Use of gut microbiota modulator in preparation of drug for preventing and / or treating brain neurological disease

Abstract

The present invention relates to use of a gut microbiota modulator in the preparation of a drug for preventing and / or treating a brain neurological disease. It is found in the present invention that an oral formulation of the gut microbiota modulator can modulate the microbiota type and abundance of an intestinal site, and can also inhibit Th17 cells of the intestinal site, thereby achieving safe and efficient prevention and / or treatment of the brain neurological disease.

Description

Use of intestinal flora regulators in the preparation of drugs for preventing and / or treating brain neurological diseases Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the use of an intestinal flora regulator in the preparation of medicines for preventing and / or treating brain neurological diseases. Background Art

[0002] Brain neurological diseases are diseases that occur in the central nervous system of the brain and are characterized by disorders of sensation, consciousness, movement, etc., including Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, depression and many other types.

[0003] It has been reported that there is a certain connection between the intestinal system and the central nervous system, and changes in the intestinal system may cause changes in brain function. For example, immune regulatory factors can cause sleep disorders by mediating the pathway of glial cells-intestinal-neurons, thereby affecting lifespan (see, Xu et al., Neuron, August 7, 2023: S0896-6273 (23) 00543-3). However, as of now, the relevant mechanisms of the brain-gut axis have not been fully revealed and further exploration and discovery are needed.

[0004] The existing technologies for treating brain neurological diseases are relatively limited. Taking multiple sclerosis as an example, there is currently no cure. The main treatment methods include high-dose hormone shock therapy in the acute phase and disease-modifying therapy (DMT) in the remission phase. Among them, the types of drugs available for DMT treatment are relatively limited, and the treatment costs are high.

[0005] Therefore, there is an urgent need in the art to develop drugs that can safely and effectively prevent and / or treat brain neurological diseases.

[0006] Summary of the Invention

[0007] The object of the present invention is to provide a drug and its use for safely and effectively preventing and / or treating brain neurological diseases of a subject.

[0008] The first aspect of the present invention provides a use of an intestinal flora regulator, characterized in that it is used to prepare a drug for preventing and / or treating brain neurological diseases, wherein the drug is an oral preparation;

[0009] Furthermore, the intestinal flora regulator is selected from the following group:

[0010] (a) dehydrocorybulbine (DHCB) or a pharmaceutically acceptable salt thereof, or an extract containing DHCB;

[0011] (b) haloperidol or a pharmaceutically acceptable salt thereof;

[0012] (c) a combination of components (a) and (b);

[0013] (d) Dimethylglycine (DMG) or a pharmaceutically acceptable salt thereof.

[0014] In another preferred embodiment, the drug is an enteric-coated preparation.

[0015] In another preferred embodiment, the intestinal flora regulator is dehydrocorybulbine (DHCB) or a pharmaceutically acceptable salt thereof.

[0016] In another preferred embodiment, the pharmaceutically acceptable salt of DHCB or haloperidol is a salt of DHCB or haloperidol and an anion selected from the group consisting of chloride anion, bromide anion, iodide anion, sulfate anion, hydrogen sulfate anion, phosphate anion, hydrogen phosphate anion, dihydrogen phosphate anion, methanesulfonate anion, maleate anion, tartaric acid anion, acetate anion, formate anion, citrate anion, camphorsulfonate anion, fumarate anion, succinate anion, lactate anion, methanesulfonate anion, malate anion, benzoate anion, gluconic acid anion, or a combination thereof; preferably, the anion is selected from the group consisting of acetate anion, formate anion, chloride anion, or a combination thereof.

[0017] In another preferred embodiment, the DHCB-containing extract is an extract of a Corydalis plant.

[0018] In another preferred embodiment, the DHCB-containing extract is an extract of the Corydalis yanhusuo plant.

[0019] In another preferred embodiment, the DHCB-containing extract is an aqueous extract of Corydalis yanhusuo or an extract of a water / alcohol solvent.

[0020] In another preferred embodiment, the intestinal flora regulator also includes the traditional Chinese medicine Corydalis yanhusuo.

[0021] In another preferred embodiment, the brain neurological disease is selected from the group consisting of depression, multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), brain inflammation, and neuromyelitis optica spectrum disorders.

[0022] In another preferred embodiment, the intestinal flora regulator includes a dopamine D2 receptor inhibitor.

[0023] In another preferred embodiment, the drug is administered to a subject with high or normal expression of intestinal dopamine D2 receptors.

[0024] In another preferred embodiment, the dosage form of the oral preparation is selected from the following group: tablets, capsules, granules, pills, powders, solutions, emulsions, and suspensions.

[0025] In another preferred embodiment, after the intestinal flora regulator is administered, the concentration reached in the intestinal tract is 1-200 μM, preferably 10-100 μM, and more preferably 20-80 μM.

[0026] In another preferred embodiment, the unit dose of the oral formulation is 0.1 to 1000 mg / kg, preferably, 1 to 500 mg / kg, more preferably, 5 to 100 mg / kg, more preferably, 10 to 50 mg / kg, based on the weight of the subject.

[0027] In another preferred embodiment, the intestinal flora regulator is used for:

[0028] Upregulates the abundance of Lactobacillus in the intestine;

[0029] Upregulates the metabolite N2-acetyl-L-lysine in the intestine.

[0030] In another preferred embodiment, the intestinal flora regulator is used to downregulate the amount of Th17 cells in the intestinal tract.

[0031] In another preferred embodiment, the subject is female or male; preferably, the subject is female.

[0032] The second aspect of the present invention provides a pharmaceutical composition, which comprises:

[0033] (i) a first active ingredient, wherein the first active ingredient is an intestinal flora regulator selected from the group consisting of:

[0034] (a) dehydrocorybulbine (DHCB) or a pharmaceutically acceptable salt thereof, or an extract containing DHCB;

[0035] (b) haloperidol or a pharmaceutically acceptable salt thereof;

[0036] (c) the combination of components (a) and (b) is selected from the group consisting of dehydrocorybulbine (DHCB), haloperidol or a pharmaceutically acceptable salt thereof; or a combination thereof;

[0037] (d) dimethylglycine (DMG) or a pharmaceutically acceptable salt thereof;

[0038] (ii) optionally, a second active ingredient selected from the group consisting of fingolimod, siplatimud, or any combination thereof;

[0039] (iii) a pharmaceutically acceptable carrier.

[0040] In another preferred embodiment, in the pharmaceutical composition, the content of the first active ingredient is 0.01-99 wt %, preferably 0.1-90 wt %, based on the total weight of the pharmaceutical composition.

[0041] In another preferred embodiment, in the pharmaceutical composition, DHCB or a pharmaceutically acceptable salt thereof is the sole active ingredient for treating multiple sclerosis.

[0042] In another preferred embodiment, the multiple sclerosis is selected from the following group: relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, and progressive relapsing multiple sclerosis.

[0043] The third aspect of the present invention provides a use of the pharmaceutical composition of the second aspect of the present invention, wherein the pharmaceutical composition is used to prepare a drug for treating, preventing or alleviating depression and / or multiple sclerosis-related diseases.

[0044] In another preferred embodiment, DHCB is used as a single component in combination with other effective multiple sclerosis treatment methods (surgery, radiotherapy, etc.) in the comprehensive treatment of multiple sclerosis.

[0045] A fourth aspect of the present invention provides a method for preventing and / or treating depression and / or multiple sclerosis, comprising: orally administering to a subject in need thereof a therapeutically effective amount of a gut flora modulator selected from the group consisting of:

[0046] (a) dehydrocorybulbine (DHCB) or a pharmaceutically acceptable salt thereof, or an extract containing DHCB;

[0047] (b) haloperidol or a pharmaceutically acceptable salt thereof;

[0048] (c) a combination of components (a) and (b);

[0049] (d) Dimethylglycine (DMG) or a pharmaceutically acceptable salt thereof.

[0050] In another preferred embodiment, the subject is female or male; preferably, the subject is female.

[0051] In another preferred embodiment, the subject includes rodents (such as mice and rats) and primates (such as humans).

[0052] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 shows that oral administration of DHCB to female mice has a significant anti-EAE effect. Figure 1A. Flowchart of mouse dosing and modeling; Figure 1B. EAE scoring chart, with the control group (blue) and the DHCB-treated group (30 mg / kg) (red); Figure 1C. Spinal cord LFB myelin staining and HE staining, showing that oral administration of DHCB significantly alleviated spinal cord pathology; Figure 1D. Spinal cord pathology scoring statistics.

[0054] Figure 2 shows that DHCB can antagonize intestinal epithelial dopamine D2 receptors at the cellular and in vivo levels. Figure 2A. FHC cells stimulated with 1 μM haloperidol, 10 μM haloperidol, 50 μM DHCB, and 100 μM PEA; Figure 2B. Statistical graph of panel A; Figure 2C. Western blotting of D2 downstream p-AKT in large intestinal epithelial cells from EAE-gavaged mice and control mice; Figure 2D. Statistical graph of panel C.

[0055] Figure 3 shows that oral administration of DHCB significantly reduces intestinal epithelial LYZ1 expression and affects the intestinal microbiota. Figure 3A. RNA sequencing volcano plot shows that the downregulated genes are mainly lysozyme and defensin; Figure 3B. 16S principal component analysis shows that oral administration of DHCB significantly changes the intestinal microbiota of mice.

[0056] FIG4 shows the effects of haloperidol and DHCB against EAE model.

[0057] FIG5 shows the results of flow cytometric analysis of T cells in the peripheral immune organs spleen and mesenteric lymph nodes after oral administration of DHCB.

[0058] FIG6 shows the effect of oral administration of DHCB on the EAE model in male mice.

[0059] Figure 7 shows the antidepressant effect of DHCB gavage in mice. B: DHCB administration and mouse modeling experimental scheme. C: Forced swimming behavior. All data are expressed as mean ± SEM, n = 8-10. Unpaired t-test, *p < 0.05, ns not significant. D: Tail suspension behavior. All data are expressed as mean ± SEM, n = 8-10. Unpaired t-test, *p < 0.05, ns not significant. E: Three-box social behavior-social novelty preference assessment. All data are expressed as mean ± SEM, n = 8-10. Unpaired t-test, *p < 0.05, ***p < 0.001, ns not significant. G: Representative heat map of the mouse stay area in three-box social behavior (n = 8-10). H: Representative open field track map. F: Open field behavior. All data are expressed as mean ± SEM, n = 8-10. Unpaired t-test, *p < 0.05; ns not significant.

[0060] Figure 8 shows that DHCB exerts its antidepressant effect through intestinal flora. A: Principal coordinate analysis (n=7) and relative abundance bar graph (B) of 16s rRNA sequencing results of female and male mice given DHCB by gavage. C: IBA1 in the hippocampus of female and male mice after CRS modeling. + Representative images of microglial immunofluorescence staining. Scale bar = 50 μm. B: IBA1 + Quantitative statistical graph of microglial immunofluorescence staining, n = 4-6. All data are expressed as mean ± SEM, unpaired t-test, *p < 0.05, ***p < 0.001, ns, not significant.

[0061] Figure 9 shows that dimethylglycine levels in feces were significantly upregulated after oral administration of DHCB. A: Metabolomics principal component analysis shows that the control and DHCB oral administration groups can be significantly differentiated along the horizontal axis. Volcano plot (B) and heat map (C) of differential metabolites in feces of the DHCB oral administration group compared to the control group. D: Dimethylglycine was significantly upregulated in the feces of mice in the DHCB oral administration group. All data are expressed as mean ± SEM, unpaired t-test. *p < 0.05.

[0062] Figure 10 shows the effect of oral administration of dimethylglycine on the EAE model. A: Experimental design for the anti-EAE effect study of oral administration of DMG. B: Clinical pathological scores of EAE after oral administration of DMG. N = 10 female mice. Two-way ANOVA followed by post hoc test. ****p < 0.0001. MOG 35-5521 days after immunization. C: Immunomic analysis of infiltrating cells by H&E staining (red) and myelin LFB staining (blue). Scale bar = 100 μm. DETAILED DESCRIPTION

[0063] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that intestinal flora modulators can prevent and / or treat brain neurological diseases through intestinal regulation, with significant inhibitory effects. This invention provides a new approach for the clinical therapeutic application of intestinal flora modulators. Based on this, the inventors completed the present invention.

[0064] Corydalis yanhusuo

[0065] Corydalis yanhusuo, the tuber of the poppy family plant Corydalis yanhusuo, is used as a traditional Chinese medicine to treat heart, abdomen, waist, and knee pain, menstrual irregularities, abdominal masses, metrorrhagia, postpartum blood loss, lochia retention, and traumatic injuries. In clinical use, Corydalis yanhusuo is often roasted with vinegar to enhance its blood circulation and analgesic effects. Its main components include alkaloids, among which tetrahydropalmatine has a strong analgesic effect.

[0066] Active ingredient

[0067] Dehydrocorydalis bulbine (DHCB), also known as dehydrocorydalis and dehydrocorydalis, has a CAS number of 59870-72-3. It is a small molecule alkaloid extracted from traditional Chinese medicines such as Corydalis yanhusuo and the whole herb of Corydalis cuneiformis, or synthesized artificially. Studies have shown that it can be used as an analgesic ingredient. DHCB typically exists in various salt forms, and its cationic portion has the following structure:

[0068] The counter anion is any pharmaceutically acceptable anion, including one or more of chloride anion, bromide anion, iodide anion, sulfate anion, hydrogen sulfate anion, phosphate anion, hydrogen phosphate anion, dihydrogen phosphate anion, methanesulfonate anion, maleate anion, tartaric acid anion, acetate anion, formate anion, citrate anion, camphorsulfonate anion, fumarate anion, succinate anion, lactate anion, methanesulfonate anion, malate anion, benzoate anion, and gluconic acid anion. The anion exchange can be achieved by a commonly used ion exchange method in chemistry.

[0069] There are currently no reports on the use of DHCB as an active ingredient acting on the intestinal tract to treat brain neurological diseases.

[0070] Dimethylglycine (DMG) is a derivative of the amino acid glycine with the structural formula (CH3)2NCH2COOH and can be used as a dietary supplement.

[0071] Extracts and preparation methods

[0072] As used herein, the term "extract" includes water-soluble and / or fat-soluble extracts. The method for preparing the Corydalis yanhusuo extract of the present invention is not particularly limited. Conventional methods can be used to obtain water-soluble and / or fat-soluble extracts using Corydalis yanhusuo as a raw material. These can be performed by solvent extraction, extraction, and / or chromatography.

[0073] In the present invention, the solvent used in the solvent extraction method is not particularly limited. Representative examples include (but are not limited to): water, ethanol, methanol, acetone, ethyl acetate, or a mixture of several solvents. The extraction can be performed once or multiple times.

[0074] In the present invention, the solvent used in the solvent extraction method is not particularly limited. Representative examples include (but are not limited to): n-butanol, ethyl acetate, dichloromethane, chloroform, cyclohexane, petroleum ether, or a mixture of several of these solvents. The extraction can be performed once or multiple times.

[0075] In the present invention, there is no particular limitation on column chromatography, and representative examples include (but are not limited to): activated carbon, silica gel, reversed-phase silica gel, macroporous resin, dextran gel, or a combination of several thereof.

[0076] Multiple sclerosis

[0077] Multiple sclerosis (MS) is a chronic, disabling, immune-mediated inflammatory demyelinating disease of the central nervous system that often affects the periventricular, juxtacortical, optic nerves, spinal cord, brainstem, and cerebellum. Lesions are characterized by spatial and temporal multiplicity. Spatial multiplicity refers to the presence of multiple lesions, with the cerebrum, brainstem, cerebellum, and spinal cord affected simultaneously or sequentially. Temporal multiplicity refers to a relapsing-remitting course. Due to the widespread involvement, repeated attacks can ultimately lead to disability or even death. Clinical manifestations vary, with common symptoms including decreased vision, diplopia, limb sensory and motor impairments, ataxia, and bladder or rectal dysfunction.

[0078] depression

[0079] Major depressive disorder (MDD) is a common psychiatric disorder characterized by low mood, decreased interest or pleasure in previously pleasurable activities (anhedonia), and recurrent thoughts of death. Due to the complexity of the pathophysiology of depression, several hypotheses have been proposed to explain its pathogenesis, including hypothalamic-pituitary-adrenal axis dysfunction, monoamine neurotransmitter activity, neuroinflammation, genetic and epigenetic abnormalities, and sociopsychological factors. It is important to note that no single hypothesis can fully explain the pathology of depression; rather, depression results from the interplay of these factors.

[0080] Pharmaceutical compositions and methods of administration

[0081] Since the intestinal flora regulator of the present invention has excellent activity in inhibiting the onset of brain neurological diseases, the compound of the present invention and its various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compound of the present invention as the main active ingredient can be used to treat, prevent and alleviate brain neurological diseases.

[0082] The pharmaceutical composition provided by the present invention preferably contains an active ingredient in a weight ratio of 0.001-99wt%, preferably a ratio in which the active compound of the present invention as the active ingredient accounts for 0.1wt% to 90wt% or 1wt% to 50wt% of the total weight, with the remainder being a pharmaceutically acceptable carrier, diluent or solution or saline solution.

[0083] The pharmaceutical composition of the present invention comprises a safe and effective amount of a compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0084] "Pharmaceutically acceptable carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0085] The pharmaceutical composition is in the form of tablets, capsules, granules, pills, powders, solutions, emulsions, and suspensions.

[0086] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0087] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a certain portion of the digestive tract. Examples of useful encapsulating components are polymeric substances and waxes. If desired, the active compound can also be microencapsulated with one or more of the above-mentioned excipients.

[0088] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0089] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0090] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0091] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0092] The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

[0093] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0094] Compared with the prior art, the present invention has the following main advantages:

[0095] (1) The intestinal flora regulating agent of the present invention has unexpectedly excellent activity in alleviating brain neurological diseases as an oral preparation.

[0096] (2) The intestinal flora regulator of the present invention can be a traditional Chinese medicine extract, which is safe for oral administration and has few side effects.

[0097] (3) Some patients with multiple sclerosis often suffer from pain. The intestinal flora regulator of the present invention has both anti-inflammatory and analgesic effects (Current Biology, 2014, doi: 10.1016 / j.cub.2013.11.039). This dual effect is not possessed by general drugs for the treatment of multiple sclerosis.

[0098] (4) The intestinal flora modulator of the present invention has an inhibitory effect on the neurotoxic Th17 cells of the immune system, while having no significant effect on other immune cells. It can be used alone or in combination with commonly used anti-inflammatory drugs for brain neurological diseases, on the one hand, enhancing the therapeutic effect, and on the other hand, potentially reducing the potential risk of commonly used anti-inflammatory drugs for brain neurological diseases excessively interfering with the immune system function.

[0099] (5) The intestinal flora regulator of the present invention has a price advantage. The reference price of siplatimud in the United States is US$88,000 per year. DHCB is derived from an extract of Corydalis yanhusuo, which has abundant raw materials and relatively low cost.

[0100] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0102] Experimental Materials

[0103] Adult (2-month-old) C57BL / 6J mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0104] Depression modeling method

[0105] Chronic unpredictable mild stress (CUMS)

[0106] Mice undergoing CUMS modeling were housed individually in cages and fed a normal diet unless specifically stimulated. The modeling protocol was as follows: Strong stimulation included restraint for 6 hours, flashing lights overnight, ice water stimulation for 5 minutes, and a wet cage for 6 hours. Weak stimulation included an empty cage for 12 hours, a 45° tilted cage for 12 hours, a reversed day / night cycle, and water and food deprivation. One strong stimulus plus two weak stimuli were administered daily, with no repetition of stimulation within three days. Behavioral testing was performed after four consecutive weeks of modeling.

[0107] Chronic restraint stress model (CRS)

[0108] Mice undergoing CRS modeling were housed in single cages and fed a normal diet at other times. Mice were placed in 50 ml centrifuge tubes with holes around the sides and bottom to prevent free movement. The modeling was continued for 6 hours daily for 21 consecutive days, and behavioral testing was performed after the modeling was completed.

[0109] Antidepressant Dosage: DHCB was dissolved in DMSO at a stock concentration of 50 mg / kg, diluted in sterile PBS, and administered orally at a rate of 20 mg / kg, with a total volume of 200 μL per mouse. Three weeks after pre-protection, mice were induced into a CRS model, and this dosing regimen was continued until the mice were retrieved. A control group received an equal volume of DMSO dissolved in sterile PBS and administered orally.

[0110] Administration for anti-EAE symptoms: DHCB was dissolved in DMSO at a stock concentration of 50 mg / kg, diluted with sterile PBS, and administered by gavage at 30 mg / kg, with a total gavage volume of 200 μL per mouse. Three weeks after pre-protection, the mice underwent EAE modeling. During this period, the DHCB stock solution was dissolved in the mouse drinking water at a concentration of 0.15 mg / ml and replaced every 2 days until the mice were sampled. In the control group, an equal amount of DMSO was dissolved in sterile PBS and gavage was performed. After EAE modeling, the same amount was dissolved in the mouse drinking water and replaced every 2 days until the mice were sampled.

[0111] Example 1

[0112] 1.1 Oral administration of DHCB can significantly alleviate EAE symptoms in mice

[0113] Female mice were first pretreated with DHCB by oral gavage for 3 weeks, and then MOG 35-55The mice were treated with a peptide (Myelin Oligodendrocyte glycoprotein (MOG) 33-55 peptide (Gill Biochemical Company), amino acid sequence: Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys) to establish an EAE model. Figure 1A shows the entire process of mouse dosing, modeling, and sampling. The results showed that the clinical symptoms of EAE were significantly improved in female mice gavaged with DHCB compared with control mice, as shown by a significant decrease in the incidence score of DHCB-treated mice, suggesting that DHCB has a significant inhibitory effect on the onset of the EAE model in female mice (Figure 1B). Next, the spinal cords of mice in the DHCB-treated group were pathologically analyzed. HE and LFB staining of the mouse spinal cord revealed that the degree of demyelination in the spinal cord of mice gavaged with DHCB was greatly improved, and the degree of central infiltration of peripheral immune cells was significantly reduced, which was consistent with the improvement of EAE clinical symptoms observed in mice gavaged with DHCB (Figure 1C and Figure 1D). In contrast, as shown in Figure 6, gavage with DHCB did not significantly alleviate the pathological symptoms of the EAE model in male mice.

[0114] These results suggest that pre-protective enteral administration of DHCB significantly alleviates EAE symptoms in female mice, accompanied by reduced demyelinating pathology in the spinal cord and decreased peripheral immune cell infiltration.

[0115] 1.2 DHCB targets intestinal dopamine D2 receptors

[0116] Previous results have shown that DHCB's primary target is the dopamine D2 receptor and that it is a dopamine D2 receptor antagonist. To investigate whether DHCB exerts its anti-EAE effects by antagonizing intestinal epithelial dopamine D2 receptors in this experiment, normal human colon epithelial cell lines (FHC cells) were stimulated with 50 μM DHCB. Haloperidol, a classic D2 receptor antagonist, was added as a positive control, and PEA, a D2 receptor agonist, was added as a negative control. The results showed that after DHCB stimulation, the phosphorylation level of AKT (p-AKT), a downstream protein of DRD2, was significantly upregulated in FHC cells, with an effect comparable to that of haloperidol (Figures 2A and 2B). This suggests that DHCB can antagonize the dopamine D2 receptor in FHC cells in vitro.

[0117] The same results were also observed in vivo: p-AKT levels were significantly upregulated in the colon epithelial cells of mice orally administered DHCB. These results suggest that DHCB can effectively antagonize intestinal epithelial dopamine D2 receptors, which may be the key target for DHCB's anti-EAE effects.

[0118] 1.3 Oral administration of DHCB regulates intestinal flora by reducing lysozyme (LYZ1)

[0119] To explore the specific mechanism by which DHCB affects the pathogenesis of EAE, we first performed transcriptome sequencing (RNA-seq) on the intestinal epithelial cells of mice treated with DHCB. The mRNA levels of lysozyme lyz1 and multiple defensins were significantly downregulated in the intestinal epithelium of mice receiving DHCB orally (Figure 3A).

[0120] Based on this, further 16s rRNA sequencing of the feces of mice gavaged with DHCB revealed changes in multiple gut microbiota, including an increase in the abundance of Lactobacillus. Furthermore, the DHCB gavage group and the control group were significantly separated into two groups on the principal component analysis plot (Figure 3B). These results suggest that gavage with DHCB may significantly alter the composition of the host's gut microbiota, and that metabolites produced by the gut microbiota may be key to DHCB's anti-EAE effects.

[0121] 1.4 Preventive and therapeutic effects of DHCB and haloperidol

[0122] DHCB is a novel natural small molecule compound derived from the traditional Chinese medicine Corydalis yanhusuo. To further investigate its efficacy in treating EAE compared to classic D2 receptor antagonists, female mice were simultaneously administered DHCB and haloperidol via oral gavage for three weeks before establishing an EAE model. The results showed that both DHCB and haloperidol demonstrated efficacy against EAE, as assessed by clinical scores (Figure 4), with DHCB showing a greater therapeutic effect in the third week.

[0123] 1.5.DHCB can inhibit the proportion of Th17 cells in mesenteric lymph nodes

[0124] In order to explore the effect of DHCB on the peripheral immune system of mice after oral administration, flow cytometry was used to analyze T cells in the peripheral immune organs spleen and mesenteric lymph nodes. Different T cell surface molecules were used to represent different T cell subsets, including total T cells (CD4 + ), Th17 cells (RORγt + ), Treg cells (Foxp3 + ), Th1 cells (T-bet +Flow cytometry results showed that oral administration of DHCB had no significant effect on T cell immunity in the spleen, but significantly inhibited the differentiation of Th17 cells in the mesenteric lymph nodes, while having no effect on other types of T cells. These results suggest that the protective effect of DHCB in EAE mice may have two mechanisms: one is the neuroprotective effect mediated by the intestinal flora metabolite NAL, and the other is the indirect effect of DHCB, namely the inhibition of Th17 cells in the mesenteric lymph nodes.

[0125] Example 2

[0126] Oral administration of DHCB exerts antidepressant effects in a mouse depression model

[0127] In male and female mice, 20 mg / kg DHCB was administered orally for pre-protection for 3 weeks, and then CRS modeling was performed to explore whether DHCB has an antidepressant effect (Figure 7B). Behavioral results showed that after CRS modeling, the immobility time of female mice gavaged with DHCB was significantly reduced compared with the control female mice in the forced swimming and tail suspension tests (Figures 7C and D), and the total distance explored in the open field was significantly increased (Figures 7F and H). There was no significant difference in social behavior between male DHCB-gavaged mice and male control mice after CRS modeling. However, after CRS modeling, male mice administered with DHCB showed no significant changes in forced swimming, tail suspension tests, open field and three-box social interaction compared with the male CRS control group, indicating that the antidepressant effect of DHCB on male mice was not significant (Figures 7C-H). Behavioral results showed that DHCB had a significant antidepressant effect on female mice.

[0128] 16S rRNA sequencing of feces from DHCB-treated female and male mice revealed that DHCB administration significantly altered the composition of the intestinal microbiota in female mice, whereas DHCB administration had minimal effect on the composition of the intestinal microbiota in male mice (Figures 8A and B). Furthermore, the abundance of Parabacteroides distasonis in the intestinal microbiota of DHCB-treated female mice was significantly upregulated compared to that in control female mice (Figure 8E), indicating that Parabacteroides distasonis is a key microbiota in the DHCB-treated female mice.

[0129] Example 3

[0130] Dimethylglycine (DMG), a metabolite of intestinal flora, plays an anti-EAE role

[0131] Metabolomics analysis was performed on the feces of mice that were orally administered with DHCB and subjected to EAE modeling. The metabolomics analysis results (Figure 9) showed that metabolites derived from multiple bacterial communities were significantly upregulated after orally administering DHCB, such as isobutyric acid, isovaleric acid, dimethylglycine (DMG), and methylhistidine.

[0132] Dimethylglycine (DMG) was dissolved in the drinking water of mice at a dose of 0.36 mg / ml. After 3 weeks of pre-protective administration, EAE modeling was performed to investigate whether oral administration of DMG could inhibit the pathological progression of the mouse EAE model ( FIG10A ).

[0133] The results of EAE pathological scoring showed that oral administration of DMG had a significant protective effect on the EAE model (Figure 10B). Further HE staining of the spinal cord pathological sections of the mice showed that oral administration of DMG could inhibit the infiltration of peripheral immune cells into the spinal cord. In addition, myelin LFB staining was used to observe the effect of oral administration of DMG on spinal cord myelin damage in mice. The results showed that oral administration of DMG alleviated the demyelination phenomenon in the EAE model of mice (Figure 10C).

[0134] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A use of an intestinal flora regulator, characterized in that: For preparing a drug for preventing and / or treating a subject's brain neurological disease, wherein the drug is an oral preparation; Furthermore, the intestinal flora regulator is selected from the following group: (a) dehydrocorybulbine (DHCB) or a pharmaceutically acceptable salt thereof, or an extract containing DHCB; (b) haloperidol or a pharmaceutically acceptable salt thereof; (c) a combination of components (a) and (b); (d) Dimethylglycine (DMG) or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, characterized in that The drug is an enteric-coated preparation.

3. The use according to claim 1, characterized in that The intestinal flora regulator is dehydrocorybulbine (DHCB) or a pharmaceutically acceptable salt thereof.

4. The use according to claim 1, characterized in that The DHCB-containing extract is an extract of Corydalis plants.

5. The use according to claim 1, characterized in that The DHCB-containing extract is an extract of Corydalis yanhusuo plant.

6. The use according to claim 1, characterized in that The pharmaceutically acceptable salt of DHCB or haloperidol is a salt of DHCB or haloperidol and an anion selected from the group consisting of chloride anion, bromide anion, iodide anion, sulfate anion, hydrogen sulfate anion, phosphate anion, hydrogen phosphate anion, dihydrogen phosphate anion, methanesulfonate anion, maleate anion, tartaric acid anion, acetate anion, formate anion, citrate anion, camphorsulfonate anion, fumarate anion, succinate anion, lactate anion, methanesulfonate anion, malate anion, benzoate anion, gluconic acid anion, or a combination thereof; preferably, the anion is selected from the group consisting of acetate anion, formate anion, chloride anion, or a combination thereof.

7. The use according to claim 1, characterized in that The brain neurological disease is selected from the group consisting of depression, multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), brain inflammation, and neuromyelitis optica spectrum disorder.

8. The use according to claim 1, characterized in that After the intestinal flora regulator is administered, the concentration reached in the intestinal tract is 1-200 μM, preferably 10-100 μM, and more preferably 20-80 μM.

9. The use according to claim 1, characterized in that The intestinal flora regulator is used for: Upregulates the abundance of Lactobacillus in the intestinal tract; Upregulation of the metabolite N2-acetyl-L-lysine in the intestine; Downregulate the amount of Th17 cells in the intestine.

10. The use according to claim 1, characterized in that The subject is female or male; preferably, the subject is female.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) a first active ingredient, wherein the first active ingredient is an intestinal flora regulator selected from the following group: (a) dehydrocorybulbine (DHCB) or a pharmaceutically acceptable salt thereof, or an extract containing DHCB; (b) haloperidol or a pharmaceutically acceptable salt thereof; (c) the combination of components (a) and (b) is selected from the group consisting of dehydrocorybulbine (DHCB), haloperidol or a pharmaceutically acceptable salt thereof; or a combination thereof; (d) dimethylglycine (DMG) or a pharmaceutically acceptable salt thereof; (ii) optionally a second active ingredient, wherein the second active ingredient is selected from the group consisting of fingolimod, siplatim, or any combination thereof; (iii) a pharmaceutically acceptable carrier.

12. Use of the pharmaceutical composition according to claim 11, characterized in that: The pharmaceutical composition is used for preparing a drug for treating, preventing or alleviating depression and / or multiple sclerosis-related diseases.

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