Application of guaiaolide in preparation of medicine for treating fragile X chromosome syndrome

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of guaiaolide in preparation of medicine for treating fragile X chromosome syndrome. The invention discloses the application of guaiacol lactone in preparing the medicine for treating the fragile X syndrome for the first time, specifically, 8-deoxy-11, 13-dihydroxy guaiacol lactone is used for treating FXS, and the 8-deoxy-11, 13-dihydroxy guaiacol lactone serving as a metabolite of a human body is low in toxicity, small in side effect and high in safety when being used for treating the FXS; the 8-deoxy-11, 13-dihydroxy guaialactone can be used as a metabolite of a human body and can penetrate through a blood brain barrier to reach a treatment part in the brain, so that the 8-deoxy-11, 13-dihydroxy guaialactone possibly has an excellent advantage in the aspect of treating the nervous system diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of guaiac lactone in preparing a medicine for treating fragile X chromosome syndrome. Background Art

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

[0003] Fragile X syndrome (FXS) is not only a common hereditary intellectual and cognitive dysfunction disease, but also the most common single gene defect disease causing autism spectrum disorder. It is mainly manifested as intellectual disability, social interaction disorder and cognitive impairment, which greatly affects the patient's behavioral ability and quality of life. Effective clinical treatment plans are urgently needed.

[0004] FXS is mainly caused by mutations in the fragile X mental retardation gene (Fmr1) on chromosome X, which results in a functional deficiency of fragile X mental retardation protein (FMRP). The exact etiology of the disease remains unclear.

[0005] Basic research has shown that the loss of FMRP can cause abnormal brain development and a variety of neuronal cell functional defects, including abnormalities in neurogenesis, neuronal dendritic morphogenesis, circuit integration, and axon targeting. However, since the clinical phenotype of FXS may be affected by a dynamic and complex combination of interactions between different neurobiological mechanisms, there is still a lack of effective treatments for FXS in the clinic, and most treatments for FXS are based on its specific symptoms, which also leads to a lack of sufficient controlled experiments to prove the effectiveness of these methods. Only by combining psychopharmacological intervention with other supportive strategies, including speech therapy, sensory integration occupational therapy, personalized education plans, and tailored behavioral interventions, can the therapeutic effect be maximized.

[0006] Although a series of drug studies targeting metabotropic glutamate receptor 5 (mGluR5), glycogen synthase kinase-3β (GSK-3β) and γ-Aminobutyric Acid (GABA) receptors have been conducted, the therapeutic effect of this single drug has only been shown in animal experiments, but not in clinical trials. The results of single drug treatment indicate that there is a gap between animal experiments and clinical trials, suggesting that the existing single drug treatment strategy does not meet the needs of clinical treatment. In addition, selective serotonin reuptake inhibitors (SSRIs) are used as drugs to treat mood disorders, anxiety and compulsive behaviors associated with FXS. This type of drug is effective in relieving behaviors such as social anxiety, temper tantrums and aggression. In addition, antipsychotic drugs are also used to treat FXS, but the side effects associated with antipsychotic drugs, such as weight gain, diabetes, nausea, constipation and tardive dyskinesia, lead to poor results. Atypical antipsychotics are also used to treat self-injury, aggressive behavior, and autism. Although the atypical antipsychotic aripiprazole has shown improvements in mood stability, attention, and academic performance, it should be used at low doses to avoid the agitation caused by high doses, and its side effects should not be underestimated.

[0007] To date, there is still a lack of effective therapeutic drugs that can cure or alleviate the main adverse symptoms of patients with this disease. Therefore, it is urgent to explore the etiology of fragile X syndrome and develop new therapeutic drugs. Studying the genes and proteins that are abnormally expressed after FMRP loss and examining the specific effects of abnormal expression of related targets on the structure, morphology and function of neurons can provide positive clinical application value for the comprehensive treatment of FXS.

[0008] Guaiac lactone and its derivatives have multiple activities such as insecticide, anti-inflammatory, anti-tumor, and anti-hyperuricemia, but there is no existing technology to prove that they can be used to treat fragile X syndrome. Summary of the invention

[0009] The primary purpose of the present invention is to address the problems existing in the above-mentioned prior art, to provide a new application of guaiac lactone or its derivatives in treating hereditary intellectual and cognitive dysfunction diseases, especially a new application in drugs for fragile X syndrome, and to provide a new medicinal use of guaiac lactone or its derivatives (especially 8-deoxy-11,13-dihydroxyguaiac lactone).

[0010] The purpose of the first aspect of the present invention is to provide the use of guaiac lactone or its derivatives in the preparation of products.

[0011] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted is:

[0012] The first aspect of the present invention provides the use of guaiac lactone or its derivatives in preparing products, wherein the products are used to treat and prevent hereditary intellectual and cognitive dysfunction diseases.

[0013] The hereditary intelligence and cognitive dysfunction diseases include at least one of 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-chromosome linked intellectual disability comprises at least one of fragile X chromosome syndrome, Lesch-Nyhan syndrome, alpha thalassemia intellectual disability, X-linked lissencephaly syndrome, epilepsy with periventricular translocation, cerebral dysplasia hydrocephalus, XLMR with Rett-like syndrome, Nance-Horan syndrome, microcephaly-paleocytic-facial anomaly 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. The 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 Xchromosome gene (ARX), Serine-threonine kinase 9 (STK9), Polyglutamine tract binding protein (PQBP1), K1AA1202 protein, and Midline 1 (MID1).

[0016] Further preferably, the X-chromosome linked intellectual disability is fragile X syndrome.

[0017] Preferably, the guaiac lactone or its derivative includes at least one of 8-deoxy-11,13-dihydroxyguaiac lactone, 12,6-guaiac lactone, 12,8-guaiac lactone and pseudoguaiac lactone.

[0018] More preferably, the guaiac lactone or its derivative is 8-deoxy-11,13-dihydroxyguaiac lactone. The molecular formula of the 8-deoxy-11,13-dihydroxygrosheimin (8D11,13d) is C 15 H 20 O5, Cas number is 83551-03-5. The structural formula is shown in Formula 1:

[0019]

[0020] Preferably, the pharmaceutically acceptable derivatives include pharmaceutically acceptable salts and pharmaceutically acceptable chemical modifications.

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

[0022] Preferably, the metal salt comprises an alkali metal salt or an alkaline earth metal salt.

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

[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 a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N,N'-dibenzylethylenediamine.

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

[0027] Preferably, the salt formed with the organic acid includes a salt formed with the following organic acids: at least one of 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 a basic amino acid includes a salt formed with the following basic amino acids: at least one of arginine, lysine and ornithine.

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

[0030] Preferably, the pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation. For example, lipophilicity can be enhanced by introducing fluorine atoms (fluoroethyl) to improve blood-brain barrier permeability. The corresponding modification method can be selected according to the actual use of the drug.

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

[0032] Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0033] The present invention provides a drug for treating fragile X chromosome syndrome, which uses guaiac lactone or its derivatives (especially 8-deoxy-11,13-dihydroxyguaiac lactone) as an active ingredient and a corresponding drug dosage form.

[0034] Wherein, the drug exists in the form of oral preparation, injection or local administration preparation.

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

[0036] The pharmaceutical preparation contains the guaiac lactone or its derivatives (especially 8-deoxy-11,13-dihydroxyguaiac lactone) as effective active ingredients, and includes other pharmaceutically acceptable carrier components.

[0037] The carriers in drugs include excipients, such as starch, water, etc.; lubricants, such as magnesium stearate, etc.; disintegrants, such as microcrystalline cellulose, etc.; fillers, such as lactose, etc.; binders, such as pregelatinized starch, dextrin, etc.; sweeteners; antioxidants; preservatives; flavoring agents; spices, etc.

[0038] The carrier selected when preparing oral preparations can be conventional pharmaceutical excipients such as starch, dextrin or cyclodextrin and various chemically modified cyclodextrins, sucrose, stearate, etc. When preparing freeze-dried powder injections, they can be prepared by aseptic spray drying, low-temperature vacuum drying, freeze drying, etc. The later preparation processes and equipment of each preparation belong to conventional techniques in the pharmaceutical field, and the present invention does not limit this.

[0039] The drugs described in the present invention are in the form of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, and cataplasms, that is, pharmaceutical preparations include tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, cataplasms and the like, but are not limited to the above forms.

[0040] Furthermore, for the convenience of medication, the active ingredient guaiac lactone or its derivatives can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch slurry and syrup, etc.), disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol and micropowder silica gel, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue and medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium pyrosulfite and sodium thiosulfate, etc.), antibacterial agents (such as phenol, benzyl alcohol and thimerosal, etc.), and isotonic regulators (such as sodium chloride and glucose, etc.).

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

[0042] The guaiac lactone or its derivative (especially 8-deoxy-11,13-dihydroxyguaiac lactone) described in the present invention can be used alone or in the form of a pharmaceutical composition containing guaiac lactone or its derivative (especially 8-deoxy-11,13-dihydroxyguaiac lactone) when used to treat fragile X syndrome.

[0043] Preferably, the dosage form of the product includes a dosage form for enteral administration or a dosage form for parenteral administration.

[0044] Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0045] Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

[0046] Preferably, the product is administered to a mammal.

[0047] Preferably, the mammal comprises a human.

[0048] The beneficial effects of the present invention are:

[0049] 1. The present invention explores new medicinal value of guaiac lactone or its derivatives, uses them to treat fragile X syndrome, and can be prepared into drugs for treating fragile X syndrome, opening up a new application field for the application of secondary metabolites such as guaiac lactone or its derivatives (such as 8-deoxy-11,13-dihydroxyguaiac lactone).

[0050] 2. The present invention specifically uses 8-deoxy-11,13-dihydroxyguaiac lactone to conduct an in vitro neural stem cell experiment. The results show that 8-deoxy-11,13-dihydroxyguaiac lactone significantly promotes the differentiation of adult neural stem cells in Fmr1 KO mice into neurons, but has no effect on the differentiation of adult neural stem cells in WT mice.

[0051] 3. The present invention uses 8-deoxy-11,13-dihydroxyguaiac lactone to conduct an in vitro neural stem cell test to conduct an in vitro newborn neuron dendrite complexity test. The results show that the dendrite complexity of hippocampal newborn neurons in Fmr1 KO mice is reduced compared with that in WT mice, and the dendrite length, dendrite nodes and dendrite terminals are reduced. 8-deoxy-11,13-dihydroxyguaiac lactone can restore neuronal morphology after intervention; significantly promote the dendrite complexity of hippocampal newborn neurons in Fmr1 KO mice cultured in vitro, increase the dendrite complexity of hippocampal newborn neurons in Fmr1 KO mice; increase the dendrite length, dendrite nodes and dendrite terminals of hippocampal newborn neurons in Fmr1 KO mice, so that the dendrite length of newborn neurons is restored to a similar level as the dendrite length of hippocampal newborn neurons in WT mice, as well as a similar number of dendrite nodes.

[0052] 4. The present invention uses different animal models and conducts a large number of animal tests through injection. The test results show that 8-deoxy-11,13-dihydroxyguaiac lactone promotes the maturation of new neurons of adult neural stem cells in Fmr1 KO mice. 8-deoxy-11,13-dihydroxyguaiac lactone promotes the differentiation of neural stem cells in the dentate gyrus (DG) of the hippocampus caused by FMRP deficiency into neurons, reduces hippocampal-dependent learning damage, and corrects cognitive dysfunction caused by FMRP deficiency.

[0053] 5. The 8-deoxy-11,13-dihydroxyguaiac lactone of the present invention has a strong pharmacological effect, is effective in treating fragile X syndrome, has a rapid effect, has little toxicity and side effects, is safe, can be taken for a long time, and has a good medicinal prospect.

[0054] 6. The raw materials of the product of the present invention are derived from natural metabolites, are safe for clinical use, have a simple preparation process, can be made into various dosage forms, and have a small dosage and are easy to use, so they are easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0056] Figure 1 This is a diagram of the experimental scheme for 8-deoxy-11,13-dihydroxyguaiac lactone to regulate the differentiation of newborn neurons from neural stem cells in the adult mouse brain.

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

[0058] Figure 3 This is a diagram showing the data analysis results of newborn and mature neurons in a neural stem cell differentiation experiment.

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

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

[0061] Figure 6 This is a diagram of the experimental scheme for 8-deoxy-11,13-dihydroxyguaiac lactone to regulate the morphology and function of hippocampal neurons at P0 in vitro.

[0062] Figure 7 This is a microscopic observation of hippocampal neurons cultured in vitro on day P0.

[0063] Figure 8 Sholl analysis of dendritic complexity in primary hippocampal neurons.

[0064] Fig. 9 This is a statistical graph of the Sholl analysis of the dendrite length of primary hippocampal newborn neurons.

[0065] Fig.10 This is a Sholl analysis statistical chart of the number of dendritic nodes of primary hippocampal newborn neurons.

[0066] Fig.11 This is a Sholl analysis statistical chart of the number of dendritic terminals of primary hippocampal newborn neurons.

[0067] Fig.12 This is a graph showing the average discharge frequency of the primary hippocampal newborn neuron network.

[0068] Fig.13 This is a statistical chart of the average discharge frequency of the primary hippocampal newborn neuron network.

[0069] Fig.14 This is a statistical chart of the sustained discharge time of the primary hippocampal newborn neuron network.

[0070] Fig.15 Schematic diagram of the mouse novel location recognition experiment.

[0071] Fig.16 Statistical graph of the discrimination index for the mouse novel location recognition experiment.

[0072] Fig.17 Schematic diagram of the novel object recognition experiment for mice.

[0073] Fig.18 This is a statistical chart of the discrimination index in the mouse novel object recognition experiment.

[0074] Fig.19 Schematic diagram of the mouse social interaction experiment.

[0075] Fig. 20 This is a statistical chart of the discrimination index in the mouse social interaction experiment. DETAILED DESCRIPTION

[0076] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

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

[0078] Male wild-type (WT, or Fmr1 + / y ) mice, and female Fmr1 knockout (KO) mice (Fmr1 KO, or Fmr1 - / - ) were purchased from Jackson Laboratory, USA.

[0079] Fmr1 gene knockout (Knock out, KO), can be represented by Fmr1 KO, or Fmr1 - / - (female mice) / Fmr1 - / y (Male mouse). Male wild-type (WT) can be represented by WT or Fmr1 + / + (female mice) / Fmr1 + / y (Male rat).

[0080] All mice were bred and raised at the Animal Center of the Chinese Medical Research Institute of the University of Macau, in a specific pathogen-free (SPF) animal breeding room with a breeding temperature of 22-25°C and a circadian rhythm cycle of 12 h light / 12 h dark. The mice had free access to water and food, which was approved and recognized by the Animal Ethics Committee of the University of Macau.

[0081] As mentioned above, the occurrence of fragile X syndrome is mainly caused by the mutation of the Fmr1 gene on the X chromosome, which leads to the functional loss of FMRP. In order to avoid the deviation of experimental data and conclusions caused by the influence of the physiological cycle of female mice and the different expressions of the Fmr1 gene on the two X chromosomes on physiological functions, the present invention only selects adult wild-type and Fmr1 gene-deficient male mice as experimental samples.

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

[0083] Table 1

[0084]

[0085]

[0086] Example 1 In vivo neural stem cell differentiation experiment

[0087] 1. Methods for breeding mice for neural stem cell differentiation experiments

[0088] Male mice (Fmr1 + / y ) and Fmr1 HET female mice (Fmr1 + / - ) can be mated to produce the following genotype mice, which are marked as: ①Fmr1 + / y (male); ②Fmr1 - / y(male); ③Fmr1 - / + (female); ④Fmr1 + / + (female).

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

[0090] 2. Grouping, drug administration and sampling of neural stem cell differentiation experiments

[0091] After 7-8 weeks of feeding, the male mice marked as ① and ② were divided into 4 groups, 3 mice in each group, specifically:

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

[0093] Each group of mice was intraperitoneally injected with drugs, where the dosage of 8-deoxy-11,13-dihydroxyguaiac lactone was 20 mg / kg; the volume of phosphate buffer was calculated according to the dosage concentration of 8-deoxy-11,13-dihydroxyguaiac lactone, and the same volume of phosphate buffer was given; the drugs were intraperitoneally injected at the same time every day, once a day, for a total of 7 days.

[0094] 8-Deoxy-11,13-dihydroxyguaiac lactone and phosphate buffer were administered by intraperitoneal injection at the same time every day, once a day, for 7 days; on the 3rd and 4th days during the administration of 8-deoxy-11,13-dihydroxyguaiac lactone and phosphate buffer, each mouse was intraperitoneally injected with BrdU, twice a day, for two days, with a BrdU dose of 200 mg / kg.

[0095] 8-Deoxy-11,13-dihydroxyguaiac lactone was administered for 7 consecutive days, and BrdU administration was terminated on the 3rd and 4th days. After waiting for four weeks, the mice were anesthetized and the brains were removed by perfusion. The entire hippocampus of the mouse brain was sliced ​​continuously at a thickness of 40 μm, and the hippocampus was systematically randomly sampled by selecting one slice every 6 slices. This was used for subsequent neural stem cell differentiation experiments. The experimental process is as follows Figure 1 .

[0096] The measurement range of neurons in the dentate gyrus region of the hippocampus is relative to the anterior fontanelle, the hippocampus region with a width of -0.94mm to -2.30mm on the dorsal side, and the hippocampus region with a width of -2.30mm to -3.80mm on the ventral side. The frozen brain sections of mice in each experimental group were stained with immunohistochemistry (frozen sections) (IHC-Fr), and sealed with anti-fluorescence quenchers to obtain stained brain sections of mice in each experimental group; then the neurosterological quantitative analysis method was used, that is, the stained brain sections were placed under an upright microscope, the Stereo Investigator neurosterological software was opened, the fluorescence channel was set, and the DG area in the stained brain sections was scanned in the order of the brain sections. After the scanning, the fluorescence staining through different channels was used to determine NeuN + and BrdU+ and other positive targets, and after scanning, NeuN + and BrdU + Cell counting, quantification of positive targets in the stained mouse brain sections, including green-NeuN + ; Red-BrdU + .

[0097] Scanning brain slices under a microscope, the newborn and mature neurons in the DG region of mice (NeuN + BUD + Representative micrographs of 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 experiment

[0099] NeuN is a marker for mature stem cells, and BrdU marks neural stem cells in the division phase, that is, stem cells that are in a proliferative state. + and BrdU + Double positive cells represent neural stem cells that have newly differentiated into mature neurons. NeuN was counted using Stereo Investigator neurostereometry software. + and BrdU + The number of cells. NeuN + and BrdU + The number of cells 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 in the phosphate buffer group (NeuN + BUD + / BrdU + ) accounted for 77.99%; Group B: Fmr1 - / y +Mature newborn neurons in the phosphate buffer group (NeuN + BUD + / BrdU + ) accounted for 58.56%; Group C: Fmr1 + / y +8-deoxy-11,13-dihydroxyguaiac lactone in mature newborn neurons (NeuN + BUD + / BrdU + ) accounted for 79.53%; Group H: Fmr1 - / y +8-deoxy-11,13-dihydroxyguaiac lactone group (NeuN + BUD + / BrdU + ) accounts for 77.63%.

[0101] The results of the neural stem cell differentiation experiment showed that Figure 3 From the above data analysis, we can see that the number of mature newborn neurons in group B is less than that in group A, but after the administration of 8-deoxy-11,13-dihydroxyguaiac lactone, the proportion of newborn neurons in group D can be seen to be almost restored to a level similar to that in group A. This indicates that 8-deoxy-11,13-dihydroxyguaiac lactone can promote the maturation of newborn neurons in adult neural stem cells in Fmr1 KO mice.

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

[0103] According to NeuN + BUD + / BrdU + The percentage shows that 8-deoxy-11,13-dihydroxyguaiac lactone promotes the differentiation and maturation of neurons. Therefore, it can be seen that 8-deoxy-11,13-dihydroxyguaiac lactone can promote the differentiation of neural stem cells in the brain of Fmr1 KO mice into neurons and can correct the neurogenesis disorder of Fmr1 KO mice.

[0104] As mentioned above, FMRP deficiency leads to abnormal reduction of differentiation of neural stem cells into neurons in the DG region and impairs hippocampal-dependent learning. After administration of 8-deoxy-11,13-dihydroxyguaiac lactone, the differentiation of neural stem cells into neurons can be restored to a level similar to normal, promoting normal neurogenesis and reducing the damage caused by FMRP deficiency.

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

[0106] Adult DG neural stem cells of wild-type (WT) mice and Fmr1 gene-deficient (Fmr1 KO) mice were cultured in vitro, and quantitative analysis was performed using immunofluorescence staining combined with neurosterological quantitative analysis after cell administration. The results showed that 8-deoxy-11,13-dihydroxyguaiac lactone 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 regions of the mice under a stereomicroscope, and then primary adult neural stem cells were 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, namely WT stem cells and Fmr1 KO stem cells, and there were three stem cells of each type (ie, n=3), which were marked as WT1, WT2, WT3; KO1, KO2, KO3, respectively.

[0110] 2. Experimental methods

[0111] 2.1 Preliminary preparation:

[0112] 1) Configuration of related reagents

[0113] Neural basal medium: Neural basal medium;

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

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

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

[0117] Glutamine L-Glutamine (Gibco, 200 mM), using 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 adult neural stem cells+0.005% phosphate buffer; Group B: KO+DMSO, Fmr1KO adult neural stem cells+0.005% phosphate buffer; Group C: WT+8D11,13d, WT adult neural stem cells+8D11,13d with a final concentration of 0.5μM; Group D: KO+8D11,13d, i.e. Fmr1KO adult neural stem cells+8D11,13d with a final concentration of 0.5μM.

[0120] The two types of WT and Fmr1 KO adult neural stem cells were subjected to blank and drug treatments, respectively, wherein: in the blank treatment (blank group), 0.005% phosphate buffer was added to the culture medium, i.e., WT+phosphate buffer group, WT adult neural stem cells+0.005% phosphate buffer; in the KO+DMSO group, Fmr1 KO adult neural stem cells+0.005% phosphate buffer; in the drug treatment (drug treatment group), 8D11,13d with a final concentration of 0.5 μM was added to the culture medium, i.e., WT+8D11,13d group, WT adult neural stem cells+8D11,13d with a final concentration of 0.5 μM; in the KO+8D11,13d group, Fmr1 KO adult neural stem cells+8D11,13d with a final concentration of 0.5 μM.

[0121] 3) Slide coating treatment

[0122] A cell slide with a diameter of 14 mm is placed in a 24-well plate, and then an appropriate volume (200-300 microliters) of poly-l-ornithine is added and coated overnight; after recovering the poly-ornithine, an appropriate amount (200-300 microliters) of laminin (0.01068 mg / mL) is added to the 24-well plate containing the cell slide and coated again until the laminin is recovered before the cells are plated; wherein, laminin with a concentration of 0.01068 mg / mL is prepared according to the following method: 2.67 mg / mL laminin stock solution is diluted 250 times with sterile ultrapure water to obtain; the recovered poly-l-ornithine and laminin can be reused repeatedly.

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

[0124] Table 2 Stem cell experimental groups

[0125]

[0126] A 24-well plate was prepared according to the above method for differentiation assay.

[0127] 4) Stem cell digestion and counting

[0128] a) 8 mL of adult neural stem cell suspension was taken from each of the stem cells numbered WT1, WT2, WT3; KO1, KO2, KO3 from their respective culture dishes, and then transferred to their respective centrifuge tubes (15 mL), centrifuged (3 min, 1200 r), and the supernatant was discarded, retaining the cell pellet;

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

[0130] b) Add 1×Trypsin (trypsin solution, 1 mL) at a concentration of 0.05% to each centrifuge tube and incubate for 1-2 minutes;

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

[0132] d) Add stem cell culture medium (neural basal culture medium + B27 + L-Glu + double antibody + EGF + FGF, 1 mL, wherein the final concentration of EGF and FGF is 10 ng / mL) into each centrifuge tube respectively, pipette and beat evenly, filter with a 40-micron mesh after resuspending the stem cells, hit all 1 mL of stem cell suspension on the mesh, and filter into a 50 mL centrifuge tube, so that the adult stem cells are in a single cell state, that is, obtain an adult stem cell suspension in a single cell state.

[0133] e) Take 6 EP tubes (1.5 mL), add 90 μL of neural basal culture medium to each EP tube, accurately pipette 10 μL of the adult stem cell suspension in single-cell state filtered in step 3D) into the corresponding EP tube, mix well, pipette 10 μL from each, drop them on a blood cell counting plate, and observe and count under a microscope.

[0134] The purpose of cell counting is to keep the number of cells in each duplicate well of each group consistent when the cells are plated. Each type of cell of WT1 / WT2 / WT3; KO1 / KO2 / KO3 is counted separately and the average value is taken, and then plated separately.

[0135] 2.2 Differentiation

[0136] 1) After digestion and counting in the preliminary preparation steps, the single-cell adult stem cells were counted at 2×10 5 The inoculation volume was added to the coated 24-well plate containing the coated glass slide. When the cells were plated, the number of stem cells seeded in one well of the 24-well plate was 200,000 (2×10 5 ), the volume of culture medium in each well was 2 mL, and the cells were cultured in a cell incubator for 18 h;

[0137] 2) After 18 h of culture, 500 μL of the culture medium in each well of the 24-well plate was aspirated and 500 μL of new culture medium was added, wherein the new culture medium contained forskolin (final concentration 5 μM); retinoic acid (final concentration 1 μM); blank group: maintained at 0.005% DMSO; drug group: 8-deoxy-11,13-dihydroxyguaiac lactone maintained at a final concentration of 0.5 μM, and then incubated in an incubator for 3 days, and the medium was changed and the drug was administered for three consecutive days.

[0138] 2.3 Cell immunofluorescence

[0139] 1) After incubation, the 24-well plate was taken out from the cell culture room, and 0.5 mL of culture medium was aspirated from each well, leaving 0.5 mL of culture medium in each well. Then, 4 wt% paraformaldehyde (0.5 mL) was added to each well and placed at room temperature. After 20 min, all the liquid in the well was aspirated, and 1 mL of 4 wt% paraformaldehyde was added to each well and placed at room temperature for 15 min. Then, the liquid in the well was aspirated and the wells were washed with PBS for 5 min / times, for a total of three times.

[0140] 2) Antibody preparation and incubation

[0141] a) Prepare antibody diluent: add 1×TBS to 0.5 g BSA, 2 mL Trionx-100 and 2 mL goat serum until the volume reaches 40 mL, vortex until dissolved, and store at -20°C;

[0142] b) Primary antibody, prepare Tuj1 antibody: use the antibody diluent prepared in "3. Cell immunofluorescence (a)" to prepare Tuj1 antibody, add primary antibody Tuj1 at a dilution of 1:3000 to the well plate treated in "3. Cell immunofluorescence (1)", add 300 μl to each well, incubate overnight at room temperature; then wash with PBS, 5 min / time, for a total of three times;

[0143] c) Secondary antibody, Alexa Fluor 568goat anti-rat IgG (H+L), use the antibody diluent prepared in step (b) of "3. Cell immunofluorescence" to prepare fluorescent secondary antibody (1:2000), then add 300 μl to each well of the well plate, incubate at room temperature for 2 h to obtain fluorescent secondary antibody;

[0144] d) DAPI solution can be incubated with the fluorescent secondary antibody for the last 15 minutes of incubation time, or after the fluorescent secondary antibody incubation time is over, the secondary antibody is aspirated cleanly, and then 500 microliters of DAPI solution is added to each well. After incubating the fluorescent secondary antibody, DAPI needs to be incubated: 4',6-diamidino-2-phenylindole is a fluorescent dye that can strongly bind to DNA and is commonly used in fluorescence microscopy observations to display blue fluorescence.

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

[0146] 2.4 Sealing, fixation, microscopic examination, and counting

[0147] Take an ordinary glass slide (long), and drop 15 μl (usually 10-20 μl) of anti-fluorescence quencher at two different positions on it, then take out the cell slide incubated in step 2 from the 24-well plate, fix it on the glass slide with the cell side facing down, and then observe and count it under an upright microscope; adjust the microscope fluorescence to the red channel, observe and count the cells with red fluorescence, and the red fluorescent cells are Tuj1-labeled cells representing differentiated neural stem cells; adjust the microscope fluorescence to the DAPI channel, observe and count the blue fluorescent cells, and the blue fluorescent DAPI labels all cells; calculate the number of Tuj1 and DAPI respectively, and calculate the ratio of Tuj1 / DAPI.

[0148] Microscopic observation of neural stem cells after differentiation Figure 4 , where Tuj1-labeled differentiated cells are red; DAPI-labeled all cell nuclei are 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 the final concentration of 8D11,13d was 0.5 μM.

[0149] 2.5 Data Analysis and Results

[0150] The neurostereometry software Stereo Investigator was used for counting analysis, and the average of the three analysis data of each group was taken. The statistical analysis results are shown in Figure 5 .

[0151] Group A: WT+Veh group is in the process of adult neural stem cell differentiation (Tuj1 + / Tuj1 + DAPI + ) accounted for 13.08%; Group B: KO+Veh group was in the process of adult neural stem cell differentiation (Tuj1 + / Tuj1 + DAPI + ) accounted for 4.44%; Group C: WT+8D11,13d group was in the process of adult neural stem cell differentiation (Tuj1 + / Tuj1 + DAPI + ) accounted for 13.61%; Group D: KO+8D11, 13d group was in the process of adult neural stem cell differentiation (Tuj1 + / Tuj1 + DAPI + ) accounts for 11.18%.

[0152] Tuj1 + Marked neurons, Tuj1 + / DAPI +Represents the proportion of neural stem cells converted into neurons. The above data show that 8-deoxy-11,13-dihydroxyguaiac lactone significantly promoted the neuronal differentiation of adult neural stem cells in Fmr1 KO mice in vitro. And the differentiation level of Fmr1 KO neural stem cells was increased to a level similar to that of WT, and 8-deoxy-11,13-dihydroxyguaiac lactone had no effect on the differentiation of adult neural stem cells in WT mice in vitro. Therefore, 8-deoxy-11,13-dihydroxyguaiac lactone can be used to treat diseases caused by mutations in the fragile X mental retardation gene (Fmr1) during the formation of X chromosomes.

[0153] Example 3 Effects of 8-deoxy-11,13-dihydroxyguaiac lactone on the complexity and function of dendrites of newborn neurons in the hippocampus of Fmr1 KO mice

[0154] The DG neurons of P0 day wild-type (WT) mice and Fmr1 gene-deficient (Fmr1 KO) mice were cultured in vitro, and the morphology of newborn neurons was analyzed by sholl analysis after cell administration by transfection of GFP plasmid; the function of neurons was analyzed by electrophysiology. The results showed that 8-deoxy-11,13-dihydroxyguaiac lactone significantly promoted the dendritic maturation of Fmr1 gene-deficient (Fmr1 KO) newborn neurons in vitro, including dendritic length, number of dendritic nodes and number of dendritic terminals; and 8-deoxy-11,13-dihydroxyguaiac lactone significantly increased the hypodischarge frequency and neuronal discharge duration of Fmr1 gene-deficient (Fmr1 KO) newborn neurons.

[0155] 1. Experimental Materials

[0156] 1.1 Primary culture method of mouse newborn neurons

[0157] When male wild-type mice and Fmr1 knockout mice reached maturity after eight weeks of growth, female Het(Fmr1 - / + ) and male WT (Fmr1 + / y ) were co-bred at a ratio of 1:2. P0 day male mice were selected, and the hippocampal tissues of P0 day mice were isolated under a stereomicroscope, and then primary newborn neurons were cultured in a cell culture room. The neurons were stored in an incubator at a temperature of 37°C and 5% CO2.

[0158] Two types of neurons were obtained, namely WT neurons and Fmr1 KO neurons, and there were three neurons of each type (ie, n=3), which were marked as WT1, WT2, WT3; KO1, KO2, KO3, respectively.

[0159] 1.2 Preparation method of related reagents

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

[0161] 2) Preparation of LB solid culture medium: Take 10g of tryptone powder, 10g of yeast powder, 10g of NaCl, and 10-20g of agar powder, dissolve in 1L of ultrapure water, and use after autoclaving. When the culture medium liquid cools to 50-60℃, add 1mL of 50mg / mL ampicillin to fully dissolve it. Before the culture medium solidifies, quickly dispense it into culture dishes (10mL / each). After cooling and solidification, cover the culture dish lid, invert it (to prevent water from dripping on the solid culture medium), and store it in a refrigerator at 4℃ for later use.

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

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

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

[0165] 6) Preparation of ampicillin solution (50 mg / mL): Weigh 2.5 g of ampicillin into a 50 mL plastic centrifuge tube, add nearly 50 mL of sterile ultrapure water and mix thoroughly to dissolve, then make up to 50 mL, filter through a 0.22 μm filter membrane for sterilization, divide into small portions (1 mL / tube), and store at -20°C.

[0166] 7) 4 wt% paraformaldehyde (PFA): Take 40 g of PFA powder and place it in a 1 L beaker, then add 1 L of PBS phosphate buffer, stir with a stirrer, and add 6 mg of NaOH during stirring until it is completely dissolved, then adjust the pH to 7.0-7.4 with hydrochloric acid, cool, divide into packages, and store in a -20°C refrigerator.

[0167] 8) Preparation of polylysine: Dilute 10× polylysine to 1× polylysine with sterilized ultrapure water at a dilution ratio of 1:9, filter and sterilize with a 0.22μM microporous filter membrane, and then store at 4°C. Note that polylysine can be recycled, but it must be filtered before reuse. If it is turbid or contaminated, it should be discarded.

[0168] 9) Neuron-related culture medium

[0169] ① Culture medium, material culture medium: DMEM-F12 + 1% penicillin-streptomycin;

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

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

[0172] Digestion solution: Trypsin-EDTA (0.25%), containing phenol red, used directly.

[0173] 2. Experimental Grouping and Drug Administration

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

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

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

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

[0178] The two types of WT and Fmr1 KO neuronal cells were subjected to blank treatment and drug treatment, respectively, among which: WT+VEH group (blank group, group A), 0.005% phosphate buffer was added to the culture medium, WT neuronal cells+0.005% phosphate buffer; KO+VEH group (group B), Fmr1 KO neuronal cells+0.005% phosphate buffer; drug treatment (drug treatment group, groups C and D) added 8D11,13d with a final concentration of 10 μM to the culture medium, that is, WT+8D11,13d group, WT neuronal cells+8D11,13d with a final concentration of 10 μM; KO+8D11,13d group, Fmr1 KO neuronal cells+8D11,13d with a final concentration of 10 μM.

[0179] 3. Plasmid Transformation

[0180] 1) Place DH5α competent E. 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), flick the tube to mix the plasmid and competent cells thoroughly; then place 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 the centrifuge tube to an ice bath for 2-3 minutes. Be careful not to shake the centrifuge tube during this process. Introduce the Rv-GFP plasmid into E. coli to amplify it in large quantities.

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

[0184] 5) Pipette the culture mixture (100 μL) from "Step 4)" and add it to LB solid medium containing ampicillin (final concentration 50 μg / mL), and gently spread the cells evenly with a sterile inoculation loop; then invert the culture dish and culture in a 37°C incubator for 12-16 hours, and colonies can be observed.

[0185] 6) Add 200 mL of LB liquid culture medium containing ampicillin (50 μg / mL) to a 250 mL conical flask that has been sterilized by high pressure and set aside.

[0186] 7) Use a sterilized pipette tip to pick up the single bacterial pellet cultured in "3. Plasmid transformation step (5)", add it to the conical flask in "3. Plasmid transformation step (6)", and culture at 37°C, 200 rpm, and shake for 12-16 hours.

[0187] 8) After shaking, the bacterial solution was centrifuged (15 min, 4500 rpm, 4°C), the supernatant was discarded, and the bacterial pellet was kept. The pellet was stored at -40°C for plasmid extraction.

[0188] 4. Plasmid Extraction

[0189] Use a high-purity plasmid extraction kit and perform plasmid extraction according to the kit's instructions and experimental methods, as follows:

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

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

[0192] 3) Add solution P4 of the plasmid extraction kit to the centrifuge tube and mix thoroughly immediately until white dispersed flocculent precipitates appear in the solution; after standing at room temperature for 4-5 minutes, centrifuge (7800 rpm, 15 minutes) to allow the white precipitate to centrifuge to the bottom of the tube; then carefully pour all the solution into the filter CS1 (be careful to avoid pouring a large amount of precipitate to block 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 times the volume of NaCl to the filtrate, and mix thoroughly by inverting; centrifuge at 4°C and 7800 rpm for 90 min, gently pour off the supernatant, invert the centrifuge tube on absorbent paper, absorb the liquid, and add 70% ethanol to the centrifuge tube to fully rinse the precipitate; centrifuge at 4°C and 7800 rpm for 15 min, gently pour off the supernatant, invert the centrifuge tube on absorbent paper, repeat 1-2 times to fully rinse the precipitate.

[0194] 5) Leave the centrifuge tube open at room temperature for 30 minutes to allow the ethanol to fully evaporate, add DEPC water and vortex to mix, and extract the RV-GFP plasmid.

[0195] The concentration and purity of RV-GFP plasmid were tested using a DNA micrometer. The results are as follows:

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

[0197] The extracted plasmids were marked (date, plasmid type, concentration) and stored in a -20°C refrigerator for future use.

[0198] 5. Slide coating

[0199] 1) Place a 14 mm diameter cell slide into a 24-well plate, add an appropriate volume (200-300 μl) of 1× poly-lysine coating solution, and coat (for more than 8 hours). Recover the coating solution before the experiment and wash the slide 2-3 times with sterile ultrapure water; 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-lysine coating solution to the 24-well microelectrode array cell culture plate for coating (more than 8 hours). Recover the coating solution before the experiment and wash the slides 2-3 times with sterile ultrapure water. This experiment is used for functional analysis of primary neurons cultured in vitro.

[0201] 6. Primary Neuron Culture

[0202] 1) Dispense medium No. ① for neuron culture into culture dishes and EP tubes with corresponding numbers and pre-cool them on ice;

[0203] 2) Take out the P0 day (newly born mice within 0-24h), disinfect with alcohol, take out the tail tissue of the mice and store it in a 1.5mL EP tube; then cut off the head of the mice, among which: male WT and Fmr1 KO mice were divided into 4 groups, namely WT+VEH, KO+VEH, WT+8D11,13d, KO+8D11,13d groups, each group had three mice, namely n=3, and there were 12 mice in total in the four groups. The specific grouping is shown in Table 3, where the DMSO concentration was 0.005%; the 8D11,13d concentration was 10μM.

[0204] Table 3 Neuronal cell experimental grouping

[0205]

[0206]

[0207] 3) After removing the brainstem, the whole brain was divided into two hemispheres from the middle. The hippocampus was located on the ventral inner side of the hemisphere. The whole process was performed under a stereoscope. The meninges were peeled off with microtweezers, and then the hippocampi on both sides were peeled off. The brain was quickly placed in culture medium No. ①, the hippocampus was separated, and the hippocampi on both sides of the brain were transferred to the corresponding numbered EP tubes. The hippocampi were cut into small segments of 1-2 mm in the cell operation room. 3 The small pieces were collected into 15 mL centrifuge tubes, 300 μL digestion solution (GIBCO, 25200056) was added, and then incubated in a 37°C incubator containing 5% CO2 for 5 min.

[0208] 4) After incubation, centrifuge (1500r / 1min) and remove the supernatant; then add 2mL of No. ② culture medium to each 15mL centrifuge tube, gently blow until there are no tissue clumps, then pass the cell suspension through a 40μm sieve, collect the cell suspension in a new 15mL centrifuge tube, make the above cells in a single cell state, continue to blow evenly, and inoculate (3-5)×10 5The inoculated amount of cells is added to a 24-well plate containing a coated cell slide or a 24-well microelectrode array cell culture plate for seeding. When the cells are seeded, the volume of the culture medium in each well is 1 mL, and the cells are cultured in a cell incubator for 4 hours.

[0209] 5) For neuronal morphological analysis experiments: 4 hours after seeding, the culture medium in each well of the 24-well plate was replaced with medium No. ③; the day of seeding was day 0, and half of the medium was replaced every other day, that is, half of the medium No. ③ was replaced, and the plate was transfected and treated with drugs according to the grouping in Table 3 on the 4th day of seeding. The plate was fixed on the 7th day. The specific experimental process is as follows: Figure 6 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 No. ③; the day of seeding was day 0, and half of the medium was replaced every other day, that is, half of the medium No. ③ was replaced. On the 4th day of seeding, the drug treatment was carried out according to the grouping in Table 3. On the 13th day of seeding, the MEA neuroelectrophysiological monitoring system was used to detect the discharge frequency of neurons and the network burst frequency. The specific experimental process is as follows: Figure 6 shown.

[0211] 7. Cell transfection and drug treatment

[0212] 1) Preparation:

[0213] High-pressure sterilize the materials required for the experiment and sterilize them by ultraviolet irradiation in a clean bench to ensure aseptic operation; place culture medium No. ③ at room temperature for later use.

[0214] Prepare transfection reagent (the amount of reagent 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, replace the culture medium in the well plate with new medium No. ③ 1 hour before transfection.

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

[0217] 4) The DNA-CaCl2-HBS mixed system was added dropwise to each well of the 24-well plate after seeding, and then the 24-well plate was placed 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 (room temperature) (to remove the precipitate and reduce cell death).

[0219] 6) Replace medium ③ according to the grouping: Group A and Group B: add 1 mL of medium ③ + 0.005% DMSO to each well; Drug-treated groups (Group C and Group D): add 1 mL of medium ③ + puerarin (final concentration of 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 is performed.

[0221] 8. Fixation, sealing and measurement of neurons

[0222] 1) After the seventh day of neuron culture, the 24-well plate was taken out from the cell culture room, 0.5 mL of culture medium was aspirated from each well, and 4 wt% paraformaldehyde (0.5 mL) was added to each well and allowed to stand at room temperature for 20 min. All the liquid in the well was aspirated, and 4 wt% paraformaldehyde (1 mL) was added to each well and allowed to stand at room temperature for 15 min. The liquid in the well was then aspirated, and the wells were washed 3 times with PBS, 5 min / time.

[0223] 2) Take a common glass slide (long) and drop 15 μL (usually 10-20 μL) of anti-fluorescence quencher at two different positions on it. Then take out the cell slide with neurons fixed and washed in step 7-1) from the 24-well plate, fix it on the glass slide with the cell side facing down, and then observe it under an upright microscope and measure tracing (real-time depiction); wherein: adjust the microscope fluorescence to the green channel, randomly select neurons expressing green fluorescence, and measure the dendritic length, dendritic nodes, dendritic ends, etc.

[0224] Observe GFP using fluorescence field under an upright microscope + Changes in neuronal cell morphology ( Figure 7 ). To analyze the effect of PR on the dendritic complexity of newborn neurons in the hippocampus of WT or Fmr1 KO mice, GFP was analyzed using a stereological analysis system. + The newborn neurons were depicted in real time and the neuronal morphology was quantified using the neuromorphic analysis system. + The length of dendrites of newborn neurons ( Fig. 9 ), the number of dendritic nodes ( Fig.10 ), the number of dendrite terminals ( Fig.11 ) quantification.

[0225] 9. Data results and analysis

[0226] GraphPad Prism software was used for two-way ANOVA or SPSS statistical analysis software was used for multivariate analysis of variance (MANOVA) for threshold analysis. The data processing results were expressed as Mean ± SEM. When P < 0.05, it was considered to be statistically significant.

[0227] 1) Effects of 8-deoxy-11,13-dihydroxyguaiac lactone on the morphology of newborn neurons in the hippocampus of WT and Fmr1 KO mice

[0228] Microscopic observation of the morphology of newborn neurons Figure 7 ,Depend on Figure 8-11 It can be seen that compared with WT+VEH, the dendritic complexity of KO+VEH neurons was low, the dendritic length was short, and the dendritic branches were few; after administration of 8D11 and 13d, compared with KO+VEH, the dendritic complexity of KO+8D11 and 13d neurons increased, the dendritic length increased, and the dendritic branches increased; compared with WT+VEH, the morphology of KO+8D11 and 13d neurons can be restored to a level similar to that of the newborn neurons in the hippocampus of WT mice.

[0229] In the neurons cultured in vitro, the neuronal cells were transfected by using the calcium phosphate transfection method, and 8-deoxy-11,13-dihydroxyguaiac lactone was added during the culture process for drug intervention. The morphological analysis of neurons expressing green GFP fluorescence using the neurostereoscopic analysis method found that the hippocampal newborn neurons of Fmr1 KO mice were significantly different in morphology compared with WT, that is, the dendritic complexity network complexity of Fmr1 KO neurons was lower than that of WT neurons. After the intervention of 8-deoxy-11,13-dihydroxyguaiac lactone, the morphology of Fmr1 KO hippocampal newborn neurons could be restored to a level similar to that of WT hippocampal newborn neurons ( Figure 7 ).

[0230] The experimental results showed that 8-deoxy-11,13-dihydroxyguaiac lactone improved the dendritic morphology of newborn neurons in the hippocampus of Fmr1 KO mice: 8-deoxy-11,13-dihydroxyguaiac lactone could significantly promote the dendritic complexity of newborn neurons in the hippocampus of 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-dihydroxyguaiac lactone on the complexity of newborn neurons in the hippocampus of WT and Fmr1 KO mice

[0232] The Sholl analysis method was used to examine the complexity of newborn neurons in the hippocampus of WT or Fmr1 KO mice. The statistical analysis results of the complexity of newborn neurons in the hippocampus identified by the Sholl analysis method are shown in the figure. Figure 8 .

[0233] Sholl Analysis starts with the neuron cell body as the center (excluding the cell body) and draws a series of concentric circles to obtain the number of intersections of neuron processes that varies with the distance from the cell body, thereby reflecting the complexity of neurons. By calculating the number of branches that intersect with each circle, the branching patterns of neuron dendrites and axons in different regions are obtained, thereby quantitatively characterizing the morphological characteristics of the imaged neurons. The analysis results are shown in Figure 8 .

[0234] The minimum mean-maximum mean of the number of intersections at equal distances from the cell body for WT+VEH; KO+VEH; WT+8D11,13d; KO+8D11,13d were [2.7, 7.5]; [1.59, 4.53]; [3.23, 7.78]; [2.56, 7.48], respectively. The experimental results showed that the complexity of newborn neurons in Fmr1 KO mice was lower than that of newborn neurons in WT mice, and 8-deoxy-11,13-dihydroxyguaiac lactone could significantly increase the dendritic complexity of newborn neurons in the hippocampus of Fmr1 KO mice.

[0235] a) Effects of 8-deoxy-11,13-dihydroxyguaiac lactone on the dendrite length of newborn neurons in the hippocampus of WT and Fmr1 KO mice

[0236] The Sholl analysis method was used to analyze the length of dendrites of newborn neurons in the hippocampus. The statistical analysis results of the Sholl analysis method for identifying the length of dendrites of newborn neurons in the hippocampus are as follows: Fig. 9 .

[0237] Depend on Fig. 9 It can be seen that the average length of dendrites in the WT+VEH; KO+VEH; WT+8D11,13d; KO+8D11,13d groups are: 2994.35μm; 1101.84μm; 3528.39μm; 3604.87μm. The experimental results show that the length of dendrites of newborn neurons in Fmr1 KO mice is shorter than that of newborn neurons in WT mice, and 8-deoxy-11,13-dihydroxyguaiac lactone can significantly increase the length of dendrites of newborn neurons in Fmr1 KO mice. Compared with WT+VEH, the length of dendrites of newborn neurons in Fmr1 KO mice given 8-deoxy-11,13-dihydroxyguaiac lactone was restored to the level of dendrites of newborn neurons in the hippocampus of WT mice.

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

[0239] The Sholl analysis method was used to analyze the dendritic nodes of newborn neurons in the hippocampus. The statistical analysis results of the Sholl analysis method for identifying the number of dendritic nodes of newborn neurons in the hippocampus are as follows: Fig.10 .

[0240] Depend on Fig.10 It can be seen that the average number of dendritic nodes in the WT+VEH; KO+VEH; WT+8D11,13d; KO+8D11,13d groups is 17.43; 6.17; 19.36; 20.74; The experimental results show that the newborn neurons of Fmr1 KO mice have fewer dendritic nodes than those of WT mice. The intervention of 8-deoxy-11,13-dihydroxyguaiac lactone can significantly increase the number of dendritic nodes of newborn neurons in Fmr1 KO mice. Compared with WT+VEH, the number of dendritic nodes of newborn neurons in the hippocampus of Fmr1 KO mice given 8-deoxy-11,13-dihydroxyguaiac lactone was restored to the level of the number of dendritic nodes of newborn neurons in the hippocampus of WT mice.

[0241] c) Effects of 8-deoxy-11,13-dihydroxyguaiac lactone on the number of dendritic terminals of newborn neurons in WT and Fmr1 KO mice

[0242] The Sholl analysis method was used to identify the dendritic terminals of newborn neurons in the hippocampus. The results of the statistical analysis of the number of dendritic terminals of newborn neurons in the hippocampus were as follows: Fig.11 .

[0243] Depend on Fig.11 It can be seen that the average number of dendritic terminals in the WT+VEH; KO+VEH; WT+8D11,13d; KO+8D11,13d groups is 22.66; 9.94; 24.75; 25.48 respectively. The experimental results show that the newborn neurons of Fmr1 KO mice have fewer dendritic terminals than those of WT mice. The intervention of 8-deoxy-11,13-dihydroxyguaiac lactone can significantly increase the number of dendritic terminals of newborn neurons in Fmr1 KO mice. Compared with WT+VEH, the number of dendritic terminals of Fmr1 KO mice given 8-deoxy-11,13-dihydroxyguaiac lactone was restored to the level of the number of dendritic terminals of newborn neurons in the hippocampus of WT mice.

[0244] d) Using the MEA neuroelectrophysiological monitoring system, primary cultured hippocampal newborn neurons were cultured on MEA chips to explore the differences in their spontaneously formed neural networks.

[0245] Depend on Fig.12 , 13 It can be seen that the average firing frequencies of the neuronal networks in the WT+VEH; KO+VEH; WT+8D11,13d; KO+8D11,13d groups are: 0.54; 0.19; 0.47; 0.48; Fig.14 It can be seen that the average discharge duration of the neuronal network in the WT+VEH; KO+VEH; WT+8D11,13d; KO+8D11,13d groups is: 0.12; 0.07; 0.11; 0.11; The experimental results show that the average discharge frequency and discharge duration of the newborn neuronal network of Fmr1 KO mice are lower than those of WT mice. After intervention with 8-deoxy-11,13-dihydroxyguaiac lactone, the average discharge frequency and discharge burst duration of the newborn neuronal network of Fmr1 KO mice given 8-deoxy-11,13-dihydroxyguaiac lactone can be significantly increased. Compared with WT+VEH, the average discharge frequency and discharge burst duration of the newborn neuronal network of Fmr1KO mice given 8-deoxy-11,13-dihydroxyguaiac lactone were restored to similar levels of newborn neurons in the hippocampus of WT mice.

[0246] Example 4 Cognitive function regulation test

[0247] Animal cognitive function detection methods have been used to prove that 8-deoxy-11,13-dihydroxyguaiac lactone can correct cognitive dysfunction and social interaction disorders in Fmr1 gene-deficient mice (Fmr1 KO). In order to further study whether the improvement effect of 8-deoxy-11,13-dihydroxyguaiac lactone on cognitive impairment functions by promoting adult neurogenesis, the present invention uses temozolomide (TMZ), a neurogenesis-specific blocker, for intervention. The results showed that TMZ can block the therapeutic effect of 8-deoxy-11,13-dihydroxyguaiac lactone on cognitive dysfunction in Fmr1 gene-deficient mice (Fmr1 KO). The results of this study show that 8-deoxy-11,13-dihydroxyguaiac lactone can treat cognitive dysfunction in mice with fragile X syndrome by regulating neurogenesis.

[0248] 1. New location recognition experiment

[0249] The new location recognition experiment is a classic behavioral test for detecting the spatial memory ability of mice. In order to explore whether 8-deoxy-11,13-dihydroxyguaiac lactone can correct the spatial memory ability of mice with fragile X syndrome, mice were treated with 8-deoxy-11,13-dihydroxyguaiac lactone by intraperitoneal injection, and then the new location recognition experiment was tested. In order to further study whether the improvement of spatial memory by 8-deoxy-11,13-dihydroxyguaiac lactone functions by promoting adult neurogenesis, neurogenesis-specific blocker temozolomide (TMZ) was used to block neurogenesis in advance, and 8-deoxy-11,13-dihydroxyguaiac lactone was co-administered, and then the new location recognition experiment was tested.

[0250] 1.1 Materials:

[0251] A square open field: object A, object B, facial tissue, 75% alcohol, and two timers.

[0252] 1.2 Experimental 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 Carbosons: XD170631;

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

[0256] 1.2 Experimental groups and drug administration

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

[0258] 1.2.2 Administration:

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

[0260] ②: WT+8D11,13d, KO+8D11,13d groups: 7-8 week old Fmr1 KO and WT mice were intraperitoneally injected with 8-deoxy-11,13-dihydroxyguaiac lactone once a day for 7 consecutive days at a dose of 20 mg / kg.

[0261] ③: WT+8D11,13d / TMZ, KO+8D11,13d / TMZ groups: 6-week-old Fmr1 KO and WT mice were intraperitoneally injected with temozolomide (TMZ) once a day in the 6th, 7th, 8th and first three days of the 9th week, and with 8-deoxy-11,13-dihydroxyguaiac lactone (8D11,13d) once a day in the 8th week for 7 consecutive days. The dosage on 8D11 and 13d was 20 mg / kg.

[0262] 1.3 Experimental procedures

[0263] The 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 brought to the behavioral testing laboratory and adapted for at least 1 hour. In the training phase, each animal was placed individually in a square field, and two identical objects were placed at the same distance from a side of colored wallpaper. The mice were allowed to explore freely for 6 minutes, and the training process was repeated 3 times. In the test phase, the location of one of the familiar objects was moved to another new location. The exploration time of the experimental animals on the new and old objects in the 6 minutes was recorded. A normal animal should spend more time exploring the objects 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. In the test phase, the exploration of the object was defined as any exploratory behavior within 1 cm of the animal's nose from the object, including head direction, climbing, sniffing, etc. The calculation method of the discrimination index is: discrimination index = (new location object recognition time / total recognition time × 100)-(original location object recognition time / total recognition time × 100).

[0265] The schematic diagram of the new position recognition experiment is as follows Fig.15 After each mouse test, the field needs to be wiped clean with 75% alcohol to eliminate the influence of the same odor interference on the test behavior of the next experimental mouse.

[0266] 1.4 Experimental Results

[0267] The data were analyzed by two-way analysis of variance (ANOVA) using GrapaPad Prism software. The data processing results were expressed as Mean±SEM. When P<0.05, it was considered to be statistically significant.

[0268] The discrimination indexes of WT+Vehicle; KO+Vehicle; WT+8D11,13d; KO+8D11,13d; WT+8D11,13d / TMZ; KO+8D11,13d / TMZ groups were: 38.60%; -29.77%; 20.70%; 19.40%; -20.50%; -22.30%. The statistical results of the mouse novel location recognition experiment are shown in Figure 2. Fig.16 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 old objects, which indicates that the spatial memory ability of Fmr1 KO mice is defective, and 8-deoxy-11,13-dihydroxyguaiac lactone can correct this defect after administration, while TMZ can block the improvement of spatial memory of Fmr1 KO mice by 8-deoxy-11,13-dihydroxyguaiac lactone. This indicates that 8-deoxy-11,13-dihydroxyguaiac lactone can improve the spatial memory of 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 objects. This experiment examined the novel object recognition ability of mice and was used to explore whether 8-deoxy-11,13-dihydroxyguaiac lactone could correct the learning and memory impairment of fragile X syndrome mice by regulating adult neurogenesis.

[0272] 2.1 Experimental Materials

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

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

[0275] 2.2 Experimental groups and drug administration

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

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

[0278] 2.3 Experimental procedures

[0279] The test measures novel object recognition, a form of memory task that does not rely on spatial cues. In this task, animals are trained to recognize specific objects.

[0280] like Fig.17 First, the experimental animals were brought to the behavioral testing laboratory and adapted for at least 1 hour. During the training phase, each animal was placed individually in an L-shaped field with corridors of the same length. Two identical objects were placed at the end of the corridors on both sides and were allowed to explore for 10 minutes. During the test phase, one of the familiar objects was replaced with a new object, and the exploration time of the experimental animals on the new and old 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 test phase, the detection of objects was defined as any exploratory behavior within 1 cm of the animal's nose from the object, including head direction, climbing, sniffing, etc. The calculation method of the discrimination index is: discrimination index = (strange 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 pure water to avoid affecting the results of subsequent mouse experiments.

[0282] 2.4 Experimental Results

[0283] The data were analyzed by two-way analysis of variance (ANOVA) using GrapaPad Prism software. The data processing results were expressed as Mean±SEM. When P<0.05, it was considered to be statistically significant.

[0284] The results of the mouse novel object recognition experiment are as follows Fig.18 The discrimination indexes of WT+Vehicle; KO+Vehicle; WT+8D11,13d; KO+8D11,13d; WT+8D11,13d / TMZ; KO+8D11,13d / TMZ groups were: 25.50%; -23.9%; 16.57%; 20.38%; -9.53%; -26.78%, respectively.

[0285] The results showed that WT mice were more inclined to explore new objects, while Fmr1 KO mice showed the opposite behavior, indicating that Fmr1 KO mice had defects in learning and memory, which could be corrected by administration of 8-deoxy-11,13-dihydroxyguaiac lactone. TMZ could block the learning and memory improvement effect of 8-deoxy-11,13-dihydroxyguaiac lactone on Fmr1 KO mice.

[0286] From the above experiments, it can be seen that 8-deoxy-11,13-dihydroxyguaiac lactone can improve the memory of fragile X syndrome model mice by regulating adult neurogenesis.

[0287] 3. Social interaction experiment

[0288] Social interaction disorder is one of the most prominent manifestations of autism. This study used a three-box interactive social behavior test to test whether 8-deoxy-11,13-dihydroxyguaiac lactone can correct the social behavior defects of fragile X syndrome model mice by regulating neurogenesis.

[0289] 3.1 Experimental Materials

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

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

[0292] 3.2 Experimental groups and drug administration

[0293] 3.2.1 Experimental groups: WT+Vehicle: WT mice were intraperitoneally injected with phosphate buffered saline; KO+Vehicle: Fmr1KO mice were intraperitoneally injected with phosphate buffered saline; WT+8D11,13d: WT mice were intraperitoneally injected with 8-deoxy-11,13-dihydroxyguaiac lactone; KO+8D11,13d: Fmr1KO mice were intraperitoneally injected with 8-deoxy-11,13-dihydroxyguaiac lactone; WT+8D11,13d / TMZ: WT mice were intraperitoneally injected with temozolomide and 8-deoxy-11,13-dihydroxyguaiac lactone; KO+8D11,13d / TMZ: Fmr1KO mice were intraperitoneally injected with temozolomide and 8-deoxy-11,13-dihydroxyguaiac lactone, 8 mice in each group (i.e., 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] The test is to detect changes in the social behavior of rodents, which are naturally gregarious and have a tendency to explore new objects. The social behavior test device is a three-chamber box with a partition wall made of black plexiglass and a small round door with a diameter of 7 cm inside. The animals can move freely in the three boxes.

[0297] like Fig.19 First, the experimental animals were brought to the behavioral testing laboratory and adapted for at least 1 hour. The experiment consisted of three phases: habituation, social interaction test, and social novelty recognition. In the test phase, the experimental animals were first placed in the middle box and allowed to freely explore the three chambers of left, middle, and right for 10 minutes; next, a strange animal was placed in the wire cage on the left side of the chamber, and a toy was placed in the wire cage on the right side of the chamber. In the social interaction test, the exploration time of the experimental animals on the toy and the strange animal was recorded within 10 minutes. In the social novelty recognition test, the animal that had interacted before remained unchanged (left side), and the toy in the right side of the chamber was replaced by a new strange animal (right side). The exploration time of the experimental animals on the familiar animal (left side, from the previous social interaction phase) and the strange animal (right side, the newly replaced animal) was recorded within 10 minutes. The discrimination criterion was calculated based on the difference between the percentage of time spent recognizing the strange animal and the time spent recognizing the toy (or recognizing the familiar animal). During the test phase, detection of an object or another animal was defined as any exploratory behavior in which the animal's nose was 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 pure water to avoid affecting the results of subsequent mouse experiments.

[0299] 3.4 Experimental Results

[0300] The data were analyzed by two-way analysis of variance (ANOVA) using GrapaPad Prism software. The data processing results were expressed as Mean ± SEM. When P < 0.05, it was considered to be statistically significant.

[0301] The results of the mouse social interaction experiment are statistically shown as follows Fig. 20 The discrimination indexes of WT+Vehicle; KO+Vehicle; WT+8D11,13d; KO+8D11,13d; WT+8D11,13d / TMZ; KO+8D11,13d / TMZ groups were: 29.15%; -16.06%; 38.71%; 33.80%; -15.55%; -17.85%, respectively.

[0302] The experimental results showed that WT mice were more inclined to interact with new mice, while Fmr1 KO mice were more inclined to interact with old mice, indicating that Fmr1 KO mice had defects in social ability, and 8-deoxy-11,13-dihydroxyguaiac lactone administration could correct this defect. TMZ could block the improvement of social interaction of Fmr1 KO mice by 8-deoxy-11,13-dihydroxyguaiac lactone. It was concluded that 8-deoxy-11,13-dihydroxyguaiac lactone could correct the social interaction disorder of Fragile X syndrome model mice by regulating neurogenesis.

[0303] From the above experiments, it can be seen that 8-deoxy-11,13-dihydroxyguaiac lactone can correct the social defects of fragile X syndrome model mice by regulating neurogenesis.

Claims

1. Use of guaiac lactone or its derivatives in the preparation of products for the treatment and prevention of hereditary intellectual and cognitive dysfunction diseases; The hereditary intelligence and cognitive dysfunction diseases include at least one of autism spectrum disorder, X-chromosome linked intellectual disability, global developmental delay, global mental retardation, attention deficit hyperactivity disorder, and epilepsy.

2. The use according to claim 1, characterized in that: The guaiac lactone or its derivative includes at least one of 8-deoxy-11,13-dihydroxyguaiac lactone, 12,6-guaiac lactone, 12,8-guaiac lactone and pseudoguaiac lactone.

3. The use according to claim 2, characterized in that: The derivatives include pharmaceutically acceptable salts or pharmaceutically acceptable chemical modifications.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid; and / or The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

5. The use according to claim 1, characterized in that: The X-chromosome linked intellectual disability includes fragile X syndrome.

6. The use according to claim 1, characterized in that: The products include pharmaceuticals.

7. The use according to claim 6, characterized in that: The drug includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

9. The use according to claim 8, characterized in that: The dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration; Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

10. The use according to any one of claims 1 to 9, characterized in that: The product is administered to a mammal; Preferably, the mammal comprises a human.

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