Use of taurocholic acid in the preparation of a drug for preventing or treating alzheimer's disease

Abstract

The application belongs to the field of neuropharmacology and medicine, and particularly relates to application of taurine cholic acid in preparation of a drug for preventing or treating Alzheimer's disease. Through animal model experiment verification, the A beta lateral ventricle injection is used to prepare an AD mouse model, and after oral administration of TCA for 2 weeks, cognitive behavior and pathological indexes are significantly improved.

Description

Technical Field

[0001] This invention belongs to the fields of neuropharmacology and medicine, specifically relating to the application of taurocholic acid (TCA) or its pharmaceutically acceptable salts, hydrates or solvates in the preparation of drugs that improve cognitive dysfunction and neuropathology by regulating the metabolism of glial cells, and is particularly suitable for the prevention and treatment of Alzheimer's disease (AD) and other neurodegenerative diseases. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive impairment and neuropathology, for which there is currently no effective treatment. Recent studies have found that metabolic disorders in brain glial cells (such as microglia) (e.g., abnormal lipid droplet accumulation, autophagy dysfunction) are an important pathological mechanism in AD. Existing drugs often target amyloid-beta (Aβ) or tau protein, but their clinical efficacy is limited and they cannot reverse metabolic-immune imbalances. Taurocholic acid (TCA) is an endogenous bile acid known to have anti-inflammatory and metabolic regulatory effects, but its role in the central nervous system (especially glial cell metabolism and AD pathology) remains unclear. Summary of the Invention

[0003] In one aspect, the present invention provides the use of taurocholic acid (TCA) or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases.

[0004] In some implementations, the neurodegenerative disease is Alzheimer's disease.

[0005] In some implementations, the drug works by activating the TGR5-cAMP-AMPK signaling pathway, thereby regulating lipid metabolism and autophagy in microglia.

[0006] In some embodiments, the drug is formulated as a dosage form suitable for oral or injectable administration.

[0007] In some embodiments, the TCA or its pharmaceutically acceptable salt, hydrate, or solvate is administered orally at a dose of 500 mg / kg / day to 2000 mg / kg / day or by injection at a dose of 5 mg / kg / day to 25 mg / kg / day.

[0008] On the other hand, the present invention provides a pharmaceutical composition for the prevention or treatment of Alzheimer's disease, comprising:

[0009] 1) TCA or its pharmaceutically acceptable salts, hydrates, or solvates; and 2) Pharmaceutically acceptable carriers.

[0010] In some implementations, the pharmaceutically acceptable carrier is water.

[0011] In some implementations, the TCA or its pharmaceutically acceptable salt, hydrate, or solvate is administered orally at a dose of 1000 mg / kg / day.

[0012] In some embodiments, the pharmaceutical composition further includes bile acid metabolism regulators, such as probiotics.

[0013] In some embodiments, the pharmaceutical composition further includes an autophagy enhancer, such as rapamycin.

[0014] On the other hand, the present invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases.

[0015] In some implementations, the neurodegenerative disease is Alzheimer's disease.

[0016] On the other hand, the present invention provides a method for preventing or treating neurodegenerative diseases, comprising administering to a subject in need a preventive or therapeutically effective amount of TCA or a pharmaceutically acceptable salt, hydrate or solvate thereof or a pharmaceutical composition thereof.

[0017] In some implementations, the neurodegenerative disease is Alzheimer's disease.

[0018] On the other hand, the present invention provides a functional food or beverage, comprising: 1) TCA or its pharmaceutically acceptable salts, hydrates, or solvates; and 2) An acceptable carrier for food or beverages. Attached Figure Description

[0019] Figure 1 Oral TCA can alleviate cognitive impairment and brain pathological changes induced by Aβ1-42 injection. (A) Experimental setup and timeline. (BD) Results of the Morris water maze experiment, where, (B) Learning curve, where # Comparison between Model and Vehicle, p < 0.05; # Comparison between Model and TCA, p < 0.05. (C) Platform crossing times; (D) Target area dwell time; (E) New object recognition index; (F) Statistical analysis of Aβ1-42 positive areas in the hippocampus; (G) Representative immunofluorescence staining images of GFAP (red) and Aβ1-42 (green) in the hippocampus, scale bar: 100 μm; (H) Representative immunofluorescence staining images of Iba-1 (red) in the hippocampus, scale bar: 100 μm; (I) Representative immunofluorescence staining images of GFAP (red) and Aβ (green) in the cortex, scale bar: 100 μm; (J) Representative immunofluorescence staining images of Iba-1 (red) in the cortex, scale bar: 100 μm; (K) Statistical analysis of GFAP positive areas in the hippocampus; (L) Statistical analysis of GFAP positive areas in the cortex. (M) Statistical analysis of Iba1-positive regions in the hippocampus. (N) Statistical analysis of Iba1-positive regions in the cortex. bar = 100 μm. Behavioral experiments n = 12. Immunofluorescence experiments n = 3, with 6 slides selected from each mouse for staining and statistical analysis. All values ​​are expressed as mean ± standard error, and p < 0.05 indicates statistical significance.

[0020] Figure 2Oral TCA improves lipid accumulation and autophagy disorder induced by Aβ1-42 injection by activating the TGR5-AMPK pathway. (A) Western blot (WB) detection of TGR5 protein expression. (B) Western blot detection of PLIN2 protein expression. (C) TGR5 quantification results are expressed as a percentage of the sham group. (D) PLIN2 quantification results are expressed as a percentage of the sham group. (E) Western blot detection of AMPK activation levels in the hippocampus (upper) and cortex (lower). (FG) Western blot detection of AMPK activation levels in the hippocampus (F) and cortex (G) are expressed as a percentage of the sham group. (H) Western blot detection of ACC activation levels in the hippocampus (upper) and cortex (lower). (IJ) Western blot detection of ACC activation levels in the hippocampus (I) and cortex (J) are expressed as a percentage of the sham group. (K) Western blot detection of mTOR activation (upper) and P62 protein expression (lower) in the hippocampus. (L) Western blotting (WB) was used to detect mTOR activation (top) and P62 protein expression (bottom) in the cortex. (MP) Quantitative results of mTOR activation in the hippocampus (M) and cortex (O) are expressed as a percentage of the Sham group; quantitative results of P62 in the hippocampus (N) and cortex (P) are expressed as a percentage of the Sham group. (Q) Western blotting (WB) was used to detect Beclin-1 protein expression in the cortex. (R) Western blotting (WB) was used to detect Beclin-1 protein expression in the hippocampus. (ST) Quantitative results of Beclin-1 in the hippocampus (S) and cortex (T) are expressed as a percentage of the Sham group. β-actin was used as an internal control in all WB experiments. n=3, and all experiments were performed in duplicate. All data are expressed as mean ± standard error (mean ± SEM), and p < 0.05 was considered statistically significant.

[0021] Figure 3 TCA activates TGR5. (A) Representative immunofluorescence staining image of TGR5 (red fluorescence) in BV2 cells, scale bar: 100 μm. (B) Statistical analysis results of TGR5 positive signal. All data are expressed as mean ± standard error (mean ± SEM), p < 0.05 indicates statistical significance.

[0022] Figure 4 TCA improves lipid accumulation in microglia via the AMPK pathway. (A) Representative immunofluorescence staining image of boron-dipyrrole methylene (BODIPY, green fluorescence) in BV2 cells, scale bar: 100 μm. (B) Statistical analysis results of the proportion of BODIPY positive areas. All data are expressed as mean ± standard error (mean ± SEM), p < 0.05 indicates statistical significance.

[0023] Figure 5 TCA activates AMPK through the TGR5-cAMP-LKB1 signaling pathway. (A) Western blot results of TGR5 protein in BV2 cells. (B) Statistical analysis of the relative expression level of TGR5 protein in BV2 cells. (C) Western blot results of TGR5 (top), AMPK activation level (middle), and ACC activation level (bottom). (DF) Statistical analysis of the relative expression levels of TGR5 (D), AMPK activation level (E), and ACC activation level (F). (G) Detection of cyclic adenosine monophosphate (cAMP) content by enzyme-linked immunosorbent assay (ELISA). (H) Western blot results of AMPK activation level (top) and ACC activation level (bottom). (IJ) Statistical analysis of the relative expression levels of AMPK activation level (I) and ACC activation level (J). (K) Western blot results of LKB1 protein. (L) Statistical analysis of the relative expression level of LKB1 protein. All data are expressed as mean ± standard error (mean ± SEM), and p < 0.05 indicates a significant difference. Detailed Implementation

[0024] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0025] "Comprising" means including the stated elements, integers, or steps, but does not exclude any other elements, integers, or steps. In this document, when the term "comprising" is used, it also covers situations consisting of the stated elements, integers, or steps, unless otherwise specified. For example, when a pharmaceutical composition is referred to as "comprising" component A and component B, it is also intended to cover pharmaceutical compositions consisting of component A and component B.

[0026] "Prevention" refers to avoiding, reducing, or delaying the onset of a specific disease or disease-related symptoms in a subject, provided that the disease or disease-related symptoms have not yet appeared before the administration of the relevant drug. "Prevention" does not necessarily require completely preventing the onset of the disease or disease-related symptoms. For example, reducing the risk of a subject developing a specific disease or disease-related symptoms after administration of the relevant drug, or lessening the severity of subsequently occurring related symptoms, can be considered "prevention" of the onset or development of the disease. "Treatment" refers to alleviating, reducing, improving, or inhibiting (e.g., preventing the development) a disease that a subject has exhibited or has previously experienced. For a specific disease, "treatment" can include "curing" the disease, but in most cases, it does not require the complete elimination of all symptoms. For example, if administration of the relevant drug results in a reduction or elimination of at least one symptom in the subject, this can be considered treatment.

[0027] When referring to pharmaceutical compositions, a "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that is safe for administration to animals or humans, suitable for administration to humans and / or animals without excessive adverse side effects, and suitable for maintaining the activity of the drug or active ingredient contained therein. Depending on the route of administration, various carriers well-known in the art can be used. Commonly used routes of administration include oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or inhalation and transdermal administration. Commonly used carriers include, but are not limited to, sugars, starch, cellulose and their derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils (e.g., castor oil), synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and / or water. Accordingly, the pharmaceutical composition can be prepared in any clinically acceptable dosage form, such as tablets, granules, powders, capsules, injectable preparations, suppositories, eye drops, topical ointments, ointments, medicated oils, or sprays, etc.

[0028] "Pharmaceutically acceptable salts" refer to inorganic or organic acid addition salts that are substantially harmless to animals or humans, such as hydrochlorides, hydrobroms, nitrates, perchlorates, phosphates, sulfates, formates, acetates, aconates, ascorbic acid salts, benzenesulfonates, benzoates, cinnamates, citrates, heptanoates, fumarates, glutamates, glycolate, lactates, maleates, malonates, mandelates, methanesulfonates, naphthalene-2-sulfonates, phthalates, salicylates, sorbates, stearates, succinates, tartrates, and p-toluenesulfonates. These salts can be formed by methods well known to those skilled in the art.

[0029] When this article refers to "taurocholic acid (TCA)," it includes not only "pharmaceutically acceptable salts" but also its hydrates, solvates, and derivatives. "Hydrate" refers to a complex formed by taurocholic acid or its salts with water. "Solvate" refers to an association or complex formed by one or more solvent molecules with taurocholic acid or its salts. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and ethanolamine. Examples of taurocholic acid derivatives may include ester derivatives of taurocholic acid, such as methyl taurocholate, ethyl taurocholate, etc.

[0030] "Subject" refers to an individual (preferably) who has or is suspected of having a disease (e.g., AD), or, for example, when predicting or preventing the risk of disease, "subject" may also include healthy individuals. This term is often used interchangeably with "patient," "subject to testing," and "subject to treatment."

[0031] "Effective dose for prevention or treatment" refers to an amount sufficient to elicit a biological or medical response in a subject that is expected by a clinician, and is typically determined by a person skilled in the art based on factors such as route of administration, subject weight, age, and disease condition. For example, for TCA, a typical daily dose range can be from 0.01 mg (or less) to 1000 mg (or more) of the active ingredient per kg of body weight, e.g., 1-500 mg / kg. For example, for mice, the dose range of TCA is 10-1000 mg / kg / day, preferably 100-500 mg / kg / day; for rats, the dose range of TCA is 10-1000 mg / kg / day, preferably 50-500 mg / kg / day; and for humans, the dose range of TCA is 0.1-100 mg / kg / day, preferably 3-20 mg / kg / day.

[0032] Pharmaceutical compositions may contain two or more active ingredients to facilitate co-administration of multiple drugs. Besides the coexistence of two or more active pharmaceutical ingredients in the same pharmaceutical formulation (e.g., a pharmaceutical composition), those skilled in the art will recognize that these two or more active pharmaceutical ingredients may exist as separate pharmaceutical formulations in the same pharmaceutical kit, or even as separate pharmaceutical formulations in different pharmaceutical kits. In fact, co-administration is achieved whenever administration allows two or more active pharmaceutical ingredients to be simultaneously present in the subject's body at some point. In most cases, co-administration of two active pharmaceutical ingredients occurs because they produce a synergistic effect in the subject's body. This synergy includes the combined administration producing a greater efficacy than either active pharmaceutical ingredient administered alone, one active pharmaceutical ingredient reducing the side effects of another, or the combined administration producing a new efficacy, such as for treating a condition different from the original indication of either active pharmaceutical ingredient. These active pharmaceutical ingredients are also optionally formulated with pharmaceutically acceptable carriers. In some embodiments, these active pharmaceutical ingredients may be delivered via carriers such as exovesicles or nanoparticles. In other words, this invention covers not only pharmaceutical composition forms but also other forms in which different active pharmaceutical ingredients may coexist in the body of a subject. In some embodiments, TCA is used in combination with components that regulate bile acid metabolism (such as probiotics). In other embodiments, TCA is used in combination with autophagy enhancers (such as rapamycin).

[0033] "Functional foods" or "functional beverages" may also be referred to as "health foods" or "health drinks" in this article. They refer to foods that, in addition to having the functions of ordinary foods or beverages (such as providing the energy and nutrients needed by animals (including humans)), also have other functions, such as preventing or treating certain diseases, such as Alzheimer's disease (AD).

[0034] The inventors have discovered for the first time that oral administration of TCA can significantly improve cognitive function, reduce Aβ deposition, and regulate microglial lipid metabolism and autophagy in AD model mice by activating the TGR5-cAMP-AMPK signaling axis, thus providing a novel AD treatment strategy. For example, the inventors found that adding TCA to drinking water (e.g., 1000 mg / kg / day) significantly improved cognitive function in Aβ-induced AD model mice (Morris water maze, new object recognition experiment); TCA upregulated cAMP levels by activating the TGR5 receptor in microglia, thereby activating the LKB1-AMPK pathway, promoting lipid droplet clearance (reducing PLIN2 expression) and autophagy recovery (increasing Beclin-1, reducing p62); and TCA inhibited the release of pro-inflammatory factors (TNF-α, IL-1β) from microglia, alleviating neuroinflammation.

[0035] In some embodiments, TCA or its salts (such as sodium taurocholate) are used to prepare medicaments for the prevention or treatment of Alzheimer's disease (AD) and other neurodegenerative diseases. In other embodiments, TCA or its salts (such as sodium taurocholate) are used to prepare functional foods or beverages (such as cognitive health drinks with added TCA) for the prevention or treatment of AD and other neurodegenerative diseases.

[0036] In some implementations, the administration method used is, for example, oral (e.g., added to drinking water, capsules, tablets), injection (e.g., intravenous administration) or targeted delivery system (e.g., nanocarriers).

[0037] In some implementation schemes, the subjects are high-risk groups for AD (such as patients with mild cognitive impairment) or patients with metabolic syndrome combined with neurodegenerative diseases.

[0038] This invention utilizes TCA to prevent or treat Alzheimer's disease (AD). It works through multiple targets, simultaneously improving metabolic abnormalities, protein pathology, and neuroinflammation associated with AD. TCA is an endogenous substance with good biocompatibility and no significant toxic side effects (IC50). 50 (>3.9 mM), with high safety. Furthermore, it is effective orally, crosses the blood-brain barrier, and is superior to traditional neurological drugs.

[0039] In some preferred embodiments, the pharmaceutical formulation including TCA is to dissolve TCA in drinking water for administration (1000 mg / kg / day), or to form tablets with pharmaceutical excipients (such as starch, lactose) (daily dose 0.1-100 mg / kg).

[0040] The present invention was verified through animal model experiments: An AD mouse model was prepared by injecting Aβ into the lateral ventricle. After orally administering TCA for 2 weeks, cognitive behavior (water maze) and pathological indices (Aβ deposition, glial cell activation) were significantly improved. The AMPK inhibitor (Compound C) could completely block the efficacy of TCA, thus confirming its dependence.

[0041] The following specific examples are used to further illustrate the present invention.

[0042] Example 1 Experimental Animals Preparation of AD model mice: 8-week-old male C57BL / 6 wild-type mice (Spebefu (Beijing) Biotechnology Co., Ltd., license number: SYXK (Beijing) 2023-0011) were adaptively housed in a SPF-class experimental animal room for 3-5 days, with a 12 h light / 12 h dark alternating cycle, and free access to food and water. The experiment was approved by the Medical and Experimental Animal Ethics Committee of Beijing University of Chinese Medicine (approval number: BUCM-2024092402-3230). The above mice were anesthetized with 1.5-2% isoflurane in oxygen at a flow rate of 0.8 LPM. An Aβ1-42 oligomer solution (GL Biotech, 052487) was injected at a rate of 0.25 μL / min using a microsyringe (Hamilton, 33GA) and a microinjection pump (Harvard Apparatus). The coordinates were as follows: The Aβ1-42 oligomer solution was unilaterally injected into the lateral ventricle (anteroposterior (AP): -0.58 mm, mediolateral (ML): +1.14 mm, dorsoventral (DV): -1.77 mm). A total of 3 μL of the Aβ1-42 oligomer solution was delivered to each site, and the needle remained at the site for at least 10 minutes to allow diffusion.

[0043] Experimental intervention and grouping: The above AD model mice and sham-operated mice were acclimated to the environment for 72 hours in a SPF-class laboratory. Throughout the experiment, each group of mice had free access to food, but their water intake was strictly controlled, i.e., the water intake of each group of mice was the same. The mice were divided into a sham-operated group, a model group, and a model + TCA intervention group. Among them, the TCA intervention group was given sodium taurocholate (MCE, HY-N0545) dissolved in sterile water (dosage: 1000 mg / kg / day), while the sham-operated group and the model control group were given high-pressure sterilized water and drank continuously for 7 days. Subsequently, the following tests were conducted.

[0044] Example 2 Novel Object Recognition Test (NOR) The new object recognition test was conducted in a rectangular open arena (50×50×50 cm) under dim lighting conditions. Habituation: Mice were allowed free exploration for 10 minutes 24 hours before the test. Familiarization phase: Two identical objects (objects A1 and A2) were placed in opposite corners of the arena, 10 cm from the walls. Each mouse was placed in the center and allowed to explore the object for 10 minutes. Testing phase: After a 24-hour test interval, a familiar object was replaced with a new object (object B) of similar size but different shape / texture. Mice were again allowed to explore for 10 minutes. Exploration was defined as the mouse's nose pointing towards the object and within 2 cm. The exploration time for each object was recorded (Tn = new object; Tf = familiar object). The recognition index = (Tn-Tf) / (Tn+Tf) reflects the mouse's ability to recognize the new object; an increase in the recognition index indicates improved memory.

[0045] The results show Figure 1 In the study, the model group showed a significant difference compared to the sham group (p<0.05), indicating successful model establishment; the TCA intervention group showed a significant difference compared to the model group (p<0.001), indicating that the addition of TCA improved the memory ability of the model mice.

[0046] Example 3: Water Maze Test (MWM) The Morris water maze utilizes the natural swimming ability and fear of water in mice, driving them to swim and find a way out. In the experiment, climbing onto a platform hidden underwater is the only way for the mice to escape the aquatic environment. By observing and recording the time it takes for the mice to find the underwater platform, their swimming trajectory, and search strategies, we can analyze and infer the mice's learning, memory, and spatial cognition abilities.

[0047] The maze consisted of a circular pool (90 cm in diameter, 50 cm in height) filled with water (22±1°C), made opaque with non-toxic white tempera paint. A hidden platform (10 cm in diameter) was submerged 1 cm below the water surface in the target quadrant. Different geometric visual cues were placed at equal intervals around the room. The platform was made invisible by adding milk powder. For 24 hours prior to the experiment, mice were acclimatized to the testing room and given free access to food and water under a standard 12:12 light-dark cycle. Animals were trained for four consecutive days, with each mouse undergoing two trials per day. At the start of the experiment, the mouse was placed in the water, facing the pool wall, in one of the four quadrants. The order of entry into each quadrant was randomized each day, so that all four quadrants were used once every two days. The mouse was allowed a maximum of 60 seconds to swim to find the hidden platform. If unsuccessful within the allotted time, the mouse scored 60 seconds and was guided to the platform and remained there for 10 seconds. Training was conducted twice daily, the average of the two trials was taken, and the results were recorded for four consecutive days to plot the learning curve. Exploration Test: On day 5, an exploration test was conducted by removing the platform from the pool. Mice were then allowed to swim freely in the pool for 60 seconds. A circular track around the previously identified target location was drawn on a computer screen, and the number of times the mice crossed the track was analyzed, along with the number of times they crossed the platform.

[0048] The results show Figure 1 From B to D. From the learning curve ( Figure 1 (B) Platform traversal times ( Figure 1 (C) and time spent in the target area ( Figure 1 As can be seen from Figure D, the addition of TCA improved the learning, memory, and spatial cognition abilities of the model mice.

[0049] Example 4: Immunofluorescence detection of brain tissue Brains from the three mouse groups (Sham, Model, and TCA groups) were isolated, embedded in OCT compounds (Sakura, Finetek), frozen, and stored at -20°C. Coronal or sagittal frozen sections (40 μm thick) were prepared and stored in 24-well culture plates containing phosphate-buffered saline (PBS). The culture plates containing the sections were stored at 4°C until immunofluorescence staining. For cell slides, fixation with 4% paraformaldehyde (PFA) was performed at room temperature for 20 minutes, followed by washing three times with PBS to remove residual PFA. The fixed cell slides were then subjected to subsequent immunofluorescence staining.

[0050] Brain tissue sections were washed three times with PBS, then permeated with PBS solution containing 0.5% Triton X-100 (PBST) for 30 minutes, and blocked with 3% donkey serum PBST for 1 hour at room temperature. The tissue sections were then incubated overnight at 4°C with antibodies against glial fibrillary acidic protein (GFAP) (1:1000, 80788S, CST, USA), ionized calcium binding adapter molecule 1 (IBA1) (1:500, 011-27991, Fujifilm, Japan), and 6E10 (1:1000, 803004, BioLegend, USA). The sections / cells were then washed three times with 0.5% PBST and incubated with different secondary antibodies for 2 hours (1:1000, Alexa Fluor™ 488, A21206, Invitrogen, USA; Alexa Fluor™ 594, A11058, Invitrogen, USA). The sections / cells were washed three times with 0.5% PBST and stained with 4',6-diamidino-2-phenylindole (DAPI) (5 μM, DAPI02, LABLEAD, China) for 10 minutes at room temperature according to the manufacturer's instructions. After washing once with PBS, the sections were mounted with mounting medium (S2100, Solarbio, China) and stored in the dark at -20°C. Images were acquired and subsequently analyzed using a confocal microscope (Olympus, Japan).

[0051] The results show Figure 1 From F to N. From the proportion of plaques ( Figure 1 (F), percentage of astrocytes ( Figure 1 China G, Figure 1 Middle I, Figure 1 Zhong K, Figure 1 The proportion of mid-L and microglia ( Figure 1 H in the middle Figure 1 J, Figure 1 M, Figure 1 As can be seen from the N), the addition of TCA improved the amount of amyloid protein (Aβ) pathological products in the brain of model mice and alleviated the neuroinflammatory response in the mouse brain.

[0052] Example 5: Detection of Brain Tissue Proteins by Western Blotting (WB) The brains of the three groups of mice (Sham, Model, and TCA groups) were isolated. Mouse brain tissue (cortex or hippocampus, surgically dissected) was lysed in RIPA lysis buffer containing a mixture of protease inhibitors (Selleck, B14002, USA), and protein concentration was determined using the BCA assay. Equal volumes of protein were separated by 10% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature for 2 hours, the membrane was incubated overnight at 4°C with the following primary antibodies: bile acid receptor (Takeda G Protein-Coupled Receptor 5, TGR5) (1:1000, ab72608, abcam, UK), PLIN2, AMP-Activated Protein Kinase α (AMPKα) (1:1000, F0269, Selleck, USA), Phosphorylated AMP-Activated Protein Kinase α (pAMPKα) (1:1000, 2535S, Cell Signaling Technology, USA), Phosphorylated Acetyl-CoA Carboxylase (pACC) (1:1000, 11818, Cell Signaling Technology, USA), and Acetyl-CoA Carboxylase (pACC). Carboxylase (ACC) (1:1000, 3676, Cell Signaling Technology, USA), Mammalian Target of Rapamycin (mTOR) (1:1000, 2972, Cell Signaling Technology, USA), Phosphorylated Mammalian Target of Rapamycin (pmTOR) (1:1000, 2971, Cell Signaling Technology, USA), ubiquitin-binding protein P62 (1:20000, A19700, ABclonal, China), Beclin-1 (1:1000, 3738, Cell Signaling Technology, USA), GAPDH (1:100000, A19056, ABclonal, China), β-actin (1:100000, AC026, ABclonal, China).After incubation with primary antibody, the membrane was washed three times with TBST for 5 minutes each time. Following washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution) at room temperature with gentle agitation for 2 hours, followed by washing three times with TBST for 5 minutes each time. Protein bands were visualized using ECL substrates (Themofisher, USA) and quantified using ImageJ software.

[0053] The results show Figure 2 From A to T. From TGR5 protein expression level ( Figure 2 China A Figure 2 As can be seen from (C), TCA significantly increases the expression of bile acid receptors in mouse brain tissue; from the expression level of PLIN2 protein ( Figure 2 B, Figure 2 As can be seen from D), TCA significantly inhibits the expression of lipid droplet formation-related proteins.

[0054] AMPK is a key regulatory pathway for lipid droplet formation; activation of the AMPK pathway inhibits lipid droplet formation. The protein expression levels of its phosphorylated state (p-AMPK) and its downstream factor (p-ACC) are compared. Figure 2 E, Figure 2 China F, Figure 2 China G, Figure 2 H in the middle Figure 2 Middle I, Figure 2 As can be seen from the results (J), the addition of TCA significantly activated the AMPK pathway in the cerebral cortex and hippocampus of the model mice, reducing lipid droplet formation.

[0055] Another pathological feature of autophagy disorder is the elevated expression of autophagy-regulating proteins mTOR and p62, and the decreased expression of Becline-1. The expression levels of key autophagy-regulating proteins mTOR, p62, and Becline-1... Figure 2 Zhong K, Figure 2 L in the middle, Figure 2 M, Figure 2 N, Figure 2 China O, Figure 2 China P, Figure 2 China Q, Figure 2 R in the middle, Figure 2 S in the middle Figure 2 As can be seen from the results (T), TCA significantly improved the autophagy function of the cerebral cortex and hippocampus in the model mice.

[0056] Example 6: Cell immunofluorescence detection INT-777 is a known TGR5 agonist and was used as a positive control to observe whether TCA could mimic the effect of INT-777. INT-777 (MCE, catalog number HY-15677) was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 50 mmol / L, which was stored at -20°C for later use.

[0057] BV2 cells (cell model: SCSP-5208) were treated with INT-777 and TCA for 12 hours, respectively, followed by immunofluorescence detection. Cells were washed three times with PBS, permeabilized with PBS containing 0.5% Triton X-100 for 30 minutes, and blocked with 3% donkey serum PBST for 1 hour at room temperature. Cells were then incubated overnight at 4°C with TGR5 antibody (1:1000, ab72608, abcam, UK). Cells were then washed three times with 0.5% PBST and incubated with different secondary antibodies for 2 hours (Alexa Fluor™ 594, A11058, Invitrogen, USA). Cells were washed three times with 0.5% PBST and stained with DAPI (5 μM, DAPI02, LABLEAD, China) for 10 minutes at room temperature according to the manufacturer's instructions. Cells were washed once with PBS, mounted with mounting medium (S2100, Solarbio, China), and stored in the dark at -20°C. Images were acquired and subsequently analyzed using a confocal microscope (Olympus, Japan).

[0058] The results show Figure 3 From A to B. From TGR5 protein expression levels ( Figure 3 China A Figure 3 As shown in Figure B, TCA significantly increased bile acid receptor expression in BV2 cells; among them, the activation effect of 100 μm TCA on TGR5 was consistent with that of the positive control INT-777, and both showed significant differences from the control. Figure 3 (B)

[0059] Example 7: Cell BODIPY Staining LKB1 is an upstream regulated kinase of the AMPK signaling pathway. Using an LKB1 inhibitor (Pim1) can specifically block the LKB1-AMPK signaling axis, thereby clarifying whether the TCA activation of AMPK depends on LKB1-mediated pathway activation.

[0060] In the AD model, abnormal accumulation of lipid droplets in microglia is one of the core markers of their dysfunction, and the regulation of lipid droplet metabolism is directly related to the activation state of the AMPK pathway. When AMPK is activated, it can inhibit lipid droplet accumulation by regulating downstream lipid metabolism-related targets; conversely, when the LKB1-AMPK signaling axis is blocked, lipid droplet accumulation increases significantly. Therefore, the level of lipid droplet accumulation in microglia can directly reflect the activation effect of the AMPK pathway. Combined with quantitative analysis using BODIPY fluorescence staining, the mechanism by which TCA regulates microglia lipid metabolism via the LKB1-AMPK pathway can be verified.

[0061] LKB1 inhibitor Pim1 (MCE, catalog number HY-10371) was dissolved in DMSO to prepare a stock solution with a concentration of 50 mmol / L, which was stored at -20°C for later use. BV2 cells (cell model: SCSP-5208) were treated with Pim1 and TCA for 12 hours, followed by BODIPY staining. BV2 cells were washed three times with PBS, and then incubated with 5 μM BODIPY493 / 503 (HY-W090090, MCE, USA) at room temperature for 10 minutes according to the manufacturer's instructions. Afterwards, the cells were washed twice with PBS and mounted with anti-fluorescence quenching mounting medium (S2100, Solarbio, China), and stored at -20°C in the dark. Images were acquired using a confocal microscope (Olympus, Japan) for subsequent analysis.

[0062] Lipid droplet accumulation is a marker of microglial dysfunction in Alzheimer's disease (AD), and BODIPY staining provides visual evidence for the neuroprotective effect of transcatheter adenocarcinoma (TCA). Results showed... Figure 4 China A and Figure 4 In B1. From the amount of BODIPY staining ( Figure 4 China A Figure 4 As shown in Figure B), TCA significantly reduces Aβ-induced lipid droplet accumulation in microglia, and this process is related to the AMPK pathway.

[0063] Example 8: Cell Western Blotting Detection BV2 cells (cell model: SCSP-5208) were lysed in RIPA lysis buffer containing a mixture of protease inhibitors (Selleck, B14002, USA), and protein concentration was determined using the BCA assay. Equal volumes of protein were separated by 10% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature for 2 hours, the membrane was incubated overnight at 4°C with primary antibodies: TGR5 (1:1000, ab72608, abcam, UK), AMPKα mAb (1:1000, F0269, Selleck, USA), pAMPKα (1:1000, 2535S, Cell Signaling Technology, USA), pACC (1:1000, 11818, Cell Signaling Technology, USA), ACC (1:1000, 3676, Cell Signaling Technology, USA), LKB1 (1:1000, 3047, Cell Signaling Technology, USA), and β-actin (1:100000, AC026, ABclonal, China). After incubation with primary antibodies, the membrane was washed three times with TBST for 5 minutes each time. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution) at room temperature with gentle agitation for 2 hours, followed by washing three times with TBST for 5 minutes each. Protein bands were visualized using ECL substrate (Themofisher, USA) and quantified using ImageJ software.

[0064] The results show Figure 5 China A and Figure 5 In B, the expression level of TGR5 protein was determined. Figure 5 China A Figure 5 As shown in Figure B), TCA significantly increases bile acid receptor expression in BV2 cells, and the effect is consistent with that of the TGR5 agonist (INT-777).

[0065] AMPK is a key regulatory pathway for lipid droplet formation; activation of the AMPK pathway inhibits lipid droplet formation. The protein expression levels of its phosphorylated state (p-AMPK) and its downstream factor (p-ACC) are compared. Figure 5 C, Figure 5 D, Figure 5 Chinese E, Figure 5 China F, Figure 5 As can be seen from the data (G), the addition of TCA significantly activated the AMPK pathway in BV2 cells, which was blocked by the TGR5 inhibitor (SBI-115). Using the AMPK pathway inhibitor (Pim1 / AKK1-IN-1)... Figure 5H, Figure 5 Middle I, Figure 5 As can be seen from J), the TCA-activated AMPK effect is blocked.

[0066] AMPK phosphorylation is regulated by LKB1, a downstream gene of TGR5. The expression level of LKB1... Figure 5 K Figure 5 As can be seen from the data (L), the addition of TCA significantly activated LKB1 expression in BV2 cells, which was blocked by the TGR5 inhibitor (SBI-115).

[0067] Statistical analysis Data are expressed as mean ± standard error (SEM) of at least three independent experiments. Tukey post-hoc tests were performed after either a t-test (two groups) or a one-way ANOVA. p < 0.05 was considered statistically significant.

Claims

1. The use of taurocholic acid (TCA) or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases, preferably, the neurodegenerative disease being Alzheimer's disease.

2. The application as described in claim 1, wherein the drug works by activating the TGR5-cAMP-AMPK signaling pathway to regulate lipid metabolism and autophagy in microglia.

3. The application as described in claim 1 or 2, wherein the drug is formulated as a dosage form suitable for oral or injectable administration.

4. The application as described in claim 3, wherein the TCA or its pharmaceutically acceptable salt, hydrate or solvate is administered orally at a dose of 500 mg / kg / day to 2000 mg / kg / day orally or by injection at a dose of 5 mg / kg / day to 25 mg / kg / day.

5. A pharmaceutical composition for the prevention or treatment of Alzheimer's disease, comprising: 1) TCA or its pharmaceutically acceptable salts, hydrates or solvates; as well as 2) Pharmaceutically acceptable carriers.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable carrier is water.

7. The pharmaceutical composition of claim 5 or 6, wherein: The pharmaceutical composition also includes bile acid metabolism regulators, such as probiotics; and / or The pharmaceutical composition also includes autophagy enhancers, such as rapamycin.

8. The use of the pharmaceutical composition according to any one of claims 5-7 in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases, preferably, the neurodegenerative disease being Alzheimer's disease.

9. A method for preventing or treating a neurodegenerative disease, comprising administering to a subject in need a preventive or therapeutically effective amount of TCA or a pharmaceutically acceptable salt, hydrate or solvate thereof or a pharmaceutical composition according to any one of claims 5-7, preferably, the neurodegenerative disease being Alzheimer's disease.

10. A functional food or beverage, comprising: 1) TCA or its pharmaceutically acceptable salts, hydrates or solvates; as well as 2) An acceptable carrier for food or beverages.

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