Application of biphenyl lactone compound in treatment of Alzheimer's disease

Abstract

The invention discloses application of biphenyl lactone compounds in treatment of Alzheimer's disease, and belongs to the field of medicine. According to the present invention, in the oleic acid treatment combined APOE4 point mutation microglial cells, the benzene lactone compound Graphislactone A can improve the abnormal lipid droplet accumulation, inhibit the microglial cells from generating inflammatory factors, and further protect the neuronal function; meanwhile, in-vivo AD mouse model experiments prove that the benzolactone compound Graphislactone A can improve cognitive impairment of AD mice and reduce abnormal accumulation of lipid droplets in brain and expression of inflammatory factors, can be used as an active ingredient of medicines for treating AD, and has wide application.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically relates to the application of a biphenyl lactone compound in the treatment of Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease mainly characterized by impaired memory and cognitive function. The main pathological features of AD include extracellular amyloid protein deposition, abnormal phosphorylation of intracellular Tau protein, neuroinflammation, and mitochondrial dysfunction. At present, the pathogenesis of AD is complex, and the existing drugs for treating AD cannot reverse the disease condition and have certain side effects. Therefore, it is of great significance to deeply understand the pathological mechanism of AD and find key AD regulatory targets to develop new therapeutic drugs.

[0003] In recent years, the regulatory role of microglia in AD has become increasingly prominent. Single-cell transcriptomics studies have shown that during brain aging, a type of microglia rich in lipid droplets, namely lipid-droplet-accumulating microglia, can produce high levels of reactive oxygen species and secrete pro-inflammatory factors, thereby affecting the function of neurons. Research reports have shown that this type of microglia has unique transcriptomic characteristics and exists in various neurodegenerative diseases. In AD, carrying the APOE4 genotype is more likely to induce microglia to produce lipid droplets and reduce the ability of microglia to phagocytose Aβ, releasing inflammatory factors. Therefore, regulating the lipid droplet metabolism of microglia is expected to improve the pathological symptoms of AD and is of great significance for the development of new drugs for AD.

[0004] The biphenyl lactone compound Graphislactone A (GPA) is a natural product first isolated from the secondary metabolites of lichen (Graphis scripta var. pulverulenta) in 1997. After 7 months of fermentation culture of lichen, 36.7 mg of GPA was isolated from 51.74 g of fermentation extract. The isolation of GPA has also been reported in the fermentation product of the endophytic fungus Cephalosporium sp. IFB-E001 of the Trachelospermum jasminoides, a plant of the genus Trachelospermum in the family Apocynaceae. 13 mg of GPA was isolated from the 30-day culture fermentation product (750 g) of the fungus. GPA can also be obtained by artificial total synthesis through 11-step chemical reactions. There is no report in the prior art that GPA has an effect on Alzheimer's disease. Summary of the Invention

[0005] The object of the present invention is to provide an application of a biphenyl lactone compound in the treatment of Alzheimer's disease. Experimental studies on in vivo AD mouse models have shown that the biphenyl lactone compound Graphislactone A (GPA) can improve cognitive dysfunction in AD mice and reduce the abnormal accumulation of lipid droplets and the expression of inflammatory factors in the brain.

[0006] The present invention first provides an application of a biphenyl lactone compound Graphislactone A in the preparation of a drug for preventing and / or treating Alzheimer's disease.

[0007] Second, the present invention provides an application of a biphenyl lactone compound Graphislactone A in the preparation of any one of the following drugs:

[0008] (1) A drug for reducing the expression of inflammatory factors IFNG and IL17;

[0009] (2) A drug for promoting the expression of anti-inflammatory factor IL10;

[0010] (3) A drug for improving cognitive dysfunction;

[0011] (4) A drug for reducing the abnormal accumulation of lipid droplets in the brain;

[0012] (5) A drug for reducing the expression of inflammatory factors in the brain;

[0013] (6) A drug for protecting neurons.

[0014] In the above applications, the structural formula of the biphenyl lactone compound Graphislactone A is shown as formula (I):

[0015]

[0016] Finally, the present invention provides a drug for preventing and / or treating Alzheimer's disease, and the active ingredient of the drug is the biphenyl lactone compound Graphislactone A.

[0017] In the present invention, in oleic acid-treated microglia with APOE4 point mutation, it is found that GPA can improve the abnormal accumulation of lipid droplets, inhibit microglia from producing inflammatory factors, and thus protect neuron function. At the same time, experimental studies on in vivo AD mouse models have confirmed that GPA can improve cognitive dysfunction in AD mice and reduce the abnormal accumulation of lipid droplets and the expression of inflammatory factors in the brain, and can be used as the active ingredient of a drug for treating AD, with wide applications. Description of the Drawings

[0018] Figure 1 Results of GPA improving inflammatory lesions in HMC3 human microglia.

[0019] Figure 2 These are the results of GPA inhibiting microglial activation based on lipid droplet metabolism.

[0020] Figure 3 These are the results of GPA improving cognitive function impairment in AD model mice.

[0021] Figure 4 These are the effects of GPA on mouse body weight, blood biochemical indexes and hippocampal pathological tissue changes. Specific Embodiments

[0022] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention.

[0023] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0024] In the following embodiments, quantitative tests are set up with three repeated experiments and the results are averaged unless otherwise specified.

[0025] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0026] The percentages in the following embodiments refer to volume percentages unless otherwise specified.

[0027] The structural formula of Graphislactone A used in the following embodiments is shown in Formula (I) (its CAS number is 52179-44-9):

[0028]

[0029] Example 1. Detection of changes in the gene levels of inflammatory factors in microglia induced by Aβ by GPA

[0030] 1. RNA Extraction and RT-qPCR

[0031] Take HMC3 cells in the logarithmic growth phase and use 1×10 5Cells were evenly seeded at a density of [[ID=]] cells / well into a 6-well plate and cultured until the density reached 70%. Then the medium was changed. The experimental groups included a blank control group (Control), an amyloid-β model group (Aβ), a compound GPA treatment group (Aβ+GPA), a donepezil positive drug treatment group (Aβ+Donepezil), and a dexamethasone positive drug treatment group (Aβ+DEX). The blank control group was added with 100 μL of MEM complete medium; the amyloid-β model group was added with 100 μL of MEM complete medium containing 10 μM amyloid-β; the compound GPA treatment group was added with 100 μL of compound GPA at a concentration of 20 μM diluted with MEM complete medium containing 10 μM amyloid-β; the donepezil positive drug treatment group and the dexamethasone positive drug treatment group were respectively added with 100 μL of compound donepezil or dexamethasone at a concentration of 20 μM diluted with MEM complete medium containing 10 μM amyloid-β, and cultured statically at 37 °C and 5% (v / v) CO2 for 24 hours. Among them, the MEM complete medium was MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. After the culture, RNA extraction and RT-qPCR experiments were performed on each group to detect the expression levels of inflammatory factors.

[0032] HMC3 cells were digested, centrifuged and collected into a centrifuge tube, RNA was extracted and reverse transcribed into cDNA. The primers used for RT-qPCR are shown in Table 1.

[0033] Table 1 Primers for HMC3 microglial cell samples used in RT-qPCR

[0034]

[0035] 2. Detection of the effect of GPA administration on cell viability by CCK8

[0036] A blank control group and a drug administration group were set up in a 96-well plate. Only MEM complete medium was added to the blank control group; MEM complete medium containing compounds GPA at concentrations of 2.5, 5, 10, 20, and 40 μM was added to the drug administration group, and three replicates were made for each sample. Cells were not cultured in the outermost circle of the 96-well plate to avoid edge effects. On the first day, a cell suspension was prepared, counted, and plated. The cell suspension was inoculated into the 96-well plate, about 100 μL per well, and 10,000 cells were inoculated per well. The culture plate was placed in an incubator at 37 °C and 5% (v / v) CO2 until the density reached about 70% and then the drug was administered, that is, the medium was replaced. 100 μL of MEM complete medium containing compounds GPA at concentrations of 2.5, 5, 10, 20, and 40 μM was added to each well of the 96-well plate, and 3 replicate wells were set. Among them, the MEM complete medium was MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The culture plate was placed in the incubator at 37 °C and 5% (v / v) CO2 and incubated for 24 h. After the incubation ended, the old medium was removed, and 10 μL of CCK-8 solution was added to 100 μL of medium in each well. The culture plate was placed in an incubator at 37 °C and 5% (v / v) CO2 and incubated for 4 h. The absorbance of each well was detected by an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0037] Viability calculation: Cell viability (%) = [A (drug added) - A (blank)] / [A (control) - A (blank)] × 100

[0038] A (drug added): OD value of the well with cells, CCK-8 solution, and drug solution

[0039] A (control): OD value of the well with cells and CCK-8 solution but without drug solution

[0040] A (blank): OD value of the well without cells.

[0041] The results are shown in Figure 1 ; Figure 1 In which A is the structural diagram of compound GPA. Figure 1 In which B is the effect of GPA on the viability of HMC3 cells. The CCK8 detection results showed that the viability of HMC3 cells gradually decreased with the increase of GPA concentration. In subsequent experiments, the drug administration concentration was set at 10 μM. Figure 1 In which C is the effect of GPA on the viability of SY5Y cells. The CCK8 detection results showed that the viability of SY5H cells gradually decreased with the increase of GPA concentration. Figure 1 From E to G in which, after GPA administration, the expression of inflammatory factors IFNG and IL17 was significantly reduced, and at the same time, the expression of anti-inflammatory factor IL10 could be promoted. Thus, it can be seen that GPA can improve the inflammatory lesions in OA combined with APOE4 point mutant HMC3 cells, thereby playing a protective role on neurons.

[0042] Experimental Example 2: Effects of GPA on Lipid Droplet Accumulation and Polarization of APOE4 Microglia Induced by Oleic Acid

[0043] 1. Construction of a Stable Cell Line with APOE4 Point Mutation

[0044] One day before transfection, digest the cells and passage them into a 100-mm dish, controlling the cell density at 70 - 80% at the time of transfection. Place them in a 37°C, 5% (v / v) CO₂ incubator and continue culturing for 24 hours. Take out the cell culture dish one hour before transfection, remove the original cell culture medium, add 10 mL of Opti-MEM medium, and put the cells back into the incubator.

[0045] Prepare the transfection complex of the transfection reagent and the plasmid. The specific steps are as follows:

[0046] a. Dissolve 32 μg of the viral vector plasmid to be transfected (the mass ratio of the backbone plasmid to the shuttle plasmid is 1:1) in Opti-MEM medium with a total volume of 500 μL, gently mix, and let stand for 5 minutes to obtain a plasmid dilution;

[0047] b. Dissolve 20 μL of the transfection reagent lip3000 in Opti-MEM medium with a total volume of 500 μL, gently mix and let stand for 5 minutes to obtain a transfection reagent dilution;

[0048] c. Drop the transfection reagent dilution into the plasmid dilution, gently mix while adding, and then let stand at room temperature for 20 minutes to allow the DNA and the transfection reagent to fully combine to form a stable transfection complex.

[0049] Take out the cell culture plate, add the above-prepared transfection complex to the cell culture plate, make marks, and put it back into the incubator. After 6 - 8 hours, aspirate the medium, wash once with PBS, add 10 mL of fresh complete DMEM medium, and culture for 48 hours after transfection. Collect the supernatant after culture into a 50-mL centrifuge tube, make marks, and replace the cells with fresh complete medium (the first virus harvest). 72 hours after transfection, collect the medium again, combine it with the supernatant collected at 48 hours, and dispose of the remaining cell culture as waste (the second virus harvest).

[0050] 2. Lentivirus Infection of HMC3 Cells

[0051] Collect the harvested lentivirus supernatant, centrifuge it at 3500 rpm for 10 minutes, and discard the precipitate. Filter the centrifuged supernatant with a 0.22 μm filter membrane, aliquot the filtered supernatant into ultracentrifuge tubes, and centrifuge at 30,000 rpm at 4°C for 2 hours. Open the centrifuge tubes in a biosafety cabinet, carefully aspirate the supernatant, invert the centrifuge tubes on sterilized absorbent paper, and discard the residual liquid. Resuspend the precipitate in 100 - 200 μL of pre-cooled PBS (Seville, G4202-500ML) per tube and place it in a 4°C refrigerator overnight. Collect the lentivirus resuspension, centrifuge it at 6000 rpm for 5 minutes, filter it with a 0.22 μm filter membrane, transfer it to a new EP tube, aliquot it as needed, and store it in an -80°C refrigerator. Draw 1 sample to infect cells and perform titer detection.

[0052] When the cell confluence reaches 30 - 40%, select HMC3 cells in good condition for infection, set the MOI to 5, 10, and 20 for lentivirus infection, explore the optimal MOI value, and observe the cell status in real time. If the cell status is poor 12 hours after infection, replace the medium with a new one in a timely manner. If the cell status is good, replace the medium again 24 hours later, and continue to culture it in a 37°C, 5% (v / v) CO2 incubator. Observe the fluorescence expression under a fluorescence microscope at 48 hours and 72 hours after lentivirus infection of cells. At the same time, collect cells to extract proteins and RNA to detect the lentivirus infection efficiency. Obtain HMC3 cells overexpressing the APOE4 gene. Overexpression of the APOE4 gene will increase the deposition of amyloid proteins, exacerbate the pathological phosphorylation of tau proteins, and induce cell lipid metabolism disorders.

[0053] Lentivirus containing the APOE4 gene (Gene ID: 348, updated on 7-Apr-2025) and capable of expressing the APOE4 protein can also be prepared by conventional methods.

[0054] 3. Cellular immunofluorescence staining

[0055] (1) Cell culture

[0056] Take HMC3 cells and HMC3 cells overexpressing the APOE4 gene in the logarithmic growth phase, at a density of 1×10 4Seed evenly at a density of 1×10⁶ cells / well into a 24-well plate. Set up blank control group, oleic acid model group and compound GPA treatment group, with six replicates in each group; Blank control group: When the cell density reaches 70%, add only MEM complete medium (MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin); Oleic acid model group: When the cell density reaches 70%, change the medium to MEM serum-free medium containing oleic acid (500 μM) (MEM medium containing 1% penicillin-streptomycin); Compound GPA treatment group: When the cell density reaches 70%, change the medium to compound GPA at a concentration of 20 μM diluted with MEM serum-free medium containing oleic acid (500 μM) (MEM medium containing 1% penicillin-streptomycin); Dexamethasone positive drug treatment group: When the cell density reaches 70%, change the medium to dexamethasone at a concentration of 20 μM diluted with MEM serum-free medium containing oleic acid (500 μM) (MEM medium containing 1% penicillin-streptomycin); Incubate statically at 37 °C and 5% (v / v) CO₂ for 24 hours. After 24 hours, perform immunofluorescence staining experiments on each group.

[0057] (2) Lipid droplet BODIPY staining

[0058] Culture the cells in a 24-well plate with a cell slide at the bottom. After discarding the liquid, take 500 μL of PBS solution and rinse the slide at room temperature for two times. Take 500 μL of 4% PFA solution to fix the cells for 20 minutes. Take 1 mL of PBS solution and let it stand and rinse at room temperature for 5 minutes, repeating three times. Add 500 μL of blocking solution to each well and block at room temperature for 60 minutes. Take 200 μL of BODIPY solution diluted with blocking solution and incubate at room temperature for 30 min. And take 1 mL of PBS solution and rinse at room temperature for 10 minutes, repeating three times. Take out the slides in the 24-well plate, air-dry them in the dark at room temperature, drop the mounting medium (containing DAPI), invert them on the glass slide, air-dry them in the dark and store for subsequent experiments.

[0059] 4. Cell immunofluorescence staining

[0060] Take HMC3 cells in the logarithmic growth phase and HMC3 cells overexpressing the APOE4 gene, at a density of 1×10⁶ 4Cells were evenly seeded at a density of

[0061] cells / well into a 24-well plate. The experiment was set up with a blank control group, an oleic acid model group, a compound GPA treatment group, and a dexamethasone positive drug treatment group, with six replicates in each group. Blank control group: When the cell density reached 70%, only MEM complete medium (MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) was added. Oleic acid model group: When the cell density reached 70%, the medium was replaced with serum-free MEM medium containing oleic acid (500 μM) (MEM medium containing 1% penicillin-streptomycin). Compound GPA treatment group: When the cell density reached 70%, the medium was replaced with compound GPA at a concentration of 20 μM diluted with serum-free MEM medium containing oleic acid (500 μM) (MEM medium containing 1% penicillin-streptomycin). Dexamethasone positive drug treatment group: When the cell density reached 70%, the medium was replaced with compound dexamethasone at a concentration of 20 μM diluted with serum-free MEM medium containing oleic acid (500 μM) (MEM medium containing 1% penicillin-streptomycin). The cells were cultured statically at 37 °C and 5% (v / v) CO2 for 24 hours. After 24 hours, an immunofluorescence staining experiment was performed on each group (iNOS antibody (abcam, ab178945), Bodipy antibody (Invitrogen, D3922)). The experimental data were expressed as Mean ± SEM, and statistical difference analysis was performed using GraphPad Prism 8 software. A P value less than 0.05 was considered to be statistically significant.

[0061] The cells were cultured in a 24-well plate with a cell slide at the bottom. After discarding the liquid, 500 μL of PBS solution was used to rinse the slide at room temperature twice. Then, 500 μL of 4% PFA solution was used to fix the cells for 20 minutes. 1 mL of PBS solution was used to rinse the cells statically at room temperature for 5 minutes, and this was repeated three times. 500 μL of blocking solution was added to each well and incubated at room temperature for 60 minutes. 200 μL of primary antibody solution (iNOS antibody (abcam, ab178945), Bodipy antibody (Invitrogen, D3922)) diluted with the blocking solution was incubated overnight at 4 °C. 1 mL of PBS solution was used to rinse the cells statically at room temperature for 10 minutes, and this was repeated three times. 200 μL of secondary antibody solution (Alexa fluor 594, abcam, ab150080) diluted with the blocking solution was incubated on a shaker at room temperature for 1 - 2 hours, and the plate was wrapped with tin foil to avoid light. 1 mL of PBS solution was used to rinse the cells statically at room temperature for 10 minutes, and this was repeated three times, with the plate wrapped with tin foil to avoid light. The slides in the 24-well plate were taken out, air-dried at room temperature in the dark, a mounting medium (containing DAPI) was dropped, and the plate was inverted on a glass slide and air-dried in the dark for storage for subsequent experiments.

[0062] Figure 2 This is the result of GPA inhibiting microglial activation based on lipid droplet metabolism.Figure 2 In Figure 2 , A is that GPA can improve the morphological changes of HMC3 cells induced by oleic acid (OA) combined with APOE4 point mutation. In the model group, HMC3 cells showed obvious shrinkage and vacuolization, and after GPA administration, the cell morphology was improved. Under normal physiological conditions, microglia are in a resting state, and under pathological conditions, microglia will release anti-inflammatory factors or pro-inflammatory factors to protect or damage neurons. Figure 2 In Figure 2 , B is that GPA administration can effectively inhibit the up-regulation of iNOS expression induced by APOE4 point mutation and the activation of microglia induced by OA combined with APOE4 point mutation, but cannot effectively inhibit the activation of microglia and the up-regulation of iNOS expression induced by OA. Figure 2 In Figure 2 , C is that GPA effectively reduces the lipid droplet accumulation induced by OA, while the positive dexamethasone administration group has no significant effect on lipid droplet accumulation. It shows that GPA can inhibit microglial activation based on lipid droplet metabolism.

[0063] Experimental Example 3: Cognitive behavioral tests of GPA on AD mouse models

[0064] 1. Establish an Alzheimer's disease mouse model and administer the compound

[0065] Sixty 6-month-old male C57BL / 6J mice (weighing 18 - 20 g) were selected, with 12 mice in each group. The experiment was set up with a wild-type mouse control group, an AD model group, a low-dose administration group of 10 mg / kg, a high-dose administration group of 30 mg / kg, and a positive control group of 2 mg / kg donepezil. The start of modeling was the first day. Wild-type mouse control group: On the first day, 1 μL of normal saline was stereotactically microinjected into each of the two sides of the mouse lateral ventricle area. From the second day, the same dose of normal saline was intraperitoneally injected once a day for two consecutive weeks; AD model group: On the first day, 1 μL of β-amyloid protein (Sigma, A9810) with a concentration of 100 μM was stereotactically microinjected into each of the two sides of the mouse lateral ventricle area. From the second day, the same dose of normal saline was intraperitoneally injected once a day for two consecutive weeks; 10 mg / kg low-dose administration group: Starting from the first day, 1 μL of β-amyloid protein (Sigma, A9810) with a concentration of 100 μM was stereotactically microinjected into each of the two sides of the mouse lateral ventricle area. From the second day, GPA (solvent: normal saline) was intraperitoneally injected once a day for two consecutive weeks; 30 mg / kg high-dose administration group: On the first day, 1 μL of β-amyloid protein (Sigma, A9810) with a concentration of 100 μM was stereotactically microinjected into each of the two sides of the mouse lateral ventricle area. From the second day, GPA (solvent: normal saline) was intraperitoneally injected once a day for two consecutive weeks; 2 mg / kg donepezil positive control group: On the first day, 1 μL of β-amyloid protein (Sigma, A9810) with a concentration of 100 μM was stereotactically microinjected into each of the two sides of the mouse lateral ventricle area. From the second day, donepezil (solvent: normal saline) was intraperitoneally injected once a day for two consecutive weeks. During the experiment, the mice were allowed to eat and drink freely, and the body weight changes were recorded. The experiment lasted for 15 days. Starting from the 16th day of the experiment, the mice were allowed to eat and drink freely without other treatments.

[0066] The experimental data were expressed as Mean±SEM, and statistical difference analysis was performed using GraphPad Prism 8 software. A P value less than 0.05 was considered to have a statistical difference.

[0067] 2. Open field test

[0068] On the 16th day of the experiment, the mice in each experimental group were placed in the center of the test box, and the staying time of the mice in the center of the open field and the number of rearing behaviors within 10 minutes were recorded.

[0069] 3. Novel object recognition test

[0070] On the 17th day of the experiment, the novel object recognition experiment was carried out. The experimental process was divided into an adaptation stage, a familiarization stage, and a testing stage. The first day was the adaptation stage: The mice in each experimental group were placed in the center of the test box and allowed to familiarize themselves with the environment for 10 minutes. The second day was the familiarization stage. Two objects with the same color and shape were placed at equal distances on the diagonal of the test box. The mice were placed in the center of the test box, and the exploration time of the mice around the test objects within 10 minutes was recorded. The third day was the testing stage. One of the test objects was replaced with another object with a different color and shape, and the exploration time of the mice for the new and old objects within 10 minutes was recorded. The calculation formula for the discrimination index (DI) of novel object recognition is:

[0071] Discrimination index (DI)% = New object time × 100% / (New object time + Old object time)

[0072] 4. Morris water maze test

[0073] Spatial memory training stage: Starting from the 18th day of the experiment, the mice in each experimental group were placed in the water pool from different quadrants at the same time every day for 60 s each time, and trained once a day. The time required for the mice to climb onto the circular platform with their four limbs from entering the water was recorded, and the swimming trajectories and escape latencies of the mice after entering the water were continuously recorded for 5 days. If the mice could find the platform and stay for more than 2 s, it was regarded as finding the platform; if the mice could not find the platform within 60 s after entering the water, the mice could be manually guided to the platform to stay for 10 s, and the latency was calculated as 60 s.

[0074] Spatial memory test stage: An exploration test was carried out on the 6th day. The platform was removed, and the mice were randomly placed into the water from one quadrant, and the exploration data within 1 minute were recorded, including swimming trajectories, the time to reach the hidden platform for the first time, the total time of staying in the target quadrant, and the number of times of crossing the platform, etc.

[0075] Figure 3 The results showed that GPA improved the cognitive function impairment in AD model mice. Figure 3 In A and B, after administration of low-dose and high-dose GPA, the time of staying in the central area and the number of rearing times of AD model mice were increased, and the anxiety-like behavior was improved. Figure 3 In C, after administration of high-dose GPA, the novel object recognition ability of AD model mice could be improved. Figure 3 In D, after administration of high-dose GPA, the performance and exploration trajectories of AD model mice in the water maze could be effectively improved. The results showed that GPA could effectively restore the cognitive behavior disorders induced by intracerebroventricular injection of amyloid protein in mice.

[0076] Example 4. Physiological and pathological detection of GPA on AD mouse models

[0077] 1. Biochemical analysis of mouse blood samples

[0078] On the day after the water maze test, the collected mouse blood samples were centrifuged at 3000 rpm for 10 min, and the supernatant was taken. Alanine aminotransferase, aspartate aminotransferase, creatinine, urea and other indicators in the serum were tested to detect drug metabolic toxicity.

[0079] 2. HE staining of brain slices

[0080] (1) Preparation of brain slices

[0081] 2% sodium pentobarbital was injected into the mouse intraperitoneally. After the mouse was anesthetized, the mouse was fixed on the dissecting board with the abdomen facing up and the limbs spread out. The mouse chest was opened with surgical scissors, the heart was exposed, and the right atrial appendage was cut. PBS was injected into the mouse's systemic blood circulation system from the left ventricle with an intravenous syringe needle. The liver was perfused until the color turned white, and then 4% paraformaldehyde was replaced and injected into the mouse's systemic blood circulation system to fix the whole body tissue. The liver was perfused until it became hard, and the perfusion was terminated. The whole brain of the mouse was placed in 4% paraformaldehyde and placed in a 4°C refrigerator for post-fixation.

[0082] (2) Whole brain gradient dehydration

[0083] The whole brain of the mouse was placed in 10 mL of 0.2 g / mL sucrose solution (prepared with PBS solution) and placed in a 4°C refrigerator for 12 hours until the whole brain sank to the bottom. After sinking to the bottom, the whole brain of the mouse was placed in 10 mL of 0.3 g / mL sucrose solution (prepared with PBS solution) and placed in a 4°C refrigerator for 12 hours until the whole brain sank to the bottom for subsequent experiments.

[0084] (3) Frozen sections

[0085] Use filter paper to absorb the residual solution on the whole brain, place it in a freezing slicer at -20℃ for precooling, stick the whole brain to the base with OCT embedding agent, and place it in a -20℃ slicer for 30 minutes. Take the base and insert it into the slicer plug, adjust the position and angle of the blade and base. Adjust the slice thickness of the slicer to 20μm, and start slicing after the OCT is frozen as a whole. If the brain slices are used immediately, transfer them to PBS first. If they are stored for a long time, freeze them in a brain slice preservation solution at -20℃.

[0086] (4) HE staining

[0087] After the frozen sections are rehydrated, they are placed in hematoxylin staining solution for 10 minutes, washed with water for 1 minute, differentiated with 0.5-1% hydrochloric acid alcohol for 10 seconds, and rinsed with tap water for 15 minutes. They are then stained with 1% eosin alcohol for 1 minute and washed with water for another 1 minute. Finally, they are dehydrated, transparentized, and sealed.

[0088] Figure 4The effects of GPA on the body weight, blood biochemical indexes and hippocampal pathological tissue changes in mice. Figure 4 In A, GPA had no significant effect on the body weight of mice, and there were no obvious changes after continuous administration of GPA for 15 days compared with other experimental groups. Figure 4 In B are the results of blood biochemical indexes of mice in each group after 15 days of administration. The results show that there are no significant differences in the indexes such as alanine aminotransferase, aspartate aminotransferase, creatinine, and urea in each group, indicating that continuous administration of GPA for 15 days has little effect on liver and kidney functions. The hippocampus is a crucial brain region for regulating learning and memory, and the damage is particularly obvious in AD. Figure 4 In C are the changes of hippocampal neurons in each group detected by HE staining. The results show that the neurons in the CA1, CA3 and DG brain regions of the control group are arranged neatly and closely, with many cell layers, while the neurons in the AD model group are arranged sparsely, the cell gaps become larger, and obvious pathological damage phenomena appear. The administration group can effectively improve the damage of hippocampal neurons in mice.

Claims

1. Use of a biphenyl lactone compound, graphislactone A, in the preparation of a drug for preventing and / or treating Alzheimer's disease.

2. Use of a biphenyl lactone compound, Graphislactone A, in the preparation of any of the following drugs: (1) Drugs that reduce the expression of inflammatory factors IFNG and IL17; (2) drugs that promote the expression of the anti-inflammatory factor IL10; (3) drugs that improve cognitive dysfunction; (4) drugs that reduce abnormal accumulation of lipid droplets in the brain; (5) drugs that reduce the expression of inflammatory factors in the brain; (6) Drugs that protect neurons.

3. The use according to claim 1 or 2, characterized in that: The structural formula of the biphenyl lactone compound Graphislactone A is shown in formula (I):

4. A drug for preventing and / or treating Alzheimer's disease, characterized in that: The active ingredient of the medicine is graphislactone A, a biphenyl lactone compound.

5. The drug according to claim 4, characterized in that: The structural formula of the biphenyl lactone compound Graphislactone A is shown in formula (I):

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