Use of short-chain fatty acids in the preparation of a drug for preventing or / and treating ischemic stroke

Abstract

This invention discloses the application of short-chain fatty acids in the preparation of drugs for the prevention or treatment of ischemic stroke, belonging to the field of pharmaceutical technology. Specifically, the short-chain fatty acid is acetate, a novel drug for treating stroke that can be administered via intravenous injection and oral administration. Acetate improves stroke-induced brain atrophy by altering histone acetylation levels, enhances motor function in mice, promotes neuronal survival, promotes astrocyte activation, enhances glial scar formation, and improves cognitive function. This invention provides a safe and effective new treatment strategy for stroke.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of short-chain fatty acids (acetates) in the preparation of drugs for the prevention or / or treatment of ischemic stroke. Background Technology

[0002] Globally, stroke is the second leading cause of death, accounting for 11.6% of all deaths in 2019. Ischemic stroke is the most common type, accounting for 62.4% of all stroke cases worldwide in 2019. This type of stroke leads to neurological death and long-term disability in adults, placing a significant burden on health and the economy. In 2019, 77.19 million people worldwide suffered ischemic strokes, resulting in 63.48 million disabilities and 3.29 million deaths. China has the highest number of stroke patients in the world. Although the incidence rate of stroke in my country decreased from 222 per 100,000 in 2005 to 201 per 100,000 in 2019, the prevalence rate is still on the rise. Data from the 2019 “Stroke High-Risk Population Screening and Intervention Project” shows that the population-standardized prevalence of stroke among people aged 40 and above in my country increased from 1.89% in 2012 to 2.58% in 2019. By 2019, the number of people aged 40 and above in my country who currently have or have had stroke was approximately 17.04 million.

[0003] The goal of acute ischemic stroke treatment is to restore cerebral perfusion as quickly as possible through intravenous thrombolysis, endovascular thrombectomy, or a combination of both. Currently, clinical treatments still have significant limitations, such as the narrow time window (4.5-6 hours) for intravenous thrombolysis (rt-PA) and endovascular thrombectomy (EVT), and reperfusion injury (free radical bursts, inflammatory responses exacerbating secondary brain injury). Furthermore, despite timely restoration of cerebral perfusion, the duration of tissue viability under pre-perfusion ischemic conditions is a decisive factor in achieving good clinical outcomes. Factors that may affect tissue viability include advanced age and diseases (such as diabetes), as well as factors affecting collateral blood flow and cascade injuries caused by ischemia.

[0004] Following the initial ischemic injury, a cascade of reactions immediately erupts in the central nervous system (CNS), including excitotoxicity, calcium overload, free radical bursts, and inflammatory responses. Post-stroke metabolic changes exhibit dynamic evolution. The hyperacute phase (<6 hours) is dominated by energy crisis, the acute phase (6-72 hours) shows significant inflammatory metabolic reprogramming, and the subacute phase shifts towards restorative metabolic adaptation. In clinical monitoring, brain microdialysis can detect the lactate / pyruvate ratio (LPR) in real time; an LPR > 40 indicates an energy metabolic crisis.

[0005] Therefore, exploring novel neuroprotective strategies, especially therapies based on metabolic regulation, anti-inflammation, and epigenetic modification, has become a current research hotspot. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to prevent and / or treat stroke.

[0007] In a first aspect, the present invention provides the use of acetate in the preparation of drugs for the prevention or / or treatment of stroke, or in the prevention or / or treatment of stroke.

[0008] In the above applications, the prevention and / or treatment of stroke is manifested in at least one of the following aspects: (a1) Improves neurological function in stroke patients or animal models; (a2) Improve motor function in stroke patients or animal models; (a3) Improves memory ability in stroke patients or animal models; (a4) Inhibits brain atrophy in stroke patients or animal models; (a5) Promotes the activation of astrocytes in the brain tissue of stroke patients or animal models; (a6) Enhances the formation of glial scars after stroke in stroke patients or animal models; (a7) Enhances the activity of astrocytes in stroke patients or animal models.

[0009] In some embodiments of the present invention, the stroke includes ischemic stroke and hemorrhagic stroke.

[0010] In some embodiments of the present invention, the stroke is ischemic stroke.

[0011] In a specific embodiment of the present invention, the stroke is an acute ischemic stroke.

[0012] In a specific embodiment of the present invention, the drug is used for the treatment of acute ischemic stroke in the acute phase.

[0013] Secondly, the present invention provides the application of acetate in any of the following aspects: (b1) To prepare products for improving neurological function in stroke patients or animal models; (b2) To prepare a treatment for improving neurological function recovery in stroke patients or animal models; (b3) To prepare products for improving memory in stroke patients or animal models; (b4) Prepare products for inhibiting brain atrophy in stroke patients or animal models; (b5) Prepare products that enhance the activity of astrocytes in the brain tissue of stroke patients or animal models; (b6) To prepare products that enhance the formation of glial scars after stroke in stroke patients or animal models; (b7) Prepare products that enhance the activity of astrocytes in the brain tissue of stroke patients or animal models; (b8) Products used to improve neurological function in stroke patients or animal models; (b9) Used to improve the recovery of neurological function in stroke patients or animal models; (b10) Used to improve memory in stroke patients or animal models; (b11) Used to inhibit brain atrophy in stroke patients or animal models; (b12) Increase the activity of astrocytes in the brain tissue of stroke patients or animal models; (b13) Enhances the formation of glial scars after stroke in stroke patients or animal models; (b14) Increase the activity of astrocytes in the brain tissue of stroke patients or animal models.

[0014] In the above applications, the improvement in neurological function recovery in stroke patients or animal models is reflected in at least one of the following aspects: 1) improved motor coordination, 2) improved balance, 3) improved limb coordination, and 4) improved exercise endurance.

[0015] In the above applications, the inhibition of brain atrophy in stroke patients or animal models is manifested in the inhibition of neuronal apoptosis in brain tissue; 2) inhibition of neuronal apoptosis in the M2 region of the motor cortex.

[0016] In a second aspect, the present invention provides a method for preventing and / or treating at least one symptom or pathological manifestation of stroke in a subject.

[0017] The method provided by the present invention for preventing and / or treating any symptom or pathological manifestation of stroke in a subject includes the following steps: administering to the subject in need a therapeutic agent capable of modulating changes in acetylation modification levels caused by stroke onset (hereinafter, the therapeutic agent is an acetate, a type of short-chain fatty acid).

[0018] In some embodiments of the present invention, the stroke includes ischemic stroke and hemorrhagic stroke.

[0019] In some embodiments of the present invention, the stroke is ischemic stroke.

[0020] In a specific embodiment of the present invention, the stroke is an acute ischemic stroke.

[0021] In a specific embodiment of the present invention, the method is used for the treatment of acute ischemic stroke in the acute phase.

[0022] In some embodiments of the present invention, at least one symptom or pathological feature of the stroke includes: ① extremely low density resembling cerebrospinal fluid on CT, and low signal on T1 and high signal on T2 on MRI. Widening of peripheral sulci, enlargement of the ipsilateral ventricle, manifesting as localized brain tissue atrophy; ② motor behavioral disorders; ③ decreased histone acetylation levels; ④ energy depletion leading to ischemic necrosis of brain cells, accompanied by a series of cascade reactions such as cytotoxic edema and inflammation.

[0023] In some embodiments of the present invention, the subject is a mammal, such as a human.

[0024] In this article, "gene expression" refers to the process by which the information contained in a gene is transformed into a gene product. Gene products can be direct transcription products of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozymes, structural RNA, shRNA, RNAi, miRNA, or any other type of RNA) or proteins produced by the translation of mRNA. Gene products also include modified RNA and modified proteins. RNA modification processes include capping, polyadenylation, methylation, and editing; protein modification processes include methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, tetradecylation, and glycosylation.

[0025] In some embodiments of the present invention, the therapeutic agent is an acetate.

[0026] The acetate is a pharmaceutically acceptable acetate or its ester, prodrug, stereoisomer, hydrate, solvate, crystal form, their metabolite form, or any combination or mixture thereof.

[0027] In a specific embodiment of the present invention, the acetate is sodium acetate.

[0028] In some embodiments of the present invention, the dosage form of the drug or therapeutic agent may be tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions and lyophilized powders), inhalers, sprays, etc. It may be present in the pharmaceutical composition in unit dose form to facilitate administration by any suitable method known in the art, including but not limited to oral, intravenous, rectal, parenteral, or topical administration.

[0029] In some embodiments of the invention, the medicament further includes the use of other carrier materials and routes of administration known in the pharmaceutical field. Pharmaceutical compositions comprising the acetates described herein can be prepared by any known pharmaceutical process, such as effective formulations and methods of administration. The above considerations regarding effective formulations and methods of administration are well known in the art and described in standard textbooks. Formulations of the medicament are described, for example, in Hoover, John E., Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients (3rd edition), American Pharmaceutical Association, Washington, 1999.

[0030] In some embodiments of the present invention, the acetate therapeutic agent is an activator capable of complementing modified changes, which can regulate gene expression changes by increasing the acetylation level of histone modifications.

[0031] Compared with the prior art, the present invention has the following beneficial effects: Through in-depth research, this invention has discovered therapeutic targets for ischemic stroke and further developed a class of drugs targeting these targets, which can significantly improve symptoms such as neuronal morphological and functional changes, motor behavior disorders, ipsilateral ventricular enlargement, local brain tissue atrophy, and cell ischemia and necrosis caused by ischemic stroke. Attached Figure Description

[0032] Figure 1 A shows the TTC staining and infarct volume of cerebral infarction in mice after tMCAO in Example 1 (where the red area is the non-infarcted area and the white area is the infarcted area).

[0033] Figure 1 B represents the changes in cerebral blood perfusion in mice after tMCAO detected by laser speckle blood flow imaging in Example 1 (tMCAO (Surg): transient middle cerebral artery occlusion model; Sham: sham-operated group; LIS: infarct area; NIA: non-infarct area).

[0034] Figure 1 C represents the KEGG analysis of mouse brain transcriptomes at different pathological stages after tMCAO in Example 1.

[0035] Figure 1 D represents the volcano plot analysis of differential gene expression in the mouse brain transcriptome at different pathological stages after tMCAO in Example 1.

[0036] Figure 2 A represents the histone deacetylase expression at different time points in mice after tMCAO in Example 1, as analyzed by transcriptome analysis.

[0037] Figure 2 B is a graph showing the changes and statistics of panacetylated protein expression levels in the striatum, cortex, and hippocampus of the brain 24 hours after tMCAO in Example 1.

[0038] Figure 2 CD represents the drug half-life in acetate mice.

[0039] Figure 3 A represents the mNSS score of mice in each group after tMCAO surgery. The bar chart data represent the mean ± standard deviation; **p<0.01, ***p<0.001.

[0040] Figure 3 Figure B shows the results of measuring the dwell time of mice in each group on the rotarod after tMCAO surgery in the rotarod experiment.

[0041] Figure 3 CD represents the results of the number of sliding steps and the time taken by mice in each group on the balance beam test on day 28 after tMCAO surgery. The bar chart data represent the mean ± standard deviation; **p<0.01, ***p<0.001.

[0042] Figure 3 EG shows the results of total walking distance and central region activity ratio measurements in mice in each group after tMCAO surgery in the open field experiment. The bar chart data represent the mean ± standard deviation; **p<0.01, ***p<0.001.

[0043] Figure 3 HI represents the results of running distance and time to exhaustion in mice in each group after tMCAO surgery in the treadmill experiment. The bar chart data represent the mean ± standard deviation; ** p < 0.01, *** p < 0.001.

[0044] Figure 3 JM is a graph showing the results of the walking distance and latency to first reach the target in mice in the Barnes maze experiment after surgery. The bar chart data represent the mean ± standard deviation; **p<0.01, ***p<0.001.

[0045] Figure 3NP represents the results of the number of times mice passed through the target hole and the time spent in the target area after tMCAO surgery in each group of mice in the Barnes maze experiment. The bar chart data represent the mean ± standard deviation; **p<0.01, ***p<0.001.

[0046] Figure 3 QR represents the spontaneous alternation rate and motor ability of mice in each group after tMCAO surgery in the Y-maze experiment. The bar chart data represent the mean ± standard deviation; **p<0.01, ***p<0.001.

[0047] Figure 4 The brain atrophy in different tMCAO-treated groups and the control group was observed after 28 days of tissue multispectral imaging in Example 3. The bar chart data represent mean ± standard deviation; **p<0.01, ***p<0.001. LIS: infarct area, NIA: non-infarct area.

[0048] Figure 5 The images show the survival of neurons (Neun), and the activation of astrocytes (GFAP) and microglia (Iba1) in different tMCAO-treated groups and the control group after day 28, as determined by immunofluorescence staining in Example 3. The bar chart data represent mean ± standard deviation; **p<0.01, ***p<0.001.

[0049] Figure 6 This image shows the survival of neurons (Neun) and activation of astrocytes (GFAP) in the M2 region of the motor cortex in different tMCAO-treated groups and the control group after 28 days of immunofluorescence staining in Example 3. The bar chart data represent mean ± standard deviation; **p<0.01, ***p<0.001.

[0050] Figure 7 A represents the effect of different concentrations of acetate on the viability of astrocytes as determined by CCK8 in Example 4. The bar chart data represent the mean ± standard deviation; **p<0.01, ***p<0.001.

[0051] Figure 7 B represents the effect of different concentrations of acetate on the viability of astrocytes as determined by OGD in Example 4. The bar chart data represent the mean ± standard deviation; ** p < 0.01, *** p < 0.001. Detailed Implementation

[0052] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0054] The experimental materials and methods involved in the following examples are as follows: 1. Laboratory animals Focal cerebral ischemia was induced using transient intracavitary middle cerebral artery occlusion (tMCAO) with a single filament. Animals were first anesthetized with 2.5 μl avertin (20 μl / g, ip). A midline ventral neck incision was made, the temporalis muscle was retracted, and the left carotid artery was exposed and isolated. After ligation of the common carotid and external carotid arteries, a silicone-coated suture plug (RWD, CHN) was inserted into the left internal carotid artery through the common carotid incision until it reached the origin of the left MCA (left middle cerebral artery), encountering mild resistance (9–10 mm). One hour after MCA occlusion, the single filament was removed to restore blood flow, and the skin was sutured. Laser Doppler flow imaging (PeriMed) was used to monitor regional cerebral blood flow (CBF) and ensure successful ischemia-reperfusion. MCA occlusion was determined by a reduction of more than 70% in CBF compared to baseline. Throughout the procedure, each mouse's body temperature was maintained at 37°C ± 0.5°C using an electric heating pad, and the mice were returned to their cages after regaining consciousness.

[0055] Sham surgery group: Animals were anesthetized with only 2.5 Avertin (20 μl / g, ip). A midline abdominal-neck skin incision was made, the temporalis muscle was retracted, the left carotid artery was exposed and isolated, and then sutured. Ligation of the common carotid and external carotid arteries and insertion of silicone-coated suture plugs (RWD, CHN) into the left internal carotid artery through the common carotid incision were not performed; the remaining steps were the same as in the surgical group.

[0056] In animal experiments, n≥8 mice were used in each group for model establishment and treatment.

[0057] 2. Behavioral Analysis Mice were housed in a temperature-controlled (23°C) room with a regular 12-hour light / dark cycle. Behavioral analyses were performed using male mice aged 2 to 3 months. Mice were transferred to the testing room 24 hours prior to behavioral testing for acclimatization.

[0058] The rotundus test is a behavioral assessment method commonly used to evaluate the motor coordination, balance, and endurance of rodents. During the test, the animal is placed on a rotundus that is rotating at a constant or accelerating speed. The core indicators of the test are the time it takes for the animal to maintain balance on the rotundus without falling (latency) and the rotational speed at which it falls. To complete this task, the animal needs to actively adjust its posture to counteract the movement of the rotundus, thus continuously engaging its motor coordination and balance functions.

[0059] Balance beam experiment: To quantitatively assess the motor coordination, balance, and limb motor control of rodents (such as rats and mice). The experimental setup typically consists of a narrow wooden beam of a certain length, at a certain height off the ground, with a square or circular cross-section. During the test, the animal must independently walk from one end of the balance beam to a refuge cage at the other end. Researchers record quantitative indicators such as the time taken to cross the balance beam, the number of slips, and the number of hind limb slips, and then comprehensively evaluate the animal based on a pre-set gait scoring system (e.g., grading based on limb usage on the beam and trunk stability).

[0060] Treadmill Experiment: This experiment is a commonly used behavioral paradigm for assessing the motor function, endurance, and motor learning ability of rodents. Animals are forced to run continuously on an electric track, and their speed can be kept constant or gradually increased through a programmed sequence.

[0061] Researchers quantify an animal's endurance level by recording the time it takes to reach exhaustion, the total running distance, and its ability to cope with increasing speed. Furthermore, this experiment allows for detailed observation of the animal's gait characteristics (such as stride length and paw prints) and can be combined with electrical or airflow stimulation to force the animal to run harder to assess its maximal athletic ability. This model is widely used in exercise physiology, fatigue research, and for evaluating the recovery of motor function in animal models of neuromuscular diseases, stroke, or spinal cord injury.

[0062] 3. Memory ability assessment Barnes Maze Experiment: The Barnes Maze Experiment utilizes the natural aversion of rodents to light and darkness and their dislike of open, high places. Several holes are evenly distributed on a circular elevated platform with a diameter of 92-100 cm (mice) or 122 cm (rats). Only one hole is connected to a detachable dark box as the escape target. Spatial cues are placed around the box at a distance, and strong light and white noise are applied from above. During training, an adaptation period of 1 day is given to guide the animal into the dark box and stay for 1-2 minutes. Then, a 5-7 day acquisition training is conducted. The animal is released from the center of the platform multiple times a day, and the latency to find the target hole and the number of errors in exploring blind holes are recorded. After the training, the retention of spatial reference memory is evaluated by a detection test after the dark box is removed, with a focus on the proportion of time spent exploring the target quadrant or the target hole.

[0063] Y-maze experiment: The Y-maze experiment has two commonly used paradigms. The spontaneous alternation experiment uses three identical arms at 120-degree angles to each other. The animal is placed at the end of any arm and allowed to explore freely for 5-8 minutes. The order in which the animal enters each arm is recorded. Entering three different arms consecutively is counted as a correct alternation. Spatial working memory is tested by the alternation rate. The novel arm recognition experiment first allows the animal to explore two open arms for 10 minutes during the sampling phase. After an interval of 1-2 hours or 24 hours, a third novel arm is opened and tested for 5 minutes. The recognition index is calculated by comparing the exploration time of the novel arm with that of the familiar arm to evaluate recognition memory.

[0064] 4. Drug treatment in animals Sodium acetate was purchased from Sigma-Aldrich (catalog number A13184). Mice were administered acetate (20 mg / kg, 200 mg / kg, or 2 g / kg) (dissolved in PBS) via intraperitoneal injection, or phosphate-buffered saline (PBS) (200 μl / mice) as a control, with the first dose given 1 hour after modeling, followed by doses every 24 hours for seven consecutive days. Behavioral tests were performed one month after treatment.

[0065] 5. Immunohistochemistry (IHC) Brain tissue was fixed with 4% paraformaldehyde and serially sectioned into 40 μm thick coronal sections. Sections were post-fixed in 4% PFA for 15 min, treated with blocking buffer (2% BSA, 0.3% Triton X-100) for 2 h, and incubated overnight with primary antibodies in blocking buffer at 4°C. The primary antibodies used in our study were: rabbit anti-Iba1 (1:1000, catalog number HPA005456, Sigma), porcine anti-Neun (1:1000, catalog number ab5905, Millipore), and rabbit anti-GFAP (1:1000, catalog number ab4729, Abcam). Sections or coverslips were washed in PBS and incubated with secondary Alexa Fluor conjugate antibody (Invitrogen) at room temperature for 1.5 h, washed three times in PBS, and fixed with adhesion-resistant anti-fading medium. Images were acquired using confocal laser scanning microscopy, and the results were analyzed using ImageJ.

[0066] 6. Western Blot (WB) Brain tissue was homogenized in RIPA buffer (P0013B, Beyotime) containing PMSF. Protein concentration for each sample was determined using a BCA kit (Thermo Fisher), and equal volumes of total protein were loaded onto SDS-PAGE gels. After electrophoresis, proteins were transferred to PVDF membranes. The membranes were blocked in 5% milk in TBST (TBS + 0.05% Tween-20) at room temperature for 1 hour and incubated overnight at 4°C with primary antibody. The following antibodies were used: histone H3 panacetylation antibody (1:5000, BE3312, EASYBIO), and mouse anti-β-actin (1:10000, A5441, Sigma). Secondary antibody conjugated with HRP was incubated at room temperature for 1 hour. Protein bands were visualized using an enhanced chemiluminescence reagent (ECL, Pierce) and quantified using ImageJ.

[0067] 7. Cell isolation, culture and processing Primary astrocytes were prepared from 1-day-old newborn mice. In short, the cerebral cortex of newborn mice was dissected and placed in a calcium-free environment. 2 + and Mg 2 + The cortex was placed in ice-cold PBS. After thorough removal of the meninges from the cortical surface, the cortex was ground and digested with 0.125% trypsin and DNase I at 37°C for 20 minutes. After digestion, the enzyme digestion was stopped with complete culture medium, and the cell suspension was filtered through a 70 μm cell filter. For primary astrocyte culture, the cells were centrifuged and resuspended in DMEM / F12 supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin / streptomycin (Gibco), and 5 ng / ml M-CSF (Sigma), and seeded into flasks pre-coated with poly-D-lysine (Solarbio, China). After the mixed glial cell culture reached complete confluence on day 10, the astrocytes were separated from the mixed glial cell culture by rotating the flask at 200 rpm at 37°C for 4 hours in an orbital shaker. The culture supernatant enriched with astrocytes was collected and cultured for 24 hours to provide adhesion for subsequent experiments. The purity of the isolated astrocytes was quantified using FACS.

[0068] 8. CCK8 Experiment The CCK-8 assay is a colorimetric method widely used to detect cell proliferation and cytotoxicity. Its principle is that WST-8 in the reagent can be reduced to highly water-soluble orange-yellow formazan by dehydrogenases in the mitochondria of living cells. The amount of formazan generated is directly proportional to the number of living cells and inversely proportional to cytotoxicity. In the experiment, cells in the logarithmic growth phase are digested, resuspended, counted, and seeded at an optimized density into 96-well plates (usually 100 μL of cell suspension per well, with the edge wells sealed with sterile PBS). The plates are incubated at 37°C in a 5% CO2 incubator until the cells adhere and reach the desired state. Then, drug treatment or continued culture is performed according to the experimental design. At the preset detection time point, the old culture medium is discarded, or 10 μL of CCK-8 reagent is added directly to each well (avoiding air bubbles). The mixture is gently mixed and returned to the incubator for 1-4 hours in the dark. Finally, the absorbance at 450 nm is measured using a microplate reader. If the culture medium contains phenol red or higher precision is required, 630 nm can be read simultaneously as a reference wavelength for calibration. During data processing, the OD values ​​of the blank wells (containing only cell-free culture medium and CCK-8) are subtracted from the OD values ​​of each well. The cell viability percentage of each treatment group is then calculated with the OD value of the control group (untreated cells) set at 100%, or a proliferation curve of OD values ​​at different time points is plotted. Additionally, the absolute number of viable cells can be calculated based on the standard curve. Aseptic technique must be maintained throughout the experiment. Each experiment should have 3-5 replicates. The seeding density needs to be determined through preliminary experiments to ensure it remains within the linear range during detection. When inoculating, the solution should be added at a unidirectional tilt to ensure uniformity. After adding CCK-8, avoid light exposure and closely observe color changes to prevent oversaturation. Furthermore, interference from the light absorption of the phenol red-containing culture medium and the drug itself needs to be eliminated by setting appropriate background subtraction.

[0069] 9. Oxygen Deprivation Test (OGD) Ischemia was simulated in vitro via oral glucose-induced decomposition (OGD), which was induced by replacing the growth medium with pre-warmed DMEM containing no serum and glucose (Gibco, Invitrogen, 11966-025). Cells were then transferred to a hypoxic incubator with 95% N2 and 5% CO2 and maintained at 37 °C. To simulate the in vivo ischemia / reperfusion process, intact culture medium was recovered, and cells were returned to a normoxic incubator at appropriate times according to cell type.

[0070] 10. Statistical Analysis We applied the Shapiro-Wilk and Kolmogorov-Smirnov tests to assess the normality of the datasets. All datasets passed the normality test in this study. Statistical analyses of the data were generated using GraphPadPrism 8 software with Student's t-test (t-test), one-way or two-way analyses of covariance, as specified in the charts for each graph. All data are expressed as mean ± standard deviation, and statistical significance was defined as *p < 0.05; **p < 0.01; ***p < 0.001.

[0071] Example 1: Bioinformatics Analysis To demonstrate the feasibility of acetate therapy for stroke, we first constructed a tMCAO model to simulate pathological ischemic stroke. Laser speckle fluorescence imaging results showed that, compared to the sham surgery group, the tMCAO group had significantly reduced cerebral blood flow, proving that the suture embolism blocked the middle cerebral artery, forming an ischemic area. Figure 1 A). Homogeneity verification was then performed, and TTC staining results showed that the infarct area ratio was approximately 60% (calculated as: infarct area percentage = (infarct area / total area of ​​the ipsilateral hemisphere) × 100%), demonstrating good homogeneity. Figure 1 B), and immunofluorescence staining results showed that, compared with the undamaged area, microglia were significantly activated in the ischemic injury area, and a large number of neurons died (B). Figure 3 These results all demonstrate the success of tMCAO modeling.

[0072] Subsequently, to verify the feasibility of the experiment, we analyzed RNA-seq transcriptome data from the brain tissue of tMCAO mice at 1d, 3d, 7d, 14d, and 28d, and performed KEGG enrichment analysis. Figure 1 C) found that lipid metabolism pathways were significantly enriched after tMCAO, and the levels of acetate metabolism-related enzymes acetyl-CoA synthase 1 (ACSS1), acetyl-CoA synthase 2 (ACSS2) and acetyltransferase (Ep300) were significantly increased. ACSS2 is involved in epigenetic modifications (such as histone acetylation) in the cell nucleus.

[0073] Furthermore, we analyzed the expression of histone deacetylase (HDAC) and found that it showed an overall upward trend. Figure 2 A), followed by Western blotting to verify the H3 panacetylation levels in the striatum, hippocampus, and cortex of tMCAO mice 24 hours later. The results showed that the panacetylation levels in the striatum and cortex were significantly reduced, but there was no significant difference in the panacetylation levels in the hippocampus region. Figure 2B). To clarify the plasma pharmacokinetic characteristics of exogenous acetate in mice, we performed time-series analysis on plasma acetate concentrations following intraperitoneal (IP) administration of 200 mg / kg acetate. Figure 2 C): Plasma acetate concentration rapidly increased from 0 to 20 min after administration, reaching a peak concentration (approximately 20 mM) at 20 min. After 20 min, acetate was gradually cleared, and the plasma concentration returned to a lower level by 120 min. Error bars at each time point indicated small fluctuations in the intra-group test data, and the overall absorption, peak, and elimination trends were clear. Based on the elimination phase data after peak concentration, a standard pharmacokinetic (PK) curve of plasma acetate was fitted (Figure 2D), and the plasma elimination half-life of acetate was calculated to be approximately 24.1 min. The fitted curve matched the measured sample concentration points well, indicating that the body clears acetate rapidly after intraperitoneal injection, and the plasma acetate concentration cannot be maintained at a high level for a long period. This provides a pharmacokinetic basis for the timing of exogenous acetate regulation of histone acetylation and the design of dosing regimens.

[0074] Example 2: Effect of acetate on motor function in tMCAO mice We then used tMCAO mice as a model and divided them into PBS administration group, acetate administration group, 200 mg / kg acetate administration group, and 2 g / kg acetate administration group. We conducted rotarod test, balance beam test, and treadmill test to evaluate the improvement of tMCAO mouse motor ability after administration of different concentrations of acetate.

[0075] Experiments showed that on day 1 after tMCAO surgery, mice in all groups exhibited severe neurological deficits. Compared with the PBS group, acetate treatment significantly reduced mid-to-late-stage mNSS scores, with the 200 mg / kg group showing the most significant improvement, suggesting that acetate can accelerate neurological function recovery after stroke. Figure 3 A). Rotarod test showed no significant differences among groups in the early postoperative period, but from day 7 onwards, acetate treatment significantly prolonged the time mice spent on the rotarod, with the 200 mg / kg group showing the best performance on days 14 and 28. Figure 3 B).

[0076] In the balance beam test on day 28 post-surgery, acetate significantly reduced the number of slip steps and shortened the time to pass through, with the 200 mg / kg group showing the strongest effect. Figure 3 CD). In the open field test, acetate treatment significantly increased the total walking distance and the proportion of activity in the central region of mice, with the 200 mg / kg group showing the most significant improvement. However, due to the reduced motor function of mice after tMCAO, it cannot be concluded that acetate can alleviate post-stroke anxiety-like symptoms. Figure 3EG (extracorporeal membrane oxygenation) to some extent demonstrates that 200 mg / kg acetate administration does indeed enhance the exercise capacity of tMCAO mice. Treadmill test results showed that acetate treatment significantly increased running distance and prolonged time to exhaustion, suggesting it can enhance post-stroke exercise endurance; however, there was no significant difference between the two doses. Figure 3 HI).

[0077] In addition, to assess the effect of acetate on memory ability, we conducted Barnes maze and Y-maze experiments. The results showed that during the Barnes maze training phase, the acetate-treated group exhibited a more efficient target-finding strategy. Compared with the PBS group, acetate significantly shortened the walking distance and the latency to first reach the target; the 200 mg / kg group showed the fastest learning speed, while there was no significant difference in the number of major errors. Figure 3 JM). In the Barnes maze memory test, acetate treatment significantly shortened the time to first reach the target hole and increased the number of times the target hole was traversed and the time spent in the target area, with the 200 mg / kg group showing the most significant improvement (JM). Figure 3 NP). The Y maze test showed that acetate treatment significantly increased the spontaneous alternation rate in mice, while there was no significant difference in motor function among the groups, suggesting that acetate can improve working memory after stroke, rather than simply enhancing motor function. Figure 3 QR).

[0078] In summary, acetate improved neurological function, motor coordination, and cognitive function in stroke-affected mice, and the 200 mg / kg dose showed stronger therapeutic effects in most behavioral indicators, supporting the possibility of acetate as a potential stroke intervention strategy.

[0079] Example 3: The effect of acetate on the brain of tMCAO mice To further investigate the effect of acetate administration on tMCAO, we performed immunofluorescence staining. The results showed that, overall, the 200 mg / kg acetate group significantly reduced brain atrophy in tMCAO mice, and the brain integrity was relatively stronger. Figure 4 Neun, Gfap, and Iba1 staining results indicated that acetate administration inhibited neuronal apoptosis and significantly activated astrocytes, enhancing glial scar formation after stroke. Figure 5 Furthermore, immunofluorescence staining results for the motor cortex indicated that acetate administration inhibited apoptosis of neurons in the M2 region of the motor cortex. Figure 6 It is speculated that this may inhibit the spread of toxic tissue fluid formed by liquefied necrosis behind the infarct core due to ischemia in stroke, thereby further protecting the surrounding brain tissue.

[0080] Example 4 To further demonstrate the feasibility of acetate supplementation for stroke patients, we first conducted in vitro validation. Under simulated normal physiological conditions, a drug toxicity test (CCK8) showed that, compared to the control group (without additional acetate), acetate dosage ≤1.25 mg / ml had no effect on astrocyte viability, while acetate concentration ≥2.5 mg / kg inhibited astrocyte activity. Figure 7 A). Subsequently, the ischemia-reperfusion process of ischemic stroke was simulated in vitro using an oxygen deprivation (OGD) assay. OGD was performed by replacing the growth medium with preheated DMEM without serum and glucose. Cells were then transferred to a hypoxic incubator with a gas mixture of 95% N2 and 5% CO2. To simulate the in vivo ischemia / reperfusion process, cells were transferred to a conventional incubator after 2 hours. Results showed that astrocyte viability was significantly reduced under in vitro ischemic conditions (OGD) compared to cells in normal physiological conditions (cor normoxia). When acetate was applied (at a concentration less than 0.625 mg / ml), cell viability recovered to varying degrees compared to the control group, with the greatest recovery observed at a concentration of 0.156 mg / ml. Figure 7 B).

[0081] The pathophysiological mechanisms of ischemic stroke are not fully understood, and current clinical treatment strategies are limited. Although many neuroprotective strategies have been developed, clinical trial results have been less than optimistic due to a lack of efficacy or adverse side effects. In this study, we first demonstrated through bioinformatics analysis and Western blotting experiments that ischemic stroke leads to a decrease in histone acetylation levels. Furthermore, we used acetate as a drug targeting these levels and found that it significantly improved symptoms such as neuronal morphological and functional changes, motor behavioral disorders, ipsilateral ventricular enlargement, local brain tissue atrophy, and cell ischemia and necrosis caused by ischemic stroke. These results suggest that acetate may be a potential therapeutic drug for ischemic stroke.

[0082] Glial scars are barrier structures formed during the repair process of neurological damage following ischemic stroke. They are primarily composed of activated astrocytes, along with a small number of microglia, oligodendrocyte precursor cells (OPCs), and extracellular matrix (ECM) components. They have both protective and negative effects, inhibiting nerve regeneration. After ischemic stroke, acetate, as a key short-chain fatty acid and energy substrate in the brain, significantly affects the activation state of astrocytes and the formation of glial scars. Once in the brain, acetate is mainly taken up by astrocytes and converted to acetyl-CoA via acetyl-CoA synthetase 2 (ACSS2). Acetyl-CoA can enter the tricarboxylic acid cycle (TCA cycle) for energy metabolism and is also an acetyl donor for histone acetylation. Therefore, acetate not only regulates cellular metabolism but also influences the gene expression profile of reactive astrocytes through epigenetic mechanisms. Our study found that acetate injection after ischemic stroke promotes astrocyte activation, thereby enhancing glial scar formation and inhibiting the spread of liquefied necrotic fluid formed by ischemia in necrotic tissue, thus protecting other neurons.

[0083] In summary, acetate promotes astrocyte activation and extracellular matrix deposition through both metabolic reprogramming and epigenetic modification pathways, thereby enhancing glial scar formation after ischemia. During the acute phase of stroke, moderate scar formation helps limit the spread of inflammation and protect residual neurons; however, during the recovery phase, excessive scarring may inhibit axonal regeneration and neural network reconstruction. Therefore, the role of acetate in stroke treatment is time-dependent, and future research needs to further clarify its bidirectional regulatory mechanisms at different stages to achieve a balance between protective repair and functional recovery.

[0084] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of acetate in the preparation of drugs for the prevention or / or treatment of stroke.

2. The application according to claim 1, characterized in that: The prevention or / or treatment of stroke is manifested in at least one of the following aspects: (a1) Improves neurological function in stroke patients or animal models; (a2) Improve motor function in stroke patients or animal models; (a3) Improves memory ability in stroke patients or animal models; (a4) Inhibits brain atrophy in stroke patients or animal models; (a5) Promotes the activation of astrocytes in the brain tissue of stroke patients or animal models; (a6) Enhances the formation of glial scars after stroke in stroke patients or animal models; (a7) Enhances the activity of astrocytes in stroke patients or animal models.

3. The application according to claim 1 or 2, characterized in that: The stroke includes ischemic stroke and / or hemorrhagic stroke.

4. The application according to claim 3, characterized in that: The stroke mentioned is ischemic stroke; Furthermore, the stroke is an acute ischemic stroke; Furthermore, the drug is used for the treatment of acute ischemic stroke in the acute phase.

5. The application according to any one of claims 1-4, characterized in that: The acetate is sodium acetate.

6. The application according to any one of claims 1-5, characterized in that: The dosage form of the drug includes at least one of the following: tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections, inhalers, and sprays.

7. Applications of acetates in any of the following aspects: (b1) To prepare products for improving neurological function in stroke patients or animal models; (b2) To prepare a treatment for improving neurological function recovery in stroke patients or animal models; (b3) To prepare products for improving memory in stroke patients or animal models; (b4) Prepare products for inhibiting brain atrophy in stroke patients or animal models; (b5) Prepare products that enhance the activity of astrocytes in the brain tissue of stroke patients or animal models; (b6) To prepare products that enhance the formation of glial scars after stroke in stroke patients or animal models; (b7) Prepare products that enhance the activity of astrocytes in the brain tissue of stroke patients or animal models.

8. The application according to claim 7, characterized in that: The improvement in neurological function recovery in stroke patients or animal models is reflected in at least one of the following aspects: 1) improved motor coordination, 2) improved balance, 3) improved limb coordination, and 4) improved exercise endurance.

9. The application according to claim 7, characterized in that: The inhibition of brain atrophy in stroke patients or animal models is manifested in the inhibition of neuronal apoptosis in brain tissue; 2) inhibition of neuronal apoptosis in the M2 region of the motor cortex.

10. The application according to any one of claims 7-9, characterized in that: The stroke includes ischemic stroke and / or hemorrhagic stroke; Furthermore, the stroke is an ischemic stroke; Furthermore, the stroke is an acute ischemic stroke.

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