Application of gingerol compound in preparation of product for treating hereditary intellectual and cognitive dysfunction diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of gingerol compounds in preparation of products for treating hereditary intellectual and cognitive dysfunction diseases. The invention proves that the 6-gingerol can improve the neurogenesis of the fragile X-chromosome syndrome and correct the cognitive deficit, enriches the application of the 6-gingerol in neurodevelopment disorder diseases and the scientific connotation of the 6-gingerol in the aspect of disease treatment, and is suitable for industrial production and easy to popularize. The invention provides a new drug source for treating fragile X chromosome syndrome and related diseases caused by change and mutation of in-vivo X chromosome in a genetic process, and provides a new medicinal application of the gingerol compound.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of gingerol compounds in preparing products for treating hereditary intelligence and cognitive dysfunction diseases. Background Art

[0002] Fragile X syndrome (FXS) 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 6 Gotein, FMRP) encoded by it.

[0003] The absence of FMRP can lead to abnormal neurogenesis and cognitive impairment, and neural stem (progenitor) cells change in the developing brain. Patients with fragile X syndrome mainly show abnormal mental development, reproductive system abnormalities, short-term memory, executive function, visual memory, visual spatial processing disorders and impaired social skills. The IQ of patients is only 40% of that of normal people, which seriously affects patients and families who provide lifelong care.

[0004] The loss of FMRP can cause neurons to show impaired dendritic morphogenesis, reduced synapse formation, and abnormal circuit integration. Studies on FXS patients and Fmr1 KO mice or Drosophila animal models have found that the number of dendritic branches and dendritic spine density of cortical neurons in Fmr1 KO mice are abnormal, the newborn hippocampal neurons of Fmr1 KO mice show defects in synaptic plasticity and neurite extension, and the adult hippocampal new neurons of Fmr1 KO mice also show impaired dendritic maturation and spine maturation. The dendritic spines of neurons in FXS patients are immature, and the neurons show 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, 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] Today, stimulants are the most common medications used to treat FXS, but the effectiveness of these medications and their side effects vary from person to person. The most commonly used treatment is stimulants. Although stimulants are effective for treating symptoms of hyperactivity, impulsivity, and inattention in young males with FXS, adult males with FXS are less responsive to stimulants.

[0007] The physiological and pathological processes of neurological diseases are extremely complex, with poor prognosis, and there is still a lack of effective treatment measures. FXS is a single gene genetic disease that has attracted great attention in recent years. Its incidence is second only to Down syndrome in hereditary mental retardation. The intellectual disability caused by FXS brings great physical and psychological pain to patients and their families, and the long course of the disease brings a heavy burden to the family and society. The treatment effect can be maximized by combining psychopharmacological intervention with other supportive strategies, including speech therapy, sensory integration occupational therapy, personalized education plans, and tailored behavioral interventions.

[0008] The most famous classical theory of the pathophysiology of FXS is the mGluR theory. The various dysfunctions noted in the behavioral, electrophysiological, and molecular systems of FXS are speculated to be due to excessive glutamatergic signaling through mGluRs. The abnormal development of dendritic spines in fragile X syndrome is associated with excessive activation of mGluR5 signaling. At the same time, mGluRs can increase FMRP synthesis, that is, FMRP, as a downstream molecule of mGluR, can negatively regulate the activity of mGluR. Therefore, the classical mGluR theory of FXS believes that the loss of FMRP increases the synthesis of related proteins, leading to excessive internalization of AMPA receptors, making mGluR-mediated LTD overactive, and thus leading to the characteristics associated with FXS. It has been shown that in mouse and Drosophila models, inhibition of the mGluR5 signaling pathway can rescue protein synthesis and some FXS manifestations.

[0009] A large amount of preclinical data suggests that antagonists of metabotropic glutamate receptor 5 (mGluR5) may be effective in treating patients with FXS, and multiple preclinical pharmacology studies have used mGluR5 antagonists for treatment. Fenobam is a non-benzodiazepine anxiolytic that has been studied in FXS due to its effects on negative allosteric regulation of mGluR5. But it has hallucinations, dizziness, paresthesias, and insomnia in normal people. Mavoglurant / AFQ056 is a non-competitive mGluR5 antagonist used for therapeutic blockade of mGluR5 and is being evaluated for the treatment of behavioral symptoms of FXS. There were significant improvements in stereotyped behaviors, hyperactivity, and inappropriate speech, but these findings were not adequately measured on any standardized scale. In addition, in a clinical study of 30 male FXS who were given mavoglurant for 28 days, it was found that 24 people experienced some form of adverse events, most of which were mild or moderate in severity. Finally, the development of mavoglurant for FXS was discontinued after 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 and improved wakefulness and anxiolysis. Results from a Phase II preclinical trial showed a trend toward clinical improvement.

[0010] However, 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.

[0011] Ginger is the fresh rhizome of ginger, a plant of the ginger family, while dried ginger is the dried rhizome. 6-gingerol is the active ingredient and indicator ingredient of ginger. 6-gingerol has been reported to have a good effect on cholesterol metabolism, regulating the content of plasma cholesterol, mediating the endocytosis of cholesterol by cells, and promoting the decomposition of cholesterol in cells, thereby reducing internal cholesterol. 6-gingerol can also regulate cholesterol metabolism by activating SREBP2 to upregulate LDLR and upregulating cholesterol efflux-related genes LXRα and ABCA1. In addition, 6-gingerol can pass through the blood-brain barrier well and has a wide biological safety window. It has been used in the study of brain diseases such as brain injury, Alzheimer's disease, Parkinson's disease and epilepsy.

[0012] However, the application of 6-gingerol in the treatment of FXS has not been reported yet. Summary of the invention

[0013] The primary purpose of the present invention is to provide a gingerol compound with the performance and efficacy of treating hereditary intellectual and cognitive dysfunction diseases, especially a new application in medicine for fragile X syndrome, and a new medicinal use of gingerol compounds (especially 6-gingerol) in view of the problems existing in the above-mentioned prior art.

[0014] The purpose of the first aspect of the present invention is to provide the use of gingerol compounds in preparing products.

[0015] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted is:

[0016] The first aspect of the present invention provides the use of gingerol compounds in preparing products, wherein the products are used for treating and preventing hereditary intellectual and cognitive dysfunction diseases.

[0017] 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.

[0018] 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.

[0019] 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).

[0020] Further 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 C 17 H 26 O4, Cas number is 23513-14-6.

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

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

[0025] Preferably, the gingerol compound comprises a pharmaceutically acceptable derivative.

[0026] Preferably, the pharmaceutically acceptable derivatives include pharmaceutically acceptable salts and pharmaceutically acceptable chemical modifications.

[0027] 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.

[0028] Preferably, the metal salt comprises an alkali metal salt or an alkaline earth metal salt.

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

[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 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.

[0032] 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.

[0033] 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.

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

[0035] 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.

[0036] 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.

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

[0038] 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.

[0039] The invention provides a medicine for treating fragile X chromosome syndrome, which takes gingerol compounds (especially 6-gingerol) as active ingredients and a corresponding medicine dosage form.

[0040] Wherein, the drug exists in the form of oral preparation, injection or local administration preparation.

[0041] 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.

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

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] When the 6-gingerol described in the present invention is used to treat fragile X syndrome, it can be used alone or in the form of a pharmaceutical composition containing 6-gingerol.

[0048] Preferably, the dosage form of the product includes a dosage form for enteral administration or a dosage form for parenteral administration.

[0049] 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.

[0050] 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.

[0051] Preferably, the product is administered to a mammal.

[0052] Preferably, the mammal comprises a human.

[0053] The beneficial effects of the present invention are:

[0054] 1. The present 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 disease treatment effect of active small molecules of traditional Chinese medicine, and provides ideas for the research of mining target drugs in the treasure house of traditional Chinese medicine.

[0055] 2. The present invention uses 6-gingerol to conduct in vitro newborn neuron dendrite complexity test and neural stem cell proliferation and differentiation experiment. The results show that: after intervention, 6-gingerol can significantly promote the dendritic complexity of newborn neurons in the hippocampus of Fmr1 KO mice cultured in vitro, increase the dendritic length, the number of dendritic nodes and the number of dendritic terminals, and can also increase the differentiation of neural stem cells and correct their abnormal proliferation.

[0056] 3. The cognitive function experiments on Fmr1 gene-deficient animals (Fmr1 KO mice) of the present invention prove that 6-gingerol can correct the cognitive dysfunction and social interaction disorder of Fmr1 KO mice; improve the spatial memory ability of Fmr1 KO mice and improve the pattern separation cognitive function defect of Fmr1 KO mice by regulating neurogenesis; improve the learning and memory of Fmr1 KO mice by regulating adult neurogenesis; and correct and improve the social interaction disorder of Fmr1 KO mice by regulating neurogenesis.

[0057] 4. The 6-gingerol of the present invention has strong pharmacological effects, is effective in treating fragile X syndrome, has rapid effects, few toxic and side effects, is safe, can be taken for a long time, and has good medicinal prospects.

[0058] 5. The product of the present invention has abundant raw material sources, is inexpensive, safe for clinical use, has a simple preparation process, can be made into various dosage forms, has a small dosage, is easy to use, and is therefore easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Diagram of the experimental scheme for 6-gingerol regulating the abnormal proliferation of adult neural stem cells in the brain of adult mice.

[0060] Figure 2 The newborn and mature neurons (NeuN) in the DG region of WT / Fmr1 KO mice + BUD + ) Representative micrographs of the 247 cells of the Fmr1 KO mice; the effect of 6-gingerol on neuronal differentiation in Fmr1 KO mice was assessed by immunostaining for the neuronal markers NeuN (green) and BrdU (red); scale bar: 20 μm.

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

[0062] Figure 4 Diagram of the experimental scheme for 6-gingerol regulating the differentiation of newborn neurons from neural stem cells in the brain of adult mice.

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

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

[0065] Figure 7 Schematic diagram of the experimental protocol for Sholl analysis of primary hippocampal neurons.

[0066] Figure 8 This is a microscopic observation of hippocampal neurons cultured in vitro on day P0.

[0067] Fig. 9 Sholl analysis of dendritic complexity in primary hippocampal neurons.

[0068] Fig.10 This is a statistical graph of the Sholl analysis of the dendrite length of primary hippocampal newborn neurons.

[0069] Fig.11 This is a Sholl analysis statistical chart of the number of dendritic nodes of primary hippocampal newborn neurons.

[0070] Fig.12 This is a Sholl analysis statistical chart of the number of dendritic terminals of primary hippocampal newborn neurons.

[0071] Fig.13 Schematic diagram of the mouse behavioral experimental protocol.

[0072] Fig.14 Schematic diagram of the mouse novel location recognition experiment.

[0073] Fig.15 Statistical graph of the discrimination index for the mouse novel location recognition experiment.

[0074] Fig.16 Schematic diagram of the L-maze experiment for mice.

[0075] Fig.17 This is a statistical chart of the discrimination index of the mouse L-maze test.

[0076] Fig.18 Schematic diagram of mouse pattern separation experiment.

[0077] Fig.19 This is a statistical graph of the discrimination index of the mouse pattern separation experiment.

[0078] Fig. 20 Schematic diagram of the mouse social interaction experiment.

[0079] Fig.21 This is a statistical chart of the discrimination index in the mouse social interaction experiment. DETAILED DESCRIPTION

[0080] 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.

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

[0082] Male wild-type (WT, or Fmr1 + / y ) mice and female Fmr1 knockout (KO) mice (Fmr1 KO, or Fmr1 - / - ), and Nestin-GFP transgenic mice were purchased from Jackson Laboratory, USA.

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

[0084] Fmr1 HET (Fmr1 + / - ), female mice, heterozygous is Fmr1 HET also uses Fmr1 + / - express.

[0085] Male Nestin-GFP transgenic mice and Fmr1 HET female mice (Fmr1 + / - ) were mated and bred to obtain wild-type or Fmr1 gene-deficient transgenic mice marked with nestin green fluorescent protein (GFP) (Fmr1 + / y ::Nestin-GFP or Fmr1 - / y: :Nestin-GFP).

[0086] All mice were housed in a specific pathogen-free (SPF) animal breeding room at a temperature of 22-25°C with a circadian cycle of 12 h light / 12 h dark, and mice had free access to water and food, which was approved and recognized by the Animal Ethics Committee of the University of Macau.

[0087] The occurrence of fragile X syndrome is mainly caused by 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, this study only selected adult wild-type and Fmr1 gene-deficient male mice as experimental samples.

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

[0089] Table 1 Source information of drugs and reagents

[0090]

[0091] Table 2 Instrument and equipment information

[0092]

[0093] Example 1 In vivo proliferation and differentiation experiment of neural stem cells

[0094] 1. Experimental Methods

[0095] 1.1 Experimental animals

[0096] Proliferation experiments were performed using male wild-type or Fmr1-deficient transgenic mice labeled with nestin green fluorescent protein (GFP). + / y ::Nestin-GFP or Fmr1 - / y: :Nestin-GFP).

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

[0098] Male Nestin-GFP transgenic mice and Fmr1 HET female mice (Fmr1 + / - ) were mated and bred to obtain male wild-type or Fmr1 gene-deficient transgenic mice marked with nestin green fluorescent protein (GFP) (Fmr1 + / y ::Nestin-GFP or Fmr1 - / y: : Nestin-GFP). The mice were given the drug when they were 7-8 weeks old for subsequent cell activation experiments.

[0099] 1.3 Experimental groups, medication and material collection

[0100] 1.3.1 Neural stem cell proliferation experiment

[0101] Male wild-type and Fmr1 gene-deficient transgenic mice (Fmr1) tagged with nestin green fluorescent protein (GFP) at 7-8 weeks of age were raised.+ / y ::Nestin-GFP;Fmr1 - / y: : Nestin-GFP) were randomly divided into 4 groups, 3 mice in each group. Each group of mice was intraperitoneally injected with a dose of 10 mg / kg; the volume of blank solvent was calculated according to the concentration of 6-gingerol, and the same volume of blank solvent (PBS) was given; intraperitoneal injection was performed at the same time every day, once a day, for a total of 30 days. The four groups were 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 sliced ​​using a freezing microtome to obtain frozen sections for subsequent neural stem cell proliferation experiments. The experimental process is as follows: Figure 1 shown.

[0104] 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 + Positive targets such as blue-DAPI are used to determine the location of cells. After scanning, NeuN + and BrdU + Cell counting, quantification of positive targets in the stained mouse brain sections, including green-NeuN + ; Red-BrdU + ; Blue-DAPI.

[0105] 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, Figure 5 DAPI in the middle represents all cells, BrdU marks proliferating newborn cells, and NeuN marks mature neurons; green - NeuN + ; Red-BrdU + ; Blue - DAPI + .

[0106] GFP + and GFAP + Double positive cells represent neural stem cells, MCM2 + , GFP + and GFAP + The triple-positive cells represent activated neural stem cells, and the MCM2 + , GFP + GFAP + The number of triple-positive cells and GFP + GFAP + The ratio of the number of double-positive cells indicates the proportion of activated cells (the average value of 3 times is taken).

[0107] 1.3.2 Neural stem cell differentiation experiment

[0108] Male wild-type mice (Fmr1 + / y ) and Fmr1 KO mice (Fmr1 - / y ) were randomly divided into 4 groups, 3 mice in each group. Each group of mice was intraperitoneally injected with 10 mg / kg of 6-gingerol; the blank solvent was calculated according to the concentration of 6-gingerol, and the same volume of PBS was given; intraperitoneally injected at the same time every day, once a day, for 30 days, the 4 groups were specifically:

[0109] Group E: Fmr1 + / y +Blank solvent group (WT+Veh); Group F: 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 the 15th and 16th days of 6-gingerol administration, BrdU was injected intraperitoneally to mark neural stem cells in the division stage, that is, to mark stem cells in the proliferative state. The dose was 200 mg / kg, twice a day, for two days. Four weeks after the last BrdU injection, the mice were anesthetized and the brains were perfused. The entire mouse brain hippocampus region was sliced ​​continuously at a thickness of 40 μm, and the hippocampus was systematically randomly sampled by selecting one slice every six slices. This was used for subsequent neural stem cell differentiation experiments. The process is as follows Figure 4 shown.

[0111] The frozen brain sections of the four groups of mice were stained with immunohistochemistry (frozen sections) (IHC-Fr), and the sections were mounted in the order of the hippocampus from small to large, and sealed with anti-fluorescence quencher to obtain the stained brain sections of each group of mice; then the neurostereoscopic quantitative analysis method was used, that is, the stained brain sections were placed under an upright microscope, and the StereoInvestigator neurostereoscopic software was opened. For the DG area in the stained brain sections, the fluorescent channels were set as follows: blue: DAPI; green: GFP; red: MCM2; gray: GFAP, and the brain sections were scanned in the order on the slide. After the scan, the fluorescence staining through different channels was used to determine the MCM2 + , GFP + and GFAP + Positive targets such as cells are counted and blue-DAPI is used to determine the location and status of cells. Positive targets are measured on the mouse brain sections that have been stained.

[0112] Scanning brain slices under a microscope, representative images of neural stem cell activation are shown below. Figure 5 , In the figure, blue: DAPI; green: GFP; red: MCM2; gray: GFAP; scale bar: 20 μm.

[0113] NeuN is a marker for mature stem cells, and BrdU marks neural stem cells in the division phase, that is, neural stem cells that are in a proliferative state. + and BrdU + Double-positive cells represent neural stem cells that have newly differentiated into mature neurons. In order to further study the effect of 6-gingerol on the differentiation of neural stem cells in FXS model mice, this study used neurostereometry software to count NeuN + and BrdU + The number of cells in each group reflects the degree of differentiation of neural stem cells into newborn neurons. + BUD + / BrdU +), 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 newborn and mature neurons.

[0114] 2. Experimental results

[0115] 2.1 Results of neural stem cell proliferation experiments

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

[0117] The experimental results show that the proportion of activated stem cells in FXS model mice is higher than that in wild-type mice (p<0.05), but after administration, the proportion of activated stem cells in FXS model mice is similar to that in wild-type mice (p>0.05). Therefore, 6-gingerol inhibits the abnormal activation of adult neural stem cells in Fmr1 KO mice.

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

[0119] 2.2 Results of neural stem cell differentiation experiments

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

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

[0122] 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 6-gingerol can correct this disease phenotype and increase the number of mature neurons differentiated from adult neural stem cells in Fragile X syndrome. Therefore, 6-gingerol has a certain therapeutic significance for Fragile X syndrome and can be used to treat Fragile X syndrome.

[0123] The research of neural stem cells has become a hot topic in the field of life sciences. The directional differentiation of neural stem cells into neurons is of great significance for the treatment and rehabilitation of nervous system diseases. This example illustrates from the whole animal level that 6-gingerol promotes the differentiation of stem cells into neurons by reducing proliferation, thereby improving the abnormal manifestation of neurogenesis in FXS model mice. Moreover, under this administration method, neurogenesis in normal mice is not affected.

[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 hippocampal-dependent learning. After administration of 6-gingerol, neural stem cells can restore normal proliferation and differentiation, promote normal neurogenesis, and reduce the damage caused by FMRP deficiency.

[0125] Example 2 Effect of 6-gingerol on the dendritic complexity of newborn neurons in the hippocampus of Fmr1 KO mice

[0126] Dendrites are the branch-like protrusions that extend outward from the cytoplasm of nerve cells. Dendrites are one or more protrusions that extend from neurons. They are thick at the beginning, branch repeatedly, and gradually become thinner, forming a branch-like shape. There are many receptors on the cell membrane outside the dendrites. Dendrites are important structures between neurons. The complexity of dendrite branches affects the ability of neurons to receive and integrate synaptic information. Therefore, changes in the total length of dendrites and changes in structural complexity may affect signal transmission between neurons and cell function, and thus affect cognitive function.

[0127] 1. Experimental Materials

[0128] 1.1 Experimental animals

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

[0130] The occurrence of fragile X syndrome is mainly due to 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, this example only selected adult wild-type and Fmr1 gene-deficient male mice as experimental samples.

[0131] 1.2 Cell culture

[0132] Hippocampal newborn neurons from WT and Fmr1 KO mice were cultured in a 5% CO2, 37°C incubator.

[0133] 2. Experimental Methods

[0134] 2.1 Reagent preparation

[0135] 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.

[0136] 2) 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 adjust the volume to 50 mL, filter through a 0.22 μm filter membrane to sterilize, and store at -20°C.

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

[0138] 4) Preparation of poly-lysine: 10x poly-lysine was diluted to 1x poly-lysine in a ratio of 1:9 using sterile double distilled water, and then sterilized by filtration using a 0.22 μm microporous filter membrane, and then stored at 4°C.

[0139] 5) Preparation of 6-gingerol: DMSO was used to prepare a 20 mM 6-gingerol mother solution, which was filtered through a 0.22 μm microporous filter membrane to sterilize and then stored at -20°C.

[0140] 6) 2×HEBS: accurately weigh 1.6000 g of NaCl, 0.0740 g of KCl, 0.0213 g of Na2HPO4, 0.2000 g of D-Glucose, and 1.1900 g of Hepes (4-hydroxyethylpiperazineethanesulfonic acid), dissolve them in sterile ultrapure water, adjust the pH to 7.05 with NaOH or HCl, add sterile ultrapure water to make up to 100 mL, filter and sterilize with a 0.22 μm microporous filter membrane, and then store at room temperature.

[0141] 7) CaCl2: Accurately weigh 29.4 g of CaCl2.2H2O and dilute to 100 mL with sterilized ultrapure water to prepare a 2 M calcium chloride solution. Filter and sterilize with a 0.22 μm microporous filter membrane before use, and then store at room temperature.

[0142] 8) 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.

[0143] 9) Preparation of SOC (Super Optimal broth with Catabolite re6Gession) medium: Take 20g Tryptone, 5g Yeast extract, 0.5g NaCl, 0.186g KCl, 12g MgSO4, and 0.95g MgCl2 and dissolve them in nearly 1L of ultrapure water, adjust the pH to 7.0, and make up to 1L. Sterilize it under high pressure, and then add 20mL of the sterilized glucose solution prepared in reagent preparation 3).

[0144] 10) 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.

[0145] 11) 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.

[0146] 12) Neuron-related culture medium

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

[0148] ② Seed medium: DMEM-F12 + 10v / v% FBS + 1v / v% penicillin-streptomycin;

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

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

[0151] 2.2 Experimental groups and drug administration

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

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

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

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

[0156] The two types of WT and Fmr1 KO neurons were subjected to blank treatment and drug treatment, respectively, among which: blank treatment (blank group, group A and group B): WT+Veh group (group A), PBS was added to the culture medium, WT neurons+PBS; KO+Veh group (group B), Fmr1KO neurons+PBS; drug treatment (drug treatment group, group C and group D): WT+6G group (group C), 6G with a final concentration of 1μM was added to the culture medium, WT neurons+6G with a final concentration of 1μM; KO+6G group (group D), Fmr1 KO neurons+6G with a final concentration of 1μM.

[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 plasmid Rv-GFP (1 μL) to a 1.5 mL centrifuge tube in sequence, flick the tube to mix the plasmid and competent cells thoroughly; then place it in an ice bath for 30 minutes, place the centrifuge tube in a 42°C water bath for 60-90 seconds, and 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. The Rv-GFP plasmid is introduced into the E. coli, thereby amplifying it in large quantities.

[0159] (2) 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 step (1). Mix well and place in a 37°C shaking incubator (180 rpm, 90 min) to culture the competent cells into which the plasmid was introduced.

[0160] (3) Pipette the culture mixture (100 μL) from step (2) 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 h, and then colonies can be observed.

[0161] (4) 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.

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

[0163] 2.4 Plasmid extraction

[0164] Use a high-purity plasmid extraction kit and follow the kit instructions and experimental methods to extract the plasmid, as follows:

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

[0166] (B) Add the mixed solution in step A) to the centrifuge tube with bacterial cell precipitate extracted in "3. Plasmid transformation", vortex to suspend the bacterial cell precipitate; add solution P2 of the plasmid extraction kit, mix immediately to fully lyse the bacteria, and let it stand at room temperature for 5 minutes; then add solution P4 of the plasmid extraction kit to the centrifuge tube, mix immediately until white dispersed flocculent precipitate appears in the solution; then let it stand at room temperature for 4-5 minutes, centrifuge (7800 rpm, 15 minutes) to centrifuge the white precipitate to the bottom of the tube; then carefully pour all the solution into filter CS1 (be careful to avoid pouring a large amount of precipitate to block 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 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, and invert the centrifuge tube on absorbent paper; then add 70 v / v% ethanol to the centrifuge tube to thoroughly rinse the precipitate; centrifuge at 4°C and 7800 rpm for 15 min, gently pour off the supernatant, and invert the centrifuge tube on absorbent paper, and repeat 1-2 times to thoroughly rinse the precipitate.

[0168] (D) The centrifuge tube was left open at room temperature for 30 min to allow the ethanol to fully evaporate, DEPC water was added and vortexed to mix, and the RV-GFP plasmid was extracted.

[0169] The extracted plasmids were marked (date, plasmid type, concentration) and stored in a -20°C refrigerator for future use.

[0170] 2.5 Neuron Culture

[0171] 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;

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

[0173] 3) Take out the P0 day (newly born mice within 0-24h), disinfect with alcohol for 3min, take the tail tissue of the mouse and store it in a 1.5mL EP tube; then use fine scissors to take the head of the mouse, among which: male WT and Fmr1 KO mice are divided into 4 groups, namely WT+Veh, KO+Veh, WT+6G, KO+6G groups, each group has 3 mice, that is, n=3, and the four groups have a total of 12 mice. The specific grouping is shown in Table 3, the blank solvent is PBS; the concentration of 6G is 1μm.

[0174] Table 3 Neuronal experimental groups

[0175]

[0176] 4) Take the hippocampus: Disinfect the suckling mice with alcohol, cut the tail and place it in an EP tube for genotype identification, separate the head with surgical scissors, take out the complete brain and place it in a dissection medium, peel off the cerebral cortex, clamp the hippocampus tissue and place it in a dissection medium. 5-5. Digestion: Digest with 0.25% Trypsin-EDTA for 5 minutes.

[0177] 5) Pipetting: gently pipette to form a single cell suspension. 5-7. Planting culture medium: the cell culture medium is planted in a 24-well plate containing a cell slide and cultured in a 37°C, 5% CO2 incubator.

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

[0179] 2.6 Cell transfection and drug treatment

[0180] 1) The experiment was divided into a normal mouse primary cultured hippocampal neurons given a blank solvent group (abbreviated as: WT+Veh), a FXS model mouse primary cultured hippocampal neurons given a blank solvent group (abbreviated as: KO+Veh), a normal mouse primary cultured hippocampal neurons given a 6-gingerol group (abbreviated as: WT+6G), and a FXS model mouse primary cultured hippocampal neurons given a 6-gingerol group (abbreviated as: 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, wherein 2×HEBS and CaCl2 were used to prepare calcium phosphate-DNA precipitates; the calcium phosphate particles were evenly added dropwise to the cells, and placed in a 37°C incubator for one hour; after evenly distributed granules were formed on the cell surface, the culture medium containing serum was replaced, and the cells were returned to the 37°C incubator for culture; the neurons were fixed after being cultured to the 7th day.

[0182] 2.7 Fixation, sealing and measurement of neurons

[0183] 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 placed at room temperature. After fixation for 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 fixation for 15 min; then the liquid in the well was aspirated and washed with PBS for 5 min / time, for a total of three times.

[0184] Take out the cell slide and place the cell side on a glass slide with DAPI anti-fluorescence quencher added; place it in a cool, dark place and observe it the next day.

[0185] Observe GFP using fluorescence field under an upright microscope + Changes in neuronal cell morphology ( Figure 8 ). To analyze the effect of 6G on the dendritic complexity of newborn neurons in the hippocampus of WT or Fmr1 KO mice, a stereological analysis system was used to depict GFP+ newborn neurons in real time, and a neuromorphic analysis system was used to quantify the neuronal morphological structure, namely, the dendritic length of GFP+ newborn neurons ( Fig.10 ), the number of dendritic nodes ( Fig.11 ), the number of dendrite terminals ( Fig.12 ) quantification.

[0186] 3. Experimental results

[0187] GraphPad 6Gism 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.

[0188] 3.1 Effects of 6-gingerol on the morphology of newborn neurons in the hippocampus of WT and Fmr1 KO mice

[0189] Microscopic observation of the morphology of newborn neurons Figure 8 As shown in the figure, it can be seen that compared with WT+Veh, the dendritic complexity of KO+Veh neuronal cells 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 neuronal cells is increased, the dendritic length is increased, and the dendritic branches are increased; compared with WT+Veh, the morphology of KO+6G neuronal cells can be restored to a level similar to that of the newborn neurons in the hippocampus of WT mice.

[0190] In the neurons cultured in vitro, the neuronal cells were transfected by calcium phosphate transfection method, and 6G was added during the culture process for drug intervention. The neurostereoscopic analysis method was used to analyze the morphology of neurons expressing green GFP fluorescence. Compared with WT, the hippocampal newborn neurons of Fmr1 KO mice had obvious morphological differences, that is, the dendritic complexity of Fmr1 KO neurons was lower than that of WT neurons. After the intervention of 6-gingerol, the morphology of Fmr1 KO hippocampal newborn neurons could be restored to a level similar to that of WT mice hippocampal newborn neurons ( Figure 8 ).

[0191] The experimental results showed that 6G had an improving effect on the dendritic morphology of newborn neurons in the hippocampus of Fmr1 KO mice: 6G 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. (At least 20-35 GFP + of neurons).

[0192] 3.2 Effects of 6-gingerol on the complexity of newborn neurons in the hippocampus of WT and Fmr1 KO mice

[0193] The Sholl analysis method was used to investigate the complexity of newborn neurons in the hippocampus of WT or Fmr1 KO mice. The results of the Sholl analysis experiment are shown in the figure. Figure 8 As shown; the statistical analysis results of the Sholl analysis method to identify the complexity of newborn neurons in the hippocampus are shown Fig. 9 shown.

[0194] The experimental results showed that the total length of dendrites of primary cultured hippocampal newborn neurons in Fmr1 KO mice was significantly reduced, which was significantly different from that in the normal group (p<0.001). 6-gingerol could significantly increase the dendritic complexity of primary cultured hippocampal newborn neurons in Fmr1 KO mice to normal levels, and there was no significant difference between the WT+Veh group and the KO+6G group (p>0.05). The length of neurons in the normal group was not affected under this dose, 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 newborn neurons in the hippocampus of WT and Fmr1 KO mice

[0196] 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.10 shown.

[0197] The total length of dendrites of newborn neurons in primary culture of hippocampus in different groups were: WT+Veh (1192±63.28μm); KO+Veh (566.6±43.16μm); WT+6G (1391±98.15μm); KO+6G (1392±84.62μm); The experimental results showed that the dendrite length of newborn neurons in Fmr1 KO mice was shorter than that of newborn neurons in WT mice; and 6G could significantly increase the dendrite length of newborn neurons in Fmr1 KO mice. Compared with WT+Veh, the dendrite length of newborn neurons in Fmr1 KO mice given 6G was restored to the level of dendrite length of newborn neurons in hippocampus of WT mice.

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

[0199] The Sholl analysis method was used to analyze the dendritic nodes of newborn neurons in the hippocampus. The statistical analysis results of the number of dendritic nodes of newborn neurons in the hippocampus identified by the Sholl analysis method are shown in the figure ( Fig.11 ).

[0200] As can be seen from the figure, the number of dendritic nodes of primary cultured hippocampal newborn neurons in different groups are: WT+Veh (7.03±0.48); KO+Veh (3.38±0.34); WT+6G (6.70±0.58); KO+6G (8.18±0.45). The experimental results show that the newborn neurons of Fmr1 KO mice have fewer dendritic nodes than those of WT mice, and 6G administration 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 6G was restored to the level of the number of dendritic nodes of newborn neurons in the hippocampus of WT mice.

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

[0202] 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.12 shown.

[0203] As can be seen from the figure, the number of dendritic terminals of primary cultured hippocampal newborn neurons in different groups are: WT+Veh (11.82±0.69); KO+Veh (6.57±0.39); WT+6G (11.93±0.75); KO+6G (13.30±0.60). The experimental results show that the newborn neurons of Fmr1 KO mice have fewer dendritic terminals than the newborn neurons of WT mice, and the number of dendritic terminals of newborn neurons of Fmr1 KO mice can be significantly increased after 6G administration. Compared with WT+Veh, the number of dendritic terminals of Fmr1 KO mice given 6G was restored to the level of the number of dendritic terminals of newborn neurons in the hippocampus of WT mice.

[0204] Example 3 Cognitive function regulation test

[0205] This example explores whether the effect of 6-gingerol in improving cognitive impairment in Fmr1 gene-deficient mice (Fmr1 KO) is mediated by improving neurogenesis, in order to lay the foundation for clinical application. It is explored whether the abnormal behavior of Fmr1 gene-deficient mice is improved after being given 6-gingerol, and the therapeutic effect of 6-gingerol on mice is observed. Temozolomide (TMZ), a drug that blocks neurogenesis, was used for intervention. The results show that 6-gingerol can treat cognitive dysfunction in mice with fragile X chromosome syndrome by regulating neurogenesis, as follows.

[0206] 1. New location recognition experiment

[0207] The novel location recognition test is often used to evaluate the hippocampus-dependent spatial memory ability of mice. It evaluates the spatial memory ability of mice based on the fact that mice have a stronger curiosity about new things. In order to explore whether 6-gingerol can correct the spatial memory ability of mice with fragile X syndrome model, mice were given an intraperitoneal injection of 6-gingerol and then tested in the novel location recognition test.

[0208] In order to investigate whether the improvement effect of 6-gingerol on spatial memory functions by promoting adult neurogenesis, the neurogenesis-specific blocker temozolomide was used to block neurogenesis in advance, and after co-administration with 6-gingerol, a new location recognition experiment was performed.

[0209] 1.1 Materials

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

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

[0212] 1.2. Experimental groups and drug administration

[0213] 1.2.1 Experimental groups: WT+Veh: WT mice were intraperitoneally injected with blank solvent; KO+Veh: Fmr1 KO mice were intraperitoneally injected with blank solvent; WT+6G: WT mice were intraperitoneally injected with 6-gingerol; KO+6G: Fmr1 KO mice were intraperitoneally injected with 6-gingerol; WT+6G / TMZ: WT mice were intraperitoneally injected with temozolomide and 6-gingerol; KO+6G / TMZ: Fmr1 KO mice were intraperitoneally injected 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:

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

[0216] ②: WT+6G, KO+6G group: 7-8 week old Fmr1 KO and WT mice were intraperitoneally injected with 6-gingerol 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 with 50 mg / kg temozolomide (TMZ) once a day in the 6th, 7th, 8th and first three days of the 9th week. In the 9th week, 6-gingerol (6G) was intraperitoneally injected once a day for 7 consecutive days. The dosage of 6G was 10 mg / kg. The experimental diagram of the dosing scheme for experimental mice is shown in the figure below. Fig.13 shown.

[0218] 1.3 Experimental procedures

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

[0220] First, follow the new position recognition experiment instructions Fig.14 As shown in the figure, the new location recognition experiment is divided into three stages: pre-adaptation, training, and testing. First, the numbered mice are placed in an empty field to adapt for at least 1 hour. Then the same object is placed on the same side for training. In the training stage, each animal is placed alone in a square field, and two identical objects are placed at the same distance from a side with colored wallpaper. The mice are allowed to explore freely for 6 minutes, and the training process is repeated 3 times. In the test stage, the location of one of the familiar objects is moved to another new location. The exploration time of the experimental animals on the new and old objects in 6 minutes is recorded. A normal animal should spend more time exploring the objects in the new location. At the end of the experiment, the exploration time of the mice on the new location objects and the old location objects is analyzed, and the exploration index of the new location objects is calculated. Every time the mice are put back into the cage, the operation box should be cleaned with 70v / v% alcohol to eliminate the influence of the same odor interference on the test behavior of the next experimental mouse. The photography device is installed directly above the experimental device. During the test phase, detection of objects was defined as any exploratory behavior when the animal's nose was within 1 cm of the object, including head orientation, climbing, sniffing, etc. The discrimination index was calculated as follows: discrimination index = (new position object recognition time / total recognition time × 100) - (original position object recognition time / total recognition time × 100).

[0221] 1.4 Experimental Results

[0222] The data were analyzed by two-way ANOVA using GraphPad 6Gism software. The results were expressed as Mean ± SEM. When P < 0.05, the difference was considered to be statistically significant.

[0223] The exploration index of 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)%, KO+6G+TMZ(-37.37±8.90)%. The experimental statistical results are shown in Fig.15 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 old objects, which indicates that the spatial memory ability of Fmr1 KO mice is defective. 6-Gingerol can improve the spatial memory of Fmr1 KO mice by regulating neurogenesis, while TMZ can block the improvement of spatial memory of Fmr1 KO mice by 6-Gingerol. 6-Gingerol can improve the spatial memory ability of FXS model mice, and its mechanism of action may be through improving neurogenesis.

[0225] 2. Mode separation experiment

[0226] Pattern separation is a memory process, and impaired pattern separation has been suggested to be a predictor of the development of cognitive impairment.

[0227] 2.1 Materials and methods

[0228] A square open field; object A (2 pieces); object B (2 pieces), object A and object B are different; facial tissue; 75v / v% alcohol; two timers.

[0229] 2.2 Grouping and Dosing

[0230] Same as Example 1.2.

[0231] 2.3 Experimental procedures

[0232] like Fig.18 As shown, during the training period, the numbered mice were placed in an empty field for a period of time, and then placed alone in the first square field with a specific floor pattern and two identical objects (Object A), and were allowed to explore for 10 minutes. 30 minutes later, the same animal was placed in the second square field with a different floor pattern from the first experiment and two completely different sets of objects (Object B), and was allowed to explore for 10 minutes.

[0233] During the experiment, one of the two training floor patterns was randomly selected as the background, and an object A and an object B were placed in the same positions during the test. A video recorder was used to record the interaction of each mouse with object A and object B in this scene. The test time was 10 minutes. Afterwards, a timer was used to count and analyze the interaction of the mice. During the test phase, the detection of the object was defined as any exploratory behavior when the animal's nose was within 1 cm of the object, including head direction, climbing, sniffing, etc.

[0234] After each mouse was tested, it was cleaned with pure water to avoid affecting the experimental results. The discrimination index was calculated according to the formula: discrimination index = (new object detection time / total detection time) - (old object detection time / total detection time) × 100% to determine the mouse's preference for new and old objects.

[0235] 2.4 Experimental Results

[0236] The data were analyzed by two-way ANOVA using GraphPad 6Gism software. The results were expressed as Mean ± SEM. When P < 0.05, the difference was considered to be statistically significant.

[0237] The exploration index of 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)%, KO+6G+TMZ (-30.79±8.93)%. The statistical graph of the mouse pattern separation experiment results is shown in the figure. Fig.19 shown.

[0238] The 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. The reason may be that the administration of 6-gingerol caused some stress responses in mice, which in turn affected the behavioral performance of mice. Nevertheless, there was no statistical difference between the WT+6G group and the WT+Veh group. Therefore, the administration of 6-gingerol did not cause adverse reactions in animals. The performance of Fmr1 KO mice was opposite to that of WT mice, indicating that the pattern separation of Fmr1 KO mice was defective, and this defect could be corrected after the administration of 6-gingerol. TMZ can block the improvement of pattern separation of Fmr1 KO mice by 6-gingerol. It shows that 6-gingerol can improve the pattern separation defect of Fmr1 KO mice by regulating neurogenesis. Its mechanism of action may be through improving neurogenesis, and improving the behavioral defects of fragile X syndrome by promoting neurogenesis.

[0239] 3. New object recognition experiment

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

[0241] 3.1 Experimental Materials

[0242] A square open field; object A; object B, object A and object B are different; facial tissue; 75v / v% alcohol; two timers. The experimental animals and drugs are the same as those in Example 1.1

[0243] 3.2 Experimental groups and drug administration

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

[0245] 3.3 Experimental procedures

[0246] The novel object recognition experiment was divided into three stages. First, the numbered mice were placed in an empty field for training. Second, the same objects were placed at both ends of the L-maze, and the mice were placed in the middle for object recognition training. Then, the objects on one side of the L-maze were replaced with new objects, and the mice were placed in the middle for testing. Fig.16 As shown. At the end of the experiment, analyze the interaction of mice with new objects and familiar objects, and calculate their exploration index of new objects. After the experiment, clean the experimental device with 70v / v% alcohol to prevent residual odor from affecting the activity behavior of the next experimental mouse. A normal animal should spend more time exploring unfamiliar objects. The discrimination criterion is calculated based on the difference between the percentage of time spent identifying unfamiliar objects and the time spent identifying familiar objects. During the test phase, the detection of objects is 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).

[0247] 3.4 Experimental Results

[0248] The data were analyzed by two-way ANOVA using GraphPad 6Gism software. The results were expressed as Mean ± SEM. When P < 0.05, the difference was considered to be statistically significant.

[0249] The results of the mouse novel object recognition experiment are as follows Fig.17The exploration index of different groups was 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)%, KO+6G+TMZ (-18.13±5.45)%.

[0250] The 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. The reason may be that the administration of 6-gingerol caused certain stress responses in mice, which in turn affected the behavioral performance of mice. Nevertheless, there was no statistical difference between the WT+6G group and the WT+Veh group. Therefore, the administration of 6-gingerol did not cause adverse reactions in animals. The performance of Fmr1 KO mice was opposite to that of WT mice, which indicated that the learning and memory ability of Fmr1 KO mice was defective, and this defect could be corrected after the administration of 6-gingerol. TMZ can block the learning and memory improvement effect of 6-gingerol on Fmr1 KO mice.

[0251] The above experiments show that 6-gingerol can improve the memory of fragile X syndrome model mice by regulating adult neurogenesis; 6-gingerol can correct the learning and memory disorders of fragile X syndrome model mice by regulating adult neurogenesis.

[0252] 4. Social interaction experiment

[0253] In animals, social behavior disorders are typical features of many psychiatric disorders. The three-box interaction test assesses mice's social behavior and their preference for novel objects.

[0254] 4.1 Experimental Materials

[0255] Rectangular operating box, metal cage; two mice; facial tissue; 75v / v% alcohol; two timers. The experimental animals and drugs are the same as those in Example 1.1.

[0256] 4.2 Experimental groups and drug administration

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

[0258] 4.3 Experimental procedures

[0259] like Fig. 20As shown, the photographic device was installed directly above the experimental device. First, the experimental animals were brought to the behavioral testing laboratory and adapted for at least 1 hour. The experiment consisted of three stages: habituation, social interaction test, and social novelty recognition. In the test stage, the experimental animals were placed in the middle box for the first time and allowed to freely explore the three chambers of left, middle, and right for 10 minutes; in the social interaction test, the exploration time of the experimental animals on toys and unfamiliar animals was recorded within 10 minutes. In the training stage, objects were placed in the right box, mouse C1 was placed in the left box, and numbered experimental mice A1 were placed in the middle box. In the test stage, unfamiliar mice C2 were placed in the right box, familiar mice C1 were placed in the left box, and numbered experimental mice A1 were placed in the middle box. The photographic device was used to record the interaction of the experimental mice with unfamiliar mice C2 and familiar mice C1. After each experiment, the test box and the small cage where the mice were placed were cleaned with 70v / v% alcohol to remove the residual smell of the mice. After the experiment was completed, a stopwatch was used to analyze the contact time of the experimental mice with C1 and C2. During the test phase, the detection of an object or another animal was defined as any exploratory behavior when the animal's nose was within 1 cm of the object's wire cage or the wire cage of another animal, including head direction, climbing, sniffing, etc. The calculation method of the discrimination index was: discrimination index = (strange 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 pure water to avoid affecting the results of subsequent mouse experiments.

[0261] 4.4 Experimental Results

[0262] The data were analyzed by two-way ANOVA using GraphPad 6Gism software. The results were expressed as Mean ± SEM. When P < 0.05, the difference was considered to be statistically significant.

[0263] The results of the mouse social interaction experiment are statistically shown as follows Fig.21 As shown. Social behavior disorder is a typical feature of many mental illnesses. The exploration index of different groups was 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)%, 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. The reason may be that the administration of 6-gingerol caused certain stress responses in mice, which in turn affected the behavioral performance of mice. Nevertheless, there was no statistical difference between the WT+6G group and the WT+Veh group. Therefore, the administration of 6-gingerol did not cause adverse reactions in animals. However, Fmr1 KO mice were more inclined to interact with old mice, which indicated that the social ability of Fmr1 KO mice was defective. The administration of 6-gingerol can increase their interest in new mice, but the above improvement will not be achieved after TMZ intervention.

[0265] From the above experiments, we can know that:

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

[0267] The above embodiments of the present invention are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. Application of gingerol compounds in the preparation of products, wherein the products are used to treat and prevent 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 gingerol compound includes at least one of 6-gingerol, 8-gingerol, 10-gingerol, 12-gingerol or methyl-6-gingerol; Preferably, the gingerol compound comprises a pharmaceutically acceptable derivative.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable derivatives include pharmaceutically acceptable salts and 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 product 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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