Application of echinacoside in schizophrenia treatment

Abstract

The invention discloses application of echinacoside in treatment of schizophrenia. The method belongs to the field of medicines and comprises the following operation steps: screening schizophrenia risk genes; constructing a gene editing mouse model; evaluating a behavior normal form; predicting a protein structure; molecular docking; and carrying out behavioral pharmacology experiments. According to the echinacoside disclosed by the invention, in schizophrenia treatment, a healthy control mouse model and a schizophrenia mouse model are respectively subjected to intraperitoneal injection drug treatment for two weeks, and then the influence of echinacoside treatment on the schizophrenia mouse behavior phenotype is evaluated through a behavioral experiment. The pharmaceutical composition disclosed by the invention has a good treatment effect in the aspects of negative symptoms of schizophrenia, cognitive impairment and sensory gating defect, and a new drug target and a new treatment approach are provided for the treatment of schizophrenia.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine, and relates to the application of coniferin in the treatment of schizophrenia. BACKGROUND

[0002] Schizophrenia (SCZ) is a severe mental disorder that affects a patient's thinking, emotions, and behavior. It has diverse clinical symptoms, including positive symptoms (such as hallucinations, delusions, and disorganized thinking), negative symptoms (such as blunted affect and social withdrawal), and cognitive dysfunction. The disease usually occurs in late adolescence or early adulthood, and is often chronic or recurrent. SCZ not only brings great suffering to patients, but also causes heavy economic and psychological burdens on their families, and poses a serious challenge to the medical resources and public health system of society.

[0003] Only about 8% of these patients have sought professional help, and 5% have received standard treatment, highlighting the problem of undiagnosed and untreated patients. Currently, the main clinical intervention for SCZ is antipsychotic drugs, such as clozapine, olanzapine, and risperidone. Although these drugs have achieved certain effects in alleviating the positive symptoms of SCZ patients, they have limited effects in improving negative symptoms and cognitive dysfunction, and may cause serious side effects. In addition, different patients have significant differences in response to antipsychotic drugs, and about 30% of patients have no significant improvement with existing drug treatment. This heterogeneity in treatment response poses a great challenge to clinical treatment. SUMMARY

[0004] The purpose of the present application is to provide an application of a natural phenylethanoid glycoside polyphenol compound (coniferin) for improving negative symptoms and cognitive dysfunction in schizophrenia.

[0005] The technical solution of the present application is: the application of coniferin in the treatment of schizophrenia.

[0006] Further, the experimental operation of the application of coniferin in the treatment of schizophrenia includes the following steps:

[0007] (1) Screening of schizophrenia risk genes;

[0008] (2) Screening of schizophrenia risk gene FAM65B through gene differential expression analysis;

[0009] (3) Behavioral experiment to evaluate whether the mouse model has a schizophrenia-like phenotype;

[0010] (4) Virtual screening of target risk genes to find potential therapeutic drugs;

[0011] (5) Behavioral pharmacological experiments to evaluate the therapeutic effect of Geronigroside.

[0012] Further, the schizophrenia risk gene screening in step (1) is specifically:

[0013] (a) Collect (include) postmortem brain tissue transcriptome data of schizophrenia and healthy control samples;

[0014] (b) Gene differential expression analysis, screening of schizophrenia differential expression risk gene FAM65B; When P-values <0.05, Log2(fold change)>0 is marked as up-regulated gene; Log2(fold change)<0 is marked as down-regulated gene.

[0015] Further, the gene differential expression analysis to screen schizophrenia risk gene FAM65B in step (2) is commissioned to a certain biotechnology Co., Ltd. in Shanghai to complete, and the specific steps are:

[0016] (a) Using CRISPR / Cas9 technology, the target gene is knocked in at the Gt(ROSA)26Sor gene site by homologous recombination to construct FAM65B transgenic mouse model;

[0017] (b) Design genotyping primers, PCR (Polymerase Chain Reaction) genotyping of mice;

[0018] (c) Cross with Nestin-Cre tool mouse to get brain tissue specific expression FAM65B transgenic mouse model.

[0019] Further, the behavioral experiment to evaluate whether the mouse model has schizophrenia-like phenotype in step (3) is specifically:

[0020] (a) Breeding transgenic mice to 2 months old;

[0021] (b) Move the mice to the behavioral room and adapt to the environment for one week;

[0022] (c) Weigh the mice;

[0023] (d) Complete the neuropsychiatric experiment; including open field experiment, nest building experiment, PPI experiment, etc.;

[0024] (e) Complete the learning and cognitive experiment; including Y maze and water maze, etc.

[0025] Further, the potential therapeutic drugs of the risk genes in step (4) are specifically:

[0026] (a) Protein preparation: the protein structure of the risk gene FAM65B predicted by the Alphafold website is hydrogenated using the Protein Preparation Wizard module, followed by energy optimization;

[0027] (b) Small molecule compound library preparation: the 2D format of HYL065 Traditional Chinese Medicine Active Compound Library (containing 2,900 compounds), HYL022P FDA Approved Drug Library Plus (containing 3,400 compounds), HYL001P Bioactive Compound Library Plus (containing 24,300 compounds), and HYL028CNS Penetrant Compound Library (containing 800 compounds) are hydrogenated, energy optimized, etc. by the software LigPrep Module module, and the 3D structure is output for virtual screening;

[0028] (c) Screening of molecular docking compounds: virtual screening is performed using the Virtual Screening Workflow module, the prepared compounds are imported, and molecular docking is performed using the Glide module, i.e. the receptor and ligand molecules are docked with each other through geometric matching and energy matching;

[0029] (d) Compound ranking: the higher the absolute value of the molecular docking score, the stronger the binding force of the compound to the risk gene protein.

[0030] Further, the behavioral pharmacology experiment in step (5) for evaluating the treatment effect of ombuoside is specifically:

[0031] (a) According to the body weight, age, body surface area and administration route of the mouse, the mouse is treated with drugs;

[0032] (b) Weigh the body weight of the mouse;

[0033] (c) Complete neuropsychiatric experiments, including: open field test, nest building test, PPI (pre-pulse inhibition, Prepulse Inhibition) test, etc.;

[0034] (d) Complete learning and cognitive experiments including Y maze, water maze, etc.

[0035] ​Beneficial effects: compared with the prior art, the present application has the following significant features: the present application first discovers that matatabi specifically binds to FAM65B protein, has a new use in the treatment of negative symptoms of schizophrenia, cognitive impairment and sensory gating defects, and provides a new way for the treatment of schizophrenia. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is a schematic diagram of screening schizophrenia risk gene FAM65B and constructing a transgenic mouse model in the embodiments of the present application; wherein (a), in the CMC dataset, the expression of FAM65B in the dorsolateral prefrontal cortex (DLPFC) of the schizophrenia patients is significantly up-regulated compared with the control group; (b), in the LIBD2 dataset, the expression of FAM65B in the DLPFC of the schizophrenia patients is significantly up-regulated compared with the control group; (c), in the GEO dataset, the expression of FAM65B in the brain tissue of the schizophrenia patients is significantly up-regulated compared with the control group; (d), in the GSE25673 dataset, the expression of FAM65B in the neurons induced by the hiPSCs of the schizophrenia patients is significantly up-regulated compared with the control group; (e), in the PsychENCODE dataset, the expression of FAM65B in the brain tissue of the schizophrenia patients is significantly up-regulated compared with the control group; (f), by comparing the differentially expressed genes of the four datasets, it is found that FAM65B is the only gene that is significantly and consistently up-regulated in the four datasets; (g), the schematic diagram shows the construction of the FAM65B transgenic mouse; using CRISPR-Cas9 technology and homologous recombination, the LSL-FAM65B-IRES-EGFP (annotated as FAM65B in the figure) expression frame is precisely integrated into the Gt(ROSA)26Sor(Rosa26) gene site; recombinant Nestin-cre mice are used to realize the specific expression of FAM65B in mouse neural stem cells and the neurons and glial cells differentiated therefrom; the mouse with the inserted FAM65B expression frame is called the control group (Ctrl), and the mouse recombinant with Nestin-Cre is called the transgenic group (Tg); (h), Western blot confirms the overexpression of FAM65B in the mouse brain, and the high expression of 115kDa and 150kDa is detected;

[0037] Figure 2Figure is the behavioral detection result chart of FAM65B transgenic mouse model in the embodiment of the present application; wherein, (a), mouse weight, the Tg mouse weight is significantly lower than that of the control group mice; (b-d), in the open field test, the total movement distance of Tg mice increases (b), the immobile time decreases (c), and the central region movement distance has no difference (d); (e), in the nest building behavior test, the nest quality score (1-5 points) quantitative analysis shows that: the nest building ability of Tg mice is significantly impaired; (f), in the Y maze test, the spontaneous alternation rate of Tg mice is significantly lower than that of the Ctrl group; (g), in the PPI test, the Tg mice have a significant defect in the pre-pulse inhibition ability; (a-g), the data is represented by mean ± standard deviation, and the P value is calculated by two-tailed unpaired Student's t-test;

[0038] Figure 3 Figure is the virtual screening workflow chart based on molecular docking in the embodiment of the present application;

[0039] Figure 4 Figure is the virtual screening of the small molecule compound Echinacoside targeting FAM65B protein in the embodiment of the present application; in turn, the cartoon (a), surface (b), 2D (c), and 3D (d) diagrams of the binding mode of Echinacoside and FAM65B protein are shown; in the 3D diagram, FAM65B protein forms 7 hydrogen bond interactions and 1 π-π interaction with Echinacoside;

[0040] Figure 5 Figure is a schematic diagram of Echinacoside improving schizophrenia-like behaviors of Tg mice by binding to FAM65B in the embodiment of the present application; (a), a schematic diagram of the mouse administration and behavior experiment process; (b, c), the body weight of Tg group mice before (b) and after (c) Echinacoside treatment is significantly lower than that of the control group; (d, e), in the Y maze test, the spontaneous alternation rate of the Tg group in the baseline period is significantly lower than that of the control group (d); after administration, the spontaneous alternation rate is improved to the level comparable to the control group (e); (f, g), the nest building ability of Tg mice before administration is significantly lower than that of the control group (f); after treatment, the nest building ability of some Tg mice is significantly improved (g); (h-m), in the open field test, the Tg mice have increased total movement distance (h), reduced immobile time (i), and unchanged central region movement distance (j) compared with the control group; after administration, the Tg group still shows increased total movement distance (k) and unchanged central region distance (m), but the immobile time (l) returns to the level of the control group; (n, o), in the PPI test, the Tg group shows a significant pre-pulse inhibition defect (n); after administration, the pre-pulse inhibition ability is significantly restored (o); (b-o), the data is represented by mean ± standard deviation, and the P value is calculated by two-tailed unpaired Student's t-test. DETAILED DESCRIPTION

[0041] The specific technical solution of the present invention will be further described in detail below with reference to specific examples.

[0042] As shown in the figure, this invention discloses the application of echinacoside in the treatment of schizophrenia. It is the first time that echinacoside has been found to exhibit a high binding affinity to the schizophrenia risk gene FAM65B, and has significant therapeutic effects on negative symptoms, cognitive impairment, and sensory gating deficits in a mouse model of schizophrenia. By treating healthy controls and schizophrenia model mice with echinacoside, the behavioral phenotypic changes of schizophrenia and healthy control mice before and after administration were analyzed to verify the effect of echinacoside on the phenotypic treatment of schizophrenia.

[0043] The application of echinacoside in the treatment of schizophrenia, wherein the screening of schizophrenia risk genes and the construction of gene-edited mouse models include the following steps:

[0044] a. Collect transcriptome data from brain tissues of patients with schizophrenia and healthy controls;

[0045] b. Gene expression analysis, screening to obtain the differentially expressed gene FAM65B;

[0046] c. Using CRISPR / Cas9 technology, a FAM65B transgenic mouse model was constructed through homologous recombination.

[0047] d. Crossed with Nestin-Cre tool mice to obtain a transgenic mouse model of brain tissue-specific expression of FAM65B;

[0048] e. Conducted neuropsychiatric and cognitive behavioral experiments to evaluate the results and successfully constructed a mouse model of schizophrenia; the neuropsychiatric experiments included: open field test, nesting test, PPI test, etc.

[0049] Learning and cognitive experiments include: Y-maze, water maze, etc.

[0050] The FAM65B protein structure prediction and potential therapeutic drug screening include the following steps:

[0051] a. The protein structure of gene FAM65B was predicted using the Alphafold website;

[0052] c. Preparation of small molecule compound library;

[0053] c. Molecular docking to screen compounds.

[0054] The echinacoside drug treatment and behavioral testing include the following steps:

[0055] a. Breed and prepare control group mice and FAM65B transgenic mice;

[0056] b. Baseline behavioral test;

[0057] c. Intraperitoneal injection of pycnogenol once a day for two weeks;

[0058] d. Behavioral test to evaluate the effect of drug treatment.

[0059] Specifically, the application of pycnogenol in the treatment of schizophrenia has the following test operation steps:

[0060] 1. Schizophrenia risk gene screening

[0061] Collect brain tissue transcriptome data of CMC, LIBD2, GEO and PsychENCODE; by gene differential expression analysis on postmortem brain tissue transcriptome data of schizophrenia and healthy control samples, screen schizophrenia risk genes with consistent differential expression in different data sets; meet P-value < 0.05, of which Log2(fold change) > 0 is marked as up-regulated gene; Log2(fold change) < 0 is marked as down-regulated gene; as shown in (a-f), FAM65B is the only gene that is significantly and consistently up-regulated in four data sets; Figure 1 (a-f) as shown, FAM65B is the only gene that is significantly and consistently up-regulated in four data sets;

[0062] 2. Construction of FAM65B transgenic mouse model

[0063] The FAM65B transgenic mouse model is commissioned by a certain biotechnology Co., Ltd. in Shanghai, which uses CRISPR / Cas9 technology to knock in LSL-FAM65B-IRES-EGFP expression frame at the Gt(ROSA)26Sor gene site by homologous recombination; The process is as follows: a donor vector is constructed by In-Fusion cloning method, which contains 1.087kb 5' homologous arm, CAG promoter, LSL-FAM65B-IRES-EGFP coding region, 4.259kb 3' homologous arm and MC1-DTA-polyA negative selection marker; Cas9 mRNA, gRNA and donor vector are microinjected into fertilized eggs of C57BL / 6J mice to obtain FAM65B transgenic mouse model;

[0064] 3. Genotype identification of mice

[0065] Genotype identification of FAM65B transgenic mouse model, as shown in Table 1, primers are commissioned by a certain company in Shanghai.

[0066] Table 1 PCR reaction system design for mouse genotype identification

[0067]

[0068]

[0069]

[0070] 4. FAM65B overexpression verification

[0071] FAM65B transgenic mice were crossed with Nestin-Cre tool mice to achieve specific overexpression of FAM65B in mouse neural stem cells and their differentiated neurons and glial cells. The cerebral cortex samples were taken from mouse Embryonic day 14.5 (E14.5), E16.5, Postnatal day 0 (P0), P30 and P60 for Western Blot, and the primary antibodies included FAM65B monoclonal antibody (Proteintech Cat# 17015-1-AP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Proteintech Cat# 10494-1-AP). The results are shown in Figure Figure 1 h, which shows that Tg mice highly express FAM65B at five different time periods;

[0072] 5. Mouse behavioral experiment detection

[0073] a. Experimental animals and grouping

[0074] Healthy male C57BL / 6J mice were selected for breeding to 2 months of age; all mice were raised under standard SPF conditions, with free access to food and water; after one week of environmental adaptation, the mice were divided into Ctrl and Tg groups according to genotype; and the body weight of the mice was recorded Figure 2 a);

[0075] b. Open field test

[0076] Open field test box: the device is a white opaque cubic box with a size of 50 cm x 50 cm x 50 cm, and the bottom is evenly divided into 16 equal-area squares (the central 4 squares are the central area, and the peripheral 12 squares are the peripheral area); equipment debugging: turn on the video tracking system, adjust the camera view to ensure that the entire open field area is covered, and calibrate; environmental setting: keep the light, temperature and noise level of the experimental room consistent and suitable;

[0077] Experimental procedure: Gently remove the mouse from its cage and place it in the same corner of the open field box, back facing the same direction; immediately start video recording and the timer; allow the animal to freely explore the box for 5 minutes; at the end of the 5 minutes, immediately stop recording and gently remove the animal from the box and return it to its home cage; after each animal has been tested, clean the floor and walls of the box thoroughly with 15% ethanol to remove any scent cues left by the previous animal and prevent interference with subsequent animals; repeat the above steps until all animals have been tested;

[0078] c. Nest building experiment

[0079] Nest building material: one piece of compressed cotton wool (Nestlet) measuring 5 cm x 5 cm and weighing approximately 2.5 g;

[0080] Experimental procedure: On the day of the experiment, remove all old nest material and food debris from the cage, leaving only the bedding; place one pre-weighed standard piece of compressed cotton wool (Nestlet) flat in a fixed location in the cage; gently return the animal to the cage and assess the final nest building result the next morning;

[0081] The scoring criteria are as follows:

[0082] 1 point: Nestlet not significantly disturbed (more than 90% intact);

[0083] 2 points: Nestlet partially shredded (50-90% intact);

[0084] 3 points: Nestlet mostly shredded but no clear nest site: less than 50% of the Nestlet remains intact and less than 90% of the material is distributed within the first quarter of the cage floor; i.e. the cotton wool is not gathered into a nest but is scattered throughout the cage; sometimes the material can be located within a wider nest area, but the key definition for this level is that 50-90% of the material has been shredded;

[0085] 4 points: identifiable but flat nest: more than 90% of the Nestlet is shredded and the material is gathered into a nest within the first quarter of the cage floor, but the nest is flat and more than 50% of its perimeter does not reach the height of the mouse's body when it is lying on its side;

[0086] 5 points: (almost) perfect nest: more than 90% of the Nestlet is shredded and the nest is in the form of a bowl, more than 50% of its perimeter reaching or exceeding the height of the mouse's body when it is lying on its side;

[0087] d. Y-maze experiment

[0088] Y-maze structure: The Y-maze consists of three identical arms (labelled A, B, and C) with a 120° angle between each arm; each arm is 35 cm long, 5 cm wide, and 15 cm high and is made of white opaque PVC;

[0089] Experimental procedure: Gently remove the mouse from its home cage, facing the center of the maze, and place it in the A arm of the Y maze; immediately start the video recording and timer, and allow the animal to freely explore the maze for 8 minutes; during the experiment, the experimenter should remain quiet and out of the animal's visual field, avoiding any cues or disturbances; at the end of the 8 minutes, immediately stop the recording and gently remove the animal from the maze and return it to its home cage; clean the maze to ensure no odor remains; repeat the above steps until all animals have been tested;

[0090] Spontaneous Alternation Rate (%): is an index of spatial working memory; it is calculated as "the number of sequential combinations of three different arms entered divided by the total number of possible alternations"; a high alternation rate indicates that the animal remembers which arm it has just visited;

[0091] e、PPI experiment

[0092] Test chamber: PPI testing is performed in a soundproof, ventilated, dedicated behavioral test chamber (65 cm long x 35 cm wide x 25 cm high); the chamber is made of transparent organic glass, and the bottom is a piezoelectric sensor that can sense animal movement; a speaker is installed at the top of the chamber to produce precisely controlled white noise stimuli;

[0093] Experimental procedure: Place the test mouse in a plastic holder on the gravimetric platform in the soundproof cabin, and pre-adapt for 5 minutes; maintain a background noise of 62 dB throughout the experiment; in the first stage of testing, the mouse receives 10 120 dB startle pulses (lasting 40 ms) with an average interval of 15 seconds; in the second stage of testing, the mouse receives five different test conditions: only different intensity pre-pulses (74 dB, 78 dB, 86 dB); only 120 dB startle pulses; 74 dB pre-pulse + 120 dB startle pulse; 78 dB pre-pulse + 120 dB startle pulse; 86 dB pre-pulse + 120 dB startle pulse; the pre-pulse (20 ms) is presented 100 ms before the startle pulse (40 ms); each test condition is repeated 10 times in this stage, presented in random order with an average interval of 15 seconds; at the end of the entire test session, remove the animal from the chamber and return it to its home cage; thoroughly clean the inside of the test chamber and wipe it dry to eliminate any odor residues; repeat the above steps until all animals have been tested;

[0094] Primary observation index: Pre-pulse inhibition rate (PPI %) is the most core index; the calculation formula is: PPI % = [1- (average value of startle response in pre-pulse-startle trials / average value of startle response in only startle trials)] x 100%; the higher the PPI value, the stronger the sensory-motor gating function;

[0095] 6. Virtual screening of small molecule compounds targeting FAM65B protein

[0096] a. Protein preparation: The structure of gene FAM65B predicted by Alphafold website was hydrogenated using ProteinPreparation Wizard module, followed by energy optimization (OPLS2005 force field RMSD ); The processed protein was used to generate a grid file using the Receptor Grid Generation module, with the site1 predicted by Schrodinger (key amino acids SER889 / GLN905 / LEU980 / LEU916) as the center, and the box size set to

[0097] b. Compound preparation: The 2D format of the HYL065 Chinese active compound library (containing 2,900 compounds), HYL022P FDA approved drug library (containing 3,400 compounds), HYL001P bioactive compound library (containing 24,300 compounds) and HYL028 central nervous system penetrating compound library (containing 800 compounds) were processed by software LigPrepModule module for hydrogenation, energy optimization, etc., and the output 3D structure was used for virtual screening;

[0098] c. Molecular docking: Virtual Screening Workflow module ( Figure 3 ) was used for virtual screening, the prepared compounds were imported, and Glide module was used for molecular docking, that is, the receptor and ligand molecules were docked with each other through geometric matching and energy matching; The higher the absolute value of the molecular docking score, the stronger the binding force of the compound with FAM65B protein;

[0099] d. HYL065 Traditional Chinese Medicine Active Compound Library: First, the prepared small molecule compounds in the database were screened using the standard (SP) mode in the Glide module, and the top 50% of the small molecule compounds were selected for the second round of screening using the high precision (XP) mode, and the ranking of the small molecule compounds was obtained (Table 2); HYL022P FDA Approved Drug Library: First, the prepared small molecule compounds in the database were screened using the standard (SP) mode in the Glide module, and the top 20% of the small molecule compounds were selected for the second round of screening using the high precision (XP) mode, and the ranking of the small molecule compounds was obtained (Table 3); HYL001P Bioactive Compound Library: First, the prepared small molecule compounds in the database were screened using the high-throughput screening (HTVS) mode in the Glide module, and the top 15% of the small molecule compounds were selected for the second round of screening using the standard (SP) mode; then the top 15% of the scoring values were selected for the third round of screening using the high precision (XP) mode, and the ranking of the small molecule compounds was obtained (Table 3); HYL028 Central Nervous System Penetrating Compound Library: The prepared small molecule compounds in the database were screened using the high precision (XP) mode in the Glide module, and the ranking of the small molecule compounds was obtained (Table 4).

[0100] Table 2 Top 20 small molecule compounds in HYL065 Traditional Chinese Medicine Active Compound Library for molecular docking scoring

[0101] Serial number Article number Name Docking score 1 HY-N0020 Echinacoside -15.51 2 HY-N6838 1,1,1,1-Kestohexaose -15.43 3 HY-N1968 Quercetin-3-O-β-D-glucose-7-O-β-D-gentiobiosiden -14.82 4 HY-N0468 Rebaudioside D -14.11 5 HY-N2531 Notoginsenoside Fc -14.09 6 HY-N0029 Forsythoside B -14.08 7 HY-N6837 Fructo-oligosaccharide DP7 / GF6 -14.08 8 HY-113529 Stachyose(tetrahydrate) -13.67 9 HY-111832 1,2,3,6-Tetragalloylglucose -13.59 10 HY-N2577 1F-Fructofuranosylnystose -13.17 11 HY-N0657 Pinoresinol Diglucoside -13.15 12 HY-N0669 Stevioside -12.97 13 HY-N3520 Deapi-platycodin D3 -12.969 14 HY-N6006 1,3,6-Tri-O-galloyl-beta-D-glucose -12.963 15 HY-N0636 Eriocitrin -12.936 16 HY-N1435 Oroxin B -12.935 17 HY-N2559 Maltohexaose -12.753 18 HY-N0167 Gynostemma Extract -12.676 19 HY-N2109 Macranthoidin A -12.553 20 HY-N0568 Madecassoside -12.521

[0102] Table 3 Top 20 small molecule compounds in HYL022P FDA Approved Drug Library for molecular docking scoring

[0103]

[0104]

[0105] Table 4 Top 20 small molecule compounds in HYL001P Bioactive Compound Library for molecular docking scoring

[0106] Serial number Article number Name Docking score 1 HY-N0020 Echinacoside -15.116 2 HY-129065 Nourseothricin(sulfate) -14.790 3 HY-N6265 Cauloside F -13.931 4 HY-111832 1,2,3,6-Tetragalloylglucose -13.894 5 HY-N0167 Gynostemma Extract -13.588 6 HY-N2033 Chebulinic acid -13.526 7 HY-15176A Pyridostatin(hydrochloride) -13.290 8 HY-N11729 Pyrogallol-phloroglucinol-6,6-bieckol -13.189 9 HY-18660B Ciraparantag(acetate) -12.704 10 HY-139201 Poly-D-lysine hydrobromide(MW 30000-70000) -11.854 11 HY-N0021 Verbascoside -11.781 12 HY-N2392 Kukoamine A -11.759 13 HY-N2155 Tubuloside A -11.698 14 HY-N3518 Mulberroside F -11.480 15 HY-N10592 Herbacetin-3-sophoroside-8--glucoside -11.449 16 HY-103211 L748337 -11.344 17 HY-P1075 CALP3 -11.209 18 HY-129138 Cyanidin 3,5-diglucoside(chloride) -11.089 19 HY-10133 β-Secretase Inhibitor IV -11.087 20 HY-107130 Alirinetide -10.970

[0107] Table 5 Top 20 small molecule compounds in HYL028 Central Nervous System Penetrating Compound Library for molecular docking scoring

[0108]

[0109]

[0110] 7. Behavioral pharmacology experiment to evaluate the treatment effect of acteoside

[0111] The virtual screening results of the small molecule compound targeting the FAM65B protein show that the absolute value of the molecular docking score is the highest for Echinacoside (docking score: -15.51); Echinacoside (ECH) is a phenylethanoid compound derived from the stems of Cistanche, and has a series of biological activities, including neuroprotection, anti-inflammatory and antioxidant effects, and also has significant anti-osteoporosis activity; Echinacoside can form 7 hydrogen bond interactions and 1 pi-pi interaction with the FAM65B protein: the hydroxyl group of the compound can form 6 hydrogen bond interactions with Lys977 / Glu984 / Ser889 / Gln779 / Gln864 of the target protein, the oxygen atom on the sugar ring of the compound can form 1 hydrogen bond interaction with Gln940 of the target protein, and the benzene ring of the compound can also form 1 pi-pi interaction with His894 of the target protein, and the specific distances are see Figure 4 d.

[0112] To confirm whether Echinacoside can interact with FAM65B and improve the schizophreniform phenotype, first, the open field, nesting, Y maze and PPI behavior experiments were performed in 2-month-old Ctrl and Tg mice; the experimental method is described in "5, mouse behavior experiment detection"; then the Ctrl and Tg mice were intraperitoneally injected (20 mg / kg) for two weeks, and the body weight of the Ctrl and Tg mice was measured, and the open field, nesting, Y maze and PPI behavior experiment detection was performed, the experimental method is described in "5, mouse behavior experiment detection"; the experimental results are shown in Figure 5 , wherein the Y maze and PPI experiment results show that the mice completely recover to the same level as the control group mice; the nesting experiment shows that part of the mice are recovered; in the open field experiment, the total movement distance is not recovered, and the immobility time is significantly recovered.

[0113] The present application screens schizophrenia risk genes by analyzing the differential expression of genes in the postmortem brain tissue transcriptome data of schizophrenia patients and healthy people; constructs a gene editing mouse model and completes the construction of a schizophrenia animal model through behavior evaluation; finally, through target gene protein structure prediction, molecular docking and drug treatment verification, the small molecule drug Echinacoside with potential therapeutic effect is successfully screened; the present application finds that the small molecule drug Echinacoside can specifically bind to the schizophrenia risk gene FAM65B, and has significant therapeutic effect on the negative symptoms of schizophrenia, cognitive dysfunction and sensory gating defects.

Claims

1. Application of echinacoside in the treatment of schizophrenia.

2. The application of echinacoside in the treatment of schizophrenia according to claim 1, characterized in that, The experimental procedure for the application of echinacoside in the treatment of schizophrenia includes the following steps: (1) Screening for risk genes for schizophrenia; (2) Differential gene expression analysis was used to screen for the risk gene FAM65B for schizophrenia; (3) Behavioral experiments were conducted to assess whether the mouse model exhibited a schizophrenia-like phenotype; (4) Virtual screening of potential therapeutic drugs for target risk genes; (5) Behavioral pharmacology experiments to evaluate the therapeutic effect of echinacoside.

3. The application of echinacoside in the treatment of schizophrenia according to claim 2, characterized in that, The specific steps of schizophrenia risk gene screening in step (1) are as follows: (a) Collecting transcriptome data from post-mortem brain tissue of schizophrenic and healthy control samples; (b) Differential gene expression analysis revealed the differentially expressed risk gene FAM65B for schizophrenia.

4. The application of echinacoside in the treatment of schizophrenia according to claim 3, characterized in that, In step (b), when P-values ​​< 0.05, where Log2(fold change) > 0, the gene is marked as upregulated; and when Log2(fold change) < 0, the gene is marked as downregulated.

5. The application of echinacoside in the treatment of schizophrenia according to claim 2, characterized in that, The specific steps of step (2) in the gene differential expression analysis to screen for the schizophrenia risk gene FAM65B are as follows: (a) Using CRISPR / Cas9 technology, the target gene is knocked into the Gt(ROSA)26Sor gene site through homologous recombination to construct a FAM65B transgenic mouse model. (b) Design genotype identification primers and use PCR to identify mouse genotypes; (c) Crossed with Nestin-Cre tool mice to obtain a transgenic mouse model of brain tissue-specific expression of FAM65B.

6. The application of echinacoside in the treatment of schizophrenia according to claim 2, characterized in that, The behavioral experiment assessment of whether the mouse model has a schizophrenia-like phenotype in step (3) specifically involves: (a) breeding transgenic mice to 2 months of age; (b) The mice were moved to a behavioral room for one week to acclimatize to the environment; (c) Weigh the mouse; (d) Complete neuropsychiatric experiments; (e) Complete learning and cognition experiments.

7. The application of echinacoside in the treatment of schizophrenia according to claim 6, characterized in that, In step (d), neuropsychiatric experiments include open field experiments, nesting experiments, and PPI experiments; In step (e), the cognitive learning experiments include the Y maze and the water maze.

8. The application of echinacoside in the treatment of schizophrenia according to claim 2, characterized in that, The specific steps in step (4) involving the virtual screening of potential therapeutic drugs for the target risk gene are: (a) Protein preparation: (b) Preparation of small molecule compound library; (c) Molecular docking screening of compounds; (d) Ranking of compounds.

9. The application of echinacoside in the treatment of schizophrenia according to claim 8, characterized in that, In step (a), protein preparation involves hydrogenating the protein structure of the risk gene FAM65B predicted on the Alphafold website using the ProteinPreparation Wizard module, followed by energy optimization. In step (b), the preparation of the small molecule compound library involves: converting the HYL065 Traditional Chinese Medicine Active Compound Library, HYL022P FDA Approved Drug Library Plus, HYL001PB Bioactive Compound Library Plus, and HYL028 CNS Penetrant Compound Library into 2D format via... The LigPrep Module software performs hydrogenation and energy optimization processing, and outputs 3D structures for virtual screening. In step (c), the molecular docking screening of compounds is performed by: using the Virtual Screening Workflow module to perform virtual screening, importing the prepared compounds, and using the Glide module to perform molecular docking, that is, the acceptor and ligand molecules dock with each other through geometric matching and energy matching. In step (d), the compounds are ranked as follows: the higher the absolute value of the molecular docking score, the stronger the protein binding force between the compound and the risk gene.

10. The application of echinacoside in the treatment of schizophrenia according to claim 2, characterized in that, The behavioral pharmacology experiment in step (5) to evaluate the therapeutic effect of echinacoside specifically involves: (a) Treat mice with drugs based on their weight, age, body surface area, and route of administration; (b) Weigh the mouse; (c) Complete neuropsychiatric experiments; (d) Complete learning and cognition experiments.

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