Application of small molecule compound in preparation of products for treating nervous system diseases

Abstract

The invention discloses application of a small molecule compound in preparation of a product for treating nervous system diseases. According to the application disclosed by the invention, it is determined for the first time that Chebulinic acid and 1, 1, 1-Kestohexenose are targeted PU.1 inhibitors, nerve cell activity decline caused by A beta deposition can be improved, and a new technical choice is provided for developing a treatment method for nerve injury diseases and protecting or improving nerve cell injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically, to the application of small molecule compounds in the preparation of products for treating nervous system diseases. Background Art

[0002] Nervous system diseases (such as Alzheimer's disease, AD) are closely related to nerve cell death and dysfunction. As highly differentiated terminal cells, nerve cells have limited regenerative ability, and their damage and death often lead to irreversible nerve function deficits. Therefore, developing effective nerve cell protection strategies is of great significance for the prevention and treatment of nervous system diseases. In recent years, with the in-depth study of the mechanism of nerve cell death, a variety of potential neuroprotective targets have been successively discovered, and a variety of neuroprotective strategies such as drug intervention, gene therapy, cell therapy, and physical therapy have been developed, but there are challenges such as poor drug targeting, large side effects, and limited therapeutic effects. Therefore, developing safer, more effective, and more targeted neuroprotective technologies remains the focus and difficulty of current research.

[0003] PU.1 belongs to the Ets protein family and is a transcription factor that plays a key role in the hematopoietic and immune systems, and can regulate the development, differentiation, and function of macrophages and microglia. However, the current prior art has not clearly revealed the direct relationship between PU.1 and nerve cell damage, and there is no drug that targets PU.1 to protect nerve cells or improve nerve cell damage. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides the application of small molecule compounds in the preparation of products for treating nervous system diseases.

[0005] The first object of the present invention is to provide the application of small molecule compounds in the preparation of products for treating nervous system diseases.

[0006] The second object of the present invention is to provide the application of small molecule compounds in the preparation of products for treating nerve cell damage.

[0007] The third object of the present invention is to provide the application of small molecule compounds in the preparation of products for inhibiting nerve cell death.

[0008] The fourth object of the present invention is to provide the application of small molecule compounds in the preparation of products for improving nerve cell damage caused by Aβ deposition.

[0009] The fifth object of the present invention is to provide the application of small molecule compounds in the preparation of inhibitors targeting PU.1.

[0010] The sixth object of the present invention is to provide an inhibitor targeting PU.1.

[0011] The seventh object of the present invention is to provide a preparation for treating nervous system diseases.

[0012] In order to achieve the above objects, the present invention is realized by the following solutions:

[0013] The present invention uses the MCE 50K Diversity Library database and the Natural Product LibraryPlus database to search for relevant drugs against PU.1, and uses network pharmacology and basic cell experiments for identification. It is found for the first time that Chebulinic acid and 1,1,1,1-Kestohexaose can improve the decline in cell activity caused by Aβ deposition, and it is proved that PU.1 is the target gene of Chebulinic acid and 1,1,1,1-Kestohexaose, which has broad application prospects in the field of protecting or improving nerve cells.

[0014] Therefore, the present invention requests protection for the following:

[0015] Use of a small molecule compound in the preparation of a product for treating nervous system diseases, wherein the small molecule compound has a structural formula shown in formula (1) or formula (2);

[0016]

[0017] Among them, the small molecule compound with the structural formula shown in formula (1) is Chebulinic acid, and the small molecule compound with the structural formula shown in formula (2) is 1,1,1,1-Kestohexaose.

[0018] Use of a small molecule compound in the preparation of a product for treating nerve cell damage, wherein the small molecule compound has a structural formula shown in formula (1) or formula (2);

[0019]

[0020] Use of a small molecule compound in the preparation of a product for inhibiting nerve cell death, wherein the small molecule compound has a structural formula shown in formula (1) or formula (2);

[0021]

[0022]

[0023] Use of a small molecule compound in the preparation of a product for improving nerve cell damage caused by Aβ deposition, wherein the small molecule compound has a structural formula shown in formula (1) or formula (2);

[0024]

[0025] Preferably, the small molecule compound serves as an inhibitor targeting PU.1.

[0026] Use of a small molecule compound in the preparation of an inhibitor targeting PU.1, wherein the small molecule compound has a structural formula shown in formula (1) or formula (2);

[0027]

[0028] An inhibitor targeting PU.1, the active ingredient of which comprises a small molecule compound having a structural formula shown in formula (1) or formula (2);

[0029]

[0030]

[0031] A preparation for treating nervous system diseases, the active ingredient of which comprises a small molecule compound having a structural formula shown in formula (1) or formula (2);

[0032]

[0033] Preferably, it further comprises a pharmaceutically acceptable carrier or excipient.

[0034] More preferably, the form of the preparation is an injection, a tablet, a capsule or a patch.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention for the first time determines that Chebulinic acid and 1,1,1,1-Kestohexaose are inhibitors targeting PU.1, and can improve the decline of nerve cell activity caused by Aβ deposition, providing a new technical option for developing treatment methods for nerve injury diseases and protecting or improving nerve cell injury. Description of the Drawings

[0037] Figure 1 It is a workflow diagram for virtual screening of inhibitors targeting PU.1 based on molecular docking.

[0038] Figure 2 It is a schematic diagram of the chemical structures of 10 small molecule compounds.

[0039] Figure 3Venn diagram of AD-related genes in GeneCards database and DisGeNET database; GeneCards represents AD-related genes in GeneCards database; DisGeNET represents AD-related genes in DisGeNET database.

[0040] Figure 4 Venn diagram of small molecule compounds and disease-related genes analysis; Drug represents genes related to small molecule compounds; Disease represents disease-related genes.

[0041] Figure 5 Protein-Protein (PPI) network diagram of genes shared by drugs and diseases.

[0042] Figure 6 Diagram of KEGG pathway analysis results.

[0043] Figure 7 Diagram of GO enrichment analysis results.

[0044] Figure 8 "Ingredient-Target-Disease" network diagram, circles represent target genes; triangles represent small molecule compounds.

[0045] Figure 9 Molecular docking visualization diagram of 10 small molecule compounds.

[0046] Figure 10 For different concentrations of Aβ 1-42 Effect on SY5Y cell viability ([ n = 3); compared with the blank group * P < 0.05, ** P < 0.01.

[0047] Figure 11 For different small molecule compounds on Aβ 1-42 Effect on SY5Y cell viability after treatment with oligomers ([ n = 3); compared with the blank group, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0048] Figure 12 Effect of different concentrations of Chebulinic acid (HY-N2033) and 1,1,1,1-Kestohexaose (HY-N6838) on SY5Y cell viability ([ n = 3); compared with the blank group, ns indicates P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

[0049] Figure 13 The effects of Chebulinic acid (HY-N2033) and 1,1,1,1-Kestohexaose (HY-N6838) on the expression level of PU.1 mRNA in SY5Y cells after treatment with Aβ 1-42 oligomers ( n = 3); compared with the blank group, ***P < 0.001; compared with the model group, ## P < 0.01, ### P < 0.001. Detailed implementation manners

[0050] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0051] Example 1 Determination of inhibitors targeting PU.1

[0052] 1. Virtual screening based on molecular docking

[0053] Using the 50K Diversity Library database and Natural Product Library Plus database of MCE for PU.1, and using Maestro 12.8 software for virtual screening. The process is as Figure 1 shown, and the specific steps are as follows:

[0054] Download the three-dimensional structure of Human SPI1 (PU.1) (PDB ID: 1F5N) from the PDB website. Use the ProteinPreparation Wizard module to hydrogenate the protein, delete water molecules, and delete DNA. Subsequently, perform energy optimization (OPLS2005 force field, RMSD is ). Use the Receptor Grid Generation module to create a grid file for the processed protein, and generate a grid file centered on the pocket where the DNA binding interface is located (the key amino acids are: 230R / 233L / 243K). The box size is set to

[0055] Then, the 2D formats of HY-L021P Natural Product Library Plus (containing 5,300 compounds) and HY-L901 50K Diversity Library (containing 50,000 compounds) are processed by the LigPrep Module of the software for hydrogenation, energy optimization, etc., and the 3D structures are output for virtual screening.

[0056] Virtual Screening Workflow module is used for virtual screening. The prepared compounds are imported, and the Glide module is used for molecular docking, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching. HY-L021P Natural Product Library Plus: First, the prepared small molecule compounds in the database are screened using the standard (SP) mode in the Glide module. Subsequently, the top 20% of the scoring values are selected and the third round of screening is carried out using the high-precision (XP) mode to obtain the ranking of small molecule compounds. HY-L901 50K Diversity Library: First, the prepared small molecule compounds in the database are screened using the high-throughput screening (HTVS) mode in the Glide module. The small molecule compounds with the top 20% of the scoring values are selected and the second round of screening is carried out using the standard (SP) mode; subsequently, the top 20% of the scoring values are selected and the third round of screening is carried out using the high-precision (XP) mode to obtain the ranking of small molecule compounds.

[0057] Manually review the binding force between the target and the compound, the compound structure, etc., and select and output the top 200 compounds in HY-L021P Natural Product Library Plus and the top 200 compounds in HY-L901 50K Diversity Library.

[0058] Finally, the top five small molecule compounds with the highest docking scores were obtained in each of the two databases, that is, a total of 10 small molecule compounds were obtained, namely Diadenosine pentaphosphate (pentasodium), NADPH (tetrasodium salt), Chebulinic acid, N4-Acetylcytidine triphosphate (sodium), Phytic acid (potassium), ethyl 4-hydroxy-2-methyl-6-oxo-5-(phenylthio)-1,6-dihydropyridine-3-carboxylate, 2-((4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)thio)benzoic acid, 3-(3-(4-fluorobenzoyl)thioureido)-4-methoxybenzoic acid, 2-(2-((3-cyano-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-2-yl)thio)acetamido)acetic acid, 2-Acetamido-3-(7-chloro-1H-indol-3-yl)propanoic acid, and their structural formulas are as Figure 2 shown for the next step of target prediction.

[0059] 2. Network pharmacology

[0060] (1) Prediction of small molecule compound targets

[0061] The 10 small molecule compounds screened were imported into the SwissTargetPrediction database (http: / / swisstargetprediction.ch) in Smiles format for target prediction, and 462 targets with a Probability greater than 0 as shown in Table 1 were obtained. After removing duplicates, 334 targets remained.

[0062] Table 1 Targets of small molecule compounds

[0063]

[0064] (2) Collection of disease-related genes

[0065] Alzheimer's disease is a common neurological disorder. Using the keywords "Alzheimer's disease", relevant genes were retrieved from the GeneCards database (https: / / www.genecards.org) and the DisGeNET database (http: / / www.disgenet.org / ), and the obtained genes were integrated.

[0066] A total of 26,108 Alzheimer's disease-related genes were obtained from the GeneCards database, and 3,397 Alzheimer's disease-related genes were obtained from the DisGeNET database. As Figure 3 shown, after integrating the two databases, a total of 26,266 Alzheimer's disease-related genes including PU.1 were obtained.

[0067] (3) Determination of common targets between drugs and diseases and construction of protein-protein (PPI) network diagrams

[0068] The 334 targets obtained in step (1) and the disease-related genes obtained in step (2) were uploaded to the bioinformatics platform (http: / / www.bioinformatics.com.cn) to create a Venn diagram, and the intersection of the two was taken. As Figure 4 shown, 334 common targets between drugs and diseases were obtained.

[0069] To better illustrate the relationships between the targets, the 334 common targets were uploaded to the STRING database (https: / / string-db.org / ) to obtain protein interaction information, which was then imported into cytoscape 3.7.1 to draw a PPI network diagram. Nodes represent proteins, and the size and color depth of the nodes represent the degree value; each edge represents a protein interaction relationship, and the network diagram shows the relationships between the various targets; the larger the node, the darker the node color, and the more edges indicate that the node plays an important role in protein interactions. As Figure 5 shown, the mechanism of small molecule compounds in treating Alzheimer's disease is complex, involving the synergistic action of multiple targets and not relying on a single target.

[0070] (4) Pathway enrichment analysis

[0071] The DAVID database (https: / / davidbioinformatics.nih.gov / ) was used to perform KEGG pathway analysis and GO enrichment analysis on the 334 common targets obtained in step (3).

[0072] According to the count value, the top 20 KEGG pathways were selected and plotted using the bioinformatics platform. As Figure 6As shown, the 334 common targets are mainly concentrated in neuroactive ligand-receptor interaction, cancer, platelet activation, the role of proteoglycans in cancer, lipid and atherosclerosis, cAPM signaling pathway, cellular senescence, effector cell differentiation, Rap1 signaling pathway, calcium signaling pathway, etc.

[0073] In the GO enrichment analysis, the top 20 terms with the count value of the 334 common targets in biological process, cellular component, and molecular function were analyzed. The results are as Figure 7 shown. The 334 common targets are mainly involved in phosphorylation, signal transduction, cytoplasm, mitochondria, protein binding, ATP binding, etc.

[0074] (5) Construction of the "component-target-disease" network

[0075] The 10 small molecule compounds and 334 common targets were imported into the Cytoscape 3.7.1 software to construct the "component-target" network diagram. The "component-target" network and the PPI network were merged using the Merge tool in the Cytoscape 3.7.1 software to obtain the "component-target-disease" network diagram.

[0076] As Figure 8 shown, the circles represent the targets and the triangles represent the small molecule compounds; the small molecule compounds and disease-related genes are divided into three categories, and the colors gradually deepen from light to dark as the drug-regulated targets obtained from the Natural Product Library Plus, the drug-regulated targets obtained from the 50K Diversity Library, and the common regulated targets of the two libraries. The higher the degree value, the more important the target is. There are 10 small molecule compounds, 334 nodes, and 7038 edges in total. According to the degree value ranking, the top three small molecule compounds are ethyl 4-hydroxy-2-methyl-6-oxo-5-(phenylthio)-1,6-dihydropyridine-3-carboxylate, 3-(3-(4-fluorobenzoyl)thioureido)-4-methoxybenzoic acid, and 2-(2-((3-cyano-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-2-yl)thio)acetamido)acetic acid.

[0077] (6) Molecular docking

[0078] Enter the names of 10 small molecule compounds into the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov), retrieve the relevant information of the compounds, and download the SDF format. The three-dimensional structure of the Human SPI1 (PU.1) protein has been experimentally resolved (PDB ID: 8EE9). After obtaining the small molecule ligand and the protein receptor, the following specific methods are used for processing:

[0079] First, use Chem 3D to optimize the mechanical structure of the small molecule compound, save it in mol 2 format, and then use AutDocktools to process the ligand root and rotatable bonds of the above mol 2 file, and output it as a PDBQT file. Use the Pymol software to remove water molecules, small molecule ligands, and repetitive structures from the protein receptor, save it in P BD format, and then open it with the AutDocktools software to calculate the charges, add atoms, and determine the docking region. Establish a configuration file for molecular docking. Use Pymol and the Protein-Ligand Interaction Profile r database (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index) to perform visualization processing on the docking results.

[0080] After processing the protein structure of PU.1, the docking box is determined to be (33.298, 0.248, 41.601). The docking scores of the 10 small molecule compounds from high to low are Diadenosine pentaphosphate (pentasodium) (-13.342), NADPH (tetrasodium salt) (-12.715), Chebulinic acid (-12.125), N4-Acetylcytidine triphosphate (sodium) (-11.976), 1,1,1,1-Kestohexaose (-11.748), HY-Q34174 (-7.385), HY-Q33723 (-7.181), HY-Q49486 (-7.140), HY-Q42322 (-7.118), and HY-78566 (-6.751).

[0081] For the 10 small molecule compounds, select the pose with the highest docking score for visualization processing, such as Figure 9As shown in the figure, the docking sites of PU.1 with Diadenosine pentaphosphate (pentasodium) are mainly at LYS-221, LYS-227, LYS-217, ASN-219, ANS-234, LEU-172, ARG-171; the docking sites with NADPH (tetrasodium salt) are mainly at ALA-208, ARG-212, GLN-216, PHE-175, SER-182; the docking sites with Chebulinic acid are mainly at ASN-219, LYS-227, ALA-231, ARG-230, LYS-217, YRP-213, ASN-234, LEU-172; the docking sites with N4-Acetylcytidine triphosphate (sodium) are mainly at ARG-220, ASN-219, LYS-217, TRP-213, ASN-234, TYR-235, ARG-171, ILE-170; the docking sites with Guanosine 5'-triphosphate-5'-adenosine (triammonium) are mainly at ARG-220, ASN-219, LYS-217, ILE-170, LEU-172, ARG-171, LYS-227, ASN-234, TYR-235; the docking sites with HY-Q34174 are mainly at LYS-204, SER-202, SER-203, LYS-247, LEU-248, TRP-190, GLN-200; the docking sites with HY-Q33723 are mainly at GLN-216, ILE-215, ARG-212, ILE-170; the docking sites with HY-Q49486 are mainly at ARG-212, ILE-215, GLN-216; the docking sites with HY-Q42322 are mainly at ILE-215, ARG-212; the docking sites with HY-78566 are mainly at GLN-216, ARG-212.

[0082] In summary, combining virtual screening, molecular docking and relevant literature research, Chebulinic acid (HY-N2033) and 1,1,1,1-Kestohexaose (HY-N6838) are derived from the traditional Chinese medicines Terminalia chebula Retz. and Poa gigantea (L.) Reichb. respectively. Existing studies have proved that both of these two medicines have anti-inflammatory and antioxidant pharmacological effects, and the chemical components of traditional Chinese medicines generally have less toxic and side effects. Therefore, two small molecule compounds, Chebulinic acid (HY-N2033) and 1,1,1,1-Kestohexaose (HY-N6838), are selected for subsequent experimental verification.

[0083] Example 2 Aβ 1-42 Determination of damaged cell concentration

[0084] 1. Aβ 1-42 Preparation of oligomers

[0085] Dissolve 5 mg of Aβ 1-42 (CAS No.: 107761-42-2) in 1.1 mL of hexafluoroisopropanol, let it stand at room temperature (22 - 28 °C) for 1 h, mix well and divide it into 10 portions, dry it in a fume hood to obtain Aβ 1-42 oligopeptide film, and store it at -20 °C. Before use, take one portion of Aβ 1-42 oligopeptide film, add 10 μL of DMSO solution to dissolve the oligopeptide film, and then add 1090 μL of DMEM / F12 medium. Mix well and place it in a 4 °C refrigerator for incubation for 24 h to obtain 100 μM Aβ 1-42 oligomers.

[0086] 2. Cell viability assay

[0087] Seed SY5Y cells in the logarithmic growth phase at 10 5 cells per well in a 96-well plate. After 24 h, dilute the 100 μM Aβ 1-42 oligomers obtained after incubation above to concentrations of 0 μM, 5 μM, 10 μM, 20 μM, and 40 μM Aβ 1-42 oligomers and treat the cells respectively. Among them, the cells treated with 0 μM Aβ 1-42 oligomers are recorded as the blank group, and the rest are recorded as the Aβ 1-42 modeling group.

[0088] After 24 h, remove the old medium and add fresh medium containing CCK-8; after incubation for 2 h, measure the absorbance (OD value) of each cell well at 450 nm, and calculate the cell survival rate of SY5Y cells. The calculation formula is: Cell survival rate (%) = [(OD value of each group - background OD value) ÷ (OD value of the blank group - background OD value)] × 100%.

[0089] As Figure 10 shown, compared with the blank group (0 μM), there is no significant difference in the cell survival rate of the 5 μM Aβ 1-42 modeling group (P > 0.05); the cell survival rates of the 10 μM, 20 μM, and 40 μM Aβ 1-42 modeling groups are all significantly reduced in SY5Y cells (P < 0.05, P < 0.01). Therefore, it is determined that the damaged cell concentration of Aβ 1-42 is 20 μM, and it acts on SY5Y cells for 24 h as the modeling condition for subsequent experiments.

[0090] Example 3 Different small molecule compounds on Aβ1-42 Effect of oligomers on the viability of SY5Y cells

[0091] 1. Preparation of compound stock solutions

[0092] Weigh 1 mg each of Chebulinic acid (HY-N2033) and 1,1,1,1-Kestohexaose (HY-N6838), and place them in separate EP tubes. Then, add DMSO and DMEM / F12 medium to each EP tube according to the volume ratio DMSO:DMEM / F12 medium = 1:100 to prepare a stock solution of HY-N2033 with a concentration of 1 mM and a stock solution of HY-N6838 with a concentration of 1 mM for later use.

[0093] 2. Aβ 1-42 - Preparation of compound solution mixtures

[0094] Mix the Aβ oligomers from Example 2 with the HY-N2033 stock solution to obtain a mixture with a final concentration of 20 μM Aβ and a final concentration of 10 μM HY-N2033, denoted as Aβ-HY-N2033-10 μM solution. Mix the Aβ oligomers with the HY-N2033 stock solution to obtain a mixture with a final concentration of 20 μM Aβ and a final concentration of 20 μM HY-N2033, denoted as Aβ-HY-N2033-20 μM solution. Mix the Aβ oligomers with the HY-N6838 stock solution to obtain a mixture with a final concentration of 20 μM Aβ and a final concentration of 10 μM HY-N6838, denoted as Aβ-HY-N6838-10 μM solution. Mix the Aβ oligomers with the HY-N6838 stock solution to obtain a mixture with a final concentration of 20 μM Aβ and a final concentration of 20 μM HY-N6838, denoted as Aβ-HY-N6838-20 μM solution. 1-42 oligomers and the HY-N2033 stock solution to obtain a mixture with a final concentration of 20 μM Aβ 1-42 and a final concentration of 10 μM HY-N2033, denoted as Aβ 1-42 -HY-N2033-10 μM solution. Mix the Aβ 1-42 oligomers and the HY-N2033 stock solution to obtain a mixture with a final concentration of 20 μM Aβ 1-42 and a final concentration of 20 μM HY-N2033, denoted as Aβ 1-42 -HY-N2033-20 μM solution. Mix the Aβ 1-42 oligomers and the HY-N6838 stock solution to obtain a mixture with a final concentration of 20 μM Aβ 1-42 and a final concentration of 10 μM HY-N6838, denoted as Aβ 1-42 -HY-N6838-10 μM solution. Mix the Aβ 1-42 oligomers and the HY-N6838 stock solution to obtain a mixture with a final concentration of 20 μM Aβ 1-42 and a final concentration of 20 μM HY-N6838, denoted as Aβ 1-42 -HY-N6838-20 μM solution.

[0095] 3. Cell treatment

[0096] Take SY5Y cells in the logarithmic growth phase. After digestion and centrifugation, add an appropriate amount of medium and mix well. Perform cell counting and seed 10 cells per well according to the cell density. 5They were seeded into 96-well plates and divided into a blank group (CON), a model group (MOD), a 10 μM HY-N2033 administration group (HY-N2033-10 μM), a 20 μM HY-N2033 administration group (HY-N2033-20 μM), a 10 μM HY-N6838 administration group (HY-N6838-10 μM), and a 20 μM HY-N6838 administration group (HY-N6838-20 μM).

[0097] After culturing for 24 h and waiting for the cells to adhere, the old culture medium was removed; 100 μL of complete medium without double antibiotics (i.e., DMEM / F12 medium containing 15 v / v% FBS) was added to each well of the blank group; 100 μL of 20 μM Aβ 1-42 oligomers were added to each well of the model group; 100 μL of Aβ 1-42 -HY-N2033-10 μM solution was added to each well of the 10 μM HY-N2033 administration group; 100 μL of Aβ 1-42 -HY-N2033-20 μM solution was added to each well of the 20 μM HY-N2033 administration group; 100 μL of Aβ 1-42 -HY-N6838-10 μM solution was added to each well of the 10 μM HY-N6838 administration group; 100 μL of Aβ 1-42 -HY-N6838-20 μM solution was added to each well of the 20 μM HY-N6838 administration group.

[0098] After incubating for 24 h, the old culture medium was removed, and new medium containing CCK-8 was added. After incubating for 2 h, the absorbance was measured at 450 nm, and the cell viability of SY5Y cells was calculated. The effects of different drugs on the cell viability of SY5Y cells treated with Aβ 1-42 oligomers were observed.

[0099] The results were as Figure 11 shown. Compared with the blank group, the cell viability of the model group decreased significantly, indicating that the modeling effect of damaging SY5Y cells with 20 μM Aβ 1-42 for 24 h was significant; compared with the model group, the cell viabilities of the four administration groups increased significantly. This shows that Chebulinic acid (HY-N2033) and 1,1,1,1-Kestohexaose (HY-N6838) both have significant protective effects on nerve cell damage caused by Aβ 1-42 and can effectively improve nerve cell damage.

[0100] Network pharmacology of Example 1 shows that the potential targets of small molecule compounds mainly cluster in aspects such as neuroactive ligand-receptor interaction, lipid and atherosclerosis, cellular senescence, calcium signaling pathway, mitochondrial metabolism and signal transduction, and these biological processes are all closely related to the pathogenesis of Alzheimer's disease. In this example, Aβ 1-42 induced a decrease in SY5Y cell activity, which may be due to the neurotoxicity caused by Aβ 1-42 deposition. Chebulinic acid and 1,1,1,1-Kestohexaose can improve the changes in SY5Y cell activity induced by Aβ 1-42 possibly because Chebulinic acid and 1,1,1,1-Kestohexaose inhibit the expression of PU.1 and improve the nerve damage caused by Aβ deposition.

[0101] Effect of small molecule compounds at different concentrations on SY5Y cell activity in Example 4

[0102] 1. Dilution of stock solution

[0103] To determine the safe dosing range and optimal dosing concentration of Chebulinic acid (HY-N2033) and 1,1,1,1-Kestohexaose (HY-N6838) on SY5Y cells, complete medium without antibiotics was used to dilute the 1 mM stock solution of HY-N2033 in Example 3 to concentrations of 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM respectively. The resulting solutions were denoted as HY-N2033 dilutions; according to the same method, the 1 mM stock solution of HY-N6838 was used to obtain HY-N6838 dilutions at different concentrations for standby.

[0104] 2. Cell treatment

[0105] After digesting and centrifuging SY5Y cells in the logarithmic growth phase, they were mixed with complete medium. After cell counting, 10 5 cells per well were seeded into a 96-well plate and cultured for 24 h. After the cells adhered, different concentrations of HY-N2033 dilutions were added and placed in a cell culture incubator for 24 h. The old medium was removed, and new medium containing CCK-8 was added. After incubation for 2 h, the absorbance was measured at 450 nm, and the cell survival rate was calculated. To determine the effect of different drug concentrations on SY5Y cell activity and select a suitable dosing concentration.

[0106] As Figure 12As shown, the concentrations at which two small molecule compounds are toxic to SY5Y cells are different. Cheb ulinic acid (HY-N2033) at a concentration of 5 - 100 μM has no obvious toxic effect on SY5Y cells, and 1,1,1,1-Kestohexaose (HY-N6838) at a concentration of 5 - 50 μM has no obvious toxic effect on SY5Y cells. Example 5 Inhibitory effects of Chebulinic acid and 1,1,1,1-Kestohexaose on PU.1

[0107] 1. Cell treatment

[0108] After digesting and centrifuging SY5Y cells in the logarithmic growth phase, they were mixed with complete medium. After counting the cells, 10 6 cells per well were seeded into 6-well plates, and they were set as the blank group (CON), the model group (MOD), the Chebulinic acid group, and the 1,1,1,1-Kestohexaose group.

[0109] After culturing for 24 h, after the cells adhered to the wall, 2 mL of complete medium without double antibiotics (i.e., DMEM / F12 medium containing 15 v / v% FBS) was added to each well of the blank group; 2 mL of 20 μM Aβ 1-42 oligomers of Example 2 were added to each well of the model group; Aβ of Example 3 1-42 -HY-N2033-10 μM solution was added to each well of the Chebulinic aci group; Aβ of Example 3 1-42 -HY-N6838-10 μM solution was added to each well of the 1,1,1,1-Kestohexaos group. The cells in each group were placed in a cell culture incubator and cultured for another 24 h.

[0110] 3. RNA extraction and reverse transcription

[0111] After the culture ended, the old medium was removed, 500 μL of Trizol was added to each well, and it was pipetted repeatedly and left standing at room temperature for 5 min. 300 μL of chloroform substitute was added to each well, and it was shaken for 15 s. After standing at room temperature for 5 min, it was centrifuged at 4℃ and 12000 rpm for 15 min. The upper aqueous phase was taken, 0.5 times the volume of isopropanol was added, and it was vortexed for 15 s. After standing at room temperature for 10 min, it was centrifuged at 4℃ and 12000 rpm for 15 min. The supernatant was discarded, the RNA precipitate was washed with 1 mL of 75% ethanol. After adding 75% ethanol, it was centrifuged at 4℃ and 7500 rpm for 5 min, and the washing was repeated three times. It was air-dried at room temperature for 30 min, and 20 μL of enzyme-free water was added to dissolve the RNA. The RNA concentration was detected using a spectrophotometer.

[0112] The extracted RNA was reverse-transcribed using a reverse transcription reagent (AG11706) according to an RNA content of 300 ng to obtain cDNA, which was stored at -20°C.

[0113] 3. Detection of the mRNA expression level of PU.1 by qPCR

[0114] The cDNA obtained in the previous step was diluted at a ratio of cDNA: enzyme-free water = 1:2. After mixing, it was used as a template for qPCR detection. The qPCR primers shown in Table 2 were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The qPCR reaction system was as follows: 2×SYBR Green Rro Taq HS Premix, 5 μL; primer F (10 μM), 0.2 μL; primer R (10 μM), 0.2 μL; template, 1 μL; enzyme-free water, 3.6 μL). The qPCR reaction program is shown in Table 3.

[0115] Table 2 qPCR primers

[0116] Table 3 qPCR reaction program

[0117]

[0118] As Figure 13 shown, compared with the CON group, the mRNA expression level of PU.1 in the MOD group was significantly increased; compared with the MOD group, the mRNA expression levels of PU.1 in the Chebulinic acid group and the 1,1,1,1-Kestohexaose group were significantly decreased. It was proved that both Chebulinic acid and 1,1,1,1-Kestohexaose could inhibit the expression of PU.1 and were inhibitors targeting PU.1.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of a small molecule compound in the preparation of a product for treating nervous system diseases, characterized in that, The small molecule compound has a structural formula shown in Formula (1) or Formula (2); 2. Use of a small molecule compound in the preparation of a product for treating nerve cell injury, characterized in that, The small molecule compound has a structural formula shown in Formula (1) or Formula (2); 3. Use of a small molecule compound in the preparation of a product for inhibiting nerve cell death, characterized in that, The small molecule compound has a structural formula shown in Formula (1) or Formula (2); 4. Use of a small molecule compound in the preparation of a product for improving nerve cell damage caused by Aβ deposition, characterized in that, The small molecule compound has a structural formula shown in Formula (1) or Formula (2); 5. The application according to any one of claims 1 to 4, characterized in that, The small molecule compound is used as an inhibitor targeting PU.

1.

6. Use of a small molecule compound in the preparation of an inhibitor targeting PU.1, characterized in that, The small molecule compound has a structural formula shown in Formula (1) or Formula (2); 7. An inhibitor targeting PU.1, characterized in that, Its active ingredient includes a small molecule compound having a structural formula shown in Formula (1) or Formula (2); 8. A preparation for treating nervous system diseases, characterized in that, Its active ingredient includes a small molecule compound having a structural formula shown in Formula (1) or Formula (2); 9. The preparation according to claim 8, characterized in that, It also includes a pharmaceutically acceptable carrier or excipient.

10. The preparation according to claim 9, characterized in that, The form of the preparation is an injection, tablet, capsule or patch.

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