Use of methyl-beta-cyclodextrin as a glut1 modulator in the preparation of a medicament for preventing or delaying alzheimer's disease

Abstract

The application relates to application of methyl-beta-cyclodextrin as a GLUT1 modulator in preparation of a medicine for preventing or delaying Alzheimer's disease. The application finds that, by adopting methyl-beta-cyclodextrin as a GLUT1 modulator to intervene in AD model mice, the GLUT1 expression in the brain of the AD model mice can be improved, the MbetaCD intervention has the treatment effects of improving cognitive dysfunction of the AD model mice, improving cerebral vascular lesions, restoring the brain sugar metabolism function and the like, and provides a new treatment strategy for prevention and treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of methyl-β-cyclodextrin as a GLUT1 modulator in the preparation of drugs for the prevention or delay of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease is a group of chronic, progressive cognitive decline syndromes caused by various factors. Alzheimer's disease (AD) is the most common type of dementia (accounting for 50%-70%), caused by brain degeneration (such as amyloid plaque deposition and neurofibrillary tangles) leading to diffuse cortical atrophy. The "China Alzheimer's Disease Report 2024" shows that in 2021, the number of people with AD and other dementias in my country reached 16.99 million. With the accelerating aging of the population, the incidence of Alzheimer's disease, primarily AD, is increasing year by year, seriously affecting the health and quality of life of the elderly and placing a heavy burden on families and society. Due to the complex pathogenesis of AD, although domestic and international efforts have been dedicated to developing symptomatic drugs, AD drug research has progressed slowly. Currently, existing clinical AD medications can only help control dementia symptoms; there are no specific drugs for AD.

[0003] The mechanisms of Alzheimer's disease are not fully understood, and its pathogenesis still requires further research. Current drug development is progressing slowly. To date, no specific drug has been developed globally to treat Alzheimer's disease. Clinically, limited drugs such as donepezil and memantine can only help control AD ​​symptoms. Existing treatment regimens cannot effectively alleviate or treat Alzheimer's disease, and their high cost makes them unaffordable for the general public and hinders widespread application. Therefore, it is essential to develop new treatment drugs or strategies. Summary of the Invention

[0004] This application found through research that using methyl-β-cyclodextrin (MβCD) as a GLUT1 (glucose transporter 1) regulator to intervene in AD model mice can increase GLUT1 expression in the brain of AD model mice. MβCD intervention has therapeutic effects such as improving cognitive dysfunction, improving cerebrovascular lesions, and restoring brain glucose metabolism function in AD model mice.

[0005] Based on this, the first aspect of this application provides the use of GLUT1 modulators in the preparation of drugs for the prevention or delay of Alzheimer's disease, wherein the GLUT1 modulators include methyl-β-cyclodextrin, providing a new treatment strategy for the prevention and treatment of Alzheimer's disease.

[0006] A second aspect of this application provides the use of a GLUT1 modulator in the preparation of a medicament for improving cerebrovascular function, wherein the GLUT1 modulator comprises methyl-β-cyclodextrin.

[0007] In some embodiments, the drug can increase GLUT1 expression and / or vascular density in cerebral blood vessels.

[0008] A third aspect of this application provides the use of GLUT1 modulators in the preparation of medicaments for reducing neuroinflammation in the brain, wherein the GLUT1 modulators include methyl-β-cyclodextrin.

[0009] In some embodiments, the drug can inhibit the overactivation of astrocytes and microglia.

[0010] The fourth aspect of this application provides the use of GLUT1 modulators in the preparation of medicaments for improving cognitive impairment, wherein the GLUT1 modulators include methyl-β-cyclodextrin.

[0011] In some embodiments, the drug can improve exploratory behavior and / or spatial memory retention.

[0012] The fifth aspect of this application provides the use of a GLUT1 regulator in the preparation of a medicament that regulates the glucose uptake function of brain cells, wherein the GLUT1 regulator comprises methyl-β-cyclodextrin, and the brain cells are at least one of blood-brain barrier endothelial cells, neurons, microglia, and astrocytes.

[0013] In some embodiments, the dosage of methyl-β-cyclodextrin in the drug is 250 mg / kg / day - 1000 mg / kg / day.

[0014] The sixth aspect of this application provides a drug for preventing or delaying Alzheimer's disease, or a drug for improving cerebrovascular function, or a drug for improving cognitive impairment, said drug comprising a GLUT1 modulator, said GLUT1 modulator comprising methyl-β-cyclodextrin.

[0015] In some embodiments, the drug further includes one or more pharmaceutically acceptable excipients. Attached Figure Description

[0016] Figure 1 The figure shows the results of the detection of the effect of MβCD on cognitive function in AD model mice; Figure 2 The figure shows the results of detecting the effect of MβCD on cerebrovascular function in AD model mice; Figure 3 The figure shows the results of detecting the effect of MβCD on glucose metabolism in the brain of AD model mice; Figure 4The figure shows the results of detecting the effect of MβCD on microglia activation in the brains of AD model mice; Figure 5 The figure shows the results of detecting the effect of MβCD on astrocyte proliferation in the brains of AD model mice; Figure 6 The image shows the results of detecting the effect of MβCD on Aβ deposition in the brains of AD model mice. Figure 7 Figure 1 shows the results of the MβCD effect on the mRNA expression profile of GLUTs in hCMEC / D3 cells and the results of the effect on glucose uptake capacity. Figure 8 The image shows the results of MβCD expression profile analysis of GLUTs mRNA in HT22, BV2, and SVG P12 cells. Detailed Implementation

[0017] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0018] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this application and in its specification is for descriptive purposes only and is not intended to be limiting of the application.

[0019] Unless otherwise stated or in case of conflict, the terms or phrases used in this application shall have the following meanings: The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0020] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0021] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0022] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0023] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0024] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0025] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0026] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0027] The pathogenesis of Alzheimer's disease (AD) is extremely complex and not yet fully understood. AD is the result of multiple factors, including Aβ and Tau abnormalities, combined with neuroinflammation, oxidative stress, cholinergic damage, and genetic factors. Cerebrovascular dysfunction and blood-brain barrier disruption are increasingly considered key drivers of AD pathogenesis. Studies have shown that restoring cerebrovascular integrity can alleviate AD-related pathology. Evidence suggests that alterations occur in both vascular and non-vascular glucose transporters (GLUTs) in the brains of AD patients, potentially leading to insufficient glucose supply to the brain and accelerating cognitive decline. Glucose transporter 1 (GLUT1) is an important membrane protein found on the blood-brain barrier and erythrocyte membranes. It primarily assists in the transmembrane transport of glucose, playing a crucial role in maintaining stable blood glucose levels and providing energy to the brain. The human brain accounts for only 2% of an adult's body weight, yet it requires 25% of the body's energy. More than one-fifth of the glucose in the blood is taken up by the brain, making GLUT1 particularly important for glucose uptake in the brain. Studies have shown that GLUT1 expression is downregulated in blood-brain barrier endothelial cells in the early stages of Alzheimer's disease (AD), accompanied by a decrease in brain energy metabolism. Other studies have reported that in mice with reduced GLUT1 expression in astrocytes, brain glucose metabolism is abnormally enhanced. GLUT1-deficient astrocytes exhibit increased insulin receptor-dependent ATP release, and the insulin signaling pathway in astrocytes and the brain's purine energy signaling pathway triggers metabolic reprogramming in the brain, maintaining energy demand, peripheral glucose homeostasis, and cognitive function. Disruption of GLUT1 function is closely related to blood-brain barrier rupture, microvascular degeneration, accelerated Aβ deposition, and microglia activation. GLUT1 plays a crucial role in maintaining cerebrovascular health and is essential in the pathogenesis of AD. Furthermore, excessive activation of GLUT1 may lead to continuous glucose uptake, causing energy metabolism disorders and even promoting cancer cell growth, as many cancer cells highly express GLUT1. Therefore, selecting appropriate GLUT1 modulators is of great significance for maintaining cerebrovascular health and energy metabolism.

[0028] β-Cyclodextrin (β-CD) and its derivatives have wide applications in drug modification, chiral separation, and environmental protection. Derivatives of β-cyclodextrin include methyl-β-cyclodextrin (MβCD), hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin. MβCD, a methylated derivative of β-cyclodextrin, also exhibits the intraluminal hydrophobic and extraluminal hydrophilic properties of cyclodextrin and is often used as a drug solubilizer and delivery enhancer. Several studies have reported that: 1) synthesized MβCD has been found to solubilize both β-naphthol and β-naphthylamine; 2) encapsulating anti-tuberculosis drugs in MβCD microparticles via spray drying improved the penetration of anti-tuberculosis drugs through the RPMI-2650 cell monolayer while reducing drug cytotoxicity, thus enhancing the efficacy of anti-tuberculosis drugs; 3) MβCD enhances COL I activity in age-aged skin, with Cav-1 playing a moderating role in COL I expression; 4) MβCD significantly inhibits LPS / oxLDL-induced monocyte-endothelial adhesion by downregulating adhesion molecule expression, exhibiting anti-atherosclerotic activity. Furthermore, as a commonly used pharmacological reagent, MβCD can efficiently and specifically extract cholesterol from cell membranes and disrupt lipid raft / crypt structures. For example, some studies have reported that: 1) MβCD treatment reduced cholesterol content in 3T3-L1 adipocytes by ~70%, and inhibited insulin-stimulated 2-deoxyglucose uptake by about 50%; 2) In MO7e human acute megakaryoblastic leukemia cells, similar to stem cell factors, MβCD treatment significantly increased glucose transport rate by about 60% and induced subcellular redistribution of GLUT1, recruiting intracellular GLUT1 transport molecules to the cell membrane. MβCD works through a cholesterol-dependent mechanism, ultimately leading to enhanced GLUT1 translocation; 3) In mice fed a high-fat diet... Subcutaneous injection of MβCD improved impaired glucose tolerance tests and normalized high fasting blood glucose levels. In in vitro experiments on isolated fibers from mice fed a high-fat diet, MβCD also improved insulin sensitivity and increased membrane GLUT4 content. 4) MβCD significantly improved glucose transport in mesenchymal stromal cells of horses with metabolic syndrome, influencing GLUT4 upregulation in an insulin-independent manner via a NO-dependent signaling pathway. 5) MβCD did not provide overall protection against the blood-brain barrier in acute hypertension and even caused disruption of the blood-brain barrier in normotensive animals. Currently, there are no reports on the anti-AD activity of MβCD.

[0029] Based on this, the first aspect of this application provides the use of GLUT1 modulators in the preparation of drugs for preventing or delaying Alzheimer's disease, wherein the GLUT1 modulators include methyl-β-cyclodextrin.

[0030] This application found that using methyl β-cyclodextrin (MβCD) as a glucose transporter 1 (GLUT1) regulator to intervene in AD model mice can increase GLUT1 expression in the brains of AD model mice. MβCD intervention has therapeutic effects such as improving cognitive dysfunction, improving cerebrovascular lesions, and restoring brain glucose metabolism in AD model mice, providing a new treatment strategy for the prevention and treatment of Alzheimer's disease.

[0031] It should be noted that the above-mentioned drugs may also include other GLUT1 modulators, which work synergistically with methyl-β-cyclodextrin to prevent or delay AD symptoms.

[0032] In some embodiments, the dosage of methyl-β-cyclodextrin in the medicament is 250 mg / kg / day to 1000 mg / kg / day. In some specific examples, the dosage of methyl-β-cyclodextrin is 500 mg / kg / day.

[0033] Studies have found that treatment of cancer cells (M07e human acute megakaryoblastic leukemia cells) with 10 mM MβCD for 20 min significantly increased glucose transport rate by approximately 60%. Methyl-β-cyclodextrin can act as a GLUT1 modulator. This application is the first to apply this GLUT1 modulator to the research on the preparation of drugs for the prevention or delay of Alzheimer's disease, providing a new therapeutic strategy for the prevention and treatment of Alzheimer's disease.

[0034] Targeting blood-brain barrier function and brain energy metabolism homeostasis can be applied to the prevention and / or treatment of Alzheimer's disease. Blood-brain barrier disruption exacerbates insufficient energy substrate supply, while energy metabolism disorders further weaken the blood-brain barrier's repair capacity, synergistically leading to cognitive impairment. Some studies report that: 1) Icariin significantly inhibited oxidative stress in the brain of Aβ-(arc) fruit flies, rescuing damaged energy metabolism and greatly increasing ATP supply; 2) Targeting LRP6 using the optogenetic tool OptoLRP6 can repair blood-brain barrier damage and maintain its function, reshaping the brain microenvironment homeostasis during AD, thereby alleviating endothelial cell dysfunction caused by Aβ oligomers, and can be applied to the treatment and early intervention of Alzheimer's disease.

[0035] Based on this, the second aspect of this application provides the use of a GLUT1 modulator in the preparation of a medicament for improving cerebrovascular function, wherein the GLUT1 modulator includes methyl-β-cyclodextrin.

[0036] In some embodiments, the drug can increase GLUT1 expression and / or vascular density in cerebral blood vessels.

[0037] It should be noted that the above-mentioned drugs may also include other GLUT1 modulators, which work synergistically with methyl-β-cyclodextrin to prevent or delay AD symptoms.

[0038] The experimental results of this application demonstrate that intervention using MβCD as a GLUT1 regulator not only increased the expression level of GLUT1 in the cerebral vascular endothelial cells of 5xFAD mice but also increased the density of GLUT1-expressing vessels, thus exhibiting a certain ameliorative or protective effect on cerebral vascular density in 5xFAD mice. Therefore, methyl-β-cyclodextrin can be used to prepare drugs that improve cerebral vascular function.

[0039] A third aspect of this application provides the use of GLUT1 modulators in the preparation of medicaments for reducing neuroinflammation in the brain, wherein the GLUT1 modulators include methyl-β-cyclodextrin.

[0040] In some embodiments, the drug can inhibit the overactivation of astrocytes and microglia.

[0041] In some embodiments, the dosage of methyl-β-cyclodextrin in the medicament is 250 mg / kg / day to 1000 mg / kg / day. Optionally, the dosage of methyl-β-cyclodextrin is 500 mg / kg / day.

[0042] It should be noted that the above-mentioned drugs may also include other GLUT1 modulators, which work synergistically with methyl-β-cyclodextrin to prevent or delay AD symptoms.

[0043] The experimental results of this application demonstrate that intervention using MβCD as a GLUT1 modulator significantly reduced microglial activation and astrocyte proliferation in 5xFAD mice, and can be applied to the preparation of drugs that reduce neuroinflammation in the brain.

[0044] The fourth aspect of this application provides the use of GLUT1 modulators in the preparation of medicaments for improving cognitive impairment, wherein the GLUT1 modulators include methyl-β-cyclodextrin.

[0045] In some embodiments, the drug can improve exploratory behavior and / or spatial memory retention.

[0046] In some embodiments, the dosage of methyl-β-cyclodextrin in the medicament is 250 mg / kg / day to 1000 mg / kg / day. Optionally, the dosage of methyl-β-cyclodextrin is 500 mg / kg / day.

[0047] It should be noted that the above-mentioned drugs may also include other GLUT1 modulators, which work synergistically with methyl-β-cyclodextrin to prevent or delay AD symptoms.

[0048] The experimental results of this application demonstrate that intervention using MβCD as a GLUT1 modulator partially reversed the cognitive deficits in 5xFAD mice, improved their spatial memory retention, and significantly restored their learning, memory, and exploratory behaviors. This evidence suggests that MβCD can be used to prepare drugs that improve cognitive impairment.

[0049] The fifth aspect of this application provides the use of a GLUT1 modulator in the preparation of a medicament that modulates the glucose uptake function of brain cells, said GLUT1 modulator comprising methyl-β-cyclodextrin.

[0050] In some embodiments, the brain cells are at least one of blood-brain barrier endothelial cells, neurons, microglia, and astrocytes.

[0051] Brain energy metabolism is a highly segmented and cell-specific process. Each cell type has its unique metabolic program to meet the needs of its specific role. Blood-brain barrier endothelial cells selectively control the flow of nutrients (such as glucose) from the blood into the cerebral interstitial fluid, acting as the main valve for the brain's energy supply. Neurons perform information processing (action potentials, synaptic transmission) and are the cells in the brain with the highest and most sustained energy demand. Astrocytes are the energy supporters and reserves of neurons and maintain extracellular ion / neurotransmitter balance. Microglia are responsible for immune surveillance and phagocytosis in the brain, and their metabolic state directly affects their immune function (pro-inflammatory or anti-inflammatory). Using four cell models—hCMEC / D3 (blood-brain barrier endothelial cells), HT22 (neurons), BV2 (microglia), and SVG P12 (astroglia)—we can systematically and comprehensively study the impact of MβCD on energy metabolism throughout the neurovascular unit from multiple dimensions.

[0052] Experiments have verified that methyl-β-cyclodextrin, as a GLUT1 regulator, can modulate the expression of GLUT-related mRNA in blood-brain barrier endothelial cells, neurons, microglia, and astrocytes, and can regulate the glucose uptake function of various brain cells.

[0053] The sixth aspect of this application provides a drug for preventing or delaying Alzheimer's disease, or a drug for improving cerebrovascular function, or a drug for improving cognitive impairment, said drug comprising a GLUT1 modulator, said GLUT1 modulator comprising methyl-β-cyclodextrin.

[0054] In some embodiments, the drug further includes one or more pharmaceutically acceptable excipients.

[0055] This application applies MβCD to the preparation of drugs for preventing or delaying Alzheimer's disease, or drugs for improving cerebrovascular function, or drugs for improving cognitive impairment. Using MβCD as a GLUT1 modulator for intervention can improve cognitive impairment, improve cerebrovascular lesions, and restore brain glucose metabolism, providing a new treatment strategy for the prevention and treatment of Alzheimer's disease.

[0056] The following is a specific embodiment.

[0057] Unless otherwise specified, the reagents and instruments used in the examples are conventionally selected in the art. Experimental methods not specifying particular conditions in the examples are typically performed under standard conditions, such as those described in literature, books, or recommended by the reagent kit manufacturer. All reagents used in the examples are commercially available.

[0058] Unless otherwise specified, the experimental materials and animal husbandry in the following examples are as follows: 1. Experimental Materials The methyl-β-cyclodextrin (MβCD) and GLUT1 antibodies were from Sigma-Aldrich; the CD31, Aβ, GFAP, and Iba1 antibodies were from Abcam Biotech.

[0059] 2. Laboratory animal husbandry The 5xFAD mouse is a transgenic animal model commonly used in Alzheimer's disease (AD) research, often for evaluating the efficacy of potential treatments and drugs for AD. The 5xFAD transgenic mouse model originated from the Jackson Laboratory in the United States (catalog number 34348). The 5xFAD transgenic mouse model used in the experiment, along with the control wild-type WT mice, were derived from the self-crossing of heterozygous 5xFAD mice. The animal room temperature was controlled at 22℃ ± 2℃, and the humidity at 60%–80%. Mice were given free access to standard feed, and a 12-hour light / dark cycle was implemented.

[0060] The animal experiments were divided into two main groups: Animal Experiment 1 and Animal Experiment 2. The specific groupings and experimental details for each group are as follows: The grouping and experimental details of Animal Experiment 1 are as follows: The mice were divided into six groups, each containing 8-9 six-month-old experimental mice: (1) Wild-type WT mice that were subcutaneously injected with 0.9% (w / v) NaCl injection solution were named: WT-Veh group; (2) The 5xFAD mouse group that received subcutaneous injection of 0.9% (w / v) NaCl injection solution was named: 5xFAD-Veh group). (3) The 5xFAD mouse group that received subcutaneous injection of 100 mg / kg / day MβCD solution was named: 5xFAD-MβCD100 group).

[0061] (4) The 5xFAD mouse group that received subcutaneous injection of 250 mg / kg / day MβCD solution was named: 5xFAD – MβCD250 group).

[0062] (5) The 5xFAD mouse group that received a subcutaneous injection of 500 mg / kg / day MβCD solution was named: 5xFAD-MβCD500 group).

[0063] (6) The 5xFAD mouse group that received a subcutaneous injection of 1000 mg / kg / day MβCD solution was named: 5xFAD – MβCD1000 group).

[0064] The injection volume was 0.1 ml / 20 g / day, and mice were injected subcutaneously for 7 consecutive days. Cognitive behavioral tests (novel object recognition experiment) were performed after the injections.

[0065] The grouping and experimental details of Animal Experiment 2 are as follows: The mice were divided into four groups, each containing 8-9 six-month-old experimental mice: (1) Wild-type WT mice that were subcutaneously injected with 0.9% (w / v) NaCl injection solution were named: WT-Veh group; (2) Wild-type WT mice that were subcutaneously injected with 500 mg / kg / day MβCD solution were named: WT-MβCD group). (3) The 5xFAD mouse group that received subcutaneous injection of 0.9% (w / v) NaCl injection solution was named: 5xFAD-Veh group). (4) The 5xFAD mouse group that received subcutaneous injection of 500 mg / kg / day MβCD solution was named: 5xFAD-MβCD group).

[0066] The injection volume was 0.1 ml / 20g / day, and mice were injected subcutaneously for 7 consecutive days. After the injection, cognitive function behavioral tests (new object recognition test, water maze test) were performed. After the experiment, the brain was harvested through cardiac perfusion for subsequent brain tissue immunohistochemistry and tissue analysis.

[0067] Example 1: Cognitive Function Testing in Mice The new object recognition test and the water maze test are two of the most commonly used methods in Alzheimer's disease research to assess the cognitive abilities of mice.

[0068] Novel object recognition experiment: The novel object recognition experiment mainly assesses the animal's working memory (short-term memory) and recognition memory. (1) Experimental setup: The experiment uses a white polypropylene open field device (40×40×40 cm³), with two identical cylinders (5 cm in diameter) placed symmetrically, 10 cm away from the box wall. (2) Behavioral protocol: a) Training phase: The mouse is placed facing the box wall and freely explores the object for 5 minutes. b) Testing phase: After 1 hour, one of the objects is replaced with a novel cube (5 cm on each side), and the sniffing time within 5 minutes is recorded using ANY-maze 7.1 software (nose tip ≤ 2 cm from the object). c) Data analysis: Discrimination index = (new object exploration time - familiar object exploration time) / total exploration time × 100%.

[0069] Water maze experiment: The water maze experiment is mainly used to assess spatial learning ability and spatial reference memory, and is one of the "gold standards" for studying hippocampal dependent memory. (1) Experimental equipment: A circular pool (100 cm in diameter) is filled with 25°C clean water and non-toxic white pigment is added and mixed. A transparent plexiglass platform (12 cm in diameter) is fixed in the target quadrant and submerged 1 cm below the water surface. (2) Training plan: Training for 5 consecutive days, 4 times a day (30 minutes apart). The maximum time for a single session is 60 seconds. If a mouse fails to find the platform, the experimenter will guide it to the platform and let it stay for 20 seconds. (3) Spatial exploration experiment: On the 6th day, the platform is removed and the mouse swims freely for 60 seconds. (4) Ethovision XT 15.0 software is used to analyze the time spent in the target quadrant and the number of times the platform is crossed.

[0070] Test results as follows Figure 1 As shown. Figure 1 The following figures show the results of the detection of the effect of MβCD on the cognitive function of AD model mice: a) a statistical chart of the time mice in the first group of animal experiments explored new and old objects in the new object recognition experiment; b) the time mice in the second group of animal experiments explored new and old objects in the new object recognition experiment; c) a statistical chart of the escape latency time of mice in the second group of animal experiments in the water maze experiment; d) a statistical chart of the time mice in the second group of animal experiments stayed in the target quadrant in the water maze experiment.

[0071] New object recognition experiment results Figure 1 A and Figure 1 As shown in B. Figure 1The results of the novel object recognition experiment in animal experiment A 1 showed that, compared with the WT-Veh group, the time spent exploring novel objects in the WT-Veh group was not significantly different; however, compared with the WT-Veh group, the time spent exploring novel objects in the 5xFAD-Veh group was significantly reduced; compared with the 5xFAD-Veh group, the time spent exploring novel objects in the 5xFAD-MβCD 100 group, 5xFAD-MβCD 250 group, 5xFAD-MβCD 500 group, and 5xFAD-MβCD 1000 group was significantly increased in a dose-dependent manner. Subcutaneous injection of 250 mg / kg / day - 1000 mg / kg / day of MβCD improved the exploration behavior of novel objects in 5xFAD mice, indicating that MβCD intervention partially reversed the cognitive deficits in 5xFAD mice. Figure 1 The results of the new object recognition experiment in animal experiment 2 showed that, compared with the WT-Veh group, the time spent exploring new objects in the WT-Veh group was not significantly different; however, compared with the WT-Veh group, the time spent exploring new objects in the 5xFAD-Veh group was significantly reduced; compared with the 5xFAD-Veh group, the time spent exploring new objects in 5xFAD mice was significantly increased after MβCD treatment (subcutaneous injection of 500 mg / kg / day for 7 consecutive days). MβCD treatment may have improved the exploration behavior of 5xFAD mice in exploring new objects, indicating that MβCD intervention partially reversed the cognitive deficits in 5xFAD mice.

[0072] The results of the water maze experiment are as follows: Figure 1 B and Figure 1 As shown in Figure C: Compared with the WT-Veh group, the escape latency of the WT-MβCD group did not change significantly, while the escape latency of the 5xFAD-Veh group was significantly longer than that of the WT-Veh group (impaired learning and memory). The latency of the 5xFAD-MβCD group was shorter than that of the 5xFAD-Veh group, indicating that MβCD intervention (subcutaneous injection of 500 mg / kg / day for 7 consecutive days) significantly improved the spatial learning and memory ability of 5xFAD mice. Compared with the WT-Veh group, the time spent in the target quadrant of the WT-MβCD group did not change, while the time spent in the target quadrant of the 5xFAD-Veh group was shorter than that of the WT-Veh group (impaired memory retrieval). The time spent in the target quadrant of the 5xFAD-MβCD group was longer than that of the 5xFAD-Veh group, indicating that MβCD intervention significantly improved the spatial memory retention ability of 5xFAD mice.

[0073] Example 2: Detection of cerebral vascular structure and function in mice Immunohistochemistry and tissue analysis: (1) Sample processing: a) Mouse brain tissue collection and fixation: Mice were anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection), and then perfused with pre-cooled PBS and 4% (w / v) PFA to ensure blood clearance and endothelial cell integrity. Brain tissue was fixed in 4% (v / v) PFA for 24 hours, and then dehydrated in 30% sucrose solution for 48 hours to prevent ice crystal damage during sectioning. b) Brain tissue frozen sections: After OCT embedding, coronal sections (30 μm) of brain tissue were prepared using a Leica CM3050S cryostat. Brain sections were stored in cryoprotectant (30% (v / v) glycerol + 30% (v / v) ethylene glycol / PBS) at -20°C.

[0074] (2) Immunofluorescence staining: a) Blocking and permeabilization: Brain sections were washed three times with PBS (5 minutes each time) to remove residual fixative; blocking solution (PBS containing 5% (v / v) goat serum + 0.3% (v / v) Triton X-100) was added, and incubated at room temperature for 1.5-2 hours. b) Primary antibody incubation: The blocking solution was discarded, and the diluted primary antibody mixture was added dropwise and incubated overnight in a humidified chamber at 4°C. Anti-GLUT1 primary antibody, brand: Merck, catalog number: MABS132, was used at a dilution of 1:500 to label glucose transporter 1; anti-CD31 primary antibody, brand: Sigma, catalog number: MAB1398z, was used at a dilution of 1:500 to label cerebral vascular endothelial cells. c) Secondary antibody incubation: The next day, wash the slides three times with PBS (5 minutes each time) to remove unbound primary antibody; add a mixture of fluorescein-labeled secondary antibody (protected from light to prevent fluorescence quenching), incubate at room temperature in the dark for 2 hours, and wash again with PBS three times (5 minutes each time). d) Nuclear staining and mounting: Add DAPI staining solution (blue fluorescence, specifically labeling cell nuclei), incubate at room temperature in the dark for 5 minutes, wash with PBS to remove excess staining solution; mount with anti-fluorescence quenching mounting medium (such as Fluoromount-G® mounting medium) to avoid rapid attenuation of fluorescence signal during imaging. After mounting, it can be stored at 4°C in the dark (short-term) or -20°C (long-term).

[0075] (3) Immunofluorescence quantification: Using a laser confocal microscope (such as Zeiss LSM 900), the excitation wavelengths of the corresponding fluorophores were selected (405nm for DAPI, 488nm for Alexa Fluor 488, 594nm for Alexa Fluor 594) to obtain a three-color superimposed image of "blue (nucleus) - green (GLUT1) - red (CD31)".

[0076] Vascular network morphology: CD31 green signal shows the overall distribution of cerebral blood vessels (such as capillary density and vessel diameter in the cortex and hippocampus). Blood-brain barrier integrity: the degree of colocalization of GLUT1 red signal and CD31 green signal (under normal circumstances, the two should completely overlap, indicating normal BBB endothelial function; if GLUT1 signal is absent or discrete, it indicates BBB damage). Quantitative analysis: The number / length of CD31 positive vessels (capillary density) was counted using ImageJ software, and the fluorescence intensity of GLUT1 (reflecting BBB glucose transport function) was measured.

[0077] Test results as follows Figure 2 As shown, Figure 2 Figure 1 shows the results of detecting the effect of MβCD on cerebral vascular function in AD model mice: A) Immunofluorescence staining diagram, in which CD31 labels blood vessels and GLUT1 labels glucose transporter 1; B) GLUT1 fluorescence intensity on blood vessels; C) Percentage of blood vessels expressing GLUT1; D) Blood vessel density (CD31+ length).

[0078] Results of mouse brain vascular structure and function testing, such as Figure 2 As shown in A and 2B, compared with the WT-Veh group, there was no significant change in the fluorescence intensity of GLUT1 in the blood vessels of the WT-MβCD group. The fluorescence intensity of GLUT1 in the blood vessels of the 5xFAD-Veh group was lower than that of the WT-Veh group, while the fluorescence intensity of GLUT1 in the blood vessels of the 5xFAD-MβCD group was higher than that of the 5xFAD-Veh group. This indicates that MβCD intervention significantly increased the expression of GLUT1 in the vascular endothelial cells of 5xFAD mice, which may have improved glucose supply to the brain. Figure 2 As shown in Figure C, compared with the WT-Veh group, the percentage of vessels expressing GLUT1 in the WT-MβCD group did not change significantly. The percentage of vessels expressing GLUT1 in the 5xFAD-Veh group was lower than that in the WT-Veh group, while the percentage in the 5xFAD-MβCD group was higher than that in the 5xFAD-Veh group. This indicates that MβCD intervention not only increased the expression level of GLUT1 in the cerebral vascular endothelial cells of 5xFAD mice but also increased the density of vessels expressing GLUT1. Figure 2 As shown in Figure D, compared with the WT-Veh group, there was no significant change in vascular density (CD31+ length) in the WT-MβCD group, while the vascular density (CD31+ length) in the 5xFAD-Veh group was lower than that in the WT-Veh group, indicating that the cerebral blood vessels of 5xFAD mice had some degeneration. MβCD treatment increased the cerebral blood vessel density of 5xFAD mice, indicating that MβCD intervention has a certain improving or protective effect on the cerebral blood vessel density of 5xFAD mice.

[0079] Example 3 18 F-FDG mouse PET imaging 18 F-fluorodeoxyglucose ( 18 F-FDG (F-FDG) small animal PET imaging can assess the metabolic activity of mouse organs at the molecular level, and the signal intensity directly reflects the glucose metabolism activity of cells.

[0080] (1) Imaging protocol: Mice were injected with 13.5 MBq ¹ via the tail vein. 8 F-fluorodeoxyglucose (¹) 8 F-FDG (injection volume ≤200 μL). Thirty minutes after tracer ingestion while awake, the animal was anesthetized with a 2% (v / v) isoflurane / oxygen mixture and scanned for 20 minutes using a SIAT small animal PET system (model SIAT-PET-64). Respiratory rate (80-120 breaths / min) and body temperature (37.0±0.5°C) were monitored throughout the process.

[0081] (2) Image Reconstruction and Analysis: After the scan, the original data was reconstructed to generate three-dimensional tomographic images, and the standardized uptake value (SUV) data was exported. In the experiment, Amide 1.0.4-1 software was used to select the regions of interest (ROI) in the hippocampus and cortex, and the standardized uptake value (SUV) was calculated. The SUV value (standardized uptake value) is the core indicator for quantifying metabolic activity. The calculation formula is: SUV = (tissue radioactivity concentration [Bq / g]) / (total injection dose [Bq] / mouse body weight [g]). The higher the SUV, the more active the tissue metabolism.

[0082] mouse brain 18 The PET imaging results of F-FDG mice are as follows: Figure 3 As shown, Figure 3 The figure shows the results of detecting the effect of MβCD on glucose metabolism in the brain of AD model mice.

[0083] like Figure 3 As shown: Compared with the WT-Veh group, the WT-MβCD group 18 F-FDG mouse brain PET imaging showed no significant change in SUV values. 5xFAD-Veh group mouse brain 18 F-FDG imaging SUV values ​​were lower in the WT-Veh group than in the 5xFAD-MβCD group mice. 18 The SUV value of F-FDG imaging was higher than that of the 5xFAD-Veh group, indicating that MβCD intervention significantly increased the activity of glucose metabolism in the brain of 5xFAD mice and improved the glucose supply in the brain.

[0084] Example 4: Assessment of glial cell proliferation and analysis of Aβ plaques in mouse brain Aβ plaques, microglia activation and proliferation (mediating neuroinflammation), and astrocyte proliferation (involved in blood-brain barrier repair and glial scar formation) are among the core pathological markers of Alzheimer's disease (AD). Immunofluorescence labeling of microglia-specific markers using Iba1 and astrocyte-specific markers using GFAP provide crucial evidence for assessing intracranial inflammation and repair responses. Specific immunofluorescence labeling of Aβ plaques in the brain using Aβ antibodies can assess the pathological severity of AD.

[0085] (1) Assessment of glial cell proliferation: a) Blocking and permeability: Brain sections were washed three times with PBS (5 minutes each time) to remove residual fixative; blocking solution (PBS containing 5% (v / v) goat serum + 0.3% (v / v) Triton X-100) was added and incubated at room temperature for 1.5-2 hours. b) Primary antibody incubation: The blocking solution was discarded, and the diluted primary antibody mixture was added dropwise and incubated overnight in a humidified chamber at 4°C. Rabbit anti-Iba1 primary antibody, brand: ABclonal, catalog number: A19776, was used at a 1:500 dilution to label microglia; rabbit anti-GFAP primary antibody, brand: Abcam, catalog number: ab7260, was used at a 1:500 dilution to label astrocytes. c) Secondary antibody incubation: The next day, wash the slides three times with PBS (5 minutes each time) to remove unbound primary antibody; add a mixture of fluorescein-labeled secondary antibody (protected from light to prevent fluorescence quenching), incubate at room temperature in the dark for 2 hours, and wash again with PBS three times (5 minutes each time). d) Nuclear staining and mounting: Add DAPI staining solution (blue fluorescence, specifically labeling cell nuclei), incubate at room temperature in the dark for 5 minutes, wash with PBS to remove excess staining solution; mount with anti-fluorescence quenching mounting medium (such as Fluoromount-G® mounting medium) to avoid rapid attenuation of fluorescence signal during imaging. After mounting, it can be stored at 4°C in the dark (short-term) or -20°C (long-term). Take 3-4 slides from each mouse, and select 3 fields of view from each hippocampal radiation layer to count glial cell density (cells / mm²).

[0086] (2) Aβ plaque analysis: a) Blocking and permeation: Brain sections were washed three times with PBS (5 minutes each time) to remove residual fixative; blocking solution (PBS containing 5% (v / v) goat serum + 0.3% (v / v) Triton X-100) was added, and incubated at room temperature for 1.5-2 hours. b) Primary antibody incubation: The blocking solution was discarded, and the diluted primary antibody mixture was added, and incubated overnight in a humidified chamber at 4°C. Chicken anti-Aβ primary antibody, brand: Abcam, catalog number: ab134022, was diluted 1:500 and used to label aggregated Aβ plaques in the brain. c) Secondary antibody incubation: The next day, the sections were washed three times with PBS (5 minutes each time) to remove unbound primary antibody; a fluorescein-labeled secondary antibody mixture was added (operated in the dark to prevent fluorescence quenching), and incubated at room temperature in the dark for 2 hours, followed by washing three times with PBS (5 minutes each time). d) Nuclear staining and mounting: Add DAPI staining solution (blue fluorescence, specifically labeling cell nuclei), incubate at room temperature in the dark for 5 minutes, wash with PBS to remove excess staining solution; mount with anti-fluorescence quenching mounting medium (such as Fluoromount-G® mounting medium) to avoid rapid attenuation of fluorescence signal during imaging. After mounting, it can be stored at 4°C in the dark (short-term) or -20°C (long-term). Acquire images with a 10× objective lens, and calculate the area ratio of Aβ plaques in the hippocampus using ImageJ 1.53t.

[0087] The results of mouse glial cell proliferation assessment and Aβ plaque analysis are as follows: Figures 4 to 6 As shown. Figure 4 The following figures show the results of detecting the effect of MβCD on the activation of microglia in the brains of AD model mice: a) Immunofluorescence staining results of microglia in the brains of mice, with Iba antibody used to label microglia; b) Statistical results of Iba1+ cell area percentage. Figure 5 The following figures show the results of detecting the effect of MβCD on the proliferation of astrocytes in the brain of AD model mice: a) Immunofluorescence staining results of astrocytes in the mouse brain, with GFAP antibody used to label astrocytes; b) Statistical results of GFAP+ cell area %. Figure 6 The following figures show the results of detecting the effect of MβCD on Aβ deposition in the brains of AD model mice: a) Immunofluorescence staining results of Aβ plaques in the mouse brain, with Aβ antibody used to label Aβ plaques; b) Statistical results of Aβ+ cell area %.

[0088] like Figure 4 As shown, compared with the WT-Veh group, there was no significant change in the microglial activation area in the WT-MβCD group, while the microglial activation area in the 5xFAD-Veh group was higher than that in the WT-Veh group. MβCD treatment reduced the microglial activation area in 5xFAD mice, indicating that MβCD intervention significantly alleviated neuroinflammation (microglial activation) in 5xFAD mice.

[0089] like Figure 5 As shown, compared with the WT-Veh group, there was no significant change in the activated area of ​​astrocytes in the WT-MβCD group, while the activated area of ​​astrocytes in the 5xFAD-Veh group was higher than that in the WT-Veh group. MβCD treatment reduced the activated area of ​​astrocytes in 5xFAD mice, indicating that MβCD intervention significantly alleviated neuroinflammation (astrocytosis) in 5xFAD mice.

[0090] like Figure 6 As shown, similar to the WT-Veh group, no Aβ plaques were observed in the WT-MβCD group. Compared with the WT-Veh group, obvious Aβ plaques were observed in the 5xFAD-Veh group. MβCD treatment had no significant effect on Aβ plaques in 5xFAD mice.

[0091] Example 5: In vitro experimental verification of the effect of methyl-β-cyclodextrin on glucose uptake function in different brain cells Brain energy metabolism is a highly segmented and cell-specific process. Each cell type has its unique metabolic program to meet the needs of its specific role. Blood-brain barrier endothelial cells selectively control the flow of nutrients (such as glucose) from the blood into the cerebral interstitial fluid, acting as the main valve for the brain's energy supply. Neurons perform information processing (action potentials, synaptic transmission) and are the cells in the brain with the highest and most sustained energy demand. Astrocytes are the energy supporters and reserves of neurons and maintain extracellular ion / neurotransmitter balance. Microglia are responsible for immune surveillance and phagocytosis in the brain, and their metabolic state directly affects their immune function (pro-inflammatory or anti-inflammatory). Using four cell models—hCMEC / D3 (blood-brain barrier endothelial cells), HT22 (neurons), BV2 (microglia), and SVG P12 (astroglia)—we can systematically and comprehensively study the impact of MβCD on energy metabolism throughout the neurovascular unit from multiple dimensions.

[0092] (1) GLUTs mRNA expression profiling analysis (qPCR): (a) Take logarithmically growing hCMEC / D3 cells (human brain microvascular endothelial cell line), digest and count them, and then store them at a certain density (e.g., 2×10⁻⁶). 5Cells were seeded in 12-well plates, with 500 μL of complete culture medium (hCMEC / D3 complete medium, purchased from Zhejiang Meisen Cell Technology Co., Ltd., catalog number: CTCC-003-0113-CM) added to each well. Cells were incubated at 37℃ in a 5% CO2 incubator for 24 hours to allow cell adhesion. A control group (solvent-treated, i.e., cultured and treated with the complete medium itself, with 0% methyl-β-cyclodextrin) and an MβCD experimental group (hCMEC / D3 complete medium with different concentrations of methyl-β-cyclodextrin, i.e., 1 mM, 3 mM, and 5 mM) were set up, with 3 replicates per group. The drug-containing culture medium (i.e., complete culture medium with the corresponding concentration of methyl-β-cyclodextrin; for example, the control group used complete culture medium with 0% methyl-β-cyclodextrin as the drug-containing medium, the MβCD experimental group with 1mM methyl-β-cyclodextrin used complete culture medium with 1mM methyl-β-cyclodextrin as the drug-containing medium, and so on, without further details) was replaced with drug-containing medium. After culturing for 8, 16, and 24 hours, total RNA was extracted from the cells according to the instructions of the total RNA extraction kit (TRIeasy™ Total RNA Extraction Reagent, catalog number: 10606ES60, brand: Yisheng).

[0093] (b) Take HT22 cells (mouse hippocampal neuron cell line), BV2 cells (mouse microglia cell line), and SVG P12 cells (human astrocyte cell line) in logarithmic growth phase, digest and count them, and then arrange them at a certain density (e.g., 2 × 10⁻⁶). 5Cells were seeded in 12-well plates, with 500 μL of complete culture medium (i.e., HT22 and BV2 cell complete culture medium, prepared from 90% DMEM basal medium + 10% fetal bovine serum + 1% penicillin-streptomycin solution; SVG P12 complete culture medium, purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd., catalog number: CMH8-2301) added to each well). Cells were incubated at 37℃ in a 5% CO2 incubator for 8 hours to allow cell adhesion. A control group (solvent-treated, i.e., cultured and treated with complete culture medium alone, with no added methyl-β-cyclodextrin) and an MβCD experimental group (with different concentrations of methyl-β-cyclodextrin added to the complete culture medium, at amounts of 1 mM, 3 mM, and 5 mM) were set up, with 3 replicates per group. Replace the drug-containing culture medium (i.e., complete culture medium with the corresponding concentration of methyl-β-cyclodextrin; for example, the control group used complete culture medium with 0% methyl-β-cyclodextrin as the drug-containing medium, the MβCD experimental group with 1mM methyl-β-cyclodextrin used complete culture medium with 1mM methyl-β-cyclodextrin as the drug-containing medium, and so on, without further details) for drug treatment. After culturing for 8 hours, extract total RNA from the cells according to the instructions of the total RNA extraction kit (TRIeasy™ Total RNA Extraction Reagent, catalog number: 10606ES60, brand: Yisheng).

[0094] (c) Following the kit instructions (Hifair® AdvanceFast 1st Strand cDNA SynthesisSuperMix for qPCR, catalog number: 11155ES60, brand: Yisheng), complementary DNA (cDNA) was synthesized using total RNA as a template. Following the kit instructions (Hieff UNICON® Advanced qPCR SYBR Master Mix, catalog number: 11185ES08, brand: Yisheng), real-time quantitative PCR was performed on a quantitative PCR instrument (QuantStudio 7 Flex System) using cDNA as a template in the presence of specific primers (see Table 1 for primer details) and fluorescent dye (SYBR Green) to detect the expression of the target gene mRNA.

[0095] Table 1 qPCR primers

[0096] (2) Glucose uptake function analysis (2-NBDG uptake experiment): The experiment was conducted according to the instructions of the 2-NBDG Glucose Uptake Assay Kit (catalog number: K2212-500, brand: APExBIO). The specific experimental procedures are as follows: 1) Cell culture: hCMEC / D3 cells in the logarithmic growth phase were digested and seeded into 96-well black transparent-bottomed culture plates at a density of 4 × 10⁶ cells / well. 4 1) Cells / well (100 µL per well), cultured for 24 h in an incubator. 2) Cell starvation: Once cells reach suitable confluence, remove the old culture medium. Wash cells twice with PBS, add preheated glucose-free medium (90% DMEM glucose-free medium + 10% fetal bovine serum FBS), and return cells to the incubator for starvation treatment for 2 h. 3) Drug treatment: Prepare 3 mM MβCD working solution using glucose-free medium; dilute BAY-876 (10 mM) 1:10000 in glucose-free medium and mix well to obtain 1 μM BAY-876 working solution; dilute BAY-876 (10 mM) 1:10000 in 3 mM MβCD working solution and mix well to obtain 3 mM MβCD + 1 μM BAY-876 working solution. Remove cell culture medium, add the above drug working solutions, and treat cells in an incubator for 1 h. 4) 2-NBDG staining: After drug treatment, discard the culture medium supernatant, add 100 μL of 2-NBDG working solution per well, and incubate at 37°C in the dark for 30 min. 5) Detection: After incubation, discard the supernatant, wash cells twice with Assay Buffer, then add 100 μL of Assay Buffer per well and observe and photograph using an inverted fluorescence microscope (Nikon ECLIPSE Ti2-E).

[0097] (3) Results analysis: GLUTs mRNA expression profile analysis results are as follows Figure 7 , Figure 8 As shown, Figure 7 The figures show the results of the MβCD effect on GLUTs mRNA expression profiles and glucose uptake capacity in hCMEC / D3 cells. Figure 7 Figure A shows the results of GLUT1 mRNA expression profiling analysis in hCMEC / D3 cells. Figure 7 Figure B shows the results of GLUT3 mRNA expression profiling analysis in hCMEC / D3 cells. Figure 7 Figure C shows the GLUT4 mRNA expression profile analysis results of hCMEC / D3 cells; in addition, the analysis results of glucose uptake function of hCMEC / D3 cells after 1 h of treatment with 3 mM MβCD are as follows. Figure 7 As shown in D. Figure 8This is a graph showing the results of MβCD expression profile analysis of GLUTs mRNA in HT22, BV2, and SVG P12 cells. Figure 8 Figure A shows the results of GLUT1 mRNA expression profiling analysis in HT22 cells. Figure 8 Figure B shows the results of GLUT3 mRNA expression profiling analysis in HT22 cells. Figure 8 Figure C shows the results of GLUT4 mRNA expression profiling analysis in HT22 cells. Figure 8 Figure D shows the results of GLUT1 mRNA expression profiling analysis in BV2 cells. Figure 8 Figure E shows the results of GLUT5 mRNA expression profiling analysis in BV2 cells. Figure 8 F is the result of GLUT1 mRNA expression profile analysis in SVG P12 cells. Figure 8 G represents the results of GLUT4 mRNA expression profile analysis in SVG P12 cells.

[0098] The human brain accounts for only 2% of an adult's body weight, yet it requires 25% of the body's energy. Brain energy metabolism is highly dependent on glucose; more than one-fifth of the glucose in the blood is taken up by the brain. Vascular endothelial cells, especially the endothelial cells of brain microvessels that form the blood-brain barrier, are the primary gateway and key regulatory point for glucose to enter the brain from the blood. They mediate transcellular glucose transport through glucose transporter proteins (GLUTs) expressed on their surface, particularly GLUT1.

[0099] GLUT1 is the dominant glucose transporter in brain microvascular endothelial cells, responsible for over 90% of glucose transport across the blood-brain barrier. In animal models, specific knockout of GLUT1 on endothelial cells leads to severe glucose insufficiency, reduced microvessels, neuronal loss, and premature cell death, directly demonstrating GLUT1's indispensability. GLUT1 is abundantly expressed on both the apical membrane (facing the blood) and the basement membrane (facing the brain). GLUT3 is known for its high affinity for neurons. GLUT4 is an insulin-sensitive glucose transporter involved in the insulin-sensitive pathway. In brain microvascular endothelial cells, GLUT3 may be responsible for the cells' own energy supply, while GLUT4 may be activated under specific pathophysiological conditions. hCMEC / D3 cells (human brain microvascular endothelial cells) are a classic in vitro model of the blood-brain barrier (BBB).

[0100] The results are as follows Figure 7As shown in Figure A, compared with the control group, the expression level of GLUT1 mRNA in hCMEC / D3 cells significantly increased after treatment with 3mM and 5mM MβCD for 8h and 16h, respectively, and the expression level of GLUT1 mRNA in hCMEC / D3 cells returned to normal after treatment with 3mM and 5mM MβCD for 24h. Figure 7 As shown in Figure B, compared with the control group, the expression level of GLUT3 mRNA in hCMEC / D3 cells significantly increased after treatment with 3mM and 5mM MβCD for 8h, 16h, and 24h. Figure 7 As shown in Figure C, compared with the control group, the expression level of GLUT4 mRNA in hCMEC / D3 cells was significantly reduced after 16 h and 24 h of treatment with 5 mM MβCD.

[0101] BAY-876 is a highly effective and selective GLUT1 inhibitor, often used in research to specifically inhibit glucose uptake via GLUT1. For example... Figure 7 As shown in Figure D, treatment with 3 mM MβCD for 1 h enhanced glucose uptake in hCMEC / D3 cells, while treatment with 1 μM BAY-876 inhibited glucose uptake in hCMEC / D3 cells. 1 μM BAY-876 significantly inhibited the enhancement of glucose uptake in hCMEC / D3 cells by 3 mM MβCD, suggesting that the effect of MβCD on enhancing glucose uptake in hCMEC / D3 cells may be related to GLUT1.

[0102] The most important glucose transporter in neurons is GLUT3, which has a lower Km value and higher affinity for glucose at physiological concentrations compared to GLUT1. GLUT1 (45 kDa isoform) is also ubiquitous in neurons. GLUT4 may be present in neurons in specific brain regions or in specific states, and some studies have shown that it can be mobilized as a response to sustained synaptic activity.

[0103] The results are as follows Figure 8 As shown in Figure A, compared with the control group, the expression level of GLUT1 mRNA in HT22 cells significantly increased after 8 hours of treatment with 5 mM MβCD. Figure 8 As shown in Figure B, compared with the control group, the expression level of GLUT3 mRNA in HT22 cells was significantly reduced after 8 hours of treatment with 1mM and 3mM MβCD. Figure 8 As shown in Figure C, compared with the control group, the expression level of GLUT4 mRNA in HT22 cells was significantly increased after 8 hours of treatment with 5mM MβCD.

[0104] The most important and fundamental glucose transporter in microglia is GLUT1, which plays a crucial role in both resting and activated states. GLUT5 is a hallmark transporter of microglia, primarily responsible for fructose transport, but it is highly expressed in microglia, and its expression is regulated by inflammatory states.

[0105] The results are as follows Figure 8 D and Figure 8 As shown in Figure E, compared with the control group, the expression levels of GLUT1 and GLUT5 mRNA in BV2 cells did not change significantly after treatment with 1mM, 3mM, and 5mM MβCD for 8 hours.

[0106] The most important and fundamental glucose transporter in astrocytes is GLUT1, which is responsible for maintaining the cell's basic glucose needs. GLUT4 is an insulin-sensitive transporter, and detecting it can link drugs to the insulin signaling pathway.

[0107] The results are as follows Figure 8 As shown in Figure F, compared with the control group, the expression level of GLUT1 mRNA in SVG P12 cells was significantly increased after treatment with 3mM and 5mM MβCD for 8 hours. Figure 8 As shown in G, compared with the control group, the expression level of GLUT4 mRNA in SVG P12 cells was significantly increased after 8 h of treatment with 3 mM MβCD.

[0108] In summary, methyl-β-cyclodextrin (MβCD) treatment exhibits multiple beneficial effects in 5xFAD Alzheimer's disease model mice. Its mechanism of action is likely a positive feedback loop: 1) Improved cerebrovascular function: MβCD may enhance brain energy metabolism by upregulating GLUT1 expression and improving vascular density in cerebral blood vessels through lipid raft remodeling or direct action. 2) Reduced neuroinflammation: MβCD inhibits excessive activation of astrocytes and microglia. 3) Rescue of cognitive function: The aforementioned improvements at the molecular and cellular levels ultimately lead to a significant recovery in learning, memory, and exploratory behavior. These data suggest that MβCD is a highly promising multi-target therapeutic strategy for Alzheimer's disease, simultaneously addressing the core pathological features of the disease.

[0109] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of GLUT1 modulators in the preparation of drugs for the prevention or delay of Alzheimer's disease, characterized in that, The GLUT1 modifier includes methyl-β-cyclodextrin.

2. The application of GLUT1 modulators in the preparation of drugs for improving cerebrovascular function, characterized in that, The GLUT1 modifier includes methyl-β-cyclodextrin.

3. The application according to claim 2, characterized in that, The drug can increase GLUT1 expression and / or vascular density in cerebral blood vessels.

4. The application of GLUT1 modulators in the preparation of drugs for reducing neuroinflammation in the brain, characterized in that, The GLUT1 modifier includes methyl-β-cyclodextrin.

5. The application according to claim 4, characterized in that, The drug can inhibit the excessive activation of astrocytes and microglia.

6. The application of GLUT1 modulators in the preparation of drugs for improving cognitive impairment, characterized in that, The GLUT1 modifier includes methyl-β-cyclodextrin.

7. The application according to claim 6, characterized in that, The drug can improve exploratory behavior and / or spatial memory retention.

8. The application of GLUT1 modulators in the preparation of drugs that regulate glucose uptake function in brain cells, characterized in that, The GLUT1 regulator includes methyl-β-cyclodextrin, and the brain cells are at least one of blood-brain barrier endothelial cells, neurons, microglia, and astrocytes.

9. The application according to any one of claims 1-8, characterized in that, The dosage of methyl-β-cyclodextrin is 250 mg / kg / day - 1000 mg / kg / day.

10. A drug for preventing or delaying Alzheimer's disease, or for improving cerebrovascular function, or for improving cognitive impairment, characterized in that, The drug includes a GLUT1 modifier, which includes methyl-β-cyclodextrin.

11. The medicament of claim 10, further comprising one or more pharmaceutically acceptable excipients.

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