This diagram shows the binding energy of the 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 there is strong binding activity between the molecules. The lower the energy, the stronger the binding ability. [Figure 8]Schematic diagram of docking of major active ingredients with core targets, where (A) is quercetin-Akt1, (B) is wogonin-IL6, (C) is β-sitosterol-PIK3CG, (D) is kaempferol-Akt2, and (E) is 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 that hydrogen bond to the drug and core protein. [Figure 9] Stereomicroscope images and HE staining images of normal and NTD mouse fetuses. (A) Normal group fetal morphology under a stereomicroscope (8x magnification), (B) HE staining of a neck cross section of a normal group (5x magnification), (C) HE staining of a neck cross section of a normal group (10x magnification), (D) NTD embryo under a stereomicroscope (8x magnification), (E) HE staining of a neck cross section of a model group (5x magnification), (F) HE staining of a neck cross section of a model group (10x magnification). [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; # indicates #P<0.05 compared with the folic acid group. [Figure 11] The expression results of PI3K / Akt pathway proteins in each group are shown. △ 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 INVENTION

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

[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 the JSP formula, including Zelkova, Cornus officinalis, Dahurica Root, Cinnamon Bark, Aconite Root, Poria Cocos, Rehmannia Root, and Plantago Root. Screening was performed under the constraints of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18, and the biologically active ingredients of WYP were identified.

[0027] In this example, the reported active ingredients of 10 herbal medicines in the JSP were searched through the TCMSP database using the screening criteria of OB≧30% and DL≧0.18. As a result, 27 active ingredients were found: Zelkova Root 27, Cornus Nucifera 132, Daphnia Root 44, Cauliflower Root 67, Moutan Peel 33, Cinnamon Bark 187, Aconite Root 19, Poria Cocos Root 20, Rehmannia Root 41, and Plantago Fruit 37. After removing duplicate molecules and those without corresponding targets, a total of 125 active ingredients were obtained. The specific ingredients are listed in Table 1 below.

[0028] [Table 1] JPEG0007825303000002.jpg244164 JPEG0007825303000003.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. 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 humans, and all target gene names were corrected to their official gene symbols. If the target was not found in the TCMSP database, its CAS / InChI number was confirmed and 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] JPEG0007825303000005.jpg85164

[0032] Example 3: Obtaining NTD disease targets The Gencards (http: / / www.genecards.org / ) database was searched using the keyword "Neural Tube Defects" and a median "Relevance score" of more than 2 was used as a screening criterion to collect relevant targets. This was then 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] TIFF0007825303000007.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, and a "drug-active ingredient-disease-target" graph was constructed as shown in Figure 1. It was found that this network had a total of 237 nodes and 527 edges, which provided a more intuitive display of the interrelationships between multiple ingredients and multiple targets.

[0036] Example 5: Screening for common targets between drugs and diseases and building protein interaction networks In this example, we first found the intersection between JSP 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 common targets of the JSP-NTDs were imported into the STRING (https: / / string-db.org) database and protein-protein interactions (PPI) were performed. The results were imported into Cytoscape software for visualization analysis, and a PPI network was constructed to screen for 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] Using the above drug-active ingredient-disease-target network, the common genes between drugs and disease targets were obtained using the Venn diagram. An "active ingredient-target-disease" network was then created using Cytoscape software (Figure 4). Core components were extracted based on the common genes, and their frequency values ​​were calculated using the Network Analyzer plugin. The key medicinal ingredients were analyzed and screened, and small molecule ligands were docked to the molecules. A total of 26 core components and 64 common genes were identified. Frequency values ​​were calculated to rank the JSP active ingredients. The top five active ingredients were quercetin, wogonin, beta-sitosterol, kaempferol, and stigmasterol. These were identified as the key active ingredients for JSP NTD treatment and used for molecular docking.

[0039] Example 6. GO Function and KEGG Pathway Enrichment Analysis In this example, we used the Metascape platform to perform GO and KEGG analyses 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 analyses for the core targets of JSP NTD prevention and suppression.

[0040] In this example, the GO analysis results were summarized as follows: 1,117 biological processes (BPs), 45 cellular components (CCs), and 95 molecular functions (MFs), as shown in detail in Figure 5. GO function enrichment was most commonly represented by BP entries, which are primarily involved in biological processes such as cytokine action, oxidative stress control, and apoptosis control. CCs are primarily involved in RNA polymerase II transcription regulatory complexes, cytosol, and cytoplasm. MFs are primarily involved in RNA polymerase II transcription factor binding, transcriptional coregulator binding, adenosine triphosphate assembly, and steroid binding. This suggests 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 signaling pathways such as cancer pathways, hepatitis B, phosphatidylinositol-3-kinase / protein kinase B (PI3K / Akt) signaling pathway, and hepatitis C. The most relevant pathway was the PI3K / Akt pathway, which is involved in various biological processes, and was therefore selected as the validation pathway for subsequent animal experiments.

[0042] Example 7: Validation of molecular docking In this example, the intersection of the active ingredient-disease-target network was obtained, and the core components were extracted based on the intersection genes. The core targets were then identified through protein interaction network analysis and combined with pathway analysis and literature research. As shown in Figure 7, the core active ingredients were selected as docking ligands to dock with the core targets and key pathway proteins using Autodock software. The binding energy represents the affinity and conformational stability of the active ingredient-protein.

[0043] In this example, protein structures were selected from the PDB (https: / / www.rcsb.org) database with a recent publication date, high resolution, and complete structure. Water and small molecules were removed using PyMOL software. Hydrogen was then added using AutoDock software, exported as a pdbqt file, and designated as the ligand. The active pharmaceutical ingredient files were downloaded from TCMSP, and some components were downloaded from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) to obtain sdf files. These were then converted to mol2 files using OpenBabel software. Hydrogen was added using AutoDock software, torsional bond detection and selection were performed, designated as the ligand, and exported as a pdbqt file. Docking boxes were configured, exported as gpf files, and Autogrid was run. Docking parameters (50 docking iterations) and calculation methods were set, exported as dpf files, and AutoDock was run. 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 structure tends to be stabilized by the formation of interactions such as hydrogen bonds between the component and the target.

[0045] Example 8 Animal Experiment Verification Animals: Healthy adult C57BL / 6 mice, the ratio of male to female is 2:1, body weight (23±2) g, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., animal certificate number SCXK (Beijing) 2021-0006. The breeding environment is well-ventilated, water and food can be freely ingested, lighting is carried out according to the circadian rhythm, room temperature (22±2) °C, humidity 45% - 55%.

[0046] Drugs and Reagents: JSP (consisting of Alisma orientale, Cornus officinalis, Dioscorea opposita, Achyranthes bidentata, Paeonia suffruticosa, Cinnamomum cassia, Aconitum carmichaeli, Poria cocos, Rehmannia glutinosa, and Plantago asiatica) was manufactured by the Tongrentang Pharmaceutical Factory of Beijing Tongrentang Co., Ltd. (lot number 2035248). After investigating a large number of documents, 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 (the doses were converted based on body surface area and corresponded to 0.5 times, 1 times, 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 Ya▲Mei▼ Biotechnology Co., Ltd. (lot number PG212). 1× electrophoresis transfer buffer was purchased from Solarbio (lot number D1061). The electrophoresis solution of 1× Tris-glycine electrophoresis buffer was purchased from Solarbio (lot number T1072). Ultra-high sensitivity ECL chemiluminescent instant substrate (lot number AR1197) and BCA protein concentration assay kit (lot number AR0197) were purchased from Boster Biological Technology. PVDF membrane was purchased from Millipore (lot number IPVH00010). Goat anti-rabbit (lot number ZJ2020-R) and β-actin antibody (lot number 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 model 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 model of neural tube defects C57BL / 6 male and female mice were housed in the same cage at a 1:2 ratio at 8 PM and then placed in separate cages at 8 AM the following day. Vaginal plugs were examined and mice with vaginal plugs were considered pregnant. 8 AM on the day a vaginal plug was discovered was defined as embryonic day (E) 0.5. AtRA powder was dissolved in olive oil and treated by intraperitoneal injection at a dose of 50 mg / kg into E7.5-day pregnant mice. In a preliminary study, the research team found that when E7.5-day pregnant mice were treated with ATRA, neural tube malformations began to develop in the fetuses at E9.5 days and continued until E11.5 days (when neural tube development was complete). Therefore, E11.5-day mouse fetuses were selected for this study. E11.5-day pregnant mice were dissected, and the fetuses were removed. The morphology of the neural tube in the fetuses was examined under a light microscope, using a control group as a reference. The occurrence of neural tube malformations was confirmed.

[0049] Grouping: After one week of adaptive rearing, mice in which vaginal plugs were found were divided into the following groups: normal group, model group, low-dose JSP (0.7 g / kg)-atRA co-treatment group, medium-dose JSP (1.4 g / kg)-atRA co-treatment group, high-dose JSP (2.8 g / kg)-atRA co-treatment group, and folic acid-atRA co-treatment group (see Table 4 below). Five pregnant mice were present 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 embryonic day 0.5 (E0.5d) at 8:00 AM on the day a vaginal plug was discovered. On E7.5d, all experimental pregnant mice, except for the normal control group, were intraperitoneally injected with atRA (7.5 mg / kg / d), while the normal control group received an equal volume of olive oil. From E0.5d to E11.5d, the JSP and FA groups continued to receive the drug via intragastric administration once daily, while the normal and atRA groups received an equal volume of saline. On E11.5d, the pregnant mice were anesthetized and sacrificed by cervical dislocation, and embryos were isolated by cesarean section.

[0052] Sample preparation and indicator detection On E11.5d, pregnant mice from each group were sacrificed by cervical dislocation and their limbs were fixed supine to a surgical table. The skin and muscles were cut along the abdominal midline to expose the abdominal cavity, and the uterus was then removed. The mesothelium was then cut and the uterus was removed, washed with PBS, and transferred to a glass Petri dish filled with PBS. The placenta and fetal membranes were carefully removed 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 rate of neural tube malformations (number of fetuses with neural tube malformations per pregnant mouse / total number of fetuses per pregnant mouse × 100%).

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

[0054] Western Blot Experiment Mouse fetal tissue samples were removed from the -80°C refrigerator, weighed, and placed in a grinder. Tissue lysis solution (10 μL per mg of tissue sample containing 1 μL of phosphatase inhibitor and 1 μL of 1 mM protease inhibitor PMSF) was added. The samples were thoroughly ground on ice until no visible tissue fragments remained, and then lysed for 30 minutes. The resulting tissue suspension was transferred to pre-cooled EP tubes and centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant was collected, individually packaged, and stored in a -80°C refrigerator. The resulting supernatant represents mouse fetal tissue protein. Western blot analysis 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 (Bcl-2, Bax, cleaved caspase-3) in the mouse fetal tissues from each group.

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

[0056] Example 9: Animal experiment verification results Morphological observation and pathological observation of neural tube closure in atRA-induced NTD mouse fetuses Spina bifida and anencephaly are the most common malformations of NTD. Figure 9 shows the results of stereomicroscopic observation of the appearance and development of mouse fetuses. 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 are damaged, exposing neural tissue. HE staining was used to examine the pathological changes in coronal sections of normal and NTD mouse fetuses. HE staining results showed that the neural tube in normal mouse fetuses had a regular lumen, good closure, distinct structures, and well-arranged cells. In contrast, the neural tube in NTD mouse fetuses showed impaired closure.

[0057] The 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 stereomicroscopic observation, as shown in Figure 10. Compared with the normal group, the incidence of NTD in mouse fetuses in the atRA group was significantly higher (P<0.01). Compared with the atRA group, the JSP low-dose group, JSP medium-dose group, JSP high-dose group, and FA group all reduced the malformation rate in mouse fetuses (P<0.01). Furthermore, the atRA + 2.8 g / kg / d group had a more favorable effect than the atRA + FA group (P<0.01).

[0058] JSP can promote the expression of the PI3K / Akt pathway In this example, Western blot analysis was used to detect the expression levels of PI3K / Akt pathway proteins, including Akt, p-Akt, PI3K, and p-PI3K, in JSP. The results are shown in Figure 11. 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 increased in the JSP and FA treatment groups (P<0.01). In JSP, the expression of p-Akt was significantly elevated (P<0.01), and in FA, the expression of p-Akt was elevated (P<0.05). Compared with the FA group, the expression of both p-PI3K and p-Akt was elevated in the JSP group (P<0.05).

[0059] JSP can reduce the expression of apoptotic proteins Western blot analysis was used to detect the expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins in JSP. 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 the expression levels of 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, the expression of Bcl-2 was elevated (P<0.05), the expression of both Bax and cleaved caspase-3 was decreased (P<0.05), and the Bcl-2 / Bax ratio was elevated (P<0.05) in the JSP group.

[0060] In conclusion, this study explored the mechanism of action of JSP in preventing and inhibiting neural tube defects (NTDs) in mice using network pharmacology combined with in vivo experiments. We collected JSP's active components and JSP's targets for NTD treatment through databases, constructed protein-protein interaction (PPI) networks, and performed Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Molecular docking of the main targets and active components was verified using AutoDock software. 125 active components of JSP were screened, and 172 JSP targets for NTD treatment and 64 core targets were identified. KEGG pathway enrichment analysis indicated that the phosphatidylinositol-3-kinase (PI3K) / Akt signaling pathway is one of the key mechanisms of JSP's anti-NTD effects. Molecular docking showed that the main active component of JSP has relatively stable binding activity with core action targets such as Akt1, Akt2, PIK3CG, and PI3GCA.

[0061] To verify this finding 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, and 2.8 g / kg) and folic acid (37 μg / kg) were administered for prevention and suppression. At E11.5 days, the pregnant mice were dissected and the fetuses were removed. The number of malformations in the fetuses was counted, and the malformation rate for each group was analyzed. Hematoxylin-eosin (HE) staining was used to examine pathological changes in brain tissue. Western blot analysis 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) in whole mouse embryos. Animal experiments showed that the atRA group significantly increased the incidence of NTD in mouse fetuses compared with the control group (P<0.01). Compared with the atRA group, the low-dose, medium-dose, and high-dose JSP groups, as well as the FA group, all reduced the incidence of fetal deformities in mouse fetuses (P<0.01). Furthermore, the atRA + 2.8 mg / kg JSP group had a superior effect to 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 in NTD mouse fetuses, the neural tube was not closed.Western blot analysis showed that the expression levels of both p-PI3K and p-Akt were significantly decreased in the atRA group compared with the normal group (P<0.01). Compared with the atRA group, 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 JSP (P<0.01), and the expression of p-Akt was increased in FA (P<0.05). Compared with the FA group, the expression levels of both p-PI3K and p-Akt were increased in the JSP group (P<0.05). In the atRA group, the expression levels of Bcl-2 were 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 JSP group showed increased Bcl-2 expression (P<0.01), significantly decreased Bax and cleaved caspase-3 (P<0.01), and an elevated Bcl-2 / Bax ratio (P<0.01).Compared with the FA group, the JSP group showed increased Bcl-2 expression (P<0.05), decreased Bax and cleaved caspase-3 (P<0.05), and an elevated Bcl-2 / Bax ratio (P<0.05).

[0062] As described above, the above examples of the present invention not only demonstrate the novel use and effect of JSP in drugs for preventing or treating neural tube defects, but also demonstrate 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 the purpose of clarity 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 it is not possible, 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. 1. Use of a pharmaceutical composition and its formulation in the preparation of a medicament for preventing neural tube defects, comprising: The raw herbal ingredients of the pharmaceutical composition include 20-28 parts by weight of Rehmannia glutinosa root, 4-8 parts by weight of Cornus officinalis root, 4-8 parts by weight of Daphnia serrata, 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; The pharmaceutical composition and its formulation use, characterized in that the pharmaceutical composition further comprises 4-8 parts by weight of Kallista and 4-8 parts by weight of Psyllium.

2. Use of a drug composition and its formulation in the preparation of a drug for preventing neural tube defects caused by neural tube malformations, The raw herbal ingredients of the pharmaceutical composition include 20-28 parts by weight of Rehmannia glutinosa root, 4-8 parts by weight of Cornus officinalis root, 4-8 parts by weight of Daphnia serrata, 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; The pharmaceutical composition and its formulation use, characterized in that the pharmaceutical composition further comprises 4-8 parts by weight of Kallista and 4-8 parts by weight of Psyllium.

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