Application of paeoniflorin in preparation of medicine for treating nodular sclerosis

Abstract

The invention belongs to the technical field of medicine application, discloses application of paeoniflorin in preparation of medicine for treating nodular sclerosis, and particularly discloses application of paeoniflorin or pharmaceutically acceptable salt thereof in preparation of medicine for treating and / or preventing hereditary intelligence and cognitive dysfunction diseases. The new medicinal value of the known compound paeoniflorin is explored, the paeoniflorin is used for treating the hereditary intellectual and cognitive impairment diseases (such as nodular sclerosis) and can be prepared into the medicine for treating the hereditary intellectual and cognitive impairment diseases, and a new application field is developed for application of radix paeoniae alba and radix paeoniae rubra medicinal materials.

Description

Technical Field

[0001] The invention belongs to the technical field of drug application, and particularly relates to the application of paeoniflorin in preparing a drug for treating tuberous sclerosis complex. Background Art

[0002] Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disease, and its clinical features are benign hamartomas in multiple organs and neuropsychiatric abnormalities. In addition, TSC is also the most common single gene defect disease causing autism spectrum disorder, which mainly manifests as intellectual disability, social interaction disorder and cognitive impairment, which greatly affects the patient's behavioral ability and quality of life, and an effective clinical treatment plan is urgently needed.

[0003] The occurrence of tuberous sclerosis is mainly caused by mutations in the TSC1 or TSC2 genes in the human body. Usually, the TSC1 and TSC2 proteins encoded by the TSC1 and TSC2 genes respectively form the TSC protein complex. The TSC1-TSC2 protein complex inhibits the activity of the mammalian target of rapamycin complex 1 (mTORC1) by activating GTPase and inhibiting the binding of the small G protein Rheb (Ras homologue enriched in brain) to GTP, which plays a role in cell growth and proliferation. When the TSC1 or TSC2 gene mutates, the TSC protein complex dissociates and causes excessive activation of mTORC1. Inhibiting the excessive activity of mTORC1 is now the main means of clinical treatment for tuberous sclerosis. Although the clinical use of mTORC1-specific inhibitors sirolimus (rapamycin) and sirolimus derivatives can effectively alleviate the clinical symptoms of tuberous sclerosis complex for a short time, there is still a lack of long-term effective treatment methods. Therefore, people have a strong interest in the complex causes of tuberous sclerosis, especially as it may become one of the effective ways to analyze the causes of autism, but the exact cause of the disease remains to be elucidated.

[0004] The clinical manifestations of tuberous sclerosis are complex. Currently, there is still a lack of long-term effective treatments for tuberous sclerosis complex in clinical practice. Most treatments for tuberous sclerosis complex are based on its symptoms, such as the use of anti-epileptic drugs or surgical removal of tumors. This 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.

[0005] Nowadays, sirolimus and its derivatives, as specific inhibitors of mTORC1, have been clinically approved for the treatment of renal angiomyolipoma, subependymal giant cell astrocytoma and pulmonary lymphangioleiomyomatosis in tuberous sclerosis. But the efficacy and side effects here also vary from person to person. Once patients with tuberous sclerosis stop using sirolimus or sirolimus derivatives, the symptoms of related tuberous sclerosis will relapse, and long-term use of sirolimus will have a series of adverse reactions such as hyperlipidemia, hematological abnormalities, hepatotoxicity and chronic immunosuppression. It is worth noting that the use of sirolimus in basic research can improve synaptic plasticity, social disorders and stereotyped behaviors in mice with tuberous sclerosis, but in clinical studies, the use of sirolimus cannot improve the symptoms of autism spectrum disorder in patients with tuberous sclerosis. Therefore, it is necessary to conduct in-depth research on the pathological mechanism of tuberous sclerosis and explore new treatment strategies for tuberous sclerosis.

[0006] The intellectual disability caused by tuberous sclerosis complex 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.

[0007] However, to date, there is still a lack of long-term 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 tuberous sclerosis and develop new therapeutic drugs. In addition, studying the genes and proteins that are abnormally expressed after the loss of TSC1 or TSC2 genes, and examining the specific effects of abnormal expression of related targets on the structure, morphology and function of neurons, can provide positive clinical application value and profound social significance for finding new drug intervention targets for the comprehensive treatment of tuberous sclerosis.

[0008] Peony was first recorded in Shennong's Herbal Classic. It is the dried root of Paeonia lactiflora Pall., a plant of the Ranunculaceae family. It is dug up in summer and autumn, washed, the head, tail and thin roots are removed, boiled in boiling water, and then the outer skin is removed or the skin is peeled and then boiled, and then dried. This product tastes bitter and sour, and is slightly cold in nature. It enters the liver and spleen meridians, and has the effects of nourishing blood and regulating menstruation, astringing yin and stopping sweating, softening the liver and relieving pain, and calming liver yang. In Shennong's Herbal Classic, there is no distinction between white peony and red peony, and they are collectively referred to as peony. It was not until the Ming Dynasty that a clear distinction was made between white peony and red peony. Paeoniflorin (PF) is a monoterpene glycoside compound extracted from peony, and it is also the main active ingredient in peony. At the same time, paeoniflorin is also widely present in traditional Chinese medicines such as peony bark and purple peony root. The structural formula of paeoniflorin is C 23 H28 O 11 , CAS number is 23180-57-6. Paeoniflorin has a wide range of pharmacological effects, such as anti-tumor, anti-inflammatory, analgesic, neuroprotective, anticonvulsant, hypoglycemic, and anti-thrombotic effects. In recent years, the neuroprotective effect of paeoniflorin has attracted much attention. Studies have shown that paeoniflorin can penetrate the blood-brain barrier, improve hippocampal neurogenesis in rats with depression, and correct cognitive dysfunction in Alzheimer's mice. Paeoniflorin has also been shown to have an effect on the mTOR signaling pathway in a variety of disease models. However, the application of paeoniflorin in the treatment of tuberous sclerosis has not been reported yet. Summary of the invention

[0009] The present invention aims to address the problems existing in the above-mentioned prior art, provide the performance and efficacy of paeoniflorin in treating hereditary intellectual and cognitive dysfunction diseases, especially a new application in drugs for tuberous sclerosis, and provide a new medicinal use of paeoniflorin.

[0010] The purpose of the first aspect of the present invention is to provide the use of paeoniflorin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing hereditary intellectual and cognitive dysfunction diseases.

[0011] The second aspect of the present invention aims to provide the use of paeoniflorin or a pharmaceutically acceptable salt thereof in the preparation of a product for inhibiting the abnormal proliferation of radial and / or non-radial adult neural stem cells.

[0012] The third aspect of the present invention aims to provide the use of paeoniflorin or a pharmaceutically acceptable salt thereof in the preparation of a product for promoting the maturation of newborn neurons from adult neural stem cells.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] The first aspect of the present invention provides the use of paeoniflorin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing hereditary intellectual and cognitive dysfunction diseases.

[0015] In some embodiments of the present invention, the hereditary intelligence and cognitive dysfunction diseases include autism, tuberous sclerosis, or cognitive dysfunction related to neurodevelopmental disorders.

[0016] In some embodiments of the present invention, the hereditary intelligence and cognitive dysfunction disease is tuberous sclerosis.

[0017] In some embodiments of the invention, the pharmaceutically acceptable salts include salts of free acids or bases of small molecule compounds that are non-toxic, bio-tolerated or otherwise biologically suitable for administration to a subject. See generally, SM Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without excessive toxicity, irritation or allergic response.

[0018] In some embodiments of the present invention, the pharmaceutically acceptable salt is selected from disodium salt, camphorsulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, caprate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate , butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propanesulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate and mandelate.

[0019] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0020] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, wetting agents, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors and solvents.

[0021] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose or glucose; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin or polyvinyl pyrrolidone; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked sodium carboxymethyl cellulose; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, micro-powder silica gel, talc or magnesium stearate; the suspending agent is selected from micro-powder silica gel, beeswax, cellulose, solid polyethylene glycol : The wetting agent is selected from glycerol, Tween-80, hydrogenated castor oil or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerol, propylene glycol or vegetable oil, and the vegetable oil is selected from soybean oil, castor oil, peanut oil, blending oil, etc.; the surfactant is selected from sodium dodecylbenzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavor, stevioside, acesulfame potassium, citric acid or saccharin sodium; the preservative is selected from at least one of methyl paraben or propyl paraben.

[0022] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is generally recognized for this purpose and as an inactive ingredient of the medicament. A compilation of pharmaceutically acceptable carriers 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 Press, London, 1994).

[0023] In some embodiments of the present invention, the paeoniflorin described in the present invention can be used alone or in the form of a pharmaceutical composition containing paeoniflorin when used to treat tuberous sclerosis, that is, the drug also includes a combination drug.

[0024] In some embodiments of the present invention, the combination drug includes a drug for treating and / or preventing hereditary intellectual and cognitive dysfunction diseases.

[0025] In some embodiments of the present invention, the drug is administered via the gastrointestinal tract and / or parenteral administration routes.

[0026] In some embodiments of the present invention, the non-gastrointestinal administration route is selected from injection, respiratory tract administration, skin administration, mucosal administration or cavity administration.

[0027] In some embodiments of the present invention, the drug dosage form for non-gastrointestinal administration is selected from injections, sprays, aerosols, patches, etc.; the drug dosage form for gastrointestinal administration is selected from tablets, capsules, powders, granules, pills, solutions, emulsions or syrups, etc.

[0028] In some embodiments of the present invention, the drug is in the form of an oral preparation, an injection or a local administration preparation.

[0029] In some embodiments of the present invention, 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; topical preparations include creams, ointments, sprays, aerosols, gels, cataplasms or patches.

[0030] In some embodiments of the present invention, the carrier in the drug includes 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.

[0031] In some embodiments of the present invention, the carriers that can be used in the preparation of 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.

[0032] In some embodiments of the present invention, the dosage of paeoniflorin or a pharmaceutically acceptable salt thereof is 20 to 50 mg / kg of the subject.

[0033] In some embodiments of the present invention, the dosage of paeoniflorin or a pharmaceutically acceptable salt thereof is 35-45 mg / kg of the subject.

[0034] In some embodiments of the present invention, the dosage of paeoniflorin or a pharmaceutically acceptable salt thereof is 40 mg / kg of the subject.

[0035] In some embodiments of the present invention, the purity of the paeoniflorin is greater than 60%.

[0036] The experiment proved that paeoniflorin has significant performance and efficacy in resisting hereditary intellectual and cognitive dysfunction, has little toxic side effects, is a safe, efficient, and stable drug for treating tuberous sclerosis, is suitable for industrial production, and is easy to promote. The present invention provides a new drug source for treating tuberous sclerosis and related diseases caused by changes and mutations in the TSC1 gene in the genetic process in the body, and provides a new medicinal use of paeoniflorin.

[0037] The second aspect of the present invention provides the use of paeoniflorin or a pharmaceutically acceptable salt thereof in the preparation of a product for inhibiting abnormal proliferation of radial and / or non-radial adult neural stem cells.

[0038] In some embodiments of the present invention, the product includes at least one of a reagent, a kit and a drug.

[0039] In some embodiments of the present invention, the pharmaceutically acceptable salt includes a salt of a free acid or base of a small molecule compound.

[0040] In some embodiments of the present invention, the pharmaceutically acceptable salt is selected from disodium salt, camphorsulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, caprate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate , butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propanesulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate and mandelate.

[0041] The third aspect of the present invention provides the use of paeoniflorin or a pharmaceutically acceptable salt thereof in the preparation of a product for promoting the maturation of newborn neurons from adult neural stem cells.

[0042] In some embodiments of the present invention, the product includes at least one of a reagent, a kit and a drug.

[0043] In some embodiments of the present invention, the pharmaceutically acceptable salt includes a salt of a free acid or base of a small molecule compound.

[0044] In some embodiments of the present invention, the pharmaceutically acceptable salt is selected from disodium salt, camphorsulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, caprate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate , butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propanesulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate and mandelate.

[0045] The beneficial effects of the present invention are:

[0046] The present invention discovers new medicinal value of the known compound paeoniflorin, uses it to treat hereditary intellectual and cognitive dysfunction diseases (such as tuberous sclerosis), and can be prepared into drugs for treating hereditary intellectual and cognitive dysfunction diseases, opening up a new application field for the application of white peony root and red peony root medicinal materials.

[0047] In order to explore the therapeutic effect and effect of paeoniflorin on tuberous sclerosis complex, the present invention carries out in vivo neural stem cell activation, differentiation experiment and cognitive function regulation experiment. Specifically, a large number of animal experiments were carried out by injection using different animal models, and the test results showed that paeoniflorin significantly inhibited the abnormal activation of adult neural stem cells in Tsc1 gene-deficient animals (Tsc1 cKO mice) in adult hippocampal neural stem cells; promoted the maturation of newborn neurons differentiated from adult neural stem cells in Tsc1 cKO mice. Experiments on the cognitive function of Tsc1 cKO mice have shown that paeoniflorin can correct the cognitive dysfunction of Tsc1 cKO mice; paeoniflorin improves the spatial memory ability of Tsc1 cKO mice and improves the pattern separation cognitive function defects of Tsc1 cKO mice by regulating neurogenesis; improves the learning and memory of Tsc1 cKO mice by regulating adult neurogenesis; corrects and improves the social interaction disorder of Tsc1 cKO mice by regulating neurogenesis.

[0048] The paeoniflorin of the present invention has strong pharmacological effects, is effective in treating tuberous sclerosis, has rapid effects, has little toxic and side effects, is safe, can be taken for a long time, and has good medicinal prospects. At the same time, the raw material source of paeoniflorin is abundant, inexpensive, safe for clinical use, and has a simple preparation process. It can be made into various dosage forms, has a small dosage, is easy to use, and is therefore easy to promote.

[0049] One of the most complex and critical systems in the human body is the nervous system, so the pathophysiological mechanisms of nervous system diseases are often complex and the pathogenesis is diverse. The present invention can use a single active ingredient of paeoniflorin to prepare a drug for treating tuberous sclerosis, and can also prepare a multi-target compound drug for treating tuberous sclerosis by combining paeoniflorin with other active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Diagram of the experimental scheme for the regulation of the proliferation of adult neural stem cells in the brain of adult mice by paeoniflorin.

[0051] Figure 2 These are non-radial adult neural stem cells (SOX2) in the DG region of adult WT / Tsc1 cKO mice that are in a proliferative state. + GFAP - Brdu + ) and radial adult neural stem cells in a proliferative state (SOX2 + GFAP + Brdu + ) Representative micrographs, where blue: DAPI; green: SOX2; red: Brdu; grey: GFAP; scale bar: 20 μm.

[0052] Figure 3 This is the result of data analysis of non-radial adult neural stem cells in the proliferative state in the DG region.

[0053] Figure 4 This is the result of data analysis of radial adult neural stem cells in the proliferative state in the DG region.

[0054] Figure 5 Diagram of the experimental scheme for the regulation of newborn neuron differentiation of neural stem cells in the brain of adult mice by paeoniflorin.

[0055] Figure 6 The immature neurons (DCX) differentiated from neural stem cells in the DG region of adult WT / Tsc1 cKO mice + T + )(A) and mature neurons (NeuN + T +) (B) Representative micrographs evaluating the effect of paeoniflorin on neuronal differentiation in Tsc1 cKO mice as assessed by immunostaining of immature neurons DCX (green) and mature neuronal markers NeuN (grey) and tdT (red); scale bar: 20 μm.

[0056] Figure 7 Results of neural stem cell differentiation experiment; A is the data analysis result of newborn immature neurons in the neural stem cell differentiation experiment; B is the data analysis result of newborn mature neurons in the neural stem cell differentiation experiment; in the figure, ns means no significant difference, * means p < 0.05, and ** means p < 0.01.

[0057] Figure 8 Diagram of the experimental scheme for the correction of cognitive deficits by regulating neurogenesis in the DG region of adult mice.

[0058] Fig. 9 Schematic diagram of the mouse novel location recognition experiment.

[0059] Fig.10 Statistical graph of the discrimination index for the mouse novel location recognition experiment.

[0060] Fig.11 Schematic diagram of mouse pattern separation experiment.

[0061] Fig.12 This is a statistical graph of the discrimination index of the mouse pattern separation experiment.

[0062] Fig.13 Schematic diagram of the novel object recognition experiment for mice.

[0063] Fig.14 This is a statistical chart of the discrimination index in the mouse novel object recognition experiment.

[0064] Fig.15 Schematic diagram of the mouse social interaction experiment.

[0065] Fig.16 This is a statistical chart of the discrimination index in the mouse social interaction experiment. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below through specific examples.

[0067] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0068] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0069] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0070] Example 1 In vivo proliferation and differentiation experiment of neural stem cells

[0071] 1. Experimental Materials

[0072] 1. Experimental Animals

[0073] Since systemic deletion of the Tsc1 gene causes embryonic lethality, an inducible conditional knockout mouse of the Tsc1 gene in adult hippocampal neural stem cells was constructed based on the loxp-cre system. loxP / loxp 、Nes-cre / ER T2 The Ai14 mice were purchased from Jackson Laboratory. The Tsc1 gene knockout mice (Tsc1 loxP / loxp ::Nestin-CreER T2 ::Ai14), represented by Tsc1 cKO mice; the blank control corresponding to the experimental mice was wild-type mice (Tsc1 + / + ::Nestin-CreER T2 ::Ai14), can be expressed by WT.

[0074] All mice were bred and raised at the Animal Center of the Chinese Medical Research Institute of the University of Macau, in a specific pathogen-free (SPF) animal breeding room with a breeding temperature of 22-25°C and a circadian rhythm cycle of 12 hours of light / 12 hours of darkness. The mice had free access to water and food, which was approved and recognized by the Animal Ethics Committee of the University of Macau.

[0075] 2. Experimental drugs

[0076] Paeoniflorin (PF) was purchased from MCE (MedChemExpress) with the catalog number HY-N0293; tamoxifen (TAM) was purchased from MCE (MedChemExpress) with the catalog number HY-13757A; 5-bromodeoxyuridine (BrdU) was purchased from MCE (MedChemExpress) with the catalog number 27847.

[0077] 2. Experimental Methods

[0078] 1. Breeding methods for experimental mice

[0079] 1.1. Mouse breeding for neural stem cell proliferation experiments

[0080] Female Tsc1 cells expressing Cre recombinase in neural stem cells and progenitor cells loxp / + Mouse (Tsc1 loxp / + ::Nestin-CreER T2 ) and Tsc1loxp / + male mice (Tsc1 loxp / + ) can be mated to produce the following genotype mice, which are marked as: (1) Tsc1 loxp / loxp ::Nestin-CreER T2 ⑵Tsc1 + / + ::Nestin-CreER T2 ⑶Tsc1 loxp / + ::Nestin-CreER T2 ; (4) Tsc1 loxp / loxp ; (5) Tsc1 loxp / + ; (6) Tsc1 + / + .

[0081] Mice marked as ⑴ and ⑵ among the offspring were selected and given the drug when they were 7 to 8 weeks old for subsequent cell activation experimental studies.

[0082] 1.2. Reproduction of mice for neural stem cell differentiation experiments

[0083] Female Tsc1 cells expressing Cre recombinase and inducible red fluorescent protein in neural stem cells and progenitor cells loxp / + Mouse (Tsc1 loxp / + ::Nestin-CreER T2 ::Ai14) and Tsc1 loxp / + Male mice (Tsc1 loxp / + ) can be mated to produce the following genotype mice, which are marked as: ⑺Tsc1 loxp / loxp ::Nestin-CreER T2 ::Ai14;⑻Tsc1 loxp / + ::Nestin-CreER T2 ::Ai14;⑼Tsc1 + / + ::Nestin-CreER T2 ::Ai14;⑽Tsc1 loxp / loxp ::Ai14;⑾Tsc1 loxp / + ::Ai14; ⑿Tsc1 + / + ::Ai14.

[0084] Mice marked as ⑺ and ⑻ were selected from the offspring and given the drug when they were 7 to 8 weeks old for subsequent cell differentiation experiments.

[0085] 2. Experimental grouping, medication and material collection

[0086] 2.1 Neural stem cell proliferation experiment

[0087] After 7-8 weeks of feeding, the mice marked with (1) and (2) were divided into 4 groups, 3 mice in each group, specifically:

[0088] Group A: Tsc1 + / + ::Nestin-CreER T2 + saline group (WT+Veh); Group B: Tsc1 loxp / loxp ::Nestin-CreER T2 + saline group (cKO+Veh); Group C: Tsc1 + / + ::Nestin-CreER T2 + paeoniflorin group (WT+PF); Group D: Tsc1 loxp / loxp ::Nestin-CreER T2 + paeoniflorin group (cKO+PF).

[0089] First, each group of mice was induced by intraperitoneal injection of tamoxifen (160 mg / kg) for five consecutive days. On the fifth and seventh days after the last tamoxifen injection, each group of mice was intraperitoneally injected with: the dosage of paeoniflorin was 40 mg / kg; the volume of saline was calculated according to the concentration of paeoniflorin, and the same volume of saline was given; intraperitoneal injection was performed at the same time every day; at the same time, on the seventh and eighth days after the last tamoxifen injection, each group of mice was intraperitoneally injected with BrdU (200 mg / kg) twice a day, for a total of 4 times. Two hours after the last intraperitoneal injection of BrdU, the mice were anesthetized and cardiac perfusion was performed. First, 0.9wt% saline was used for perfusion for 20 minutes, and then pre-cooled 4wt% paraformaldehyde was used for perfusion for 20 minutes. The brain was then removed and sliced ​​using a freezing microtome to obtain frozen sections for subsequent neural stem cell activation experiments. The experimental process is as follows Figure 1 .

[0090] Immunohistochemistry (frozen sections) (IHC-Fr) staining was performed on the frozen brain sections of the four groups of mice, 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 neurosterological quantitative analysis method was used, that is, the stained brain sections were placed under an upright microscope, and the StereoInvestigator neurosterological software was opened. For the DG area in the stained brain sections, the fluorescence channel was set as: green-SOX2 + Gray-GFAP + Red-Brdu + Blue-DAPI, the brain slices were scanned in the order on the slides, and SOX2 was determined by fluorescence staining in different channels after scanning. + GFAP + Brdu + (proliferating radial neural stem cells) and SOX2 + GFAP - Brdu + (non-radial neural stem cells in a proliferative state) and other positive targets, cell counting, the role of blue-DAPI is to determine the location and state of cells, and the positive targets are measured on the mouse brain sections that have been stained.

[0091] Scanning brain slices under a microscope to mark the proliferation of neural stem cells. Figure 2 As shown in the figure, blue: DAPI; green: SOX2; red: Brdu; gray: GFAP; scale bar: 20μm. The data analysis results of non-radial adult neural stem cells in the DG region in a proliferative state are shown in Figure 3 The data analysis results of radial adult neural stem cells in a proliferative state are as follows Figure 4 .

[0092] 2.2. Differentiation of neural stem cells

[0093] After 7-8 weeks of feeding, the mice marked as ⑺ and ⑻ were divided into 4 groups, 3 mice in each group, specifically:

[0094] Group E: Tsc1 + / +::Nestin-CreERT2::Ai14+normal saline group (WT-Ai14+Vehiche); Group F: Tsc1loxp / loxp::Nestin-CreERT2::Ai14+normal saline group (Tsc1 cKO-Ai14+Vehiche); Group G: Tsc1+ / +::Nestin-CreERT2::Ai14+peonidin group (WT-Ai14+PF); Group H: Tsc1loxp / loxp::Nestin-CreERT2::Ai14+peonidin group (Tsc1 cKO-Ai14+PF).

[0095] Each group of mice was induced by intraperitoneal injection of tamoxifen (160 mg / kg) for five consecutive days. On the fifth, seventh, ninth, eleventh and fourteenth days after the last tamoxifen injection, each group of mice was injected intraperitoneally: wherein: the dosage of paeoniflorin was 40 mg / kg; the volume of normal saline was calculated according to the concentration of paeoniflorin, and the same volume of normal saline was given; paeoniflorin and normal saline were administered by intraperitoneal injection at the same time each time. On the 28th day after the last administration, the mice were anesthetized and cardiac perfused, first with 0.9wt% normal saline for 20 minutes, then with pre-cooled 4wt% paraformaldehyde for 20 minutes, and then the brain was taken out. The entire hippocampus of the mouse brain was continuously sliced ​​at a thickness of 40μm, and the hippocampus was systematically randomly sampled by selecting 1 slice for every 6 slices. For subsequent neural stem cell differentiation experiments, the experimental process is as follows Figure 5 .

[0096] The measurement range of neurons in the dentate gyrus region of the hippocampus is relative to the anterior fontanelle, the hippocampus region with a width of -0.94mm to -2.30mm on the dorsal side, and the hippocampus region with a width of -2.30mm to -3.80mm on the ventral side. The frozen brain sections of mice in each experimental group were stained with immunohistochemistry (frozen sections) (IHC-Fr) and sealed with anti-fluorescence quenchers to obtain stained brain sections of mice in each experimental group; then the neurosterological quantitative analysis method was used, that is, the stained brain sections were placed under an upright microscope, the Stereo Investigator neurostereology 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 DCX was determined by fluorescent staining through different channels. + T + and NeuN + T + Positive targets such as blue-DAPI are used to determine the location of cells. After scanning, DCX + T + and DCX - NeuN+ T + The positive target cells were counted and the positive targets were measured on the mouse brain sections after staining. + ; Red-tdT + ; Gray-NeuN + ; Blue-DAPI.

[0097] Scanning brain slices under a microscope, newborn immature neurons in the DG region of mice (DCX + T + ) and newborn and mature neurons (NeuN + T + Representative micrographs of Figure 6 As shown, DAPI represents all cells, DCX marks adult newborn immature neurons, and NeuN marks mature neurons; green - NeuN + ; Red-tdT + ; Gray-NeuN + ; Blue-DAPI.

[0098] 3. Experimental Results

[0099] 1. Results of neural stem cell proliferation experiment

[0100] Brdu + and SOX2 + Double-positive cells represent proliferating radial neural stem cells, Brdu + 、SOX2 + and GFAP + The triple-positive cells represent proliferating radial neural stem cells, and the Brdu was counted using Stereo Investigator neurostereometry software. + 、SOX2 + The number of cells and BrdU + 、SOX2 + and GFAP + The ratio of the number of cells to the volume of DG indicates the density of proliferating radial stem cells and non-radial stem cells (the average value of 3 times). The experimental results are as follows:

[0101] By analyzing the non-radial stem cells in the proliferation state of each group, we found that group A: Tsc1 + / + ::Nestin-CreER T2 + Non-radiated stem cells in the proliferative state in the saline group (Brdu + SOX2 + GFAP - / mm 3 ) density is 2854 pieces / mm3 ; Group B: Tsc1 loxp / loxp ::Nestin-CreER T2 + Non-radiated stem cells in the proliferative state in the saline group (Brdu + SOX2 + GFAP - / mm 3 ) density is 5626 pieces / mm 3 ; Group C: Tsc1 + / + ::Nestin-CreER T2 + paeoniflorin group in the proliferation state of non-radial stem cells (Brdu + SOX2 + GFAP - / mm 3 ) density is 2141 / mm3; Group D: Tsc1 loxp / loxp ::Nestin-CreER T2 + paeoniflorin group in the proliferation state of non-radial stem cells (Brdu + SOX2 + GFAP - / mm 3 ) density is 2915 pieces / mm 3 .

[0102] By analyzing the radial stem cells in the proliferation state of each group, we found that group A: Tsc1 + / + ::Nestin-CreER T2 +Radial stem cells in the proliferating state in the saline group (Brdu + SOX2 + GFAP + / mm 3 ) density is 632 pieces / mm 3 ; Group B: Tsc1 loxp / loxp ::Nestin-CreER T2 +Radial stem cells in the proliferating state in the saline group (Brdu + SOX2 + GFAP + / mm 3 ) density is 1004 pieces / mm 3 ; Group C: Tsc1 + / + ::Nestin-CreER T2 + paeoniflorin group in the proliferative state of radial stem cells (Brdu + SOX2 + GFAP + / mm 3 ) density is 518 pieces / mm3 ; Group D: Tsc1 loxp / loxp ::Nestin-CreER T2 + paeoniflorin group in the proliferative state of radial stem cells (Brdu + SOX2 + GFAP + / mm 3 ) density is 561 pieces / mm 3 .

[0103] The results of the non-radial neural stem cell proliferation experiment showed that Figure 3 ; The results of radial neural stem cell proliferation experiment data analysis are as follows Figure 4 From the above data analysis, it can be seen that the density of radial stem cells and non-radial stem cells in the proliferative state in group B is higher than that in group A, but after the administration of paeoniflorin, it can be seen that the density of radial stem cells and non-radial stem cells in the proliferative state in group D is almost restored to a level similar to that in group A. This indicates that paeoniflorin significantly inhibits the abnormal proliferation of radial and non-radial adult neural stem cells in Tsc1 cKO mice.

[0104] Tsc1 cKO mice show abnormal proliferation of radial and non-radial adult neural stem cells, and the disease phenotype can be corrected after administration of paeoniflorin. It can be seen that paeoniflorin has a certain therapeutic significance for the neuropsychiatric symptoms associated with tuberous sclerosis caused by the loss of the Tsc1 gene, and paeoniflorin has a potential role in the treatment of tuberous sclerosis.

[0105] 2. Results of neural stem cell differentiation experiment

[0106] DCX is a marker for immature neurons, NeuN is a marker for mature neurons, and tdT is a marker for neural stem cells when tamoxifen is injected. Its red fluorescence will not weaken due to the proliferation and differentiation of neural stem cells, so it can track the process of neural stem cell proliferation and differentiation. + T + NeuN represents the differentiation of adult neural stem cells into immature neurons. + T + Represents newly differentiated adult neural stem cells into mature neurons. DCX was counted using Stereo Investigator Neurostereometry Software + T + and NeuN + T + The ratio of the number of cells to the volume of DG indicates the density of adult neural stem cells differentiating into immature neurons, transitional neurons and mature neurons (the average value of 3 times). The experimental results are as follows:

[0107] By analyzing the differentiation of each group of somatic neural stem cells into immature neurons, we found that group E: Tsc1 + / + ::Nestin-CreER T2 ::Adult neural stem cells in the Ai14+ saline group newly differentiated into immature neurons (DCX + T + / mm 3 ) density is 3855 pieces / mm 3 ; Group F: Tsc1 flox / flox ::Nestin-CreER T2 ::Adult neural stem cells in the Ai14+ saline group newly differentiated into immature neurons (DCX + T + / mm 3 ) density is 2284 pieces / mm 3 ; Group G: Tsc1 + / + ::Nestin-CreER T2 ::Adult neural stem cells in the Ai14+peonidin group newly differentiated into immature neurons (DCX + T + / mm 3 ) density is 3249 pieces / mm 3 ; Group H: Tsc1 flox / flox ::Nestin-CreER T2 ::Adult neural stem cells in the Ai14+peonidin group newly differentiated into immature neurons (DCX + T + / mm 3 ) density is 3494 pieces / mm 3 .

[0108] By analyzing the differentiation of adult neural stem cells into mature neurons in each group, it was found that in group E: Tsc1+ / +::Nestin-CreERT2::Ai14+ saline group, adult neural stem cells were newly differentiated into mature neurons (NeuN + T + / mm 3 ) density is 13375 pieces / mm 3 ; Group F: Tsc1 flox / flox ::Nestin-CreER T2 :: Adult neural stem cells in the Ai14+ saline group newly differentiated into mature neurons (NeuN + T + / mm 3 ) density is 8386 pieces / mm 3 ; Group G: Tsc1 + / +::Nestin-CreER T2 :: Adult neural stem cells in the Ai14+peonidin group newly differentiated into mature neurons (NeuN + T + / mm 3 ) density is 11176 pieces / mm 3 ; Group H: Tsc1 flox / flox ::Nestin-CreER T2 ::Adult neural stem cells in the Ai14+peonidin group newly differentiated into transitional neurons (NeuN + T + / mm 3 ) density is 11494 pieces / mm 3 .

[0109] The results of the neural stem cell differentiation experiment showed that Figure 7 In A and B, the above data analysis shows that the number of immature newborn neurons and mature newborn neurons in group F is less than that in group E, but after the administration of paeoniflorin, the proportion of newborn immature neurons and mature neurons in group H can be seen to be almost restored to a level similar to that in group E. This indicates that paeoniflorin can promote the differentiation of adult neural stem cells into newborn neurons in Tsc1 cKO mice.

[0110] Tsc1 cKO mice show a decrease in the number of adult neural stem cells differentiating into mature neurons, and this disease phenotype can be corrected after administration of paeoniflorin. It can be seen that the neuropsychiatric symptoms associated with tuberous sclerosis complex have certain therapeutic significance, and paeoniflorin has the potential to treat tuberous sclerosis complex.

[0111] As mentioned above, according to the quantitative analysis results of the above data, we can know that: Brdu + SOX2 + GFAP - , Brdu + SOX2 + GFAP + The density showed that paeoniflorin could reduce the abnormal proliferation of radial neural stem cells and non-radial neural stem cells in Tsc1 cKO mice, according to DCX + T + and NeuN + T + The density of peony root showed that peony root promoted the differentiation and maturation of neurons. Therefore, it can be seen that peony root can reduce the abnormal proliferation of neural stem cells in the brain of Tsc1 cKO mice, promote their neural differentiation, and correct the neurogenesis disorder of Tsc1 cKO mice.

[0112] As mentioned above, the loss of the Tsc1 gene in adult neural stem cells in the DG region will lead to abnormal proliferation of neural stem cells in the DG region, but reduce their differentiation into neurons. After administration of paeoniflorin, neural stem cells can resume normal proliferation and differentiation, promote normal neurogenesis, and reduce the damage caused by the loss of the Tsc1 gene.

[0113] Example 2 Cognitive function regulation test

[0114] Animal cognitive function detection methods have been used to prove that paeoniflorin can correct cognitive dysfunction and social interaction disorders in mice with conditional knockout of adult hippocampal neural stem cell Tsc1 gene (Tsc1 cKO). The results of this study indicate that paeoniflorin can treat cognitive dysfunction in mice with tuberous sclerosis by regulating neurogenesis.

[0115] 1. New location recognition experiment

[0116] The new location recognition experiment is a classic behavioral test for detecting the spatial memory ability of mice. In order to explore whether paeoniflorin can correct the spatial memory ability of Tsc1 cKO mice, each group of mice was induced by intraperitoneal injection of tamoxifen (160 mg / kg) for five consecutive days. On the fifth, seventh, ninth, eleventh and fourteenth days after the last tamoxifen injection, each group of mice was intraperitoneally injected with: Among them: the dosage of paeoniflorin was 40 mg / kg; the volume of normal saline was calculated according to the concentration of paeoniflorin, and the same volume of normal saline was given; paeoniflorin and normal saline were intraperitoneally injected at the same time each time. The new location recognition experiment was tested on the 28th day after the last administration. The experimental process is as follows: Figure 8 .

[0117] 1. Materials

[0118] A square open field; object A; object B; facial tissue; 75% alcohol; two timers.

[0119] Experimental animals and drugs: 7-8 weeks old Tsc1 loxp / loxp ::Nestin-CreER T2 (Tsc1 cKO) and Tsc1 + / + ::Nestin-CreER T2 The source of (WT) mice was the same as that in Example 1; paeoniflorin, purity ≥ 99%, was purchased from MCE Company with the item number HY-N0293; tamoxifen (TAM) was purchased from MCE Company with the item number HY-13757A.

[0120] 2. Experimental Grouping and Drug Administration

[0121] Experimental group: WT+Vehicle:Tsc1 + / +::Nestin-CreER T2 Mice were intraperitoneally injected with normal saline; cKO+Vehicle:Tsc1 loxp / loxp ::Nestin-CreER T2 Mice were intraperitoneally injected with normal saline; WT+PF:Tsc1 + / + ::Nestin-CreER T2 Mice were intraperitoneally injected with paeoniflorin; cKO+PF:Tsc1 loxp / loxp ::Nestin-CreER T2 Mice were intraperitoneally injected with paeoniflorin; there were 4 groups in total, with 7 mice in each group (ie, n=7).

[0122] Dosage:

[0123] ①: Each group of mice aged 7 to 8 weeks were intraperitoneally injected with tamoxifen at a dose of 160 mg / kg for five consecutive days.

[0124] ②: WT+Vehicle, cKO+Vehicle group: 7-8 weeks old Tsc1 loxp / loxp ::Nestin-CreER T2 and Tsc1 + / + ::Nestin-CreER T2 On the fifth, seventh, ninth, eleventh and fourteenth day after the last tamoxifen injection, each group of mice was intraperitoneally injected with normal saline. The volume of normal saline was calculated according to the concentration of paeoniflorin, and the same volume of normal saline was given.

[0125] ③: WT+PF, cKO+PF group: 7-8 weeks old Tsc1 loxp / loxp ::Nestin-CreER T2 and Tsc1 + / + ::Nestin-CreER T2 On the fifth, seventh, ninth, eleventh and fourteenth days after the last tamoxifen injection, each group of mice was intraperitoneally injected with paeoniflorin at a dose of 40 mg / kg.

[0126] 3. Experimental steps

[0127] The test measures the spatial memory of rodents by assessing their ability to recognize new locations of familiar objects based on spatial cues.

[0128] First, the experimental animals were brought to the behavioral testing laboratory and adapted for at least 1 hour. In the training phase, each animal was placed individually in a square field, and two identical objects were placed at the same distance from a side of colored wallpaper. The mice were allowed to explore freely for 6 minutes, and the training process was repeated 3 times. In the test phase, the location of one of the familiar objects was moved to another new location. The exploration time of the experimental animals on the new and old objects in the 6 minutes was recorded. A normal animal should spend more time exploring the objects in the new location. The discrimination criterion was calculated based on the difference between the percentage of time spent recognizing the object in the new location and the time spent recognizing the object in the original location. In the test phase, the exploration of the object was defined as any exploratory behavior of the animal's nose within 1 cm of the object, including head direction, climbing, sniffing, etc. The calculation method of the discrimination index is: discrimination index = (new location object recognition time / total recognition time × 100)-(original location object recognition time / total recognition time × 100).

[0129] The schematic diagram of the new position recognition experiment is as follows Fig. 9 After each mouse test, the test field needs to be wiped clean with 75% alcohol to eliminate the influence of the same odor interference on the test behavior of the next experimental mouse.

[0130] 4. Experimental results

[0131] The data were analyzed by two-way analysis of variance (ANOVA) using GrapaPad Prism software. The data processing results were expressed as Mean±SEM. When P<0.05, it was considered to be statistically significant.

[0132] The statistical results of the mouse novel location recognition experiment are as follows Fig.10 As shown, the discrimination indexes of the WT+Vehicle, cKO+Vehicle, WT+PF, and cKO+PF groups were 34.87%, -23.52%, 21.77%, and 46.71%, respectively.

[0133] The experimental results show that WT mice are more inclined to explore new objects, while Tsc1 cKO mice are more inclined to explore old objects, which indicates that the spatial memory ability of Tsc1 cKO mice is defective, and paeoniflorin administration can correct this defect. This shows that paeoniflorin can improve the spatial memory of Tsc1 cKO mice by regulating neurogenesis.

[0134] 2. Mode Separation Experiment

[0135] Pattern separation experiments are an advanced way to explore the ability of mice to distinguish similar events. This experiment will investigate whether paeoniflorin can correct the pattern separation defect of Tsc1 cKO model mice by promoting neurogenesis.

[0136] 1. Materials

[0137] Materials: a square open field; object A (2 pieces); object B (2 pieces), object A and object B are different; facial tissue; 75% alcohol; two timers.

[0138] Experimental animals and drugs: the same as those in “I. New location recognition experiment”.

[0139] 2. Experimental Grouping and Drug Administration

[0140] Experimental group: WT+Vehicle:Tsc1 + / + ::Nestin-CreER T2 Mice were intraperitoneally injected with normal saline; cKO+Vehicle:Tsc1 loxp / loxp ::Nestin-CreER T2 Mice were intraperitoneally injected with normal saline; WT+PF:Tsc1 + / + ::Nestin-CreER T2 Mice were intraperitoneally injected with paeoniflorin; cKO+PF:Tsc1 loxp / loxp ::Nestin-CreER T2 Mice were intraperitoneally injected with paeoniflorin; there were 4 groups in total, with 7 mice in each group (ie, n=7).

[0141] Administration method: The same as the drug administration method in “I. New location recognition experiment”.

[0142] 3. Experimental steps

[0143] like Fig.11 During the training, 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 allowed to explore for 10 minutes. After 30 minutes, the same animal was placed in a second square field with a different floor pattern from the first experiment and two completely different sets of objects (Object B) and allowed to explore for 10 minutes.

[0144] During the experiment, one of the two training wallpapers 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.

[0145] 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.

[0146] 4. Experimental results

[0147] The data were analyzed by two-way analysis of variance (ANOVA) using GrapaPad Prism software. The data processing results were expressed as Mean±SEM. When P<0.05, it was considered to be statistically significant.

[0148] The discrimination indexes of WT+Vehicle, cKO+Vehicle, WT+PF, and cKO+PF groups were 10.10%, -36.4%, 20.86%, and 35.73%, respectively. Fig.12 shown.

[0149] The experimental results showed that WT mice were more inclined to explore new objects, while Tsc1 cKO mice showed the opposite behavior, indicating that Tsc1 cKO mice had pattern separation defects, which could be corrected after administration of paeoniflorin. This indicates that paeoniflorin can improve the pattern separation defect of Tsc1 cKO mice by regulating neurogenesis.

[0150] From the above experiments, it can be seen that paeoniflorin can improve the pattern separation defect of Tsc1 cKO mice by regulating adult neurogenesis.

[0151] 3. New Object Recognition Experiment

[0152] This test is based on the natural tendency of rodents to explore novel objects rather than familiar objects. This experiment examined the novel object recognition ability of mice and was used to explore whether paeoniflorin could correct the learning and memory impairment of Tsc1 cKO mice by regulating adult neurogenesis.

[0153] 1. Experimental Materials

[0154] Materials: a square open field; object A; object B; facial tissue; 75% alcohol; two timers.

[0155] Experimental animals and drugs: Same as those in “I. New location recognition experiment”

[0156] 2. Experimental Grouping and Drug Administration

[0157] Experimental group: WT+Vehicle:Tsc1 + / + ::Nestin-CreER T2Mice were intraperitoneally injected with normal saline; cKO+Vehicle:Tsc1 loxp / loxp ::Nestin-CreER T2 Mice were intraperitoneally injected with normal saline; WT+PF:Tsc1 + / + ::Nestin-CreER T2 Mice were intraperitoneally injected with paeoniflorin; cKO+PF:Tsc1 loxp / loxp ::Nestin-CreER T2 Mice were intraperitoneally injected with paeoniflorin; there were 4 groups in total, with 7 to 9 mice in each group (ie, n = 7 to 9).

[0158] Administration method: Same as the drug administration method in "I. New location recognition experiment"

[0159] 3. Experimental steps

[0160] The test measures novel object recognition, a form of memory task that does not rely on spatial cues. In this task, animals are trained to recognize specific objects.

[0161] like Fig.13 First, the experimental animals were brought to the behavioral testing laboratory and adapted for at least 1 hour. During the training phase, each animal was placed individually in an L-shaped field with corridors of the same length. Two identical objects were placed at the end of the corridors on both sides and were allowed to explore for 10 minutes. During the test phase, one of the familiar objects was replaced with a new object, and the exploration time of the experimental animals on the new and old objects was recorded within 10 minutes. A normal animal should spend more time exploring unfamiliar objects. The discrimination criterion was calculated based on the difference between the percentage of time spent recognizing unfamiliar objects and the time spent recognizing familiar objects. During the test phase, the exploration of objects was defined as any exploratory behavior within 1 cm of the animal's nose from the object, including head direction, climbing, sniffing, etc. The calculation method of the discrimination index is: discrimination index = (strange object recognition time / total recognition time × 100%)-(familiar object recognition time / total recognition time × 100%).

[0162] After each mouse test, the scene was cleaned with pure water to avoid affecting the results of subsequent mouse experiments.

[0163] 4. Experimental results

[0164] The data were analyzed by two-way analysis of variance (ANOVA) using GrapaPad Prism software. The data processing results were expressed as Mean±SEM. When P<0.05, it was considered to be statistically significant.

[0165] The results of the mouse novel object recognition experiment are as follows Fig.14 The discrimination indexes of WT+Vehicle, cKO+Vehicle, WT+PF, and cKO+PF groups were 13.09%, -21.11%, 12.18%, and 18.91%, respectively.

[0166] The experimental results showed that WT mice were more inclined to explore new objects, while Tsc1 cKO mice showed the opposite behavior. This indicates that Tsc1 cKO mice have defective learning and memory abilities, and this defect can be corrected after administration of paeoniflorin.

[0167] From the above experiments, it can be seen that paeoniflorin can improve the memory of Tsc1 cKO model mice by regulating adult neurogenesis.

[0168] 4. Social Interaction Experiment

[0169] Social interaction disorder is one of the most prominent manifestations of autism. This experiment used a three-box interactive social behavior test to test whether paeoniflorin can correct the social behavior defects of Tsc1 cKO model mice by regulating neurogenesis.

[0170] 1. Experimental Materials

[0171] Materials: rectangular operating box, metal cage; two mice; facial tissue; 75% alcohol; two timers.

[0172] Experimental animals and drugs: Same as those in “I. New location recognition experiment”

[0173] 2. Experimental Grouping and Drug Administration

[0174] Experimental group: WT+Vehicle:Tsc1 + / + ::Nestin-CreER T2 Mice were intraperitoneally injected with normal saline; cKO+Vehicle:Tsc1 loxp / lox p::Nestin-CreER T2 Mice were intraperitoneally injected with normal saline; WT+PF:Tsc1 + / + ::Nestin-CreER T2 Mice were intraperitoneally injected with paeoniflorin; cKO+PF:Tsc1 loxp / loxp ::Nestin-CreER T2 Mice were intraperitoneally injected with paeoniflorin; there were 4 groups in total, with 7 mice in each group (ie, n=7).

[0175] Administration method: Same as the drug administration method in "I. New location recognition experiment"

[0176] 3. Experimental steps

[0177] The test is to detect changes in the social behavior of rodents, which are naturally gregarious and have a tendency to explore new objects. The social behavior test device is a three-chamber box with a partition wall made of black plexiglass and a small round door with a diameter of 7 cm inside. The animals can move freely in the three boxes.

[0178] like Fig.15 First, the experimental animals were brought to the behavioral testing laboratory and adapted for at least 1 hour. The experiment consisted of three phases: habituation, social interaction test, and social novelty recognition. In the test phase, the experimental animals were first placed in the middle box and allowed to freely explore the three chambers of left, middle, and right for 10 minutes; next, a strange animal was placed in the wire cage on the left side of the chamber, and a toy was placed in the wire cage on the right side of the chamber. In the social interaction test, the exploration time of the experimental animals on the toy and the strange animal was recorded within 10 minutes. In the social novelty recognition test, the animal that had interacted before remained unchanged (left side), and the toy in the right side of the chamber was replaced by a new strange animal (right side). The exploration time of the experimental animals on the familiar animal (left side, from the previous social interaction phase) and the strange animal (right side, the newly replaced animal) was recorded within 10 minutes. The discrimination criterion was calculated based on the difference between the percentage of time spent recognizing the strange animal and the time spent recognizing the toy (or recognizing the familiar animal). During the test phase, detection of an object or another animal was defined as any exploratory behavior in which the animal's nose was within 1 cm of the object's wire cage or another animal's wire cage, including head orientation, climbing, sniffing, etc. The discrimination index was calculated as follows: discrimination index = (unfamiliar animal recognition time / total recognition time × 100%) - (toy or familiar animal recognition time / total recognition time × 100%).

[0179] After each mouse test, the scene was cleaned with pure water to avoid affecting the results of subsequent mouse experiments.

[0180] 4. Experimental results

[0181] The data were analyzed by two-way analysis of variance (ANOVA) using GrapaPad Prism software. The data processing results were expressed as Mean±SEM. When P<0.05, it was considered to be statistically significant.

[0182] The results of the mouse social interaction experiment are statistically shown as follows Fig.16 As shown, the discrimination indexes of the WT+Vehicle, cKO+Vehicle, WT+PF, and cKO+PF groups were 21.58%, -27.63%, 42.67%, and 32.28%, respectively.

[0183] The experimental results showed that WT mice were more inclined to interact with new mice, while Tsc1 cKO mice were more inclined to interact with old mice. This indicates that Tsc1 cKO mice have defective social ability, which can be corrected by administering paeoniflorin.

[0184] From the above experiments, it can be seen that paeoniflorin can correct the social defects of Tsc1 cKO model mice by regulating neurogenesis.

[0185] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of paeoniflorin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing hereditary intellectual and cognitive dysfunction diseases.

2. The use according to claim 1, characterized in that: The hereditary intelligence and cognitive dysfunction diseases include autism, tuberous sclerosis or cognitive dysfunction related to neurodevelopmental disorders; Preferably, the hereditary intelligence and cognitive dysfunction disease is tuberous sclerosis.

3. The use according to claim 1, characterized in that: The pharmaceutically acceptable salts include salts of free acids or bases of small molecule compounds; Preferably, the pharmaceutically acceptable salt is selected from the group consisting of disodium salt, camphorsulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne- Any one of 1,4-dioic acid salt, hexyne-1,6-dioic acid salt, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propanesulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate and mandelate.

4. The use according to any one of claims 1 to 3, characterized in that: The drug also includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, wetting agents, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors and solvents.

5. The use according to any one of claims 1 to 3, characterized in that: The drug is administered by gastrointestinal or / and non-gastrointestinal routes; Preferably, the dosage of paeoniflorin or a pharmaceutically acceptable salt thereof is 20-50 mg / kg per subject.

6. The use according to any one of claims 1 to 3, characterized in that: The medicine also includes a combination drug; preferably, the combination drug includes a drug for treating and / or preventing hereditary intellectual and cognitive dysfunction diseases.

7. Use of paeoniflorin or a pharmaceutically acceptable salt thereof in the preparation of a product for inhibiting abnormal proliferation of radial and / or non-radial adult neural stem cells.

8. Use of paeoniflorin or a pharmaceutically acceptable salt thereof in the preparation of a product for promoting the maturation of newly generated neurons from adult neural stem cells.

9. The use according to claim 7 or 8, characterized in that: The product includes at least one of a reagent, a kit and a medicine.

10. The use according to claim 7 or 8, characterized in that: The pharmaceutically acceptable salts include salts of free acids or bases of small molecule compounds.

Images