Use of guaiaretic lactone in the preparation of a medicament for treating fragile x syndrome

Abstract

The application belongs to the field of biological medicine, and particularly relates to application of guaiaretic lactone in preparation of a medicine for treating fragile X chromosome syndrome. The application discloses, for the first time, application of guaiaretic lactone in preparation of a medicine for treating fragile X chromosome syndrome, and specifically uses 8-deoxy-11,13-dihydroxyguaiaretic lactone to treat FXS. The 8-deoxy-11,13-dihydroxyguaiaretic lactone is a metabolic product of human body itself, has low toxicity, small side effect, and high safety in treating FXS. Furthermore, the 8-deoxy-11,13-dihydroxyguaiaretic lactone, as a metabolic product of human body itself, can penetrate through a blood-brain barrier to reach a brain treatment site, indicating that the 8-deoxy-11,13-dihydroxyguaiaretic lactone can have excellent advantages in treating nervous system diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of a guaiaretic lactone in preparation of a medicament for treating fragile X syndrome. BACKGROUND

[0002] Fragile X syndrome (FXS, also known as Martin-Bell syndrome) is a neurodevelopmental disorder caused by point mutation, deletion mutation or repeat expansion of CGG trinucleotide in the 5' untranslated region of Fragile X mental retardation 1 (FMR1) located at Xq27.3, which leads to methylation of the adjacent CpG island and low expression or even no expression of FMRP (Fragile X mental retardation protein, FMRP) encoded by FMR1.

[0003] Fragile X syndrome (FXS) is not only a common genetic intellectual and cognitive dysfunction disease, but also the most common monogenic deficiency disease causing autism spectrum disorder. FXS mainly manifests in intellectual disability, social interaction disorder and cognitive disorder, which greatly affects the behavior ability and life quality of patients, and thus effective clinical treatment programs are urgently needed.

[0004] The occurrence of FXS is mainly caused by mutation of Fmr1 on the X chromosome, which leads to loss of function of FMRP (Fragile X mental retardation protein, FMRP). The exact pathogenesis behind the disease has not yet been elucidated.

[0005] Basic research has shown that the loss of FMRP can cause abnormal brain development and various neural cell function defects, including abnormal neurogenesis, dendritic morphogenesis of neurons, circuit integration and axon targeting. However, due to the dynamic and complex interaction combinations between different neurobiological mechanisms that may affect the clinical phenotype of FXS, there is still a lack of effective treatment methods for FXS in the clinic, and most of the treatment methods for FXS are based on its specific symptoms, which also leads to a lack of sufficient control experiments to prove the effectiveness of these methods. Only by combining psychopharmacological intervention with other supportive strategies, including speech therapy, sensory integration occupational therapy, individualized education programs and tailor-made behavior intervention, can the therapeutic effect be maximized.

[0006] Although a series of drug researches targeting metabotropic glutamate receptor 5 (mGluR5), glycogen synthase kinase-3β (GSK-3β) and γ-aminobutyric acid (GABA) receptor have been carried out, the therapeutic effect of the single drug is only obvious in animal experiments, but not obvious in clinical experiments. The research results of single drug treatment show that there is a gap between animal experiments and clinical trials, which indicates that the existing single drug treatment strategy does not meet the needs of clinical treatment. In addition, selective serotonin reuptake inhibitors (SSRIs) are also used as drugs for treating emotional disorders, anxiety and obsessive-compulsive behaviors related to FXS, which have curative effect in alleviating behaviors such as social anxiety, temper tantrums and aggression; in addition, antipsychotic drugs are also used to treat FXS, but the side effects related to antipsychotic drugs such as weight gain, diabetes, nausea, constipation and tardive dyskinesia lead to poor effect. Atypical antipsychotic drugs are also used to treat self-injury, aggressive behavior and autism, although atypical antipsychotic drug aripiprazole has improved mood stability, attention and learning performance, but it should be used at a low dose to avoid restlessness caused by a high dose, and the side effects produced by it cannot be ignored.

[0007] So far, there is still a lack of effective therapeutic drugs that can cure or alleviate the main adverse symptoms of patients with the disease. Therefore, it is urgent to explore the pathogenesis of fragile X syndrome and develop new therapeutic drugs. Research on the abnormally expressed genes and proteins after the deletion of FMRP and investigate the specific effects of abnormal expression of related target points on the structure, morphology and function of neurons can provide positive clinical application value for comprehensive treatment of FXS.

[0008] Gnetolactone and its derivatives have insecticidal, anti-inflammatory, antitumor, anti-hyperuricemia and other activities, but there is no prior art to prove that they can be used for treating fragile X syndrome. SUMMARY

[0009] The primary object of the present application is to provide a new application of gnetolactone or its derivatives in the treatment of genetic intellectual and cognitive dysfunction diseases, especially fragile X syndrome, and to provide a new pharmaceutical use of gnetolactone or its derivatives (especially 8-deoxy-11,13-dihydroxyguaiacolide).

[0010] The first aspect of this invention aims to provide the use of guaiacol lactone or its derivatives in the preparation of products.

[0011] To achieve the above-mentioned objectives of this invention, the technical solution adopted is as follows:

[0012] In a first aspect, the invention provides the use of guaiacol lactone or a derivative thereof in the preparation of products for the treatment and prevention of hereditary intellectual and cognitive impairment diseases.

[0013] The hereditary intellectual and cognitive disorders include at least one of the following: autism spectrum disorders (ASD), X-linked mental retardation (XLMR), global developmental disabilities (GDD), intellectual disabilities (ID), attention deficit hyperactivity disorder (ADHD), and epilepsy.

[0014] Preferably, the X-linked intellectual disability includes at least one of the following: Fragile X syndrome, Lesch-Nyhan syndrome, alpha-thalassemia intellectual disability, X-linked anencephalon syndrome, epilepsy with periventricular translocation, cerebral hypoplasia with hydrocephalus, XLMR with Rett-like syndrome, Nance-Horan syndrome, microcephaly-macrocephaly-facial abnormality syndrome, X-linked Angelman-like syndrome, XLMR-dwarfism-muscular dystrophy, Opitz G / BBB syndrome, Coffin-Lowry syndrome, Renpenning syndrome, Stoccos dos Santos syndrome, Turner syndrome, Goltz syndrome, and MIDAS syndrome.

[0015] X-linked intellectual disability is a type of intellectual disability caused by gene mutations on the X chromosome. Related genes include, but are not limited to, fragile X mental retardation 1 (FMR1), hypoxanthine guaningphosphoribosyl transferase (HPRT), X-linked nuclear protein, X-linked helicase2, Doublecortin (DCX), Filamin1 (FLN1), Aristaless-related X-chromosome gene (ARX), Serine-threonine kinase 9 (STK9), Polyglutamine tract binding protein (PQBP1), K1AA1202 protein, and Midline 1 (MID1).

[0016] More preferably, the X-chromosome-linked intellectual disability is Fragile X syndrome.

[0017] Preferably, the guaiacol lactone or its derivatives include at least one of 8-deoxy-11,13-dihydroxyguaiacol lactone, 12,6-guaiacol lactone, 12,8-guaiacol lactone, and pseudoguaiacol lactone.

[0018] More preferably, the guaiacol lactone or its derivative is 8-deoxy-11,13-dihydroxyguaiacol lactone. The molecular formula of 8-deoxy-11,13-dihydroxyguaiacol lactone (8D11,13d) is C2. 15 H 20 O5, CAS number 83551-03-5. The structural formula is shown in Equation 1:

[0019]

[0020] Preferably, the pharmaceutically acceptable derivative includes a pharmaceutically acceptable salt and a pharmaceutically acceptable chemical modification.

[0021] Preferably, the pharmaceutically acceptable salt includes at least one of the following: metal salt, ammonium salt, salt formed with an inorganic acid, salt formed with an organic base, salt formed with an organic acid, salt formed with a basic amino acid, and salt formed with an acidic amino acid.

[0022] Preferably, the metal salt includes alkali metal salts and alkaline earth metal salts.

[0023] Preferably, the alkali metal salt includes at least one of sodium and potassium salts.

[0024] Preferably, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0025] Preferably, the salt formed with the organic base includes at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0026] Preferably, the salt formed with the inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0027] Preferably, the salt formed with the organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0028] Preferably, the salt formed with the basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0029] Preferably, the salt formed with the acidic amino acid includes a salt formed with at least one of the following acidic amino acids: aspartic acid and glutamic acid.

[0030] Preferably, the pharmaceutically acceptable modifications include at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation. For example, lipophilicity can be enhanced and blood-brain barrier permeability improved by introducing a fluorine atom (fluoroethyl). The appropriate modification method can be selected based on the actual use of the drug.

[0031] Preferably, the product comprises a drug; the drug comprises pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

[0032] Preferably, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, and carriers.

[0033] This invention provides a medicament and corresponding dosage form for treating fragile X syndrome, using guaiacol lactone or its derivatives (especially 8-deoxy-11,13-dihydroxyguaiacol lactone) as the active ingredient.

[0034] The drug is present in the form of an oral preparation, an injection, or a topical preparation.

[0035] In particular, the oral preparations include tablets, capsules, pills, powders, granules, syrups, or solutions; the injectable preparations include injection solutions or lyophilized powder for injection; and the topical preparations include creams, ointments, sprays, aerosols, or patches.

[0036] The pharmaceutical preparation uses guaiacol lactone or its derivatives (especially 8-deoxy-11,13-dihydroxyguaiacol lactone) as the active ingredient and includes other pharmaceutically acceptable carrier components.

[0037] Carriers in pharmaceuticals include excipients such as starch and water; lubricants such as magnesium stearate; disintegrants such as microcrystalline cellulose; fillers such as lactose; binders such as pregelatinized starch and dextrin; sweeteners; antioxidants; preservatives; flavoring agents; and fragrances.

[0038] The carriers used in the preparation of oral formulations can be conventional pharmaceutical excipients such as starch, dextrin, cyclodextrin, various chemically modified cyclodextrins, sucrose, and stearates. Lyophilized powder injections can be prepared using methods such as aseptic spray drying, low-temperature vacuum drying, and freeze-drying. The subsequent preparation processes and equipment for each formulation are all conventional technologies in the pharmaceutical field, and this invention does not limit their application.

[0039] The drugs described in this invention exist in the form of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, and poultices. That is, the drug preparations include, but are not limited to, the forms of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, and poultices.

[0040] Furthermore, to facilitate medication, the active ingredient guaiacol lactone or its derivatives can be processed with one or more pharmaceutically acceptable excipients into a specific dosage form. These excipients can be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose), absorbents (e.g., calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate), wetting agents (e.g., water and ethanol), binders (e.g., hydroxypropyl methylcellulose, povidone, starch paste, and syrup), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone), and lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized powders). The following are examples of agents: silica gel, colorants (such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone), solvents (such as water for injection, ethanol, propylene glycol, and glycerin), acid-base adjusters (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol, and thimerosal), and isotonic adjusters (such as sodium chloride and glucose).

[0041] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical 6Gess, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0042] The guaiacol lactone or its derivatives (especially 8-deoxy-11,13-dihydroxyguaiacol lactone) described in this invention can be used alone or in the form of a pharmaceutical composition containing guaiacol lactone or its derivatives (especially 8-deoxy-11,13-dihydroxyguaiacol lactone) for the treatment of fragile X syndrome.

[0043] Preferably, the dosage form of the product includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0044] Preferably, the gastrointestinal dosage form includes at least one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0045] Preferably, the non-gastrointestinal dosage form includes at least one of the following: injectable dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0046] Preferably, the product is applied to mammals.

[0047] Preferably, the mammal includes humans.

[0048] The beneficial effects of this invention are:

[0049] 1. This invention discovers new medicinal value for guaiac lactone or its derivatives, enabling its use in the treatment of fragile X syndrome, and can be prepared into a drug for treating fragile X syndrome, thus opening up a new application field for the application of secondary metabolites such as guaiac lactone or its derivatives (e.g., 8-deoxy-11,13-dihydroxyguaiac lactone).

[0050] 2. Specifically, this invention uses 8-deoxy-11,13-dihydroxyguaiacolone for in vitro neural stem cell experiments. The results show that 8-deoxy-11,13-dihydroxyguaiacolone significantly promotes the differentiation of adult neural stem cells into neurons in Fmr1 KO mice, while having no effect on the differentiation of adult neural stem cells in WT mice.

[0051] 3. This invention uses 8-deoxy-11,13-dihydroxyguaiacol to conduct in vitro neural stem cell experiments to test the dendritic complexity of newly formed neurons. The results show that the dendritic complexity of newly formed hippocampal neurons from Fmr1 KO mice is lower than that from WT mice, with reduced dendritic length, number of dendritic nodes, and number of dendritic terminals. Intervention with 8-deoxy-11,13-dihydroxyguaiacol can restore neuronal morphology; significantly promote the dendritic complexity of newly formed hippocampal neurons from Fmr1 KO mice in vitro, increasing the dendritic complexity of Fmr1 KO mouse hippocampal neurons; and increase the dendritic length, number of dendritic nodes, and number of dendritic terminals of newly formed hippocampal neurons from Fmr1 KO mice, restoring the dendritic length of newly formed neurons to a similar level to that of newly formed hippocampal neurons from WT mice, as well as a similar number of dendritic nodes.

[0052] 4. This invention employs various animal models and has conducted numerous animal experiments via injection. The results show that 8-deoxy-11,13-dihydroxyguaiacol promotes the maturation of newly formed neurons from adult neural stem cells in Fmr1 KO mice. 8-deoxy-11,13-dihydroxyguaiacol promotes the differentiation of neural stem cells into neurons in the hippocampal dentate gyrus (DG) region induced by FMRP deficiency, alleviates hippocampal-dependent learning impairment, and corrects cognitive dysfunction caused by FMRP deficiency.

[0053] 5. The 8-deoxy-11,13-dihydroxyguaiacol of the present invention has strong pharmacological effects, is significantly effective in treating fragile X syndrome, has a rapid onset of action, few toxic side effects, good safety, can be taken for a long time, and has good pharmaceutical prospects.

[0054] 6. The raw materials of the product of this invention are derived from natural metabolites, which are safe for clinical use. The preparation process is simple, and it can be made into various dosage forms. Moreover, the dosage is small and the product is convenient to use, so it is easy to promote. Attached Figure Description

[0055] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0056] Figure 1 This is a schematic diagram of an experimental protocol for regulating the differentiation of newly generated neurons from neural stem cells in the adult mouse brain using 8-deoxy-11,13-dihydroxyguaiacol.

[0057] Figure 2 Representative micrographs of newly formed mature neurons (NeuN+BrdU+) in the DG region of WT / Fmr1 KO mice; the effect of 8-deoxy-11,13-dihydroxyguaiacol on neuronal differentiation in Fmr1 KO mice was assessed by immunostaining with neuronal markers NeuN (green) and BrdU (red); scale bar: 20 μm.

[0058] Figure 3 This is a graph showing the results of data analysis on newly generated and mature neurons from a neural stem cell differentiation experiment.

[0059] Figure 4 This is a microscopic observation of the in vitro differentiation of adult neural stem cells.

[0060] Figure 5 This is a statistical graph showing the in vitro differentiation of adult neural stem cells.

[0061] Figure 6 Schematic diagram of the experimental protocol for regulating the morphology and function of P0 hippocampal neurons in vitro using 8-deoxy-11,13-dihydroxyguaiacol.

[0062] Figure 7 Microscopic observation of P0-type hippocampal neurons cultured in vitro.

[0063] Figure 8 Sholll analysis statistical plot of dendritic complexity in primary hippocampal neurons.

[0064] Figure 9 Sholl analysis of dendritic length in primary hippocampal neonatal neurons.

[0065] Figure 10 Shollen analysis chart of the number of dendritic nodes in primary hippocampal neonatal neurons.

[0066] Figure 11 Sholl analysis of the number of dendritic terminals of newly formed neurons in the primary hippocampus.

[0067] Figure 12 This diagram illustrates the average firing frequency of the primary hippocampal neonatal neuronal network.

[0068] Figure 13 This is a statistical graph showing the average firing frequency of the primary hippocampal neonatal neuronal network.

[0069] Figure 14 This is a statistical graph showing the duration of continuous firing in the primary hippocampal neonatal neural network.

[0070] Figure 15 This is a schematic diagram of a mouse new location recognition experiment.

[0071] Figure 16 A statistical graph of the discriminant index for the mouse new location recognition experiment.

[0072] Figure 17 This is a schematic diagram of a novel object recognition experiment in mice.

[0073] Figure 18 Statistical graph of the discrimination index for novel object recognition experiments in mice.

[0074] Figure 19 This is a schematic diagram of a mouse social interaction experiment.

[0075] Figure 20 This is a statistical chart of the discriminant index for the social interaction experiment in mice. Detailed Implementation

[0076] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0077] The experimental animals used in the specific embodiments of this invention are as follows:

[0078] Wild-type male (WT, or Fmr1) + / y ) mice, and female Fmr1 gene knockout (KO) mice (Fmr1 KO, or Fmr1 - / - All were purchased from Jackson Laboratory in the United States.

[0079] Fmr1 gene knockout (KO) can be represented by Fmr1 KO or simply Fmr1. - / - (Female mouse) / Fmr1 - / y (Male rat). Wild-type male (WT), which can be represented by WT or Fmr1. + / + (Female mouse) / Fmr1 + / y (Male rat)

[0080] All mice were bred and housed at the Animal Centre of the Institute of Chinese Medical Sciences, University of Macau, in a specific pathogen-free (SPF) animal housing at a temperature of 22-25°C. The mice were kept in a circadian rhythm of 12 hours of light / 12 hours of darkness and had free access to water and food. The process was approved and recognized by the Animal Ethics Committee of the University of Macau.

[0081] As mentioned earlier, fragile X syndrome is mainly caused by mutations in the Fmr1 gene on the X chromosome, leading to the loss of FMRP function. To avoid bias in experimental data and conclusions caused by the influence of the female mouse menstrual cycle and the different expression of the Fmr1 gene on the two X chromosomes on physiological function, this invention only uses adult wild-type and Fmr1 gene-deficient male mice as experimental samples.

[0082] The drugs and reagents used in this invention are shown in Table 1.

[0083] Table 1

[0084]

[0085]

[0086] Example 1: In vivo differentiation experiment of neural stem cells

[0087] 1. Mouse breeding method for neural stem cell differentiation experiments

[0088] Male mice (Fmr1) + / y ) and Fmr1 HET female mice (Fmr1 + / - Mating these mice can produce the following genotypes, labeled as: ①Fmr1 + / y (Male); ②Fmr1 - / y(Male); ③Fmr1 - / + (Female); ④Fmr1 + / + (female).

[0089] Male mice marked ① and ② from the offspring were selected and administered the drug at 7-8 weeks of age for subsequent cell differentiation experiments.

[0090] 2. Grouping, drug administration, and tissue collection for neural stem cell differentiation experiments.

[0091] After being fed for 7-8 weeks, the male mice marked ① and ② were divided into 4 groups of 3 mice each, as follows:

[0092] Group E: Fmr1 + / y +Phosphate buffer group; (WT+Vehicle); F group: Fmr1 - / y +Phosphate buffer group; (KO+Vehicle); Group G: Fmr1 + / y +8-deoxy-11,13-dihydroxyguaiacol group; (WT+8D11,13d); H group: Fmr1 - / y +8-deoxy-11,13-dihydroxyguaiacolone group; (KO+8D11,13d).

[0093] Each group of mice was administered the drug via intraperitoneal injection. The dosage of 8-deoxy-11,13-dihydroxyguaiacol was 20 mg / kg. The volume of phosphate buffer was calculated based on the dosage of 8-deoxy-11,13-dihydroxyguaiacol and the same volume of phosphate buffer was administered. The intraperitoneal injection was administered once a day at the same time for 7 days.

[0094] 8-Deoxy-11,13-dihydroxyguaiacolone and phosphate buffer were administered intraperitoneally at the same time each day for 7 days. On days 3 and 4 of the administration of 8-deoxy-11,13-dihydroxyguaiacolone and phosphate buffer, each mouse was administered BrdU intraperitoneally twice a day for two days at a dose of 200 mg / kg.

[0095] Mice were administered 8-deoxy-11,13-dihydroxyguaiacol for 7 consecutive days, followed by BrdU administration on days 3 and 4. After a four-week wait, mice were anesthetized, their brains were perfused, and the entire hippocampus was serially sectioned at a thickness of 40 μm. One section from every six sections was randomly selected from the hippocampus for subsequent neural stem cell differentiation experiments. The experimental procedure is as follows: Figure 1 .

[0096] The quantification range of neurons in the dentate gyrus region of the hippocampus is defined relative to the anterior fontanelle, with the hippocampus measuring from -0.94 mm to -2.30 mm on the dorsal side and from -2.30 mm to -3.80 mm on the ventral side. Immunohistochemical staining (IHC-Fr) was performed on frozen brain sections from mice in each experimental group, followed by mounting with an anti-fluorescence quencher to obtain stained brain sections. Then, a neurostomographic quantitative analysis method was used. The stained brain sections were placed under an upright microscope, and the Stereo Investigator neurostomography software was opened. Fluorescence channels were set, and the DG region in the stained brain sections was scanned sequentially. After scanning, the NeuN region was determined by the fluorescence staining through different channels. + Positive targets such as BrdU+ were detected in NeuN after the scan. + and BrdU + Cell counting was performed on stained mouse brain slices to quantify positive targets, including green-NeuN. + Red-BrdU + .

[0097] Scanning brain slices under a microscope; newly generated and mature neurons (NeuN) in the DG region of mice. + BrdU + Representative micrographs of ) are as follows Figure 2 As shown, BrdU-labeled cells are proliferating cells, and NeuN-labeled cells are mature neurons; green-NeuN... + Red-BrdU + .

[0098] 3. Results of neural stem cell differentiation experiments

[0099] NeuN is a marker for mature stem cells, while BrdU marks neural stem cells in the division phase, i.e., stem cells that are in a proliferating state. + and BrdU + Double-positive cells represent newly differentiated neural stem cells into mature neurons. NeuN cells were counted using the Stereo Investigator neurovisualization software. + and BrdU + The number of cells. NeuN + and BrdU + Cell count and BrdU + The ratio of the number of cells indicates the proportion of activated cells. The experimental results are as follows:

[0100] Group A: Fmr1 + / y Mature newborn neurons (NeuN) in the +phosphate buffer group + BrdU + / BrdU + The percentage was 77.99%; Group B: Fmr1 - / y Mature newborn neurons (NeuN) in the +phosphate buffer group + BrdU + / BrdU + The percentage was 58.56%; Group C: Fmr1 + / y Mature newborn neurons in +8-deoxy-11,13-dihydroxyguaiacolone (NeuN) + BrdU + / BrdU + The percentage was 79.53%; Group H: Fmr1 - / y Mature newborn neurons in the +8-deoxy-11,13-dihydroxyguaiacol group (NeuN) + BrdU + / BrdU + The proportion was 77.63%.

[0101] Analysis of neural stem cell differentiation experimental data showed that... Figure 3 The data analysis above shows that group B had fewer mature new neurons compared to group A. However, after administration of 8-deoxy-11,13-dihydroxyguaiacol, the proportion of new neurons in group D almost recovered to a level similar to that in group A. This indicates that 8-deoxy-11,13-dihydroxyguaiacol can promote the maturation of new neurons from adult neural stem cells in Fmr1 KO mice.

[0102] Fragile X syndrome model mice exhibit a reduced number of adult neural stem cells differentiating into mature neurons, which in turn affects their cognitive function. Administration of 8-deoxy-11,13-dihydroxyguaiacol can correct this disease phenotype and increase the number of adult neural stem cells differentiating into mature neurons in Fragile X syndrome. Therefore, this indicates that 8-deoxy-11,13-dihydroxyguaiacol has certain therapeutic significance for Fragile X syndrome and can be used to treat it.

[0103] According to NeuN + BrdU + / BrdU + The percentages show that 8-deoxy-11,13-dihydroxyguaiacolone promotes the differentiation and maturation of neurons. Therefore, 8-deoxy-11,13-dihydroxyguaiacolone can promote the differentiation of neural stem cells in the brains of Fmr1 KO mice into neurons and can correct neurodevelopmental disorders in Fmr1 KO mice.

[0104] As mentioned above, FMRP deficiency leads to an abnormally reduced differentiation of neural stem cells in the DG region into neurons and impairs hippocampus-dependent learning. Administration of 8-deoxy-11,13-dihydroxyguaiacol lactone can restore neural stem cell differentiation into neurons to a level similar to normal, promote normal neurogenesis, and reduce the damage caused by FMRP deficiency.

[0105] Example 2: In vitro neural stem cell differentiation experiment

[0106] Adult DG region neural stem cells from wild-type (WT) mice and Fmr1 gene-deficient (Fmr1 KO) mice were cultured in vitro and quantitatively analyzed using immunofluorescence staining combined with neurosurgical quantitative analysis after cell drug administration. The results showed that 8-deoxy-11,13-dihydroxyguaiacol significantly promoted the differentiation of Fmr1 KO neural stem cells into neurons.

[0107] 1. Experimental Materials

[0108] 1.1 Cells: Three male Fmr1 KO mice and three WT mice aged 8-10 weeks were used to isolate the DG region of the mice under a stereomicroscope. Primary adult neural stem cells were then cultured in a cell culture room. The stem cells were stored in an incubator at 37°C and 5% CO2.

[0109] Two types of stem cells were obtained: WT stem cells and Fmr1 KO stem cells. There were three stem cells of each type (n=3), which were labeled as WT1, WT2, WT3 and KO1, KO2, KO3, respectively.

[0110] 2. Experimental Methods

[0111] 2.1 Preliminary preparations:

[0112] 1) Preparation of relevant reagents

[0113] Neural basal medium;

[0114] The culture medium for stem cell culture is: Neural basal medium + B27 + L-Glu + antibiotics. After the cell culture medium is prepared, growth factors EGF and FGF are added before each cell culture medium change.

[0115] B-27 TM Additive (50X), serum-free; concentration used: add 10 mL of B27 to 500 mL of Neurobasal;

[0116] Penicillin / streptomycin bispecific antibody solution (bispecific antibody, Gibco, 100X), concentration: add 8 mL of bispecific antibody to 500 mL of Neurobasal, each mL containing 10,000 units of penicillin (base) and 10,000 μg of streptomycin;

[0117] L-Glutamine (Gibco, 200mM), concentration: Add 8 mL of L-glutamic acid to 500 mL of Neurobasal.

[0118] 2) Experimental grouping and drug administration

[0119] Group A: WT + DMSO, WT-type adult neural stem cells + 0.005% phosphate buffer; Group B: KO + DMSO, Fmr1 KO-type adult neural stem cells + 0.005% phosphate buffer; Group C: WT + 8D11,13d, WT-type adult neural stem cells + 8D11,13d at a final concentration of 0.5μM; Group D: KO + 8D11,13d, i.e., Fmr1 KO-type adult neural stem cells + 8D11,13d at a final concentration of 0.5μM.

[0120] Two types of WT and Fmr1 KO-type adult neural stem cells were subjected to blank and drug treatments, respectively. The blank treatment (blank group) added 0.005% phosphate buffer to the culture medium, i.e., WT + phosphate buffer group (WT-type adult neural stem cells + 0.005% phosphate buffer); KO + DMSO group (Fmr1 KO-type adult neural stem cells + 0.005% phosphate buffer). The drug treatment (drug group) added 0.5 μM 8D11,13d to the culture medium, i.e., WT + 8D11,13d group (WT-type adult neural stem cells + 0.5 μM 8D11,13d); KO + 8D11,13d group (Fmr1 KO-type adult neural stem cells + 0.5 μM 8D11,13d).

[0121] 3) Slide wrapping treatment

[0122] Cell spreaders with a diameter of 14 mm were placed in 24-well plates, and then an appropriate volume (200-300 μL) of poly-l-ornithine was added for coating overnight. After recovering the poly-ornithine, an appropriate amount (200-300 μL) of laminin (0.01068 mg / mL) was added to the 24-well plates containing the cell spreaders, and the coating was repeated until the cells were seeded. The laminin was then recovered. The 0.01068 mg / mL laminin was prepared by diluting 2.67 mg / mL laminin stock solution 250 times with sterile ultrapure water. The recovered poly-l-ornithine and laminin can be reused.

[0123] When performing differentiation experiments on stem cells, duplicate wells are required to ensure the stability and authenticity of the data. Two duplicate wells are used for each stem cell. The specific grouping is shown in Table 2. The concentration of phosphate buffer is 0.005%; the concentration of 8D11 and 13d is 0.5μM.

[0124] Table 2. Stem cell experimental grouping

[0125]

[0126] 24-well plates were prepared using the method described above for differentiation experiments.

[0127] 4) Stem cell digestion and counting processing

[0128] a) Take 8 mL of adult neural stem cell suspension from each of the stem cells numbered WT1, WT2, WT3; KO1, KO2, KO3 respectively from their respective culture dishes, then transfer them to their respective centrifuge tubes (15 mL), centrifuge (3 min, 1200 r) and discard the supernatant, leaving the cell pellet;

[0129] Because adult neural stem cells are suspension cells, when collecting cells, the culture medium containing the cells needs to be collected into a 15mL centrifuge tube, centrifuged at 1200rpm / 3min, then the supernatant is discarded, the cell pellet is retained, and then the operation step 3B) is performed.

[0130] b) Add 1×Trypsin (1 mL) at a concentration of 0.05% to each centrifuge tube and incubate for 1-2 min.

[0131] c) After incubation, add 1 mL of Defined Trypsin Inhibitor to each centrifuge tube, pipette 20-30 times, centrifuge (2 min, 1200 rpm); discard the supernatant and keep the precipitate;

[0132] d) Add stem cell culture medium (neural basal medium + B27 + L-Glu + double antibiotics + EGF + FGF, 1 mL, where the final concentration of EGF and FGF is 10 ng / mL) to each centrifuge tube, mix well by pipetting, and filter the resuspended stem cells through a 40-micron sieve. Spread all 1 mL of stem cell suspension onto the sieve and filter it into a 50 mL centrifuge tube to make the adult stem cells appear as single cells, thus obtaining a single-cell adult stem cell suspension.

[0133] e) Take 6 EP tubes (1.5 mL), add 90 μL of neural basal culture medium to each EP tube, and accurately aspirate 10 μL of the adult stem cell suspension in single-cell state filtered in step 3D) into each corresponding EP tube. After mixing, aspirate 10 μL from each tube and drop them onto a hemocytometer for observation and counting under a microscope.

[0134] The purpose of cell counting is to ensure that the number of cells in each replicate well of each group remains consistent during cell seeding. For WT1 / WT2 / WT3 and KO1 / KO2 / KO3, each type of cell is counted separately, the average value is taken, and then the cells are seeded separately.

[0135] 2.2 Differentiation treatment

[0136] 1) After digestion and counting in the preliminary preparation steps, adult stem cells in a single-cell state are collected at a rate of 2×10⁻⁶. 5 The inoculum was added to a 24-well plate containing coated slides after the cells were seeded. At the time of seeding, the number of stem cells seeded in one well of the 24-well plate was 200,000 (2 × 10⁻⁶). 5 Each well contains 2 mL of culture medium, and the cells are incubated in a cell incubator for 18 hours.

[0137] 2) After culturing for 18 hours, 500 μL of culture medium was removed from each well of the 24-well plate, and 500 μL of fresh culture medium was added. The fresh culture medium contained guaiacol (final concentration 5 μM); retinoic acid (final concentration 1 μM); blank group: 0.005% DMSO; drug group: 8-deoxy-11,13-dihydroxyguaiacol lactone maintained at a final concentration of 0.5 μM. The plates were then incubated in an incubator for 3 days, and the medium was changed and the drugs were administered for three consecutive days.

[0138] 2.3 Cellular Immunofluorescence

[0139] 1) After incubation, remove the 24-well plate from the cell culture room, aspirate 0.5 mL of culture medium from each well, leaving 0.5 mL of culture medium in each well. Add 0.5 mL of 4 wt% paraformaldehyde to each well, incubate at room temperature for 20 min, then aspirate all the liquid from the wells. Add 1 mL of 4 wt% paraformaldehyde to each well, incubate at room temperature for 15 min, then aspirate the liquid from the wells and wash with PBS for 5 min each time, for a total of three times.

[0140] 2) Antibody preparation and incubation

[0141] a) Prepare antibody dilution solution: Add 1×TBS to 0.5g BSA, 2mL Trionx-100 and 2mL goat serum until 40mL is reached. Stir until dissolved and store at -20℃.

[0142] b) Primary antibody: Prepare Tuj1 antibody using the antibody dilution solution prepared in “3. Cell immunofluorescence (a)”. Add the primary antibody Tuj1 at a ratio of 1:3000 to the well plate treated in “3. Cell immunofluorescence (1)”, adding 300 μL to each well and incubating overnight at room temperature. Then wash with PBS for 5 min each time, for a total of three times.

[0143] c) Secondary antibody: Alexa Fluor 568goat anti-rat lgG (H+L). The fluorescent secondary antibody (1:2000) was prepared using the antibody dilution solution prepared in step (b) of “3. Cell Immunofluorescence”. Then, it was added to the well plate at a rate of 300 μL per well and incubated at room temperature for 2 h to obtain the fluorescent secondary antibody.

[0144] d) The DAPI solution can be incubated with the fluorescent secondary antibody for the last 15 minutes, or after the fluorescent secondary antibody incubation time is over, the secondary antibody can be completely aspirated, and then 500 μL of DAPI solution can be added to each well. After incubation with the fluorescent secondary antibody, DAPI (4',6-diamidinyl-2-phenylindole) needs to be incubated. DAPI is a fluorescent dye that binds strongly to DNA and is commonly used in fluorescence microscopy, displaying blue fluorescence.

[0145] e) After incubation with DAPI solution, wash with PBS for 5 min each time, for a total of three times;

[0146] 2.4 Mounting, fixing, microscopic examination, and counting

[0147] Take a regular glass slide (long) and add 15 μL (usually 10-20 μL) of antifluorescence quencher to two different positions on it. Then, remove the cell smears incubated in step 2 from the 24-well plate, with the cell side down, and fix them on the slide. Observe and count the cells under an upright microscope. Set the microscope fluorescence to the red channel and observe and count the cells with red fluorescence. The red fluorescent cells are Tuj1-labeled cells, representing the number of differentiated neural stem cells. Set the microscope fluorescence to the DAPI channel and observe and count the cells with blue fluorescence. The blue fluorescent DAPI-labeled cells are all cells. Calculate the number of Tuj1 and DAPI cells respectively, and calculate the Tuj1 / DAPI ratio.

[0148] Microscopic observation results of neural stem cell differentiation are as follows: Figure 4 In the figure, Tuj1-labeled differentiated cells appear red; DAPI-labeled cells appear blue. In the figure, "—" indicates that only 0.005% DMSO solvent was added and no drug 8D11,13d was added; "+" indicates that 8D11,13d was added and its final concentration was 0.5 μM.

[0149] 2.5 Data Analysis and Results

[0150] The counting analysis was performed using the neuro-somatometry software Stereo Investigator. The average of the three analytical data points for each group was taken. The statistical analysis results are as follows: Figure 5 .

[0151] Group A: WT+Veh group members undergoing adult neural stem cell differentiation (Tuj1) + / Tuj1 + DAPI + The proportion of 13.08% was in Group B: the KO+Veh group, which was in the adult neural stem cell differentiation stage (Tuj1). + / Tuj1 + DAPI + The proportion was 4.44%; Group C: WT+8D11,13d group was in the adult neural stem cell differentiation (Tuj1) stage. + / Tuj1 + DAPI + The proportion was 13.61%; Group D: KO+8D11,13d group was in the adult neural stem cell differentiation stage (Tuj1 + / Tuj1 + DAPI + The percentage was 11.18%.

[0152] Tuj1 + Labeled neurons, Tuj1 + / DAPI +This represents the percentage of neural stem cells that have transformed into neurons. The data above show that 8-deoxy-11,13-dihydroxyguaiacol significantly promotes the differentiation of Fmr1 KO mice into neurons from in vitro adult neural stem cells. Furthermore, it increases the differentiation level of Fmr1 KO neural stem cells to a level similar to that of WT mice, while 8-deoxy-11,13-dihydroxyguaiacol has no effect on the differentiation of WT mice into in vitro adult neural stem cells. Therefore, 8-deoxy-11,13-dihydroxyguaiacol can be used to treat diseases caused by mutations in the Fragile X intellectual disability gene (Fmr1) during the formation of the X chromosome.

[0153] Example 3: Effects of 8-deoxy-11,13-dihydroxyguaiacol on the dendritic complexity and function of newly formed neurons in the hippocampus of Fmr1 KO mice.

[0154] DG region neurons from primary cultured P0 wild-type (WT) mice and Fmr1 gene-deficient (Fmr1 KO) mice were transfected with GFP plasmid and subjected to Sholl analysis after cell drug administration for morphological analysis. Electrophysiological analysis was also performed to assess neuronal function. Results showed that 8-deoxy-11,13-dihydroxygrossheimer significantly promoted dendritic maturation in Fmr1 gene-deficient (Fmr1 KO) newborn neurons, including dendritic length, number of dendritic nodes, and number of dendritic terminals. Furthermore, 8-deoxy-11,13-dihydroxyguaiacol significantly increased the low firing frequency and duration of firing in Fmr1 gene-deficient (Fmr1 KO) newborn neurons.

[0155] 1. Experimental Materials

[0156] 1.1 Primary Culture Methods for Newborn Mouse Neurons

[0157] When male wild-type mice and Fmr1 knockout mice reached maturity at eight weeks of age, female mice with Het(Fmr1) gene knockout were... - / + ) and male rat WT(Fmr1 + / y The mice were bred in a 1:2 ratio. Male P0 mice were selected, and hippocampal tissue was isolated from them under a stereomicroscope. Primary newborn neurons were then cultured in a cell culture chamber and stored in an incubator at 37°C and 5% CO2.

[0158] Two types of neurons were obtained: WT neurons and Fmr1 KO neurons, with three neurons of each type (n=3), labeled as WT1, WT2, WT3; and KO1, KO2, KO3.

[0159] 1.2 Preparation methods of relevant reagents

[0160] 1) Preparation of LB medium: Dissolve 10g NaCl, 10g Tryptone, and 5g Yeast extract in 1L of ultrapure water and autoclave before use.

[0161] 2) Preparation of LB solid medium: Dissolve 10g of tryptone powder, 10g of yeast powder, 10g of NaCl, and 10-20g of agar powder in 1L of ultrapure water and autoclave before use. When the liquid medium cools to 50-60℃, add 1mL of 50mg / mL ampicillin and dissolve it completely. Before the medium solidifies, quickly dispense it into petri dishes (10mL / dish). After cooling and solidification, cover the petri dishes, invert them (to prevent water droplets from falling onto the solid medium), and store them in a refrigerator at 4℃ for later use.

[0162] 3) Preparation of glucose solution: Dissolve 18.0g of glucose in approximately 100mL of ultrapure water. After complete dissolution, dilute to 100mL and filter through a 0.22μm microporous membrane for sterilization before use.

[0163] 4) 5M NaCl: Weigh 292.5g of NaCl, dissolve it in sterile ultrapure water, and dilute to 1L.

[0164] 5) Preparation of SOC (Super Optimal broth with Catabolite repression) medium: Dissolve 20g Tryptone, 5g Yeast extract, 0.5g NaCl, 0.186g KCl, 12g MgSO4, and 0.95g MgCl2 in nearly 1L of ultrapure water, adjust the pH to 7.0, and bring the volume to 1L. Autoclave the solution, and then add 20mL of the sterilized glucose solution prepared in step 3.

[0165] 6) Preparation of ampicillin solution (50mg / mL): Weigh 2.5g of ampicillin and place it in a 50mL plastic centrifuge tube. Add approximately 50mL of sterile ultrapure water and mix thoroughly to dissolve. Make up to 50mL, filter through a 0.22μm filter membrane for sterilization, dispense into small portions (1mL / tube), and store at -20℃.

[0166] 7) 4wt% Paraformaldehyde (PFA): Place 40g of PFA powder in a 1L beaker, add 1L of PBS phosphate buffer, stir with a stirrer, add 6mg of NaOH during stirring until completely dissolved, adjust the pH to 7.0-7.4 with hydrochloric acid, cool, dispense, and store in a -20℃ refrigerator.

[0167] 8) Preparation of poly-L-lysine: Dilute 10× poly-L-lysine with sterile ultrapure water at a ratio of 1:9 to obtain 1× poly-L-lysine. Filter the solution through a 0.22 μM microporous membrane to remove bacteria, and then store it at 4℃. Note that poly-L-lysine can be recycled, but it should be filtered before reuse. If turbidity or bacterial contamination occurs, it should be discarded.

[0168] 9) Neuron-related culture medium

[0169] Culture medium #1, sampled from DMEM-F12 + 1% penicillin and streptomycin;

[0170] Culture medium ②, seed plate medium: DMEM-F12 + 10% FBS + 1% penicillin and streptomycin;

[0171] Culture medium #3, change medium every 4 hours - culture stage culture medium: NeuroGro + 2% B27 + 1% penicillin and streptomycin + 0.5 mM L-Glu;

[0172] Digestive fluid: trypsin-EDTA (0.25%), containing phenol red, for direct use.

[0173] 2. Experimental grouping and drug administration

[0174] Group A: WT+VEH, WT type P0 hippocampal neurons + 0.005% phosphate buffer;

[0175] Group B: KO+VEH, Fmr1 KO-type P0 hippocampal neurons + 0.005% phosphate buffer;

[0176] Group C: WT + 8D11, 13d, WT type P0 hippocampal neurons + 8D11, 13d with a final concentration of 10μM;

[0177] Group D: KO+8D11,13d, Fmr1 KO-type P0 hippocampal neurons + 8D11,13d at a final concentration of 10μM.

[0178] Two types of WT and Fmr1 KO neurons were subjected to blank treatment and drug treatment, respectively. Specifically: the WT+VEH group (blank group, group A) contained WT neurons with 0.005% phosphate buffer added to the culture medium; the KO+VEH group (group B) contained Fmr1 KO neurons with 0.005% phosphate buffer. The drug treatments (drug groups, groups C and D) contained 8D11,13d at a final concentration of 10 μM added to the culture medium; the WT+8D11,13d group contained WT neurons with 8D11,13d at a final concentration of 10 μM; and the KO+8D11,13d group contained Fmr1 KO neurons with 8D11,13d at a final concentration of 10 μM.

[0179] 3. Plasmid transformation

[0180] 1) Place DH5α competent Escherichia coli cells and Rv-GFP plasmid in an ice bath.

[0181] 2) Add 100 μL of DH5α competent cells to a 1.5 mL centrifuge tube; then add plasmid Rv-GFP (1 μL), gently tap the tube to mix the plasmid with the competent cells; then place it in an ice bath for 30 min.

[0182] 3) Place the centrifuge tube in a 42°C water bath for 60-90 seconds, then quickly transfer it to an ice bath for 2-3 minutes. Do not shake the centrifuge tube during this process. Introduce the Rv-GFP plasmid into *E. coli* for large-scale amplification.

[0183] 4) Add 900 μL of SOC medium to another 15 mL centrifuge tube, then add the DH5α competent cells introduced with plasmid in step (3) of plasmid transformation, mix well and shake in a shaker at 37°C (180 rpm, 90 min) to culture the competent cells introduced with plasmid.

[0184] 5) Take 100 μL of the culture mixture from “Step 4)” and add it to LB solid medium containing ampicillin (final concentration 50 μg / mL). Gently spread the cells evenly with a sterile inoculation loop. Then invert the culture dish and incubate at 37°C for 12-16 h. Colonies can be observed.

[0185] 6) Add 200 mL of LB liquid culture medium containing ampicillin (50 μg / mL) to a 250 mL Erlenmeyer flask that has been autoclaved, and set aside.

[0186] 7) Use the autoclaved pipette tip to pick up a single bacterial cell cultured in “3. Plasmid transformation step (5)” and add it to the conical flask in “3. Plasmid transformation step (6)”. Incubate at 37℃ and shake at 200 rpm for 12-16 hours.

[0187] 8) After shaking the bacterial culture, centrifuge (15 min, 4500 rpm, 4℃), discard the supernatant, and keep the bacterial precipitate. Store the precipitate at -40℃ for plasmid extraction.

[0188] 4. Plasmid extraction

[0189] A high-purity plasmid extraction kit was used, and plasmid extraction was performed according to the kit's instructions and experimental methods, as detailed below:

[0190] 1) Add RNase A to solution P1 of the plasmid extraction kit before use, mix well, and store at 2-8℃.

[0191] 2) Add the mixed solution prepared in step 1) of "4. Plasmid extraction" to the centrifuge tube containing the bacterial cell precipitate extracted in "3. Plasmid transformation step 8)", vortex to suspend the bacterial cell precipitate; then add solution P2 from the plasmid extraction kit to the centrifuge tube, mix immediately to fully lyse the bacterial cells, and incubate at room temperature for 5 minutes.

[0192] 3) Add solution P4 from the plasmid extraction kit to the centrifuge tube and mix thoroughly immediately until a white, dispersed flocculent precipitate appears. After standing at room temperature for 4-5 minutes, centrifuge (7800 rpm, 15 minutes) to allow the white precipitate to settle to the bottom of the tube. Then carefully pour all the solution into filter CS1 (be careful to avoid pouring in a large amount of precipitate and clogging the filter), push the push handle to filter, and collect the filtrate in a clean 50 mL centrifuge tube.

[0193] 4) Add 0.35 times the volume of isopropanol and 1 / 2 the volume of isopropanol NaCl to the filtrate, and mix thoroughly by inverting the tube. Centrifuge at 7800 rpm for 90 min at 4℃. Gently discard the supernatant, invert the centrifuge tube onto absorbent paper to dry the liquid, and then add 70% ethanol to the centrifuge tube to rinse the precipitate thoroughly. Centrifuge again at 7800 rpm for 15 min at 4℃, gently discard the supernatant, invert the centrifuge tube onto absorbent paper, and repeat 1-2 times to rinse the precipitate thoroughly.

[0194] 5) Place the centrifuge tubes open at room temperature for 30 minutes to allow the ethanol to evaporate completely. Add DEPC water and vortex to mix. Extract the RV-GFP plasmid.

[0195] The concentration and purity of the RV-GFP plasmid were detected using a DNA micro-volume analyzer. The results are as follows:

[0196] Plasmid concentration: 9489.0 ng / µL, A260 / 280 = 1.97, A260 / A230 = 2.2

[0197] The extracted plasmids should be labeled (date, plasmid type, concentration) and stored in a -20℃ refrigerator for later use.

[0198] 5. Slide wrapping treatment

[0199] 1) Place a 14 mm diameter cell smear into a 24-well plate, add an appropriate volume (200-300 μL) of 1× poly-L-lysine coating solution, and coat the smear for at least 8 hours. Before the experiment, recover the coating solution and wash the smear with sterile ultrapure water 2-3 times. This experiment is used for morphological analysis of primary neurons cultured in vitro.

[0200] 2) Add an appropriate volume (200-300 μL) of 1× poly-L-lysine coating solution to a 24-well microelectrode array cell culture plate and coat it (for more than 8 hours). Before the experiment, recover the coating solution and wash the slabs 2-3 times with sterile ultrapure water. This experiment is used for the functional analysis of primary neurons cultured in vitro.

[0201] 6. Primary neuron culture

[0202] 1) Dispense the neuronal culture medium (No. ①) into culture dishes and EP tubes that are labeled with numbers, and pre-cool them on ice;

[0203] 2) Remove mice born within 0-24 hours (P0 day), disinfect with alcohol, and preserve the tail tissue in a 1.5 mL EP tube; then cut off the mouse head. Male WT and Fmr1 KO mice were divided into 4 groups: WT+VEH, KO+VEH, WT+8D11,13d, and KO+8D11,13d groups, with three mice in each group, i.e., n=3, for a total of 12 mice in four groups. The specific grouping is shown in Table 3. The concentration of DMSO was 0.005%; the concentration of 8D11,13d was 10 μM.

[0204] Table 3. Experimental grouping of neuronal cells

[0205]

[0206]

[0207] 3) After removing the brainstem, the entire brain is divided into two hemispheres, left and right. The hippocampus is located on the ventromedial side of the hemisphere. The entire procedure is performed under a stereoscope. The meninges are dissected using micro-forceps, and then the hippocampi on both sides are dissected. The brain is quickly placed into culture medium ①, the hippocampus is separated, and the hippocampi on both sides are transferred to the corresponding numbered EP tubes. In the cell manipulation room, the hippocampus is cut into small segments of 1-2 mm. 3 Small pieces were collected into 15 mL centrifuge tubes, 300 μL of digestion solution (GIBCO, 25200056) was added, and then incubated at 37 °C in a 5% CO2 incubator for 5 min.

[0208] 4) After incubation, centrifuge (1500 rpm for 1 min) to remove the supernatant; then add 2 mL of culture medium (No. ②) to each 15 mL centrifuge tube, gently pipette until no tissue clumps remain, then pass the cell suspension through a 40 μm sieve and collect the cell suspension in a new 15 mL centrifuge tube, ensuring the cells are in a single-cell state. Continue to pipette evenly and seed at a rate of (3-5) × 10⁻⁶ cells per well. 5The cells were seeded into 24-well plates or 24-well microelectrode array cell culture plates containing coated cell spreaders. The culture medium volume in each well was 1 mL, and the cells were cultured in a cell incubator for 4 hours.

[0209] 5) For neuronal morphology analysis experiments: 4 hours after seeding, the culture medium in each well of the 24-well plate was replaced with medium ③. The day of seeding was designated as day 0. Thereafter, half the medium was replaced every other day (i.e., half the medium ③ was replaced). Transfection and drug administration according to the groupings in Table 3 were performed on day 4 after seeding. Fixation was performed on day 7 after seeding. The specific experimental procedure is as follows: Figure 6 As shown.

[0210] 6) For neuronal function analysis experiments: 4 hours after seeding, the culture medium in each well of the 24-well plate was replaced with medium ③. The day of seeding was designated as day 0. Thereafter, half the medium was replaced every other day (i.e., half the medium ③ was replaced). On day 4 after seeding, drug treatment was administered according to the groupings in Table 3. On day 13 after seeding, the firing frequency and network burst frequency of neurons were detected using the MEA neurophysiological monitoring system. The specific experimental procedure is as follows: Figure 6 As shown.

[0211] 7. Cell transfection and drug administration

[0212] 1) Preparation:

[0213] Materials required for the high-pressure sterilization experiment were sterilized by ultraviolet irradiation in a laminar flow hood to ensure aseptic operation; culture medium No. ③ was placed at room temperature for later use.

[0214] Preparation of transfection reagent (the amount of reagent to be prepared for one well in a 24-well plate): Sterile ultrapure water: 17.5 μL; RV-GFP plasmid obtained in step "4, plasmid extraction": 1 μg; 2M CaCl2: 2.5 μL; 2×HEBS: 25 μL.

[0215] 2) On the 4th day after inoculation, 1 hour before transfection, replace the culture medium in the well plate with new culture medium No. 3.

[0216] 3) Add transfection reagent to 12 EP tubes (1.5 mL each), i.e., add plasmid (1 μg), water (17.5 μL), and 2×HEBS (25 μL) to each EP tube in sequence, mix well, then add CaCl2 solution (2.5 μL), mix well, and let stand at room temperature for 10 min to obtain DNA-CaCl2-HBS mixed system.

[0217] 4) Add the DNA-CaCl2-HBS mixture to each well of the 24-well plate after seeding, and then incubate the 24-well plate in a cell incubator for 1 hour.

[0218] 5) Place the 24-well plate in a clean bench and rinse the cells twice with PBS buffer (at room temperature) (to remove the precipitate and reduce cell death).

[0219] 6) Replace medium ③ according to the groups. For groups A and B: add 1 mL of medium ③ + 0.005% DMSO to each well; for the drug treatment groups (groups C and D): add 1 mL of medium ③ + puerarin (final concentration 10 μM) to each well.

[0220] 7) Then, the 24-well plate is placed back into the cell incubator for culture and growth, i.e., neuron culture.

[0221] 8. Fixation, mounting, and dosing of neurons

[0222] 1) After the neurons have been cultured for seven days, the 24-well plate was removed from the cell culture room. 0.5 mL of culture medium was aspirated from each well, and 0.5 mL of 4 wt% paraformaldehyde was added to each well. The plate was then placed at room temperature and fixed for 20 min. All the liquid in the well was aspirated, and 1 mL of 4 wt% paraformaldehyde was added to each well. The plate was then placed at room temperature and fixed for 15 min. The liquid in the well was then aspirated, and the plate was washed three times with PBS for 5 min each time.

[0223] 2) Take a regular glass slide (long) and drop 15 μL (usually 10-20 μL) of anti-fluorescence quencher at two different positions on it. Then, take the cell smear with neurons attached, which was fixed and washed in step 7-1), out of the 24-well plate, with the cell side facing down, and fix it on the glass slide. Then observe it under an upright microscope and measure the tracing (real-time tracing). Among them, the microscope fluorescence is adjusted to the green channel, and neurons expressing green fluorescence are randomly selected to measure the dendrite length, dendrite nodes, dendrite ends, etc.

[0224] GFP was observed under an upright microscope. + Neuronal cell morphological changes ( Figure 7 The effect of PR on dendritic complexity of newly formed hippocampal neurons in WT or Fmr1 KO mice was analyzed using a stereochemical analysis system on GFP. + The newly generated neurons were depicted in real time, and their morphological structure was quantified using a neuromorphological analysis system, specifically for GFP. + Dendritic length of newly formed neurons ( Figure 9 ), number of dendritic nodes ( Figure 10 ), number of dendritic ends ( Figure 11 Quantification of ).

[0225] 9. Data Results and Analysis

[0226] Two-way ANOVA was performed using GraphPad Prism software, or multivariate ANOVA was performed using SPSS statistical analysis software. Threshold analysis was conducted, and the data were expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0227] 1) Effects of 8-deoxy-11,13-dihydroxyguaiacol on the morphology of newly formed hippocampal neurons in WT and Fmr1 KO mice

[0228] Microscopic observation results of the morphology of newly formed neurons are as follows: Figure 7 ,Depend on Figures 8-11 It can be seen that, compared with WT+VEH, the dendritic complexity of neurons in KO+VEH is lower, the dendritic length is shorter, and the dendritic branches are fewer. After administration of 8D11 and 13d, compared with KO+VEH, the dendritic complexity of neurons in KO+8D11 and 13d is increased, the dendritic length is longer, and the number of dendritic branches is increased. Compared with WT+VEH, the morphology of neurons in KO+8D11 and 13d can be restored to a level similar to that of newly formed hippocampal neurons in WT mice.

[0229] In in vitro cultured neurons, neurons were transfected using calcium phosphate transfection, and 8-deoxy-11,13-dihydroxyguaiacol was added during the culture process for drug intervention. Morphological analysis of neurons expressing green GFP fluorescence using neurosomatic analysis revealed significant morphological differences between Fmr1 KO mice and WT mice in hippocampal neonatal neurons. Specifically, the dendritic network complexity of Fmr1 KO neurons was lower than that of WT neurons. After 8-deoxy-11,13-dihydroxyguaiacol administration, the morphology of Fmr1 KO hippocampal neonatal neurons could be restored to a level similar to that of WT mouse hippocampal neonatal neurons. Figure 7 ).

[0230] Experimental results showed that 8-deoxy-11,13-dihydroxyguaiacolone improved the dendritic morphology of newly formed hippocampal neurons in Fmr1 KO mice: 8-deoxy-11,13-dihydroxyguaiacolone significantly promoted the dendritic complexity of newly formed hippocampal neurons in Fmr1 KO mice in vitro, including dendritic length, number of dendritic nodes, and number of dendritic terminals.

[0231] 2) Effects of 8-deoxy-11,13-dihydroxyguaiacol on the complexity of newborn hippocampal neurons in WT and Fmr1 KO mice

[0232] The complexity of newly formed hippocampal neurons in WT or Fmr1 KO mice was examined using Sholl analysis. The statistical analysis results of the Sholl analysis on the complexity of newly formed hippocampal neurons are as follows: Figure 8 .

[0233] Sholl analysis draws a series of concentric circles centered on the neuron cell body (excluding the cell body) to obtain the number of intersections of neuronal dendrites varying with distance from the cell body, thus reflecting the complexity of the neuron. By calculating the number of branches intersecting each circle, the branching patterns of neuronal dendrites and axons in different regions are obtained, thereby quantitatively characterizing the morphological features of the imaged neuron. The analysis results are as follows: Figure 8 .

[0234] The mean minimum and maximum values ​​of the number of intersection points from the cell body at equal intervals for WT+VEH, KO+VEH, WT+8D11,13d, and KO+8D11,13d were [2.7, 7.5], [1.59, 4.53], [3.23, 7.78], and [2.56, 7.48], respectively. The experimental results indicate that the newly formed neurons in Fmr1 KO mice have lower complexity than those in WT mice, and 8-deoxy-11,13-dihydroxyguaiacol can significantly increase the dendritic complexity of newly formed hippocampal neurons in Fmr1 KO mice.

[0235] a) Effects of 8-deoxy-11,13-dihydroxyguaiacol on dendritic length of hippocampal neoneurons in WT and Fmr1 KO mice

[0236] The dendritic length of newly formed hippocampal neurons was determined using the Sholl analysis method. The statistical analysis results of the Sholl analysis method for determining the dendritic length of newly formed hippocampal neurons are as follows: Figure 9 .

[0237] Depend on Figure 9 The average dendritic lengths of the WT+VEH, KO+VEH, WT+8D11,13d, and KO+8D11,13d groups were 2994.35 μm, 1101.84 μm, 3528.39 μm, and 3604.87 μm, respectively. The experimental results indicate that the dendritic length of newborn neurons in Fmr1 KO mice was shorter than that in WT mice, while 8-deoxy-11,13-dihydroxyguaiacol significantly increased the dendritic length of newborn neurons in Fmr1 KO mice. Compared to WT+VEH, the dendritic length of newborn neurons in Fmr1 KO mice treated with 8-deoxy-11,13-dihydroxyguaiacol significantly recovered to the level of newborn hippocampal neurons in WT mice.

[0238] b) Effects of 8-deoxy-11,13-dihydroxyguaiacol on the number of dendritic nodes in neonatal neurons of WT and Fmr1 KO mice

[0239] The Sholll analysis method was used to analyze the dendritic nodes of newly formed hippocampal neurons. The statistical analysis results of the Sholll analysis method for identifying the number of dendritic nodes in newly formed hippocampal neurons are as follows: Figure 10 .

[0240] Depend on Figure 10 The results showed that the average number of dendritic nodes in the WT+VEH, KO+VEH, WT+8D11,13d, and KO+8D11,13d groups were 17.43, 6.17, 19.36, and 20.74, respectively. These results indicate that Fmr1 KO mice had fewer dendritic nodes in their newborn neurons than WT mice. Treatment with 8-deoxy-11,13-dihydroxyguaiacol significantly increased the number of dendritic nodes in the newborn neurons of Fmr1 KO mice. Compared to WT+VEH, the number of dendritic nodes in the hippocampal newborn neurons of Fmr1 KO mice treated with 8-deoxy-11,13-dihydroxyguaiacol significantly increased the number of dendritic nodes in the hippocampal newborn neurons of WT mice.

[0241] c) Effect of 8-deoxy-11,13-dihydroxyguaiacol on the number of dendritic terminals in neonatal neurons of WT and Fmr1 KO mice

[0242] The Sholll analysis method was used to identify the dendritic terminals of newly formed hippocampal neurons. The statistical analysis results of the Sholll analysis method for identifying the number of dendritic terminals of newly formed hippocampal neurons are as follows: Figure 11 .

[0243] Depend on Figure 11 The results showed that the average number of dendritic terminals in the WT+VEH, KO+VEH, WT+8D11,13d, and KO+8D11,13d groups were 22.66, 9.94, 24.75, and 25.48, respectively. These results indicate that Fmr1 KO mice had fewer dendritic terminals in their newborn neurons compared to WT mice. Treatment with 8-deoxy-11,13-dihydroxyguaiacol significantly increased the number of dendritic terminals in the newborn neurons of Fmr1 KO mice. Compared to WT+VEH, the number of dendritic terminals in Fmr1 KO mice treated with 8-deoxy-11,13-dihydroxyguaiacol recovered to the level of newborn hippocampal neurons in WT mice.

[0244] d) Using the MEA neurophysiological monitoring system, we cultured primary cultured hippocampal neonatal neurons on MEA chips and explored the differences in their spontaneous neural network formation.

[0245] Depend on Figure 12 , 13 It can be seen that the average firing frequencies of the neural networks in the WT+VEH, KO+VEH, WT+8D11,13d, and KO+8D11,13d groups are 0.54, 0.19, 0.47, and 0.48, respectively. Figure 14 The mean firing duration of the neural networks in the WT+VEH, KO+VEH, WT+8D11,13d, and KO+8D11,13d groups was 0.12, 0.07, 0.11, and 0.11, respectively. The results indicate that the mean firing frequency and duration of the newly formed neural networks in Fmr1 KO mice were lower than those in WT mice. Treatment with 8-deoxy-11,13-dihydroxyguaiacol significantly increased the mean firing frequency and duration of the newly formed neural networks in Fmr1 KO mice. Compared to WT+VEH, the mean firing frequency and duration of the newly formed neural networks in Fmr1 KO mice treated with 8-deoxy-11,13-dihydroxyguaiacol significantly improved to levels similar to those in the hippocampal newly formed neurons of WT mice.

[0246] Example 4: Cognitive Function Modulation Experiment

[0247] Animal cognitive function testing methods demonstrated that 8-deoxy-11,13-dihydroxyguaiacolone can correct cognitive and social interaction impairments in Fmr1 gene-deficient mice (Fmr1 KO). To further investigate whether the ameliorative effect of 8-deoxy-11,13-dihydroxyguaiacolone on cognitive impairment functions by promoting adult neurogenesis, this invention used the neurogenesis-specific blocker temozolomide (TMZ) for intervention. Results showed that TMZ could block the therapeutic effect of 8-deoxy-11,13-dihydroxyguaiacolone on cognitive impairment in Fmr1 gene-deficient mice (Fmr1 KO). These results indicate that 8-deoxy-11,13-dihydroxyguaiacolone can treat cognitive impairment in Fragile X syndrome mice by regulating neurogenesis.

[0248] 1. New Location Recognition Experiment

[0249] The novel location recognition test is a classic behavioral assay used to assess spatial memory in mice. To investigate whether 8-deoxy-11,13-dihydroxyguaiacolone could correct spatial memory in a fragile X syndrome model mouse, mice were administered 8-deoxy-11,13-dihydroxyguaiacolone via intraperitoneal injection, followed by a novel location recognition test. To further investigate whether the improvement in spatial memory by 8-deoxy-11,13-dihydroxyguaiacolone functions by promoting adult neurogenesis, neurogenesis was preemptively blocked using the neurogenesis-specific blocker temozolomide (TMZ) in combination with 8-deoxy-11,13-dihydroxyguaiacolone, and then the novel location recognition test was performed.

[0250] 1.1 Materials:

[0251] A square open space: object A, object B, tissue paper, 75% alcohol, two timers.

[0252] 1.2 Laboratory animals and drugs:

[0253] 7-8 week old Fmr1 KO and WT mice, 6 week old Fmr1 KO and WT mice.

[0254] 8-Deoxy-11,13-dihydroxygrosheimin (8D11,13d), purity ≥99%, purchased from Cabotsens: XD170631;

[0255] Temozolomide (TMZ), purchased from Selleck, CAS No. 85622-93-1.

[0256] 1.2 Experimental grouping and drug administration

[0257] 1.2.1 Experimental Groups: WT+Vehicle: WT mice were injected intraperitoneally with phosphate-buffered saline (PBPS); KO+Vehicle: Fmr1KO mice were injected intraperitoneally with PBPS; WT+8D11,13d: WT mice were injected intraperitoneally with 8-deoxy-11,13-dihydroxyguaiacol; KO+8D11,13d: Fmr1 KO mice were injected intraperitoneally with 8-deoxy-11,13-dihydroxyguaiacol; WT+8D11,13d / TMZ: WT mice were injected intraperitoneally with temozolomide and 8-deoxy-11,13-dihydroxyguaiacol; KO+8D11,13d / TMZ: Fmr1 KO mice were injected intraperitoneally with temozolomide and 8-deoxy-11,13-dihydroxyguaiacol. A total of 6 groups were formed, with 8 mice in each group (n=8).

[0258] 1.2.2 Administration method:

[0259] ①: WT+Vehicle, KO+Vehicle group: 7-8 week old Fmr1 KO and WT mice were injected intraperitoneally with physiological saline once a day for 7 consecutive days.

[0260] ②: WT+8D11,13d,KO+8D11,13d group: 7-8 week old Fmr1 KO and WT mice were injected intraperitoneally once a day for 7 consecutive days with 8-deoxy-11,13-dihydroxyguaiacol. The dosage was 20 mg / kg.

[0261] ③: WT+8D11,13d / TMZ, KO+8D11,13d / TMZ group: 6-week-old Fmr1 KO and WT mice were intraperitoneally injected once daily for the first three days of weeks 6, 7, 8 and 9, and once daily for week 8 for 8-deoxy-11,13-dihydroxyguaiacolone (8D11,13d) for 7 consecutive days. The dosage for 8D11,13d was 20 mg / kg.

[0262] 1.3 Experimental Procedure

[0263] This test measures the spatial memory of rodents by assessing their ability to recognize new locations of familiar objects based on spatial cues.

[0264] First, the experimental animals were taken to the behavioral testing laboratory and allowed at least one hour to acclimatize. During the training phase, each animal was placed individually in a square area, with two identical objects placed at the same distance from a wall covered with colored wallpaper. The mice were allowed to explore freely for 6 minutes, and this training process was repeated three times. During the testing phase, the location of one of the familiar objects was moved to a new location. The time spent exploring the objects in the two different locations was recorded within 6 minutes. A normal animal should spend more time exploring the object in the new location. The discrimination criterion was calculated based on the difference between the percentage of time spent recognizing the object in the new location and the time spent recognizing the object in the original location. During the testing phase, object detection was defined as any exploratory behavior within 1 cm of the object's nose, including head orientation, climbing, and sniffing. The discrimination index was calculated as follows: Discrimination Index = (Time to recognize the object in the new location / Total recognition time × 100) - (Time to recognize the object in the original location / Total recognition time × 100).

[0265] A schematic diagram of the new location recognition experiment is shown below. Figure 15 After each mouse test, the testing area needs to be wiped clean with 75% alcohol to eliminate the influence of similar odors on the behavior of the next mouse in the test.

[0266] 1.4 Experimental Results

[0267] Data were analyzed using GrapaPad Prism software in a two-way ANOVA. The results are expressed as Mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0268] The discriminant indices for the WT+Vehicle, KO+Vehicle, WT+8D11,13d, KO+8D11,13d, WT+8D11,13d / TMZ, and KO+8D11,13d / TMZ groups were 38.60%, -29.77%, 20.70%, 19.40%, -20.50%, and -22.30%, respectively. The statistical results of the mouse novel location recognition experiment are as follows: Figure 16 As shown.

[0269] The experimental results show that WT mice are more inclined to explore new objects, while Fmr1 KO mice are more inclined to explore familiar objects. This indicates that Fmr1 KO mice have a defective spatial memory. Administration of 8-deoxy-11,13-dihydroxyguaiacol can correct this defect, while TMZ can block the effect of 8-deoxy-11,13-dihydroxyguaiacol on improving spatial memory in Fmr1 KO mice. This suggests that 8-deoxy-11,13-dihydroxyguaiacol can improve spatial memory in Fmr1 KO mice by regulating neurogenesis.

[0270] 2. New Object Recognition Experiment

[0271] This test is based on the natural tendency of rodents to explore novel objects rather than familiar ones. This experiment tested the novel object recognition ability of mice to explore whether 8-deoxy-11,13-dihydroxyguaiacol lactone could correct learning and memory impairments in a fragile X syndrome model mouse by regulating adult neurogenesis.

[0272] 2.1 Experimental Materials

[0273] 2.1.1 Materials: A square open field; object A; object B; tissue paper; 75% alcohol; two timers.

[0274] 2.1.2 Laboratory animals and drugs: Same as those used in “1. New location recognition experiment”.

[0275] 2.2 Experimental grouping and drug administration

[0276] 2.2.1 Experimental Groups: WT+Vehicle: WT mice were injected intraperitoneally with phosphate-buffered saline (PBPS); KO+Vehicle: Fmr1KO mice were injected intraperitoneally with PBPS; WT+8D11,13d: WT mice were injected intraperitoneally with 8-deoxy-11,13-dihydroxyguaiacol; KO+8D11,13d: Fmr1 KO mice were injected intraperitoneally with 8-deoxy-11,13-dihydroxyguaiacol; WT+8D11,13d / TMZ: WT mice were injected intraperitoneally with temozolomide and 8-deoxy-11,13-dihydroxyguaiacol; KO+8D11,13d / TMZ: Fmr1 KO mice were injected intraperitoneally with temozolomide and 8-deoxy-11,13-dihydroxyguaiacol, with 8 mice in each group (n=8).

[0277] 2.2.2 Administration method: Same as the drug administration method in "1. New location recognition experiment".

[0278] 2.3 Experimental Procedure

[0279] This test measures rodents' ability to recognize new objects by leveraging their tendency to explore unfamiliar objects rather than familiar ones. New object recognition is a form of memory task that does not rely on spatial cues. In this task, animals are trained to recognize specific objects.

[0280] like Figure 17 First, the experimental animals were taken to the behavioral testing laboratory and allowed at least one hour to acclimatize. During the training phase, each animal was placed individually in an L-shaped area with an equal-length corridor. Two identical objects were placed at opposite ends of the corridor, and the animal was allowed to explore them for 10 minutes. During the testing phase, one of the familiar objects was replaced with a new object, and the time the animal spent exploring the two different objects was recorded within 10 minutes. A normal animal should spend more time exploring unfamiliar objects. The discrimination criterion was calculated based on the difference between the percentage of time spent recognizing unfamiliar objects and the time spent recognizing familiar objects. During the testing phase, object detection was defined as any exploratory behavior within 1 cm of the object's nose, including head orientation, climbing, and sniffing. The discrimination index was calculated as follows: Discrimination Index = (Unfamiliar Object Recognition Time / Total Recognition Time × 100) - (Familiar Object Recognition Time / Total Recognition Time × 100).

[0281] After each mouse test, the scene was cleaned with purified water to avoid affecting the results of subsequent mouse experiments.

[0282] 2.4 Experimental Results

[0283] Data were analyzed using GrapaPad Prism software in a two-way ANOVA. The results are expressed as Mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0284] The results of the mouse novel object recognition experiment are statistically presented as follows: Figure 18 As shown, the discriminant indices for the WT+Vehicle, KO+Vehicle, WT+8D11,13d, KO+8D11,13d, WT+8D11,13d / TMZ, and KO+8D11,13d / TMZ groups were 25.50%, -23.9%, 16.57%, 20.38%, -9.53%, and -26.78%, respectively.

[0285] Experimental results showed that WT mice were more inclined to explore new objects, while Fmr1 KO mice exhibited the opposite behavior, indicating a deficiency in learning and memory in Fmr1 KO mice. This deficiency could be corrected by administration of 8-deoxy-11,13-dihydroxyguaiacol. TMZ, however, blocked the learning and memory-improving effect of 8-deoxy-11,13-dihydroxyguaiacol in Fmr1 KO mice.

[0286] The above experiments show that 8-deoxy-11,13-dihydroxyguaiacol can improve memory in mice with fragile X syndrome by regulating adult neurogenesis.

[0287] 3. Social Interaction Experiment

[0288] Social interaction impairment is one of the most prominent manifestations of autism. This study used a three-box interaction social behavior test to investigate whether 8-deoxy-11,13-dihydroxyguaiacol lactone could correct social behavioral deficits in a fragile X syndrome model mouse by regulating neurogenesis.

[0289] 3.1 Experimental Materials

[0290] 3.1.1 Materials: Rectangular operating box, metal cage; two mice; facial tissues; 75% alcohol; two timers.

[0291] 3.1.2 Laboratory animals and drugs: Same as those used in “1. New location recognition experiment”.

[0292] 3.2 Experimental grouping and drug administration

[0293] 3.2.1 Experimental Groups: WT+Vehicle: WT mice were injected intraperitoneally with phosphate-buffered saline (PBPS); KO+Vehicle: Fmr1KO mice were injected intraperitoneally with PBPS; WT+8D11,13d: WT mice were injected intraperitoneally with 8-deoxy-11,13-dihydroxyguaiacol; KO+8D11,13d: Fmr1 KO mice were injected intraperitoneally with 8-deoxy-11,13-dihydroxyguaiacol; WT+8D11,13d / TMZ: WT mice were injected intraperitoneally with temozolomide and 8-deoxy-11,13-dihydroxyguaiacol; KO+8D11,13d / TMZ: Fmr1 KO mice were injected intraperitoneally with temozolomide and 8-deoxy-11,13-dihydroxyguaiacol, with 8 mice in each group (n=8).

[0294] 3.2.2 Administration method: Same as the drug administration method in "1. New location recognition experiment".

[0295] 3 Experimental Steps

[0296] This test utilizes the natural social behavior and exploratory tendencies of rodents to detect changes in their social behavior. The social behavior testing device is a three-chamber box, with partitions made of black plexiglass and small round doors with a diameter of 7cm inside, allowing the animals to move freely within the three chambers.

[0297] like Figure 19 First, the experimental animals were taken to the behavioral testing laboratory and allowed at least one hour to acclimatize. The experiment consisted of three phases: habituation, social interaction test, and social novelty recognition. In the testing phase, the animals were initially placed in the middle chamber and allowed to freely explore the left, middle, and right chambers for 10 minutes. Next, an unfamiliar animal was placed in a wire cage in the left chamber, and a toy was placed in a wire cage in the right chamber. In the social interaction test, the time spent exploring the toy and the unfamiliar animal was recorded over 10 minutes. In the social novelty recognition test, the previously interacted animal remained unchanged (left chamber), and the toy in the right chamber was replaced with a new unfamiliar animal (right chamber). The time spent exploring the familiar animal (left chamber, from the previous social interaction phase) and the unfamiliar animal (right chamber, the newly replaced animal) was recorded over 10 minutes. The discrimination criterion was calculated based on the difference between the percentage of time spent recognizing the unfamiliar animal and the time spent recognizing the toy (or the familiar animal). During the testing phase, detection of an object or another animal was defined as any exploratory behavior by an animal within 1 cm of the object's wire cage or another animal's wire cage, including head orientation, climbing, sniffing, etc. The discrimination index was calculated as follows: Discrimination Index = (Unfamiliar animal recognition time / Total recognition time × 100%) - (Toy or familiar animal recognition time / Total recognition time × 100%).

[0298] After each mouse test, the scene was cleaned with purified water to avoid affecting the results of subsequent mouse experiments.

[0299] 3.4 Experimental Results

[0300] Data were analyzed using GrapaPad Prism software in a two-way ANOVA. The results are expressed as Mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0301] The results of the mouse social interaction experiment are statistically analyzed as follows: Figure 20 As shown, the discriminant indices for the WT+Vehicle, KO+Vehicle, WT+8D11,13d, KO+8D11,13d, WT+8D11,13d / TMZ, and KO+8D11,13d / TMZ groups were 29.15%, -16.06%, 38.71%, 33.80%, -15.55%, and -17.85%, respectively.

[0302] Experimental results showed that WT mice were more inclined to interact with new mice, while Fmr1 KO mice were more inclined to interact with older mice, indicating a deficiency in social skills in Fmr1 KO mice. This deficiency was corrected by administration of 8-deoxy-11,13-dihydroxyguaiacol. TMZ blocked the effect of 8-deoxy-11,13-dihydroxyguaiacol on improving social interaction in Fmr1 KO mice. Therefore, it can be concluded that 8-deoxy-11,13-dihydroxyguaiacol can correct social interaction impairment in a fragile X syndrome model mouse by regulating neurogenesis.

[0303] The above experiments show that 8-deoxy-11,13-dihydroxyguaiacol can correct social deficits in fragile X syndrome model mice by regulating neurogenesis.

Claims

The use of 1,8-deoxy-11,13-dihydroxyguaiacol or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a medicament, characterized in that: The drug is used to treat and prevent hereditary intellectual and cognitive impairment diseases; The hereditary intellectual and cognitive impairment is Fragile X syndrome.

2. The application according to claim 1, characterized in that: The pharmaceutically acceptable salts include at least one of the following: metal salts, ammonium salts, salts formed with inorganic acids, salts formed with organic bases, salts formed with organic acids, salts formed with basic amino acids, and salts formed with acidic amino acids.

3. The application according to claim 2, characterized in that: The drug includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that: The pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

5. The application according to claim 1, characterized in that: The dosage forms of the drug include those administered via the gastrointestinal tract or those administered outside the gastrointestinal tract.

6. The application according to claim 5, characterized in that: The gastrointestinal dosage forms include one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, emulsion, suspension, syrup, and drops; The non-gastrointestinal drug delivery dosage forms include one of the following: injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

7. The application according to any one of claims 1 to 6, characterized in that: The drug is administered to mammals.

8. The application according to claim 7, characterized in that: The mammals mentioned include humans.

Images