Use of gingerol compounds for the preparation of a product for the treatment of a genetic intellectual and cognitive function disorder

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a gingerol compound in preparation of a product for treating genetic intelligence and cognitive dysfunction diseases. The application proves that 6-gingerol can improve neurogenesis of fragile X chromosome syndrome, correct cognitive defects, enriches scientific connotation of the 6-gingerol in application of neurodevelopmental disorders and in disease treatment, and is suitable for industrial production and easy to popularize. The application provides a new drug source for treating fragile X chromosome syndrome and related diseases caused by changes and mutations of X chromosomes in a genetic process in a human body, and provides a new medicinal use of the gingerol compound.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of gingerol compounds in the preparation of products for treating hereditary intellectual and cognitive impairment diseases. Background Technology

[0002] Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by point mutations, deletions, or duplications of the 5′ untranslated region CGG trinucleotide in the fragile X mental retardation 1 (FMR1) gene located at Xq27.3, which lead to methylation of adjacent CpG islands and result in low or no expression of the encoded FMRP (fragile X mental retardation 6 Gotein, FMRP).

[0003] The absence of FMRP leads to neurological abnormalities and cognitive impairment, with alterations occurring in neural stem (progenitor) cells during brain development. Patients with Fragile X syndrome primarily exhibit intellectual and reproductive system abnormalities, and deficits in short-term memory, executive function, visual memory, visuospatial processing, and social skills. Their IQ is typically only 40% of normal, severely impacting both the patient and their family providing lifelong care.

[0004] The absence of FMRP leads to impaired dendritic morphology, reduced synaptic formation, and abnormal circuit integration in neurons. Studies on FXS patients and Fmr1 KO mice or fruit fly animal models have revealed abnormalities in the number of dendritic branches and dendritic spine density in cortical neurons of Fmr1 KO mice; defects in synaptic plasticity and neurite extension in neonatal hippocampal neurons of Fmr1 KO mice; and impaired dendritic maturation and spinal maturation in adult hippocampal neoneurons of Fmr1 KO mice. In contrast, neurons in FXS patients exhibit immature dendritic spines and impaired neurite extension.

[0005] Currently, there is still a lack of effective treatments for FXS in clinical practice, and most treatments for FXS are based on its specific symptoms. This also leads to a lack of sufficient controlled trials to prove the effectiveness of these methods. Only by combining psychopharmacological interventions with other supportive strategies, including speech therapy, sensory integration and occupational therapy, personalized education programs, and tailored behavioral interventions, can the therapeutic effect be maximized.

[0006] Stimulants are currently the most common medications used to treat FXS, but their effectiveness and side effects vary from person to person. While stimulants are effective for the symptoms of hyperactivity, impulsivity, and inattention in young boys with FXS, adult men with FXS often respond poorly to them.

[0007] Neurological diseases are characterized by extremely complex physiological and pathological processes, poor prognosis, and a lack of effective treatments. Formosan syndrome (FXS) is a single-gene genetic disorder that has attracted significant attention in recent years, second only to Down syndrome in incidence among hereditary intellectual disability disorders. The intellectual disability caused by FXS inflicts immense physical and psychological suffering on patients and their families, and the prolonged course of the disease places a heavy burden on families and society. Maximizing treatment effectiveness requires a combination of psychopharmacological interventions and other supportive strategies, including speech therapy, sensory integration and occupational therapy, personalized educational programs, and tailored behavioral interventions.

[0008] The most famous classical theory of pathophysiology in FXS is the mGluR theory. Various functional disorders observed in the behavioral, electrophysiological, and molecular systems of FXS are presumed to be due to excessive glutamatergic signaling via mGluR. Developmental abnormalities of dendritic spines in Fragile X syndrome are associated with overactivation of mGluR5 signaling. Simultaneously, mGluRs can increase FMRP synthesis, meaning that FMRP, as a downstream molecule of mGluR, can negatively regulate mGluR activity. Therefore, the classical mGluR theory of FXS posits that the loss of FMRP increases the synthesis of related proteins, leading to excessive internalization of AMPA receptors, overactivation of mGluR-mediated LTD, and thus resulting in FXS-related characteristics. It has been demonstrated in mouse and Drosophila models that inhibiting the mGluR5 signaling pathway can rescue protein synthesis and some FXS characteristics.

[0009] Numerous preclinical data suggest that antagonists of the metabolic glutamate receptor 5 (mGluR5) may be effective in treating patients with FXS, and several preclinical pharmacological studies have used mGluR5 antagonists for treatment. Fenobam, a non-benzodiazepine anxiolytic, has been studied in FXS due to its negative allosteric regulation of mGluR5. However, it has been shown to cause hallucinations, dizziness, paresthesia, and insomnia in normal individuals. Mavoglurant / AFQ056, a non-competitive mGluR5 antagonist used for therapeutic blocking of mGluR5, was evaluated for the treatment of behavioral symptoms of FXS. Significant improvements were observed in stereotyped behaviors, ADHD, and inappropriate speech, but these findings were not adequately measured on any standardized scales. Furthermore, in a clinical study of 30 men with FXS who received Mavoglurant for 28 days, 24 experienced some form of adverse event, most of which were mild to moderate in severity. Finally, development of mavoglurant for FXS was discontinued following negative results from a Phase III study. Basimglurant / RO4917523 is a novel mGlu5 negative allosteric modulator that has been studied in animal models of depression, improving arousal and anxieties. A preclinical Phase II trial showed a trend toward clinical improvement.

[0010] However, to date, there are still no effective treatments that can cure or alleviate the main adverse symptoms of this disease. Therefore, exploring the etiology and mechanisms of Fragile X syndrome and developing new therapeutic drugs is urgently needed.

[0011] Fresh ginger is the fresh rhizome of the ginger plant (Zingiber officinale), while dried ginger is the dried rhizome. 6-Gingerol is the active ingredient and indicator component of ginger. It has been reported to have beneficial effects on cholesterol metabolism, regulating plasma cholesterol levels, mediating cellular cholesterol endocytosis, promoting intracellular cholesterol breakdown, and thus lowering intracellular cholesterol. 6-Gingerol can also regulate cholesterol metabolism by activating SREBP2 to upregulate LDLR and by upregulating cholesterol efflux-related genes LXRα and ABCA1. Furthermore, 6-Gingerol can cross the blood-brain barrier well, possessing a wide biosafety window, and is currently used in research on brain diseases such as brain injury, Alzheimer's disease, Parkinson's disease, and epilepsy.

[0012] However, there are currently no reports on the use of 6-gingerol in the treatment of FXS. Summary of the Invention

[0013] The primary objective of this invention is to address the problems existing in the prior art by providing a new application of gingerol compounds in the treatment of hereditary intellectual and cognitive disorders, particularly in drugs for fragile X syndrome, and to provide new pharmaceutical uses for gingerol compounds (especially 6-gingerol).

[0014] The first aspect of this invention aims to provide the application of gingerol compounds in the preparation of products.

[0015] To achieve the above-mentioned objectives of this invention, the technical solution adopted is as follows:

[0016] In a first aspect, the invention provides the use of gingerol compounds in the preparation of products for the treatment and prevention of hereditary intellectual and cognitive impairment diseases.

[0017] 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.

[0018] 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.

[0019] 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).

[0020] More preferably, the X-chromosome-linked intellectual disability is Fragile X syndrome.

[0021] Preferably, the gingerol compound includes at least one of 6-gingerol, 8-gingerol, 10-gingerol, 12-gingerol, or methyl-6-gingerol.

[0022] More preferably, the gingerol compound is 6-gingerol. The molecular formula of 6-gingerol is C0. 17 H 26 O4, CAS number 23513-14-6.

[0023] Preferably, the purity of the gingerol compound is ≥60%, more preferably over 90%, and even more preferably over 95%.

[0024] Preferably, the purity of the 6-gingerol is ≥60%, more preferably over 90%, and even more preferably over 95%.

[0025] Preferably, the gingerol compounds include pharmaceutically acceptable derivatives.

[0026] Preferably, the pharmaceutically acceptable derivative includes a pharmaceutically acceptable salt and a pharmaceutically acceptable chemical modification.

[0027] 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.

[0028] Preferably, the metal salt includes alkali metal salts and alkaline earth metal salts.

[0029] Preferably, the alkali metal salt includes at least one of sodium and potassium salts.

[0030] Preferably, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Preferably, the salt formed with the basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0035] 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.

[0036] 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.

[0037] Preferably, the product comprises a drug; the drug comprises pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

[0038] 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.

[0039] This invention provides a drug and corresponding dosage form for treating fragile X syndrome, using gingerol compounds (especially 6-gingerol) as active ingredients.

[0040] The drug is present in the form of an oral preparation, an injection, or a topical preparation.

[0041] 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.

[0042] The pharmaceutical preparation uses gingerol compounds (especially 6-gingerol) as the active ingredient and includes other pharmaceutically acceptable carrier components.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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*.

[0047] The 6-gingerol described in this invention can be used alone or in the form of a pharmaceutical composition containing 6-gingerol when used to treat fragile X syndrome.

[0048] Preferably, the dosage form of the product includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0049] 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.

[0050] 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.

[0051] Preferably, the product is applied to mammals.

[0052] Preferably, the mammal includes humans.

[0053] The beneficial effects of this invention are:

[0054] 1. This invention clarifies that 6-gingerol can improve neurogenesis and correct cognitive deficits in fragile X syndrome, enriches the application of 6-gingerol in neurodevelopmental disorders and its scientific connotation in disease treatment, provides a reference for the therapeutic effects of small active molecules of traditional Chinese medicine, and provides ideas for the research of target drugs in the treasure trove of traditional Chinese medicine.

[0055] 2. This invention uses 6-gingerol to conduct in vitro dendritic complexity experiments of newly formed neurons and neural stem cell proliferation and differentiation experiments. The results show that 6-gingerol intervention can significantly promote the dendritic complexity of newly formed hippocampal neurons from Fmr1 KO mice cultured in vitro, increasing dendritic length, number of dendritic nodes and number of dendritic terminals. It can also increase the differentiation of neural stem cells and correct their abnormal proliferation.

[0056] 3. The cognitive function experiments of Fmr1 gene-deficient animals (Fmr1 KO mice) demonstrated in this invention that 6-gingerol can correct cognitive and social interaction impairments in Fmr1 KO mice; improve spatial memory and pattern separation cognitive deficits in Fmr1 KO mice by regulating neurogenesis; improve learning and memory in Fmr1 KO mice by regulating adult neurogenesis; and correct and improve social interaction impairments in Fmr1 KO mice by regulating neurogenesis.

[0057] 4. The 6-gingerol of this invention has strong pharmacological effects and is significantly effective in treating fragile X syndrome. It is fast-acting, has few toxic side effects, is safe, and can be taken for a long time, showing good pharmaceutical prospects.

[0058] 5. The raw materials of the present invention are abundant and inexpensive, safe for clinical use, and have a simple preparation process. They can be made into various dosage forms, and the dosage is small and convenient to use, so they are easy to promote. Attached Figure Description

[0059] Figure 1 A schematic diagram of the experimental protocol for regulating the abnormal proliferation of adult neural stem cells in the brain of adult mice using 6-gingerol.

[0060] Figure 2 Newly generated mature neurons (NeuN) in the DG region of WT / Fmr1 KO mice + BrdU + Representative micrographs; Immunostaining of neuronal markers NeuN (green) and BrdU (red) to assess the effect of 6-gingerol on neuronal differentiation in Fmr1 KO mice; Scale bar: 20 μm.

[0061] Figure 3 This is a graph showing the data analysis results of neural stem cells in the proliferating state during a neural stem cell proliferation experiment.

[0062] Figure 4 This is a schematic diagram of an experimental protocol for regulating the differentiation of new neurons from neural stem cells in the brain of adult mice using 6-gingerol.

[0063] Figure 5 Adult neural stem cells (MCM2) proliferating in the DG region of adult WT / Fmr1 KO mice + GFP + GFAP + Representative micrographs, where blue: DAPI; green: GFP; red: MCM2; gray: GFAP; scale bar: 20 μm.

[0064] Figure 6 This is a graph showing the results of data analysis on newly generated and mature neurons in a neural stem cell differentiation experiment.

[0065] Figure 7 This is a schematic diagram of the experimental protocol for Sholl analysis of primary hippocampal neurons.

[0066] Figure 8 Microscopic observation of P0-type hippocampal neurons cultured in vitro.

[0067] Figure 9 Sholll analysis statistical plot of dendritic complexity in primary hippocampal neurons.

[0068] Figure 10 Sholl analysis of dendritic length in primary hippocampal neonatal neurons.

[0069] Figure 11 Shollen analysis chart of the number of dendritic nodes in primary hippocampal neonatal neurons.

[0070] Figure 12 Sholl analysis of the number of dendritic terminals of newly formed neurons in the primary hippocampus.

[0071] Figure 13 This is a schematic diagram of a mouse behavioral experiment protocol.

[0072] Figure 14 This is a schematic diagram of a mouse new location recognition experiment.

[0073] Figure 15 A statistical graph of the discriminant index for the mouse new location recognition experiment.

[0074] Figure 16 This is a schematic diagram of the L-maze experiment in mice.

[0075] Figure 17 This is a statistical graph of the discriminant index in the L-maze test for mice.

[0076] Figure 18 This is a schematic diagram of a mouse model separation experiment.

[0077] Figure 19 This is a statistical graph of the discriminant index for the mouse model separation experiment.

[0078] Figure 20 This is a schematic diagram of a mouse social interaction experiment.

[0079] Figure 21 This is a statistical chart of the discriminant index for the social interaction experiment in mice. Detailed Implementation

[0080] 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.

[0081] The experimental animals used in the specific embodiments of this invention are as follows:

[0082] Wild-type male (WT, or Fmr1) + / y Mice and female Fmr1 gene knockout (KO) mice (Fmr1 KO, or Fmr1...) - / - The transgenic mice containing nestin green fluorescent protein (Nestin-GFP) were purchased from the Jackson Laboratory in the United States.

[0083] Fmr1 gene knockout (KO) can be represented by Fmr1 KO or simply Fmr1. - / - (Female mouse) / Fmr1 - / y (Male rat). Wild-type (WT), can be represented by WT or Fmr1. + / + (Female mouse) / Fmr1 + / y (Male rat)

[0084] Fmr1 HET(Fmr1 + / - (Female mice, heterozygotes are Fmr1; HET also uses Fmr1) + / - express.

[0085] Male nestin-green fluorescent protein (Nestin-GFP) transgenic mice and Fmr1 HET female mice (Fmr1 + / - Through mating and breeding, wild-type or Fmr1 gene-deficient transgenic mice carrying nestin green fluorescent protein (GFP) markers were obtained (Fmr1... + / y ::Nestin-GFP or Fmr1 - / y: (Nestin-GFP).

[0086] All mice were housed in a specific pathogen-free (SPF) animal housing at a temperature of 22-25°C, following a circadian rhythm of 12 hours of light / 12 hours of darkness. The mice had free access to water and food, and the process was approved and recognized by the Animal Ethics Committee of the University of Macau.

[0087] 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 due to 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 study only used adult wild-type and Fmr1 gene-deficient male mice as experimental samples.

[0088] The pharmaceuticals and reagents used in this invention are shown in Table 1; the instruments and equipment are shown in Table 2.

[0089] Table 1. Information on the Sources of Drugs and Reagents

[0090]

[0091] Table 2. Instrument and Equipment Information

[0092]

[0093] Example 1: In vivo neural stem cell proliferation and differentiation experiment

[0094] 1. Experimental Methods

[0095] 1.1 Laboratory Animals

[0096] The proliferation experiment used male wild-type or Fmr1 gene-deficient transgenic mice labeled with nestin green fluorescent protein (GFP). + / y ::Nestin-GFP or Fmr1 - / y: (Nestin-GFP).

[0097] 1.2 Mouse propagation method for neural stem cell proliferation experiment

[0098] Male nestin-green fluorescent protein (Nestin-GFP) transgenic mice and Fmr1 HET female mice (Fmr1 + / - Through mating and breeding, male wild-type or Fmr1 gene-deficient transgenic mice carrying nestin green fluorescent protein (GFP) markers were obtained (Fmr1... + / y ::Nestin-GFP or Fmr1 - / y: (Nestin-GFP). Administer the drug at 7-8 weeks of age for subsequent cell activation experiments.

[0099] 1.3 Experimental grouping, drug administration, and material collection

[0100] 1.3.1 Neural stem cell proliferation experiment

[0101] Seven- to eight-week-old male wild-type and Fmr1 gene-deficient transgenic mice marked with nestin green fluorescent protein (GFP) were fed.+ / y ::Nestin-GFP;Fmr1 - / y: (Nestin-GFP) mice were randomly divided into 4 groups, with 3 mice in each group. Each group of mice was administered the drug via intraperitoneal injection at a dose of 10 mg / kg. The same volume of blank solvent (PBS) was administered, calculated based on the 6-gingerol concentration. Intraperitoneal injections were administered once daily at the same time for 30 days. The specific details for the 4 groups are as follows:

[0102] Group A: Fmr1 + / y ::Nestin-GFP+ blank solvent group (WT+Veh); Group B: Fmr1 - / y ::Nestin-GFP+ blank solvent group (KO+Veh); Group C: Fmr1 + / y ::Nestin-GFP+6-gingerol group (WT+6G); Group D: Fmr1 - / y ::Nestin-GFP+6-gingerol group (KO+6G).

[0103] One day after the last administration, the brain was perfused and harvested. The brain sections were then prepared using a cryostat to obtain frozen sections for subsequent neural stem cell proliferation experiments. The experimental procedure is as follows: Figure 1 As shown.

[0104] 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. + and BrdU + Positive targets, the blue-DAPI marker is used to determine cell location, and NeuN is targeted after the scan. + and BrdU + Cell counting was performed on stained mouse brain slices to quantify positive targets, including green-NeuN. + Red-BrdU + Blue - DAPI.

[0105] 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, Figure 5 In the middle, DAPI represents all cells; BrdU labels represent proliferating new cells; NeuN labels represent mature neurons; green-NeuN... + Red-BrdU + Blue-DAPI + .

[0106] GFP + and GFAP + Double-positive cells represent neural stem cells, MCM2 + GFP + and GFAP + Triple-positive cells represent activated neural stem cells, and MCM2 was statistically analyzed using neuro-stereometry software. + GFP + GFAP + The number of triple-positive cells and GFP + GFAP + The ratio of double-positive cells indicates the proportion of activated cells (average of 3 tests).

[0107] 1.3.2 Neural stem cell differentiation experiment

[0108] Male wild-type mice (Fmr1) raised to 7-8 weeks of age + / y ) and Fmr1 KO mice (Fmr1 - / y Mice were randomly divided into 4 groups, with 3 mice in each group. Each group of mice received an intraperitoneal injection of 6-gingerol at a dose of 10 mg / kg. The blank solvent was prepared by calculating the volume of 6-gingerol at the administered concentration and administering the same volume of PBS. Intraperitoneal injections were administered once daily at the same time for 30 days. The specific details for the 4 groups are as follows:

[0109] Group E: Fmr1 + / y + Blank solvent group (WT+Veh); F group: Fmr1 - / y + Blank solvent group (KO+Veh); Group G: Fmr1 + / y +6-gingerol group (WT+6G); H group: Fmr1 - / y +6-gingerol group (KO+6G).

[0110] On days 15 and 16 of the 6-gingerol administration period, BrdU was injected intraperitoneally to label neural stem cells in the division phase (i.e., stem cells in a proliferative state). The dose was 200 mg / kg, twice daily for two days. Four weeks after the last BrdU injection, mice were anesthetized, and their brains were perfused and harvested. The entire hippocampus region of the mouse brain was serially sectioned at a thickness of 40 μm, and one section from every six sections was systematically and randomly selected for subsequent neural stem cell differentiation experiments. The procedure is as follows: Figure 4 As shown.

[0111] Immunohistochemical staining (IHC-Fr) was performed on frozen brain sections from four groups of mice. The sections were mounted in ascending order of hippocampal size and then sealed with an anti-fluorescence quencher to obtain stained brain sections from each group of mice. Next, a quantitative neurovisual analysis method was used. The stained brain sections were placed under an upright microscope, and the StereoInvestigator neurovisual software was used. Fluorescence channels were set for the DG region in the stained brain sections: blue: DAPI; green: GFP; red: MCM2; gray: GFAP. The brain sections were scanned according to the order on the slide. After scanning, the MCM2 was determined by the fluorescence staining through different channels. + GFP + and GFAP + Positive targets were identified, and cell counts were performed. The blue DAPI was used to determine the location and state of cells and to measure the positive targets in the stained mouse brain slices.

[0112] Representative images of neural stem cell activation obtained by scanning brain slices under a microscope are shown below. Figure 5 In the figure, blue represents DAPI; green represents GFP; red represents MCM2; gray represents GFAP; and the scale bar is 20 μm.

[0113] NeuN is a marker for mature stem cells, while BrdU marks neural stem cells in the division phase, i.e., neural stem cells that are in a proliferating state. + and BrdU + Double-positive cells represent neural stem cells newly differentiating into mature neurons. To further investigate the effect of 6-gingerol on neural stem cell differentiation in FXS model mice, this study used neurostereometry software to statistically analyze NeuN... + and BrdU + The number of cells reflects the degree of differentiation of neural stem cells into new neurons. The percentage of new neurons in different groups (NeuN) is also considered. + BrdU + / BrdU +NeuN was counted using Stereo Investigator neuro-somatic 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 newly generated mature neurons.

[0114] 2. Experimental Results

[0115] 2.1 Results of neural stem cell proliferation experiment

[0116] like Figure 3 As shown, Group A: Fmr1 + / y In the ::Nestin-GFP+ blank solvent group, the proportion of activated stem cells was 15.38%; Group B: Fmr1 - / y In the ::Nestin-GFP+ blank solvent group, the proportion of activated stem cells was 21.39%; in group C: Fmr1 + / y In the ::Nestin-GFP+6-gingerol group, the percentage of stem cells in an activated state was 13.41%; in group D: Fmr1 - / y The percentage of activated stem cells in the ::Nestin-GFP+6-gingerol group was 15.00%.

[0117] The experimental results showed that the proportion of activated stem cells in FXS model mice was increased compared with that in wild-type mice (p<0.05), but the proportion of activated stem cells in FXS model mice after drug administration was similar to that in wild-type mice (p>0.05). Therefore, 6-gingerol inhibited the abnormal activation of adult neural stem cells in Fmr1 KO mice.

[0118] Fragile X syndrome model mice exhibit abnormal activation of adult neural stem cells. 6-gingerol administration can correct this disease phenotype, indicating that 6-gingerol has certain therapeutic significance for fragile X syndrome and can be used to treat it.

[0119] 2.2 Results of Neural Stem Cell Differentiation Experiment

[0120] like Figure 6 As shown, Group E: Fmr1 + / y The percentage of newly formed mature neurons in the blank solvent group (WT+Veh) was 77.05±0.60%; in the F group: Fmr1 - / y The percentage of newly formed mature neurons in the blank solvent group (KO+Veh) was 69.42±1.38%; in the G group: Fmr1 + / yThe percentage of newly formed mature neurons in the +6-gingerol group (WT+6G) was 75.38±0.70%; in the H group: Fmr1 - / y The proportion of newly formed mature neurons in the +6-gingerol administration group (KO+6G) was 75.43±0.57%.

[0121] The data analysis above shows that the number of newly generated mature neurons in the blank solvent group (KO+Veh) of the FXS model was less than that in the wild-type blank solvent group (WT+Veh) (p<0.01). However, after treatment with 6-gingerol, the proportion of newly generated mature neurons in Fmr1 KO mice almost recovered to a level similar to that in the wild-type blank solvent group (p>0.05). This indicates that 6-gingerol can promote the generation of newly generated mature neurons in Fmr1 KO mice.

[0122] 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. 6-gingerol administration 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 6-gingerol has certain therapeutic significance for Fragile X syndrome and can be used to treat Fragile X syndrome.

[0123] Research on neural stem cells has become a hot topic in the life sciences, and the directed differentiation of neural stem cells into neurons is of great significance for the treatment and rehabilitation of nervous system diseases. This embodiment elucidates, at the whole animal level, that 6-gingerol improves the abnormal neurogenesis characteristics of FXS model mice by reducing proliferation and promoting the differentiation of stem cells into neurons. Furthermore, this administration method does not affect neurogenesis in normal mice.

[0124] As mentioned above, FMRP deficiency leads to abnormal proliferation of neural stem cells in the DG region, but reduces their differentiation into neurons and impairs hippocampus-dependent learning. Administration of 6-gingerol can restore normal proliferation and differentiation of neural stem cells, promote normal neurogenesis, and reduce the damage caused by FMRP deficiency.

[0125] Example 2: Effect of 6-gingerol on dendritic complexity of newly formed hippocampal neurons in Fmr1 KO mice

[0126] Dendrites are tree-like processes extending outward from the cytoplasm of nerve cells. Each dendrite is one or more processes originating from a neuron. They originate thicker at the beginning, branch repeatedly, and gradually taper to form a tree-like shape. The cell membrane surrounding the dendrites contains many receptors. Dendrites are important structures between neurons, and the complexity of dendritic branching affects the neuron's ability to receive and integrate synaptic information. Therefore, variations in the total length and structural complexity of dendrites may affect signal transmission and cellular function between neurons, thereby influencing cognitive function.

[0127] 1. Experimental Materials

[0128] 1.1 Laboratory Animals

[0129] When male wild-type mice and Fmr1 knockout mice reached maturity at eight weeks of age, female mice HET(Fmr1) were... - / + Mice were bred together with male WT mice at a ratio of 1:2. P0 day mice were selected, and the hippocampus of the brain was used for primary neuronal culture.

[0130] 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 due to 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 embodiment only uses adult wild-type and Fmr1 gene-deficient male mice as experimental samples.

[0131] 1.2 Cell Culture

[0132] Newly formed hippocampal neurons from WT and Fmr1 KO mice were cultured in a 5% CO2 incubator at 37°C.

[0133] 2. Experimental Methods

[0134] 2.1 Reagent Preparation

[0135] 1) Preparation of LB medium: Dissolve 10g NaCl, 10g Tryptone, and 5g Yeast extract in 1L of ultrapure water and autoclave before use.

[0136] 2) 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, and store at -20℃.

[0137] 3) 5M NaCl: Weigh 292.5g of NaCl, dissolve it in sterile ultrapure water, and dilute to 1L.

[0138] 4) Preparation of poly-Lysine: Dilute 10x poly-Lysine with sterile double-distilled water at a ratio of 1:9 to obtain 1x poly-Lysine, filter it through a 0.22μm microporous membrane to remove bacteria, and then store it at 4℃.

[0139] 5) Preparation of 6-gingerol: Prepare a 20mM 6-gingerol stock solution with DMSO, filter it through a 0.22μm microporous membrane to remove bacteria, and then store it at -20℃.

[0140] 6) 2×HEBS: Accurately weigh 1.6000g NaCl; 0.0740g KCl; 0.0213g Na2HPO4; 0.2000g D-Glucose; and 1.1900g Hepes (4-hydroxyethylpiperazine ethanesulfonic acid). Dissolve in sterile ultrapure water, adjust the pH to 7.05 with NaOH or HCl, add sterile ultrapure water to a final volume of 100mL, filter through a 0.22μm microporous membrane for sterilization, and then store at room temperature.

[0141] 7) CaCl2: Accurately weigh 29.4g CaCl2·2H2O and dilute to 100mL with sterile ultrapure water to prepare a 2M calcium chloride solution. Before use, filter the solution through a 0.22μm microporous membrane for sterilization and then store it at room temperature.

[0142] 8) 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.

[0143] 9) Preparation of SOC (Super Optimal broth with Catabolite re6Gession) 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) to the solution.

[0144] 10) 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.

[0145] 11) 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.

[0146] 12) Neuron-related culture medium

[0147] ① Sample preparation solution: DMEM-F12 + 1v / v% penicillin and streptomycin;

[0148] ② Seeding solution: DMEM-F12 + 10v / v% FBS + 1v / v% penicillin and streptomycin;

[0149] ③ Neuron culture medium: NeuroGro + 2v / v% B 27 +1 v / v% penicillin and streptomycin + 0.5 mM L-Glu;

[0150] ④ Digestive fluid: Trypsin-EDTA (0.25%), containing phenol red, to be used directly.

[0151] 2.2 Experimental grouping and drug administration

[0152] Group A: WT+Veh, WT type P0 hippocampal neurons +PBS;

[0153] Group B: KO+Veh, Fmr1 KO-type P0 hippocampal neurons +PBS;

[0154] Group C: WT+6G, WT type P0 hippocampal neurons + 6G with a final concentration of 1μM;

[0155] Group D: KO+6G, Fmr1 KO-type P0 hippocampal neurons + 6G at a final concentration of 1μM.

[0156] Two types of WT and Fmr1 KO neurons were subjected to blank treatment and drug treatment respectively. Blank treatment (blank group, A and B): WT+Veh group (group A), WT neurons + PBS were added to the culture medium; KO+Veh group (group B), Fmr1 KO neurons + PBS. Drug treatment (drug group, C and D): WT+6G group (group C), WT neurons + 6G with a final concentration of 1 μM were added to the culture medium; KO+6G group (group D), Fmr1 KO neurons + 6G with a final concentration of 1 μM.

[0157] 2.3 Plasmid Transformation

[0158] (1) Place DH5α competent E. coli cells and Rv-GFP plasmid in an ice bath; add 100 μL of DH5α competent cells and 1 μL of Rv-GFP plasmid to a 1.5 mL centrifuge tube, gently tap the tube to mix the plasmid with the competent cells; then incubate in an ice bath for 30 min, place the centrifuge tube in a 42℃ water bath for 60-90 s, and then quickly transfer the centrifuge tube to an ice bath for 2-3 min, being careful not to shake the centrifuge tube during this process. The Rv-GFP plasmid is then introduced into E. coli cells for large-scale amplification.

[0159] (2) Add 900 μL of SOC medium to another 15 mL centrifuge tube, then add the DH5α competent cells introduced with plasmid in step (1), mix well and place in a shaker at 37 °C (180 rpm, 90 min) to culture the competent cells introduced with plasmid.

[0160] (3) Take 100 μL of the culture mixture from step (2) 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 to observe colonies.

[0161] (4) 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.

[0162] (5) Pick up one single bacterial cell cultured in step (3) with a sterilized pipette tip and add it to the conical flask in step (4). Incubate at 37°C, shake at 200 rpm for 12-16 hours, then centrifuge (15 min, 4500 rpm, 4°C), discard the supernatant, keep the precipitate, and store the precipitate at -40°C for plasmid extraction.

[0163] 2.4 Plasmid Extraction

[0164] Plasmid extraction was performed using a high-purity plasmid extraction kit, following the kit's instructions and experimental methods, as follows:

[0165] (A) Add RNase A to solution P1 of the plasmid extraction kit before use, mix well, and store at 2-8℃.

[0166] (B) Add the mixed solution from step A) to the centrifuge tube containing the bacterial cell precipitate extracted in “3. Plasmid Transformation”, vortex to suspend the bacterial cell precipitate; add solution P2 from the plasmid extraction kit, mix immediately to fully lyse the bacterial cells, and incubate at room temperature for 5 min; then add solution P4 from the plasmid extraction kit to the centrifuge tube, mix immediately and thoroughly until a white, dispersed flocculent precipitate appears; then incubate at room temperature for 4-5 min, centrifuge (7800 rpm, 15 min) to allow the white precipitate to reach 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), slowly push the push handle to filter, and collect the filtrate in a clean 50 mL centrifuge tube.

[0167] (C) 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, and invert the centrifuge tube onto absorbent paper. Then add 70 v / v% ethanol to the centrifuge tube to rinse the precipitate thoroughly. Centrifuge at 7800 rpm for 15 min at 4℃, gently discard the supernatant, and invert the centrifuge tube onto absorbent paper. Repeat 1-2 times to rinse the precipitate thoroughly.

[0168] (D) Place the centrifuge tube 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.

[0169] The extracted plasmids should be labeled (date, plasmid type, concentration) and stored in a -20℃ refrigerator for later use.

[0170] 2.5 Neuron Culture

[0171] 1) Place the 14mm 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 more than 8 hours. Before the experiment, recover the coating solution and wash the smear with sterile ultrapure water 2-3 times.

[0172] 2) Dispense the sample solution from ① into labeled culture dishes and EP tubes, and pre-cool them on ice;

[0173] 3) Remove mice born within 0-24 hours (P0 day), disinfect with alcohol for 3 minutes, and then collect the tail tissue from the mice and store it in a 1.5 mL EP tube. Next, use fine scissors to remove the mouse heads. Male WT and Fmr1 KO mice were divided into four groups: WT+Veh, KO+Veh, WT+6G, and KO+6G, with 3 mice in each group (n=3), for a total of 12 mice. Specific groupings are shown in Table 3. The blank solvent was PBS; the 6G concentration was 1 Mm.

[0174] Table 3 Neuron Experimental Grouping

[0175]

[0176] 4) Obtaining the hippocampus: Disinfect the suckling mice with alcohol, cut off the tail and place it in an EP tube for genotyping. Separate the head with surgical scissors, remove the complete brain and place it in the dissection medium, and peel off the cerebral cortex. Take the hippocampal tissue and place it in the dissection medium. 5-5. Digestion: Digest with 0.25% Trypsin-EDTA for 5 minutes.

[0177] 5) Pipe: Gently pipette to form a single-cell suspension. 5-7. Seeding culture medium: Seed the cell culture medium into 24-well plates containing cell spreaders and incubate at 37°C in a 5% CO2 incubator.

[0178] 6) Medium change: After 4 hours of cell growth, change to NeuroGro neuronal basal medium containing B27, L-glutamine and penicillin and streptomycin. Then, change half of the medium every other day according to the cell growth status.

[0179] 2.6 Cell transfection and drug administration

[0180] 1) The experiment was divided into four groups: primary cultured hippocampal neurons of normal mice given blank solvent (WT+Veh), primary cultured hippocampal neurons of FXS model mice given blank solvent (KO+Veh), primary cultured hippocampal neurons of normal mice given 6-gingerol (WT+6G), and primary cultured hippocampal neurons of FXS model mice given 6-gingerol (KO+6G). The dosage of 6G was 1 μM, and it was administered once a day from the fourth day until the seventh day.

[0181] 2) The plasmid carrying GFP was transfected into mouse hippocampal neurons using calcium phosphate transfection technology. Calcium phosphate-DNA precipitate was prepared using 2×HEBS and CaCl2. The calcium phosphate particles were taken and evenly added to the cells, which were then placed in a 37°C incubator for one hour. After the uniformly distributed granular material formed on the cell surface, the culture medium containing serum was replaced, and the cells were returned to the 37°C incubator for further culture. The neurons were fixed on day 7 of culture.

[0182] 2.7 Neuron fixation, mounting, and dosing

[0183] After the neurons were cultured for seven days, the 24-well plates were 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 plates were then incubated at room temperature for 20 min to fix the cells. All liquid in the wells was aspirated, and 1 mL of 4 wt% paraformaldehyde was added to each well. The plates were then incubated at room temperature for 15 min to fix the cells. The liquid in the wells was then aspirated, and the cells were washed with PBS for 5 min each time, for a total of three times.

[0184] Remove the cell slide and place the side with cells onto a glass slide containing DAPI anti-fluorescence quencher; store in a cool, dark place and observe the next day.

[0185] GFP was observed under an upright microscope. + Changes in neuronal cell morphology ( Figure 8 The effect of 6G on the dendritic complexity of newly formed hippocampal neurons in WT or Fmr1 KO mice was analyzed. A stereoscopic analysis system was used to depict GFP+ newly formed neurons in real time, and a neuromorphological analysis system was used to quantify the neuronal morphological structure, specifically the dendritic length of GFP+ newly formed neurons. Figure 10 ), number of dendritic nodes ( Figure 11 ), number of dendritic ends ( Figure 12 Quantification of ).

[0186] 3. Experimental Results

[0187] Two-way ANOVA was performed using GraphPad 6Gism software, or multivariate ANOVA was performed using SPSS statistical analysis software. Threshold analysis was conducted. The results are expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0188] 3.1 Effects of 6-gingerol on the morphology of newly formed hippocampal neurons in WT and Fmr1 KO mice

[0189] Microscopic observation results of the morphology of newly formed neurons are as follows: Figure 8 As shown in the figure, compared with WT+Veh, the dendritic complexity of KO+Veh neurons is low, the dendritic length is short, and the dendritic branches are few. After administration of 6G, compared with KO+Veh, the dendritic complexity of KO+6G neurons is increased, the dendritic length is increased, and the number of dendritic branches is increased. Compared with WT+Veh, the morphology of KO+6G neurons can be restored to a level similar to that of WT-type mouse hippocampal neonatal neurons.

[0190] In in vitro cultured neurons, neurons were transfected using calcium phosphate transfection, and 6G was added during the culture process for drug intervention. Neurosomatic analysis was used to morphologically analyze neurons expressing green GFP fluorescence. Hippocampal neonatal neurons from Fmr1 KO mice showed significant morphological differences compared to WT mice; specifically, the dendritic network complexity of Fmr1 KO neurons was lower than that of WT neurons. After 6-gingerol 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 8 ).

[0191] Experimental results showed that 6G improved the dendritic morphology of newly formed hippocampal neurons from Fmr1 KO mice: 6G significantly promoted the dendritic complexity of newly formed hippocampal neurons from Fmr1 KO mice in vitro, including dendritic length, number of dendritic nodes, and number of dendritic terminals. (At least 20-35 GFPs from at least 3 different animals in each group) + (neurons).

[0192] 3.2 Effects of 6-gingerol on the complexity of newly formed hippocampal neurons in WT and Fmr1 KO mice

[0193] The complexity of newly formed hippocampal neurons in WT or Fmr1 KO mice was examined using Sholl analysis. A schematic diagram of the Sholl analysis results is shown below. Figure 8 As shown; the statistical analysis results of the Sholl analysis method for identifying the complexity of newly formed hippocampal neurons are as follows. Figure 9 As shown.

[0194] The experimental results showed that the total dendritic length of primary cultured hippocampal neonatal neurons in Fmr1 KO mice was significantly reduced, showing a significant difference compared with the normal group (p<0.001). 6-Gingerol could significantly increase the dendritic complexity of primary cultured hippocampal neonatal neurons in Fmr1 KO mice, reaching the normal level. There was no significant difference between the WT+Veh group and the KO+6G group (p>0.05). Under this dosage, the length of neurons in the normal group was not affected, and there was no significant difference between the WT+Veh group and the WT+6G group (p>0.05).

[0195] 3.3 Effects of 6-gingerol on dendritic length of hippocampal neonatal neurons in WT and Fmr1 KO mice

[0196] 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 10 As shown.

[0197] The total dendritic lengths of primary cultured hippocampal neonatal neurons in different groups were as follows: WT+Veh (1192±63.28 μm); KO+Veh (566.6±43.16 μm); WT+6G (1391±98.15 μm); KO+6G (1392±84.62 μm). The results showed that the dendritic length of neonatal neurons in Fmr1 KO mice was shorter than that in WT mice; however, 6G significantly increased the dendritic length of neonatal neurons in Fmr1 KO mice. Compared to WT+Veh, the dendritic length of neonatal neurons in Fmr1 KO mice treated with 6G recovered to the level of neonatal neurons in the hippocampus of WT mice.

[0198] 3.4 Effects of 6-gingerol on the number of dendritic nodes in newly formed neurons of WT and Fmr1 KO mice

[0199] The Sholl analysis method was used to identify the number of dendritic nodes in newly formed hippocampal neurons. The statistical analysis results of the Sholl analysis method are shown in the figure. Figure 11 ).

[0200] As shown in the figure, the number of dendritic nodes in primary cultured hippocampal newborn neurons of different groups were: WT+Veh (7.03±0.48); KO+Veh (3.38±0.34); WT+6G (6.70±0.58); and KO+6G (8.18±0.45). The experimental results indicate that newborn neurons from Fmr1 KO mice have fewer dendritic nodes than those from WT mice, and 6G administration significantly increases the number of dendritic nodes in Fmr1 KO mouse newborn neurons. Compared to WT+Veh, the number of dendritic nodes in hippocampal newborn neurons from Fmr1 KO mice treated with 6G recovered to the level of the number of dendritic nodes in hippocampal newborn neurons from WT mice.

[0201] 3.5 Effect of 6-gingerol on the number of dendritic terminals in newly formed neurons of WT and Fmr1 KO mice

[0202] 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 12 As shown.

[0203] As shown in the figure, the number of dendritic terminals in primary cultured hippocampal newborn neurons of different groups were: WT+Veh (11.82±0.69); KO+Veh (6.57±0.39); WT+6G (11.93±0.75); and KO+6G (13.30±0.60). The results indicate that Fmr1 KO mice have fewer dendritic terminals in their newborn neurons than WT mice, and 6G administration significantly increased the number of dendritic terminals in Fmr1 KO mice. Compared to WT+Veh, the number of dendritic terminals in Fmr1 KO mice treated with 6G recovered to the level of WT mouse hippocampal newborn neurons.

[0204] Example 3: Cognitive Function Modulation Experiment

[0205] This embodiment explores whether the effect of 6-gingerol in improving cognitive impairment in Fmr1 gene-deficient mice (Fmr1 KO) is mediated by improving neurogenesis, aiming to lay the foundation for clinical application. The study investigated whether the abnormal behavior of Fmr1 gene-deficient mice improved after administration of 6-gingerol, observing the therapeutic effect of 6-gingerol on mice. Temozolomide (TMZ), a drug that blocks neurogenesis, was used for intervention. The results showed that 6-gingerol can treat cognitive dysfunction in Fragile X syndrome mice by regulating neurogenesis, as detailed below.

[0206] 1. New Location Recognition Experiment

[0207] The novel location recognition test is commonly used to evaluate hippocampal-dependent spatial memory in mice. It assesses spatial memory based on mice's increased curiosity about novel objects. To explore whether 6-gingerol could correct spatial memory in a fragile X syndrome model mouse, mice were administered 6-gingerol via intraperitoneal injection, followed by a novel location recognition test.

[0208] To investigate whether the effect of 6-gingerol on improving spatial memory functions by promoting adult neurogenesis, neurogenesis was blocked in advance using the neurogenesis-specific blocker temozolomide, and then combined with 6-gingerol before a new location recognition experiment was conducted.

[0209] 1.1 Materials

[0210] A square open field: different objects (object A, object B), tissues, 75v / v% alcohol, and two timers.

[0211] 7-8 week old Fmr1 KO and WT mice.

[0212] 1.2. Experimental grouping and drug administration

[0213] 1.2.1 Experimental Groups: WT+Veh: WT mice were injected intraperitoneally with blank solvent; KO+Veh: Fmr1 KO mice were injected intraperitoneally with blank solvent; WT+6G: WT mice were injected intraperitoneally with 6-gingerol; KO+6G: Fmr1 KO mice were injected intraperitoneally with 6-gingerol; WT+6G / TMZ: WT mice were injected intraperitoneally with temozolomide and 6-gingerol; KO+6G / TMZ: Fmr1 KO mice were injected intraperitoneally with temozolomide and 6-gingerol. There were 6 groups in total, with 8-14 mice in each group (i.e., n = 8-14).

[0214] 1.2.2 Administration method:

[0215] ①: WT+Veh, KO+Veh group: 7-8 week old Fmr1 KO and WT mice were injected intraperitoneally once a day for 30 consecutive days with blank solvent.

[0216] ②: WT+6G, KO+6G group: 7-8 week old Fmr1 KO and WT mice were injected intraperitoneally once a day for 30 consecutive days at a dose of 10 mg / kg.

[0217] ③: WT+6G / TMZ, KO+6G / 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 at a dose of 50 mg / kg. In week 9, they were intraperitoneally injected once daily with 6-gingerol (6G) at a dose of 10 mg / kg for 7 consecutive days. A schematic diagram of the experimental mouse dosing regimen is shown below. Figure 13 As shown.

[0218] 1.3 Experimental Procedure

[0219] Spatial memory is measured by assessing the ability of rodents to recognize new locations of familiar objects based on spatial cues.

[0220] First, according to the experimental diagram of the new location recognition Figure 14 As shown, the new location recognition experiment consisted of three phases: pre-adaptation, training, and testing. First, numbered mice were placed in an open area for at least one hour to adapt. Then, identical objects were placed on the same side for training. During the training phase, each animal was placed individually in a square area, and two identical objects were 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. In the testing phase, one of the familiar objects was moved to a new location. The exploration time of the experimental animal for the two different locations was recorded within 6 minutes. A normal animal should spend more time exploring the new location. At the end of the experiment, the exploration time of the mice for the new and old object locations was analyzed, and the exploration index for the new location object was calculated. After each mouse was returned to its cage, the operating box was cleaned with 70% alcohol to eliminate the influence of similar odor interference on the behavior of the next experimental mouse. A camera was mounted directly above the experimental setup. During the testing phase, object detection was defined as any exploratory behavior of an animal within 1 cm of an object's nose, including head orientation, climbing, and sniffing. The discrimination index was calculated as follows: Discrimination Index = (Object recognition time at new location / Total recognition time × 100) - (Object recognition time at original location / Total recognition time × 100).

[0221] 1.4 Experimental Results

[0222] Data were analyzed using GraphPad 6Gism software in a two-way ANOVA. The results are expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0223] The exploration indices for different groups were WT+Veh (37.33±8.90)%, KO+Veh (-27.48±5.21)%, WT+6G (30.52±5.88)%, KO+6G (30.53±6.92)%, WT+6G+TMZ (-24.72±4.73)%, and KO+6G+TMZ (-37.37±8.90)%. The experimental statistical results are as follows: Figure 15 As shown.

[0224] 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, indicating that Fmr1 KO mice have a defective spatial memory. 6-Gingerol can improve spatial memory in Fmr1 KO mice by regulating neurogenesis, while TMZ can block the effect of 6-gingerol on improving spatial memory in Fmr1 KO mice. 6-Gingerol can improve spatial memory in FXS model mice, and its mechanism of action may be through improving neurogenesis.

[0225] 2. Pattern Separation Experiment

[0226] Pattern dissociation is a memory process, and impaired pattern dissociation function has been considered a predictor of cognitive impairment.

[0227] 2.1 Materials and Methods

[0228] A square open field; two objects A; two objects B, objects A and B are not the same; tissues; 75 v / v % alcohol; two timers.

[0229] 2.2 Grouping and Dosing

[0230] Same as in Example 1.2.

[0231] 2.3 Experimental Procedure

[0232] like Figure 18 As shown, during the training period, numbered mice were placed in an open area for a period of time. They were then placed individually in a first square area with a specific floor pattern and two identical objects (object A), and allowed to explore for 10 minutes. After 30 minutes, the same animal was placed in a second square area with a different floor pattern and two completely different sets of objects (object B), and allowed to explore for 10 minutes.

[0233] During the experiment, one of the two training floor patterns was randomly selected as the background. An object A and an object B were placed at the same location during the test. The interaction of each mouse with object A and object B in this scenario was recorded using a video recorder. The test time was 10 minutes. Afterward, the interaction of the mice was counted and analyzed using a timer. During the test phase, the detection of the object was defined as any exploratory behavior of the animal within 1 cm of the object's nose, including head orientation, climbing, sniffing, etc.

[0234] After each test, the mice were cleaned with purified water to avoid affecting the experimental results. The discrimination index was calculated using the formula: Discrimination Index = (Detection time of new object / Total detection time) - (Detection time of old object / Total detection time) × 100%, which determines the mice's preference for new and old objects.

[0235] 2.4 Experimental Results

[0236] Data were analyzed using GraphPad 6Gism software in a two-way ANOVA. The results are expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0237] The exploration indices for different groups were WT+Veh (22.49±4.96)%, KO+Veh (-20.54±4.89)%, WT+6G (19.15±5.60)%, KO+6G (24.26±6.13)%, WT+6G+TMZ (-36.73±6.28)%, and KO+6G+TMZ (-30.79±8.93)%. The statistical graph of the mouse model separation experiment results is shown below. Figure 19 As shown.

[0238] Experimental results showed that WT mice were more inclined to explore new objects, although the exploration index of the WT+6G group was slightly lower than that of the WT+Veh group. This may be due to certain stress responses induced by 6-gingerol administration, which in turn affected the mice's behavioral performance. Nevertheless, there was no statistically significant difference between the WT+6G and WT+Veh groups. Therefore, 6-gingerol administration did not cause adverse reactions in the animals. The opposite behavior was observed in Fmr1 KO mice, indicating that Fmr1 KO mice have a defective pattern separation, which can be corrected by 6-gingerol administration. TMZ blocked the pattern separation-improving effect of 6-gingerol on Fmr1 KO mice. This suggests that 6-gingerol can improve the pattern separation defect in Fmr1 KO mice by regulating neurogenesis, and its mechanism of action may be through improving neurogenesis, thereby improving the behavioral defects of Fragile X syndrome.

[0239] 3. New Object Recognition Experiment

[0240] The novel object recognition experiment is mainly used to study the ability of rodents to distinguish and remember objects in non-spatial and short-term conditions. It mainly evaluates the memory ability of mice based on their curiosity about new things.

[0241] 3.1 Experimental Materials

[0242] A square open field; object A; object B, which is different from object A; tissue paper; 75% v / v alcohol; two timers. Experimental animals and drugs are the same as in Example 1.1.

[0243] 3.2 Experimental grouping and drug administration

[0244] Same as in Example 1.2. There are 6 groups in total, with 7-14 animals in each group (i.e., n = 7-14).

[0245] 3.3 Experimental Procedure

[0246] The new object recognition experiment was conducted in three phases. First, numbered mice were placed in an open field for training. Second, identical objects were placed at both ends of an L-shaped maze, and the mice were placed in the middle for object recognition training. Then, the objects on one side of the L-shaped maze were replaced with new objects, and the mice were placed in the middle for testing. Figure 16 As shown. At the end of the experiment, the mice's interactions with new and familiar objects were analyzed, and their exploration index for new objects was calculated. After the experiment, the experimental apparatus was cleaned with 70% alcohol to prevent residual odor from affecting the behavior of the next mouse. 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).

[0247] 3.4 Experimental Results

[0248] Data were analyzed using GraphPad 6Gism software in a two-way ANOVA. The results are expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0249] The results of the mouse novel object recognition experiment are statistically presented as follows: Figure 17As shown, the exploration indices for different groups were WT+Veh (35.06±4.82)%, KO+Veh (-24.37±8.57)%, WT+6G (34.94±5.54)%, KO+6G (33.63±4.82)%, WT+6G+TMZ (-21.45±6.19)%, and KO+6G+TMZ (-18.13±5.45)%.

[0250] Experimental results showed that WT mice were more inclined to explore new objects, although the exploration index of the WT+6G group was slightly lower than that of the WT+Veh group. This may be due to certain stress responses induced by 6-gingerol administration, which in turn affected the mice's behavioral performance. Nevertheless, there was no statistically significant difference between the WT+6G and WT+Veh groups. Therefore, 6-gingerol administration did not cause adverse reactions in the animals. In contrast, Fmr1 KO mice exhibited the opposite behavior to WT mice, indicating a deficiency in learning and memory abilities in Fmr1 KO mice, which could be corrected by 6-gingerol administration. TMZ, however, blocked the learning and memory-improving effect of 6-gingerol on Fmr1 KO mice.

[0251] The above experiments show that 6-gingerol can improve memory in mice with fragile X syndrome by regulating adult neurogenesis; 6-gingerol can correct learning and memory impairments in mice with fragile X syndrome by regulating adult neurogenesis.

[0252] 4. Social Interaction Experiment

[0253] In animals, social behavioral disorders are a hallmark of many mental illnesses. A three-box interaction test assesses the social behavior of mice and their preference for novelty.

[0254] 4.1 Experimental Materials

[0255] A rectangular control box, metal cage; two mice; tissues; 75% v / v alcohol; two timers. The experimental animals and drugs are the same as in Example 1.1.

[0256] 4.2 Experimental grouping and drug administration

[0257] Same as in Example 1.2. There are 6 groups in total, with 8-12 animals in each group (i.e., n = 8-12).

[0258] 4.3 Experimental Procedure

[0259] like Figure 20As shown, the photographic device was mounted directly above the experimental setup. First, the experimental animals were brought to the behavioral testing laboratory and allowed at least one hour to acclimatize. The experiment consisted of three phases: habituation, social interaction testing, and social novelty recognition. In the testing phase, the experimental animals were initially placed in the middle box and allowed to freely explore the left, middle, and right compartments for 10 minutes. In the social interaction test, the time spent exploring the toy and the unfamiliar animal was recorded within 10 minutes. In the training phase, an object was placed in the right box, mouse C1 in the left box, and the numbered experimental mouse A1 in the middle box. In the testing phase, an unfamiliar mouse C2 was placed in the right box, a familiar mouse C1 in the left box, and the numbered experimental mouse A1 in the middle box. The photographic device recorded the interaction between the experimental mice and the unfamiliar mouse C2 and the familiar mouse C1. After each experiment, the test boxes and the cages containing the mice were cleaned with 70% alcohol to remove any residual odor from the mice. After the experiment, a stopwatch was used to analyze the contact time of the experimental mice with C1 and C2. During the testing phase, the detection of an object or another animal was defined as any exploratory behavior of 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).

[0260] After each mouse test, the scene was cleaned with purified water to avoid affecting the results of subsequent mouse experiments.

[0261] 4.4 Experimental Results

[0262] Data were analyzed using GraphPad 6Gism software in a two-way ANOVA. The results are expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0263] The results of the mouse social interaction experiment are statistically analyzed as follows: Figure 21 As shown. Social behavioral disorders are a typical feature of many mental illnesses. The exploration indices for different groups were WT+Veh (42.27±6.46)%, KO+Veh (-37.88±6.15)%, WT+6G (30.96±6.41)%, KO+6G (50.94±7.06)%, WT+6G+TMZ (-57.95±11.05)%, and KO+6G+TMZ (-46.34±10.76)%.

[0264] The experimental results showed that WT mice were more inclined to interact with new mice, although the exploration index of the WT+6G group was slightly lower than that of the WT+Veh group. This may be because 6-gingerol administration induced certain stress responses in the mice, thus affecting their behavioral performance. Nevertheless, there was no statistically significant difference between the WT+6G and WT+Veh groups. Therefore, 6-gingerol administration did not cause adverse reactions in the animals. Fmr1 KO mice, on the other hand, were more inclined to interact with older mice, indicating a deficiency in their social abilities. 6-gingerol administration increased their interest in new mice; however, TMZ intervention did not produce the same improvement.

[0265] From the above experiment, we can see that:

[0266] 6-Gingerol can improve the social abilities of FXS model mice, and its mechanism of action may be through improving neurogenesis; 6-Gingerol corrects social behavior deficits in fragile X syndrome model mice by regulating neurogenesis.

[0267] The above embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope thereof, but all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. The use of 6-gingerol or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a medicament for treating fragile X syndrome, characterized in that: The drug includes pharmaceutically acceptable excipients.

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 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.

4. 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.

5. The application according to claim 4, 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.

6. The application according to any one of claims 1 to 5, characterized in that: The drug is administered to mammals.

7. The application according to claim 6, characterized in that: The mammals mentioned include humans.

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