Application of dapsone in preparation of medicine for treating Alzheimer disease related to microglial cell dysfunction

Abstract

The invention discloses application of dapsone in preparation of a medicine for treating Alzheimer's disease related to microglial cell dysfunction. The invention discloses for the first time that normal functions of AD transgenic animal brain microglial cells are recovered through DDS, so that the phagocytic ability can be improved, A beta plaques can be effectively eliminated, and inflammatory injury caused by excessive activation can be avoided; potential obstacles of application of the DDS in a nervous system are overcome, and it is ensured that the DDS can effectively act on microglial cells in the brain of an AD transgenic animal.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of dapsone in preparing a drug for treating Alzheimer's disease associated with abnormal microglial function. Background Art

[0002] Alzheimer's Disease (AD) is the most common neurodegenerative disease. Its incidence rate has increased dramatically with the aging of the population. It is estimated that the number of patients worldwide will exceed 150 million in 2050. The disease causes progressive cognitive decline in patients, resulting in nearly $1 trillion in direct medical expenses each year, and forcing more than 55 million families to bear the physical and mental stress of long-term care. Common pathological manifestations of AD include the aggregation of amyloid proteins outside neurons to form β-amyloid (β-amyloid, Aβ) plaques, abnormal phosphorylation and aggregation of tau proteins inside neurons to form "neurofibrillary tangles", neuroinflammation, neurodegeneration characterized by progressive brain neuron / synapse loss and brain atrophy, and impaired glucose metabolism.

[0003] In recent years, the development of genome-wide association studies (GWAS) and sequencing technologies has drawn attention to the role of microglia in AD. As innate immune cells of the central nervous system, microglia exhibit dual characteristics in the progression of AD: while they exert their protective function by phagocytosing abnormal proteins, the chronic inflammatory response caused by their continued activation may accelerate neuronal damage. GWAS shows that more than 30% of AD risk genes (such as TREM2, CD33, and CR1) are specifically expressed in microglia, further highlighting the therapeutic value of targeting microglial homeostasis regulation and providing a key breakthrough for the development of new targeted therapies.

[0004] Currently, clinical treatment for AD focuses on symptom improvement and pathological intervention. Symptomatic medications (such as cholinesterase inhibitors and NMDA receptor antagonists) provide only short-term relief of cognitive symptoms and are associated with side effects such as gastrointestinal reactions and arrhythmias. Although pathological intervention drugs (such as the Aβ monoclonal antibody aducanumab) can partially clear Aβ plaques, their efficacy in Phase III clinical trials (EMERGE / ENGAGE) has been conflicting. Targeting microglia is expected to be a new approach to slow the progression of AD, but challenges remain regarding drug bioavailability, specificity, and safety. Therefore, improving microglial-specific targeting will be a key focus of new drug development.

[0005] The sulfonamide antibiotic Dapsone (DDS) has attracted attention in recent years due to its unique immunomodulatory and anti-inflammatory properties. Epidemiological cohort studies have shown that the incidence of AD in leprosy patients who use DDS for a long time is lower than that in the control group, and the annual atrophy rate of hippocampal volume is slower. It is worth noting that due to its low molecular weight (248.3Da) and high lipid solubility, DDS has a high efficiency in penetrating the blood-brain barrier. In addition, long-term clinical use has confirmed that DDS has a low incidence of adverse reactions in the nervous system and is well tolerated with long-term use. In summary, compared with other drugs, DDS has the advantages of high blood-brain barrier penetration and clear long-term drug safety, which provides strong support for the development of the first microglial function-regulating AD prevention and treatment drug. Summary of the Invention

[0006] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] One of the objectives of the present invention is to provide a use of DDS in the preparation of a medicament for treating AD associated with abnormal microglial function.

[0009] To solve the above technical problems, the present invention provides the following technical solution: the use of DDS in the preparation of a drug for treating AD associated with microglial dysfunction, wherein the DDS structural formula is shown in Formula I:

[0010]

[0011] Specifically, the compound shown in formula I is DDS (CAS No.: 80-08-0), and the molecular formula is C 12 H 12 N2O2S has a molecular weight of 284.31 g / mol and is slightly soluble in water but readily soluble in dimethyl sulfoxide (DMSO) and alkaline solutions. Its name, as specified by the International Union of Pure and Applied Chemistry (IUPAC), is 4,4'-Sulfonylbis(benzenamine). Its Chinese names include 4,4'-sulfonyldiphenylamine, dapsone, and diaminodiphenylsulfone.

[0012] As a preferred embodiment of the use of DDS of the present invention in the preparation of a drug for treating AD associated with microglial dysfunction, dapsone delays Alzheimer's disease by regulating microglial dysfunction.

[0013] As a preferred embodiment of the use of DDS of the present invention in the preparation of a drug for treating AD associated with microglial dysfunction, DDS can moderately activate microglial phagocytosis and promote the clearance of Aβ.

[0014] As a preferred embodiment of the use of the DDS of the present invention in the preparation of a drug for treating AD associated with microglial dysfunction, the microglial dysfunction includes abnormal microglial activation, phagocytic dysfunction, and metabolic disorder.

[0015] As a preferred embodiment of the application of the DDS of the present invention in the preparation of a drug for treating AD associated with abnormal microglial function, the delay includes:

[0016] (i) Improve cognitive dysfunction;

[0017] (ii) Improvement of pathological features.

[0018] As a preferred embodiment of the use of DDS of the present invention in the preparation of a drug for treating AD associated with microglial dysfunction, the cognitive dysfunction includes neurosensory motor disorder, spatial learning dysfunction, and spatial memory disorder.

[0019] As a preferred embodiment of the use of the DDS of the present invention in the preparation of a drug for treating AD associated with microglial dysfunction, the improvement of pathological characteristics includes reducing β-amyloid protein deposition, improving microglial dysfunction, and correcting brain energy metabolism disorders.

[0020] Another object of the present invention is to provide a pharmaceutical composition for regulating microglial dysfunction to delay AD, wherein the pharmaceutical composition comprises at least DDS as an active ingredient and a pharmaceutically acceptable carrier.

[0021] As a preferred embodiment of the pharmaceutical composition of the present invention, the concentration of the DDS is 10 mg / kg / d.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This study demonstrates for the first time that DDS can restore normal function in the brain microglia of AD transgenic animals, enhancing their phagocytic capacity and effectively clearing Aβ plaques while also preventing inflammatory damage caused by overactivation. This approach overcomes potential barriers to DDS application in the nervous system, such as its ability to cross the blood-brain barrier, its distribution in the brain, and its metabolic properties, ensuring its effective action on microglia in the brains of AD transgenic animals. The DDS dosing regimen and dosage are optimized to minimize side effects and improve the safety and tolerability of the treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0025] Figure 1 These are the behavioral test results of the wild-type mouse control group (C57-CON), the 3×Tg-AD mouse control group (3×Tg-CON), the wild-type mouse drug-treated group (C57-DDS), and the 3×Tg-AD mouse drug-treated group (3×Tg-DDS) in Example 1 of the present invention.

[0026] Figure 2 These are the immunofluorescence results of frozen hippocampal sections of the 3×Tg-AD mouse control group (3×Tg-CON) and the 3×Tg-AD mouse drug-treated group (3×Tg-DDS) in Example 1 of the present invention.

[0027] Figure 3 These are the single-cell transcriptome sequencing results of hippocampal microglia in the 3×Tg-AD mouse control group (3×Tg-CON) and the 3×Tg-AD mouse drug-treated group (3×Tg-DDS) in Example 1 of the present invention.

[0028] Figure 4 This is the test of Example 2 of the present invention to test the effect of DDS on the viability and phagocytic ability of in vitro cultured BV2 microglial cells, as well as the effect of DDS on the phagocytic function of primary microglial cells. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0030] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0032] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0033] Source and Description of 3×Tg-AD Mice: Purchased from Jiangsu Wukong Biotechnology Co., Ltd. 3×Tg-AD mice are a triple-transgenic mouse model widely used in AD research. They carry mutations in three AD-related genes, APP, PS1, and MAPT. They are capable of simulating multiple AD pathological features, including Aβ deposition, tau pathology, and cognitive impairment. 3×Tg-AD mice are widely used in AD pathological mechanism research, drug screening and efficacy evaluation, and disease progression studies.

[0034] Wild-type C57BL / 6J mice were purchased from Jiangsu Wukong Biotechnology Co., Ltd.

[0035] Sources of primary microglia and the BV2 microglial cell line: Primary microglia were isolated and cultured from the brains of newborn mice aged 0 to 3 days. BV2 cells, a mouse microglial cell line, were purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0036] Example 1

[0037] (1) Experimental subjects

[0038] 3×Tg-AD mice and wild-type C57BL / 6J mice

[0039] (2) Experimental instruments

[0040] Behavioral apparatus (open field test, novel object recognition, T maze and Morris water maze apparatus), freezing microtome, laser confocal microscope

[0041] (3) Experimental process

[0042] Behavioral tests were performed to assess cognitive function in wild-type C57BL / 6J mice and 3×Tg-AD mice in the control and treatment groups to evaluate the therapeutic effect of DDS. The animals were randomly divided into four groups: a wild-type mouse control group (C57-CON), a 3×Tg-AD mouse control group (3×Tg-CON), a wild-type mouse treatment group (C57-DDS), and a 3×Tg-AD mouse treatment group (3×Tg-DDS), with 14 mice in each group.

[0043] The continuous administration of DDS started from the age of 10 months. Each animal in the two treatment groups was given oral administration (10 mg / kg / d) for 6 consecutive weeks. Each animal in the two control groups was given 10% DMSO + 90% corn oil instead.

[0044] Cognitive function tests of 3×Tg-AD mice were performed sequentially after 6 weeks of continuous administration.

[0045] First, the open field test is used to assess the animals' autonomous movement, exploratory behavior and anxiety level in a new environment. This test is completed between 8 am and 8 pm. Use 75% alcohol to clean the device after each animal. The specific process is as follows: Place the experimental animals individually in the experimental device (a 50 cm × 50 cm × 50 cm white square open-top box without holes), ensure that the environmental conditions such as lighting and noise remain consistent, and give the animals a certain amount of time to adapt to the new environment (1 minute). After the animal is placed in the open field, start timing and observe its behavior immediately for 10 minutes. Record the animal's movement trajectory, activity distance, and time spent in different areas of the open field (such as the central area and the edge area) in detail.

[0046] A novel object recognition test was conducted to measure the animals' preference for novel objects and, in turn, assess their recognition memory. Each animal underwent an acclimation phase (1 day), a training phase (1 day), and a testing phase (1 day). Each phase was completed between 8:00 AM and 8:00 PM on the day of the experiment. After each phase, the apparatus was cleaned with 75% alcohol. The specific procedure was as follows: During the acclimation phase, the animals were placed in a testing box (a 50 cm × 50 cm × 50 cm white, square, open-top box without holes) and allowed to freely explore and acclimate to the environment for 5 minutes. During the training phase, two identical objects (A and A') were placed diagonally on opposite sides of the testing box. The animals were allowed to freely explore for 10 minutes, and the exploration time for each object was recorded (T1 and T1'). During the testing phase, one of the objects (e.g., A') was replaced with a novel object (B). The animals were again allowed to freely explore for 10 minutes, and the exploration time for the novel object (B) and the familiar object (A) was recorded (T2 and T2').

[0047] The T-maze test tested the animals' spatial working memory using a self-alternation test. Testing was performed between 8:00 AM and 8:00 PM on the day of the experiment, and the apparatus was cleaned with 75% alcohol after each animal's completion. The specific procedure was as follows: The T-maze was placed in the desired location in a quiet, dimly lit testing room. Visual cues outside the maze remained constant during the test. The apparatus consisted of arms 35 cm long and 7 cm wide, with a central choice area measuring 7 cm x 7 cm. The total width of the maze was 77 cm and the total length was 42 cm. The maze floor was opaque gray, with 15 cm high sidewalls. Each arm was equipped with a 16 cm high, 7 cm wide, and 2 mm thick door. At the beginning of each trial, all doors were removed from the maze, leaving no arms unobstructed. Animals were acclimated to the testing room under ambient lighting for 1 hour, after which each animal was placed in the center of the maze. The animals were allowed to explore freely, and recording began when their head turned toward the central area. When all four limbs and the tip of the tail of the animal enter the target arm, the opening and closing door behind it is closed, and the arm entered and the entry latency are recorded. The animal is confined to the selected arm for 30 seconds. At the end of the confinement period, the animal is gently removed and placed back at the distal end of the starting arm, and the timer is stopped and reset. T1 to T6 are used as markers for tracking and analyzing animal behavior in the experiment, representing different trial stages or time points. Re-entering a previously explored arm is considered an incorrect persistence, while entering a different arm is classified as a correct alternation. The alternation percentage is used as an index of working memory, and the calculation formula is as follows: (total number of correct alternations / 6)×100%.

[0048] Spatial learning and memory abilities of animals were assessed using the Morris water maze. This test was performed between 8:00 AM and 8:00 PM on the experimental day. The Morris water maze testing system for mice consists of a stainless steel, plastic-sprayed cylindrical pool and an image acquisition and analysis system. The pool is 100 cm in diameter and 30 cm high; the platform is 6 cm in diameter and 14 cm high. The pool is evenly divided into four quadrants (NE, SE, SW, and NW) in the four directions. The midpoints of the arcs on the quadrant walls serve as optional entry points for the animals, and the platform can be placed in the center of any quadrant. The specific procedure is as follows: For 3-5 days prior to the experiment, the animals were moved to the water maze testing room for 3 hours daily. The pool was filled to a height of approximately 16 cm with clean water, and the water temperature was maintained at approximately 26°C using a heating device. Non-toxic white paint was poured into the pool and the water was stirred until it turned milky white. Markers were applied to the upper portion of the pool, not touching the water surface, in four quadrants. Eight entry points for the mice were set, spaced 45° apart. The first phase consisted of a 2-day platform-visible phase, with each animal trained four times per day. The platform was placed in different locations for the first two and last two sessions, with the animals placed on either side of the quadrant opposite the platform. The animals were allowed to freely explore the water for one minute. If they failed to find the platform for more than one minute, they were guided to the platform and allowed to remain on it for 15 seconds. After training, the animals were dried with a paper towel and placed in a warm, dry cage. After two to four consecutive experiments with the animals, feces were removed from the surface of the pool. The interval between each animal's immersion in the water was no less than 20 minutes. The second phase consisted of a 5-day platform-hidden phase, with each animal trained twice per day. The platform was placed in the center of each quadrant, approximately 1 cm below the water level. The animals were placed on either side of the quadrant opposite the platform. If they failed to find the platform for more than one minute, they were guided to the platform and allowed to remain on it for 10 seconds. After training, the animals were dried with a paper towel and placed in a warm, dry cage. The interval between each animal's two immersions was no less than 20 minutes. After 2-4 consecutive experiments with animals, feces were cleaned from the pool surface. The third phase consisted of a one-day exploration period without a platform. The platform was removed, and the animal was placed at the midpoint of the edge of the quadrant opposite the platform. The animal was allowed to swim freely in the pool for 1 minute, and its swimming trajectory and the time spent in each quadrant were recorded.

[0049] The pathological process was detected by immunofluorescence technique to detect microglial activation in the hippocampus of 3×Tg-AD mice.

[0050] First, 3×Tg-AD mice in the treatment group and the control group were perfused with phosphate buffered saline (PBS) to wash away the blood. After perfusion, 4% paraformaldehyde was used for pre-fixation until the animal was stiff. The fixed brain tissue was removed and post-fixed in 4% paraformaldehyde at 4°C overnight (12-16 hours). The fixed brain tissue was placed in a 25% sucrose solution for dehydration overnight (12-16 hours). The dehydrated brain tissue was placed in a 30% sucrose solution for dehydration overnight (12-16 hours). After dehydration, the brain tissue was placed in OCT embedding medium and frozen until the OCT was completely solidified. Slices were made using a freezing microtome (40 microns in thickness) and the brain slices were floated in 1×PBS to prevent drying. The brain slices were permeabilized with a 1×PBS solution containing 0.3% Triton X-100 for 30 minutes to improve the penetration of the antibody. Nonspecific binding sites were blocked with 5% BSA for 1 hour. After incubation with microglial marker antibodies (Iba1) and anti-Aβ antibodies (6E10) at 4°C overnight, the sections were washed three times with 1× PBST for 5 minutes each. After incubation with different fluorescently labeled secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594) for 1 hour at room temperature, the sections were mounted (ProLongGold Antifade Reagent with DAPI) and microglial morphology and Aβ deposition were observed under a confocal microscope.

[0051] The effects of DDS treatment on microglia in the hippocampus of 3×Tg-AD mice were analyzed by single-cell transcriptome sequencing. The specific process is as follows: After the behavioral test, the hippocampal tissue of the animals in the control and treatment groups was removed and cryopreserved after anesthesia. The hippocampal tissue was mechanically dissociated or enzymatically dissociated to prepare a single-cell suspension, which was then sequenced using the 10x Genomics Chromium system to obtain single-cell transcriptome data. The sequencing data were quality controlled using the Seurat package to remove low-quality cells and background noise. Cluster analysis of microglia was performed to identify different microglial subpopulations. The gene expression differences of microglia in the DDS treatment group and the control group were compared, and GO enrichment analysis was performed on the differentially expressed genes to reveal the effect of DDS treatment on microglial function.

[0052] (4) Experimental results:

[0053] Figure 1 These are the behavioral test results of the wild-type mouse control group (C57-CON), the 3×Tg-AD mouse control group (3×Tg-CON), the wild-type mouse drug-treated group (C57-DDS), and the 3×Tg-AD mouse drug-treated group (3×Tg-DDS) in Example 1 of the present invention.

[0054] Figure 1A shows the schematic diagram of the experimental process of the open field test, novel object recognition test, T maze test, and Morris water maze test used in behavioral testing; Figure 1 B is the percentage of activity time of each experimental mouse in the central area in the open field test; Figure 1 C is the activity speed of each experimental mouse in the open field test (cm / s); Figure 1 D is the total activity distance of each experimental mouse in the open field test (cm); Figure 1 E is the discrimination index of each experimental mouse in the novel object recognition test; Figure 1 F is the percentage of autonomous alternation of each experimental mouse in the T maze test.

[0055] Behavioral testing revealed that DDS treatment effectively improved cognitive function and anxiety-like behaviors in 3×Tg-AD mice. The 3×Tg-AD control group (3×Tg-CON) exhibited significant learning and memory deficits in novel object recognition, T-maze, and Morris water maze tests, reflecting cognitive impairment in AD mice. The 3×Tg-AD drug group (3×Tg-DDS) showed significant improvements in the same tests, suggesting that DDS treatment effectively alleviated cognitive decline in AD mice. No significant differences were observed between the wild-type control group (C57-CON) and the drug group (C57-DDS), indicating that DDS had no significant effect on cognitive function in normal mice. The activity time of the 3×Tg-AD mouse control group (3×Tg-CON) in the central area was significantly shorter than that of the wild-type mouse control group (C57-CON), suggesting that AD model mice had anxiety-like behavior; the activity time of the 3×Tg-AD mouse drug group (3×Tg-DDS) in the central area was significantly increased compared with the 3×Tg-CON group, approaching the level of the C57-CON group, suggesting that DDS treatment alleviated the anxiety-like behavior of AD mice; there was no significant difference between the wild-type mouse drug group (C57-DDS) and the C57-CON group, indicating that DDS had no significant effect on the anxiety behavior of normal mice.

[0056] Figure 2 These are the immunofluorescence results of frozen hippocampal sections of the 3×Tg-AD mouse control group (3×Tg-CON) and the 3×Tg-AD mouse drug-treated group (3×Tg-DDS) in Example 1 of the present invention.

[0057] Figure 2 A is the microglia (Iba1) and nuclei (DAPI) labeled in the CA1 region of the hippocampus of experimental mice in immunofluorescence experiments; Figure 2 B is a representative 3D reconstruction of microglia in the CA1 region of the hippocampus of experimental mice in immunofluorescence experiments; Figure 2 C is the intersection of microglia in the CA1 region of the hippocampus of experimental mice in the immunofluorescence experiment; Figure 2D is the number of terminal points of microglial cell branches in the CA1 region of the hippocampus of experimental mice in the immunofluorescence experiment; Figure 2 E is the sum of the cumulative lengths of all branches (dendritic processes) of microglia in the CA1 region of the hippocampus of experimental mice in the immunofluorescence experiment; Figure 2 F is the average fluorescence intensity of the microglia-specific protein Iba1 labeled in the CA1 region of the hippocampus of experimental mice in the immunofluorescence experiment; Figure 2 G is the Aβ deposition marked in the CA1 region of the hippocampus of experimental mice in immunofluorescence experiments (6E10); Figure 2 H is the mean fluorescence intensity of Aβ deposition (6E10) in the CA1 region of the hippocampus of experimental mice in the immunofluorescence experiment.

[0058] Immunofluorescence results showed that DDS treatment significantly improved microglial morphology and reduced Aβ deposition, slowing the progression of AD pathology. In the 3×Tg-AD mouse control group (3×Tg-CON), microglial branching decreased; in the 3×Tg-AD mouse treatment group (3×Tg-DDS), microglial branching increased. Aβ deposition was abundant in the 3×Tg-AD mouse control group (3×Tg-CON), while Aβ deposition was significantly reduced in the 3×Tg-AD mouse treatment group (3×Tg-DDS).

[0059] Figure 3 These are the single-cell transcriptome sequencing results of hippocampal microglia in the 3×Tg-AD mouse control group (3×Tg-CON) and the 3×Tg-AD mouse drug-treated group (3×Tg-DDS) in Example 1 of the present invention.

[0060] Figure 3 A is the weighted gene co-expression network analysis of experimental mouse hippocampal microglial subtypes in single-cell transcriptome sequencing experiments; Figure 3 B is the gene expression of microglial subtypes in the hippocampus of experimental mice in the single-cell transcriptome sequencing experiment; Figure 3 C is the enrichment analysis of gene expression signaling pathways of experimental mouse hippocampal microglial subtypes in single-cell transcriptome sequencing experiments.

[0061] DDS treatment modulates the phenotype and function of microglia, enhancing their phagocytic capacity and thus alleviating AD pathology. DDS treatment may improve microglial function and phenotype by regulating the phagocytosis-related signaling pathway (NF-κB) of microglia.

[0062] In summary, behavioral testing, immunofluorescence experiments, and single-cell transcriptome sequencing analysis suggest that DDS treatment effectively improves cognitive function and pathological features in AD animals, which is associated with enhanced phagocytosis of Aβ by microglia. Furthermore, long-term DDS treatment does not exacerbate depression and anxiety in AD mice, suggesting that DDS has potential value in preventing and treating AD.

[0063] Example 2

[0064] (1) Experimental subjects

[0065] Primary microglia and BV2 microglial cell line

[0066] (2) Experimental instruments

[0067] Live cell workstation, detachable 96-well plate, clean bench, CO2 cell culture incubator, low-speed centrifuge, cell counting chamber, constant temperature water bath, autoclave and DMEM culture medium

[0068] (3) Experimental process

[0069] First, the effect of DDS (final concentrations of 0, 12.5, 25, 50, 100, and 200 μg / ml) on microglial cell viability was evaluated. The specific method was as follows: BV2 cells were first seeded on a 96-well plate at a cell number of 5×10 3 Cells were treated with various concentrations of DDS (0, 12.5, 25, 50, 100, and 200 μg / ml) for 24 and 48 hours, respectively. 10 μl of CCK-8 reagent was added to each well and the cells were returned to the incubator for a further 4 hours of incubation. The absorbance of each well was measured at 450 nm using a microplate reader. The percentage of viable cells was calculated as follows: Cell viability (%) = [(A450 treated - A450 blank) / (A450 control - A450 blank)] × 100%, with the control group used for normalization.

[0070] Next, the mechanism of synaptosome uptake by primary microglia and its role in neuroinflammation were analyzed. Synaptosomes labeled with pHrodo Red were co-incubated with primary microglia treated with DDS (100 μg / ml) to observe and record the fluorescence signal. The preparation method of synaptosomes labeled with pHrodo Red is as follows: After perfusing the mouse with 1×PBS, the whole brain was homogenized in Syn-PER reagent, and the synaptosomes were collected by centrifugation. After purification and quantification of the synaptosomes, pHrodo Red dye was used to label them. The cells were fixed with 4% paraformaldehyde, and the distribution and fluorescence intensity of pHrodo-labeled synaptosomes at different incubation time points (30 minutes and 1 hour) were observed using a fluorescence microscope, and the fluorescence intensity was quantified using image analysis software (ImageJ).

[0071] Finally, the uptake, distribution and aggregation of Aβ42 in BV2 cells treated with DDS (100 μg / ml) were observed and quantitatively analyzed in real time. TMFluor 555-labeled Aβ42 was added to the cell culture and incubated for 1 hour. The cells were fixed with 4% paraformaldehyde, and the intracellular fluorescence signal was observed using a fluorescence microscope to assess Aβ42 uptake and distribution. Fluorescence intensity was quantified using image analysis software (ImageJ) to estimate Aβ42 uptake.

[0072] (4) Experimental results

[0073] Figure 4 This is the effect of DDS on the viability and phagocytic function of in vitro cultured microglia in Example 2 of the present invention.

[0074] Figure 4 A is the percentage of BV2 microglial cell viability at different times (24 hours and 48 hours) after treatment with different concentrations of DDS in the BV2 microglial cell viability experiment; Figure 4 B is the quantitative fluorescence intensity of phagocytosis of synaptosomes at different times (0.5 h and 1 h) after treatment with DDS (100 μg / ml) in the experiment to evaluate the phagocytic function of primary microglia; Figure 4 C is the quantitative fluorescence intensity of Aβ42 uptake in the BV2 microglial cell phagocytosis experiment after DDS (100 μg / ml) treatment (24 hours).

[0075] After treating cells with different concentrations of DDS (0, 12.5, 25, 50, 100, and 200 μg / ml) for 24 and 48 hours, CCK-8 assays combined with Aβ1-42 uptake experiments revealed that DDS (100 μg / ml) enhanced Aβ42 uptake while maintaining BV2 cell viability. Co-incubation of pHrodo-labeled synaptosomes with primary microglia treated with DDS (100 μg / ml) for 1 hour revealed enhanced phagocytosis of synaptosomes by microglia.

[0076] In summary, in vitro cell culture experiments demonstrated that DDS treatment effectively improved the phagocytic function of microglia toward Aβ and synaptosomes, suggesting that DDS has the potential value to alleviate the abnormal function of microglia in AD.

[0077] Through the above examples, this patent provides a new method to regulate microglial dysfunction to delay the occurrence and development of AD, which has important clinical application prospects.

[0078] The present invention reveals for the first time that DDS can restore the normal function of microglia in the brains of AD transgenic animals, thereby enhancing their phagocytic ability to effectively remove Aβ plaques while avoiding inflammatory damage caused by over-activation.

[0079] The present invention overcomes potential obstacles to the application of DDS in the nervous system, such as its ability to penetrate the blood-brain barrier, its distribution in the brain, and its metabolic characteristics, ensuring that it can effectively act on microglia in the brains of AD transgenic animals.

[0080] The present invention optimizes the DDS administration regimen and dosage to minimize side effects and improve the safety and tolerability of treatment.

[0081] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of dapsone in the preparation of a medicament for treating Alzheimer's disease associated with microglial dysfunction, characterized in that: The structure of the dapsone is shown in Formula I:

2. The use according to claim 1, characterized in that: Dapsone delays Alzheimer's disease by regulating microglial dysfunction.

3. The use according to claim 2, characterized in that: The abnormal microglial function includes abnormal microglial activation, phagocytic dysfunction and metabolic disorder.

4. The use according to claim 2, wherein: The delay includes, (i) Improve cognitive dysfunction; (ii) Improvement of pathological features.

5. The use according to claim 4, characterized in that: The cognitive dysfunction includes neurosensory motor disorder, spatial learning dysfunction, and spatial memory ability disorder.

6. The use according to claim 4, characterized in that: The improvement of pathological characteristics includes reducing β-amyloid protein deposition, improving microglial dysfunction and correcting brain energy metabolism disorders.

7. A pharmaceutical composition for regulating microglial dysfunction to delay Alzheimer's disease, characterized by: The pharmaceutical composition at least comprises dapsone as an active ingredient and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein: The concentration of dapsone is 10 mg / kg / d.

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