Application of ginkgolide in improving neuroinflammatory cognitive impairment

Abstract

The present application relates to the technical field of cognitive impairment diseases under neuroinflammation, in particular to the application of discosoma in improving neuroinflammatory cognitive impairment in ginkgolide. The following steps are adopted: S1, screening the active monomer in the brain component of ginkgolide that may act on NKAα1 through molecular docking, and verifying the direct binding ability of the monomer with NKAα1 through CETSA; S2, in the LPS+ATP induced microglial cell inflammation model, the monomer is systematically evaluated for the regulation effect on NKAα1 endocytosis, NKAα1-P2X7R interaction, P2X7R mediated current and K+ outflow, and NLRP3 inflammasome activation; S3, the regulation effect of discosoma on improving cognitive impairment of PScDKO mice and on NKAα1-P2X7-NLRP3 signal axis is further explored through whole animal level experiment. Compared with the prior art, the molecular docking result shows that discosoma and narcissusin in the brain component of ginkgolide have good binding ability with NKAα1; discosoma and narcissusin have no obvious effect on the activity of BV2 cells within the experimental concentration range, and can inhibit the production of inflammatory factors induced by LPS+ATP.

Description

Technical Field

[0001] This invention relates to the field of cognitive impairment under neuroinflammation, specifically the application of discus extract in ginkgo biloba extract in improving neuroinflammation-related cognitive impairment. Background Technology

[0002] With social development and the accelerating aging of the population, neurodegenerative diseases have become a major public health problem posing a serious threat to national health. In recent years, the overall disease burden has shown a significant upward trend, and my country also faces severe challenges. Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common type of dementia among the elderly worldwide. Clinical manifestations include progressive cognitive decline, memory loss, and behavioral and psychological changes, with cognitive impairment being the most common clinical manifestation. Specifically, cognitive impairment refers to a syndrome primarily characterized by impairment of higher brain functions such as learning, memory, and executive function. Cognitive impairment not only severely affects patients' quality of life but also imposes a huge medical burden on society.

[0003] For a long time, the amyloid hypothesis and the tau hypothesis have been considered the core theories explaining the pathogenesis of Alzheimer's disease (AD). However, the abnormal deposition of a single protein is insufficient to fully explain the complex disease progression and clinical heterogeneity. Recent studies have shown that in addition to protein pathological changes, multiple factors such as neuroinflammation also participate in the development of AD. However, there are currently no highly effective clinical treatments for neuroinflammatory cognitive impairments, and their damage mechanisms are not yet fully understood. Therefore, further in-depth research into its pathogenesis and the development of new treatments to improve the current situation are urgent research needs. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides the application of discus extract in ginkgo biloba extract in improving neuroinflammatory cognitive impairment, which solves the problem that there is a lack of clinical treatment methods for inflammatory cognitive impairment and that its pathogenesis has not been fully elucidated.

[0005] To achieve the above objectives, an application of discus extract in ginkgo biloba extract in improving neuroinflammatory cognitive impairment is designed, characterized by the following steps:

[0006] S1. First, we screened the active monomers of ginkgo biloba extract that may act on NKAα1 through molecular docking, and verified their direct binding ability to NKAα1 through CETSA.

[0007] S2, then in an LPS+ATP-induced microglial inflammation model, the regulatory effects of this monomer on NKAα1 endocytosis, NKAα1-P2X7R interaction, P2X7R-mediated current and K⁺ efflux, and NLRP3 inflammasome activation were systematically evaluated, and its anti-inflammatory molecular mechanism was elucidated.

[0008] S3. Finally, through whole-animal level experiments, we further explored the role of discus extract in improving cognitive impairment in PScDKO mice and its regulatory effect on the NKAα1-P2X7-NLRP3 signaling axis.

[0009] The specific process of step S1 is as follows:

[0010] S1-1, Molecular docking verification of Ginkgo biloba extract for brain absorption with NKAα1:

[0011] (1) The 3D structures of 14 active ingredients of ginkgo biloba extract entering the brain were downloaded from the PubChem database as ligands and converted into mol2 format using OpenBabel.

[0012] (2) The crystal structure of NKAα1 protein was obtained from RCSB Protein Data Bank as the receptor and pretreated with PyMOL 2.6.0 for dehydration.

[0013] (3) Subsequently, the ligand was minimized and the protein activity pocket was identified in Molecular Operating Environment 2019 software to complete the ligand-receptor molecular docking. The results were visualized using PyMOL and the binding activity was evaluated based on the binding energy.

[0014] S1-2, Tetramethylazozid salt colorimetric method for detecting the effects of chondruvicin and narcisin on BV2 cell viability:

[0015] (1) Take BV2 cells in the logarithmic growth phase and seed them in 96-well plates. After the cells adhere to the plate, discard the culture medium and add culture medium containing 1.25, 2.5, 5, 10, 20, 40, and 80 μM litorin or narcissoside to treat the cells. 200 μL per well, 6 replicates per group, and a control group is also set up.

[0016] (2) After 24 hours of treatment, add 20 μL of 5 mg / mL MTT solution to each well and continue incubation for 4 hours;

[0017] (3) Then discard the culture medium, add 150 μL of dimethylsulfoxide to each well, shake on a shaker to dissolve and crystallize, and measure the OD value at a wavelength of 570 nm to evaluate cell viability;

[0018] S1-3, qRT-PCR detection:

[0019] (1) After removing the cell supernatant, add 1 mL of PBS to each well for washing, discard the PBS, add 500 μL of cell lysis buffer to each well, let stand at room temperature for 1 min, and then aspirate the lysis buffer from the well into the tube.

[0020] (2) Centrifuge at 12000 rpm for 1 min and discard the liquid inside the outer tube;

[0021] (3) Add 700 μL of wash buffer to each tube and centrifuge at 12,000 rpm for 1 min;

[0022] (4) Discard the washing solution and repeat step (3) once, that is, add 700 μL of wash buffer to each tube, centrifuge at 12000 rpm for 1 min;

[0023] (5) Discard the washing solution and centrifuge at 12,000 rpm for 2 min;

[0024] (6) Transfer the inner tube to a clean 1.5 mL EP tube, open the cap and let it stand for 3 minutes to allow the alcohol in the inner tube to evaporate completely.

[0025] (7) Add 30 μL LEPC water, let stand for 2 min, centrifuge at 12000 rpm for 1 min, discard the inner tube, and detect the RNA concentration in the EP tube;

[0026] S1-4, BV2 cell heat transfer experiment:

[0027] (1) Take BV2 cells in the logarithmic growth phase, treat them with lysis buffer and collect the supernatant protein;

[0028] (2) Add 100 μM litorin or an equal volume of DMSO control to the protein and incubate on ice for 2 h;

[0029] (3) Dispense into 1.5mL centrifuge tubes, 80μL per tube, set 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82℃, react for 3min, place at room temperature for 3min, add loading buffer and denature in 100℃ water bath for 10min for subsequent analysis;

[0030] S1-5, Western blot analysis:

[0031] (1) Extraction of cell proteins: Prepare lysis buffer in advance and keep it on ice;

[0032] (2) After the drug treatment is completed, discard the culture medium, wash with PBS 2-3 times, add 100μL of lysis buffer to each well, collect the cells with a scraper and blow them evenly, transfer them to a 1.5mL centrifuge tube, centrifuge at 12000rpm and 4℃ for 15min, and collect the supernatant as the total cell protein.

[0033] The ligand-receptor molecular docking was performed 50 times.

[0034] The specific procedure for Western blot testing is as follows:

[0035] (1) Extraction of cell proteins:

[0036] ① Prepare the lysis buffer according to the ratio of RIPA lysis buffer, protease inhibitor: PMSF: phosphatase inhibitor = 100:1:1:1;

[0037] ② Take about 30 mg of HPC or PFC tissue and add 100 µL of lysis buffer into a grinding tube. After adding steel balls, grind the tissue at 90 Hz for 60 s in a cryogenic grinder.

[0038] ③Then centrifuge at 4℃ and 12000rpm for 15min, and collect the supernatant as the total tissue protein; extract membrane proteins according to the instructions of the cell membrane protein and cytoplasmic protein extraction kit;

[0039] (2) BCA protein quantification and protein preparation:

[0040] ① Prepare standard gradients of 1 mg / mL protein standard by pipetting the corresponding volumes into EP tubes, with concentrations of 1, 0.8, 0.5, 0.4, 0.2, 0.1, 0.05, and 0 mg / mL respectively;

[0041] ②Then add 20 μL of standard or sample to a 96-well plate, with 3 replicates per group;

[0042] ③ Prepare BCA working solution according to solution A: solution B = 50:1, add 200 μL to each well, incubate at 37℃ for 30 min, measure the OD value at 562 nm, plot the standard curve and calculate the protein concentration of the sample;

[0043] ④ Based on the measured concentration, add 5× loading buffer and RIPA lysis buffer to prepare a protein sample with a final concentration of 4 μg / μL containing 1× loading buffer. Denature the sample by heating at 100℃ for 10 min and store at -20℃ for later use.

[0044] (3) Preparation of SDS-PAGE gel:

[0045] ① The experiment used a 1.5mm thick glass plate to assemble the electrophoresis gel preparation device. 10% separating gel was prepared in a 50mL centrifuge tube. After mixing, 7.8mL was added to the gel preparation device and sealed with anhydrous ethanol.

[0046] ② After the separating gel has completely solidified, discard the ethanol and allow it to dry.

[0047] (4) Western blot electrophoresis: Prepare the electrophoresis solution according to the required ratio, and place it in a magnetic stirrer to stir and dissolve it evenly;

[0048] (5) Transfer: Prepare the transfer solution in advance according to the required ratio, place it in a magnetic stirrer and stir until fully dissolved, then seal and pre-cool in a refrigerator at 4°C;

[0049] (6) Western blot blocking, incubation and color development:

[0050] ① After the transfer is complete, place the NC membrane in 5% BSA or 5% skim milk powder and seal it in a shaker at room temperature for 60-120 minutes;

[0051] ② Discard the blocking solution and rinse briefly with TBST, then add diluted primary antibody and incubate overnight at 4°C on a shaker at low speed;

[0052] ③ The primary antibody was recovered the next day, and the membrane was washed three times at room temperature using TBST.

[0053] ④Then add the corresponding secondary antibody, incubate on a shaker at room temperature for 90 minutes, and then wash the membrane 3 times with TBST at room temperature;

[0054] ⑤ Finally, add the freshly prepared ECL colorimetric solution, expose and acquire images in a gel imaging system, and use ImageJ software to analyze the gray values ​​of the target band protein.

[0055] The specific process of step S2 is as follows:

[0056] Detection of S2-1, BV2 cell membrane protein NKAα1 endocytosis:

[0057] (1) Cells were lysed after labeling the proteins on the cell surface with Biotin-SS-NHS-Sulfo provided in the kit. The lysed samples were then bound to Streptavidin Magnetic Beads.

[0058] (2) After washing to remove unbound components, elute with DTT-containing eluent to obtain cell surface proteins without biotin labeling. The cell surface proteins can then be identified by Western blot analysis.

[0059] S2-2, NKAα1 siRNA-mediated gene silencing in BV2 cells:

[0060] (1) Input Atp1a1 into the Gene database in the NCBI data platform to obtain the gene sequence of Atp1a1 of mouse species, and synthesize the siRNA of the target gene;

[0061] (2) After centrifuging the obtained siRNA powder, dissolve it in enzyme-free water to prepare a 20 μM stock solution and dispense it into 200 μL centrifuge tubes for storage at -20℃ for later use.

[0062] S2-3, siRNA transfection and drug administration in BV2 cells:

[0063] (1) Take cells in good growth condition and seed them in a six-well cell culture plate with 10% FBS antibiotic-free medium, and control the cell density to 0.5~2×105 / well for transfection after 24h;

[0064] (2) BV2 cells were transfected with NKAα1 siRNA using Lipofectamine™ 3000 transfection reagent;

[0065] S2-4, Immunoprecipitation:

[0066] (1) Take BV2 cells in the logarithmic growth phase, add pre-cooled IP lysis buffer and lyse on ice, centrifuge at 4℃ and 12000rpm for 15min, and collect the supernatant as total protein.

[0067] (2) And take a portion as the Input for comparison;

[0068] (3) The remaining protein was added to equilibrated Protein A / G magnetic beads and incubated at 4°C for 1 hour for pre-cleaning;

[0069] (4) After centrifugation or magnetic separation, take the supernatant, add the primary antibody against the target protein and an equal amount of homologous IgG, and incubate overnight at 4°C by rotation.

[0070] (5) Then add Protein A / G magnetic beads and continue incubation for 2-4 hours;

[0071] (6) After incubation, wash the magnetic beads 3-5 times with pre-cooled IP lysis buffer, discard the supernatant and add SDS loading buffer, heat at 100℃ for 5-10 min to dissociate the protein;

[0072] (7) Finally, SDS-PAGE electrophoresis was performed and the interaction of the target proteins was detected by Western blot.

[0073] Recording of P2X7R-mediated current in BV2 cells, S2-5:

[0074] (1) To record purine energy currents, cells were transferred from the culture medium to BV2 extracellular fluid;

[0075] (2) In whole-cell patch-clamp experiments, cells are exposed to a solution with variable concentrations of divalent cations, and the composition of the electrode fluid used to record the current is recorded.

[0076] (3) The data was acquired at a sampling rate of 5 kHz, filtered at 1 kHz through a 7-pole Bessel filter, and digitized using a Digidata1440A.

[0077] The specific procedure for transfecting BV2 cells with NKAα1 siRNA using Lipofectamine™ 3000 transfection reagent is as follows:

[0078] ① Before the experiment, the ultra-clean workbench was disinfected with ultraviolet light. BV2 cells were seeded in six-well plates and cultured. When the cells were growing well and the confluence reached 30%-50%, the transfection experiment was carried out.

[0079] ② Take a 250μL centrifuge tube, add 90μL of Opti-MEM serum-free medium to each tube, add 5.0μL of NKAα1 siRNA stock solution to make the final transfection concentration of siRNA in the cells 50nM, gently pipette 3-5 times to mix, and then let stand at room temperature for 5min.

[0080] ③ In another 250μL centrifuge tube, add 95μL of Opti-MEM, then add 5.0μL of Lipofectamine™ 3000 transfection reagent, and gently pipette 3-5 times to mix and dilute the transfection reagent;

[0081] ④ Slowly add the diluted transfection reagent to the centrifuge tube containing the siRNA dilution solution, gently pipette 3-5 times to mix, and incubate at room temperature for 20 minutes to form the siRNA-Lipofectamine transfection complex.

[0082] ⑤ Gently add 200 μL of the transfection complex to a six-well plate containing BV2 cells, and gently shake the plate back and forth in a cross shape to distribute the transfection complex evenly.

[0083] ⑥ After culturing the cells in a 37℃, 5% CO2 incubator for 6 hours, replace the culture medium with fresh complete medium and continue culturing for 24 hours;

[0084] ⑦ For experimental groups that were given medication at different time points, ensure that the total transfection time is consistent for each group.

[0085] The solution with variable divalent cation concentration is a solution that does not contain magnesium chloride and contains 0.1 mM calcium chloride.

[0086] In step S3, the whole animal level experiments include the open field experiment, the new object recognition experiment, the Y-maze experiment, the Morris water maze experiment, and the conditioned fear experiment.

[0087] Compared with existing technologies, this invention provides the application of discus extract in ginkgo biloba extract in improving neuroinflammatory cognitive impairment. Molecular docking results show that discus extract and narcissin, the brain-entering components of ginkgo biloba extract, have good binding ability with NKAα1. Discus extract and narcissin have no significant effect on BV2 cell viability within the experimental concentration range and can inhibit the production of LPS+ATP-induced inflammatory factors. Discus extract can directly bind to NKAα1 protein and enhance its thermal stability, while inhibiting LPS+ATP-induced NLRP3 inflammasome activation. Discus extract can inhibit NKAα1 protein endocytosis under inflammatory stimulation and enhance the interaction between NKAα1 and P2X7R. Knockdown of NKAα1 can enhance P2X7R-mediated currents and promote potassium ion efflux in BV2 cells, while discus extract can significantly inhibit this change. Discus extract improves learning and memory dysfunction in PScDKO mice. Discus extract enhances the interaction between NKAα1 and P2X7R. Discus extract inhibits NLRP3 inflammasome activation. Attached Figure Description

[0088] Figure 1 This is a schematic diagram of an open field experiment in an embodiment of the present invention.

[0089] Figure 2 This is a schematic diagram of a novel object recognition experiment in an embodiment of the present invention.

[0090] Figure 3 This is a schematic diagram of a Y-maze experiment in an embodiment of the present invention.

[0091] Figure 4 This is a schematic diagram of the Morris water maze experiment in an embodiment of the present invention.

[0092] Figure 5 This is a schematic diagram of a conditioned fear experiment in an embodiment of the present invention.

[0093] Figure 6 This is the molecular docking result of 14 brain-entering components of Ginkgo biloba extract with NKAα1 in the embodiments of the present invention.

[0094] Figure 7 The effects of discus extract and narcissin on BV2 cell viability are illustrated in the embodiments of the present invention.

[0095] Figure 8 The effects of discus extract and narcissin on LPS+ATP-stimulated IL-1β mRNA in BV2 cells are illustrated in the embodiments of the present invention.

[0096] Figure 9 This invention illustrates the effect of Clitorin on NKAα1 protein in BV2 cells at different temperatures.

[0097] Figure 10This invention relates to the effect of Clitorin on LPS+ATP-stimulated NLRP3 inflammasome protein in BV2 cells.

[0098] Figure 11 This invention illustrates the effect of Clitorin on LPS+ATP-stimulated endocytosis of NKAα1 protein in BV2 cells.

[0099] Figure 12 This invention illustrates the effect of Clitorin on the interaction between NKAα1 and P2X7R in BV2 cells stimulated by LPS+ATP.

[0100] Figure 13 This invention illustrates the effect of Clitorin on the P2X7R-mediated current in NKAα1BV2 cells knocked down.

[0101] Figure 14 This invention illustrates the effect of Clitorin on the motor function of PScDKO mice in an embodiment of the invention.

[0102] Figure 15 This invention illustrates the effect of Clitorin on the recognition and memory of PScDKO mice in this embodiment.

[0103] Figure 16 This invention illustrates the effect of Clitorin on spatial and associative memory in PScDKO mice.

[0104] Figure 17 This invention illustrates the effect of Clitorin on spatial reference memory in PScDKO mice.

[0105] Figure 18 This invention illustrates the effect of Clitorin on the interaction between NKAα1 and P2X7R in PScDKO mice.

[0106] Figure 19 This invention illustrates the effects of Clitorin on NLRP3, Caspase-1, and ASC proteins in PScDKO mice. Detailed Implementation

[0107] The present invention will now be further described with reference to the accompanying drawings.

[0108] The application of discus extract in a ginkgo biloba ester of the present invention in improving neuroinflammatory cognitive impairment comprises the following steps:

[0109] S1. First, we screened the active monomers of ginkgo biloba extract that may act on NKAα1 through molecular docking, and verified their direct binding ability to NKAα1 through CETSA.

[0110] S2, then in an LPS+ATP-induced microglial inflammation model, the regulatory effects of this monomer on NKAα1 endocytosis, NKAα1-P2X7R interaction, P2X7R-mediated current and K⁺ efflux, and NLRP3 inflammasome activation were systematically evaluated, and its anti-inflammatory molecular mechanism was elucidated.

[0111] S3. Finally, through whole-animal level experiments, we further explored the role of discus extract in improving cognitive impairment in PScDKO mice and its regulatory effect on the NKAα1-P2X7-NLRP3 signaling axis.

[0112] The specific process of step S1 is as follows:

[0113] S1-1, Molecular docking verification of Ginkgo biloba extract for brain absorption with NKAα1:

[0114] (1) The 3D structures of 14 active ingredients of ginkgo biloba extract entering the brain were downloaded from the PubChem database as ligands and converted into mol2 format using OpenBabel.

[0115] (2) The crystal structure of NKAα1 protein was obtained from RCSB Protein Data Bank as the receptor and pretreated with PyMOL 2.6.0 for dehydration.

[0116] (3) Subsequently, the ligand was minimized and the protein activity pocket was identified in Molecular Operating Environment 2019 software to complete the ligand-receptor molecular docking. The results were visualized using PyMOL and the binding activity was evaluated based on the binding energy.

[0117] S1-2, Tetramethylazozid salt colorimetric method for detecting the effects of chondruvicin and narcisin on BV2 cell viability:

[0118] (1) Take BV2 cells in the logarithmic growth phase and seed them in 96-well plates. After the cells adhere to the plate, discard the culture medium and add culture medium containing 1.25, 2.5, 5, 10, 20, 40, and 80 μM litorin or narcissoside to treat the cells. 200 μL per well, 6 replicates per group, and a control group is also set up.

[0119] (2) After 24 hours of treatment, add 20 μL of 5 mg / mL MTT solution to each well and continue incubation for 4 hours;

[0120] (3) Then discard the culture medium, add 150 μL of dimethylsulfoxide to each well, shake on a shaker to dissolve and crystallize, and measure the OD value at a wavelength of 570 nm to evaluate cell viability;

[0121] S1-3, qRT-PCR detection:

[0122] (1) After removing the cell supernatant, add 1 mL of PBS to each well for washing, discard the PBS, add 500 μL of cell lysis buffer to each well, let stand at room temperature for 1 min, and then aspirate the lysis buffer from the well into the tube.

[0123] (2) Centrifuge at 12000 rpm for 1 min and discard the liquid inside the outer tube;

[0124] (3) Add 700 μL of wash buffer to each tube and centrifuge at 12,000 rpm for 1 min;

[0125] (4) Discard the washing solution and repeat step (3) once, that is, add 700 μL of wash buffer to each tube, centrifuge at 12000 rpm for 1 min;

[0126] (5) Discard the washing solution and centrifuge at 12,000 rpm for 2 min;

[0127] (6) Transfer the inner tube to a clean 1.5 mL EP tube, open the cap and let it stand for 3 minutes to allow the alcohol in the inner tube to evaporate completely.

[0128] (7) Add 30 μL LEPC water, let stand for 2 min, centrifuge at 12000 rpm for 1 min, discard the inner tube, and detect the RNA concentration in the EP tube;

[0129] S1-4, BV2 cell heat transfer experiment:

[0130] (1) Take BV2 cells in the logarithmic growth phase, treat them with lysis buffer and collect the supernatant protein;

[0131] (2) Add 100 μM litorin or an equal volume of DMSO control to the protein and incubate on ice for 2 h;

[0132] (3) Dispense into 1.5mL centrifuge tubes, 80μL per tube, set 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82℃, react for 3min, place at room temperature for 3min, add loading buffer and denature in 100℃ water bath for 10min for subsequent analysis;

[0133] S1-5, Western blot analysis:

[0134] (1) Extraction of cell proteins: Prepare lysis buffer in advance and keep it on ice;

[0135] (2) After the drug treatment is completed, discard the culture medium, wash with PBS 2-3 times, add 100μL of lysis buffer to each well, collect the cells with a scraper and blow them evenly, transfer them to a 1.5mL centrifuge tube, centrifuge at 12000rpm and 4℃ for 15min, and collect the supernatant as the total cell protein.

[0136] The ligand-receptor molecular docking was performed 50 times.

[0137] The specific procedure for Western blot testing is as follows:

[0138] (1) Extraction of cell proteins:

[0139] ① Prepare the lysis buffer according to the ratio of RIPA lysis buffer, protease inhibitor: PMSF: phosphatase inhibitor = 100:1:1:1;

[0140] ② Take about 30 mg of HPC or PFC tissue and add 100 µL of lysis buffer into a grinding tube. After adding steel balls, grind the tissue at 90 Hz for 60 s in a cryogenic grinder.

[0141] ③Then centrifuge at 4℃ and 12000rpm for 15min, and collect the supernatant as the total tissue protein; extract membrane proteins according to the instructions of the cell membrane protein and cytoplasmic protein extraction kit;

[0142] (2) BCA protein quantification and protein preparation:

[0143] ① Prepare standard gradients of 1 mg / mL protein standard by pipetting the corresponding volumes into EP tubes, with concentrations of 1, 0.8, 0.5, 0.4, 0.2, 0.1, 0.05, and 0 mg / mL respectively;

[0144] ②Then add 20 μL of standard or sample to a 96-well plate, with 3 replicates per group;

[0145] ③ Prepare BCA working solution according to solution A: solution B = 50:1, add 200 μL to each well, incubate at 37℃ for 30 min, measure the OD value at 562 nm, plot the standard curve and calculate the protein concentration of the sample;

[0146] ④ Based on the measured concentration, add 5× loading buffer and RIPA lysis buffer to prepare a protein sample with a final concentration of 4 μg / μL containing 1× loading buffer. Denature the sample by heating at 100℃ for 10 min and store at -20℃ for later use.

[0147] (3) Preparation of SDS-PAGE gel:

[0148] ① The experiment used a 1.5mm thick glass plate to assemble the electrophoresis gel preparation device. 10% separating gel was prepared in a 50mL centrifuge tube. After mixing, 7.8mL was added to the gel preparation device and sealed with anhydrous ethanol.

[0149] ② After the separating gel has completely solidified, discard the ethanol and allow it to dry.

[0150] (4) Western blot electrophoresis: Prepare the electrophoresis solution according to the required ratio, and place it in a magnetic stirrer to stir and dissolve it evenly;

[0151] (5) Transfer: Prepare the transfer solution in advance according to the required ratio, place it in a magnetic stirrer and stir until fully dissolved, then seal and pre-cool in a refrigerator at 4°C;

[0152] (6) Western blot blocking, incubation and color development:

[0153] ① After the transfer is complete, place the NC membrane in 5% BSA or 5% skim milk powder and seal it in a shaker at room temperature for 60-120 minutes;

[0154] ② Discard the blocking solution and rinse briefly with TBST, then add diluted primary antibody and incubate overnight at 4°C on a shaker at low speed;

[0155] ③ The primary antibody was recovered the next day, and the membrane was washed three times at room temperature using TBST.

[0156] ④Then add the corresponding secondary antibody, incubate on a shaker at room temperature for 90 minutes, and then wash the membrane 3 times with TBST at room temperature;

[0157] ⑤ Finally, add the freshly prepared ECL colorimetric solution, expose and acquire images in a gel imaging system, and use ImageJ software to analyze the gray values ​​of the target band protein.

[0158] The specific process of step S2 is as follows:

[0159] Detection of S2-1, BV2 cell membrane protein NKAα1 endocytosis:

[0160] (1) Cells were lysed after labeling the proteins on the cell surface with Biotin-SS-NHS-Sulfo provided in the kit. The lysed samples were then bound to Streptavidin Magnetic Beads.

[0161] (2) After washing to remove unbound components, elute with DTT-containing eluent to obtain cell surface proteins without biotin labeling. The cell surface proteins can then be identified by Western blot analysis.

[0162] S2-2, NKAα1 siRNA-mediated gene silencing in BV2 cells:

[0163] (1) Input Atp1a1 into the Gene database in the NCBI data platform to obtain the gene sequence of Atp1a1 of mouse species, and synthesize the siRNA of the target gene;

[0164] (2) After centrifuging the obtained siRNA powder, dissolve it in enzyme-free water to prepare a 20 μM stock solution and dispense it into 200 μL centrifuge tubes for storage at -20℃ for later use.

[0165] S2-3, siRNA transfection and drug administration in BV2 cells:

[0166] (1) Take cells in good growth condition and seed them in a six-well cell culture plate with 10% FBS antibiotic-free medium, and control the cell density to 0.5~2×105 / well for transfection after 24h;

[0167] (2) BV2 cells were transfected with NKAα1 siRNA using Lipofectamine™ 3000 transfection reagent;

[0168] S2-4, Immunoprecipitation:

[0169] (1) Take BV2 cells in the logarithmic growth phase, add pre-cooled IP lysis buffer and lyse on ice, centrifuge at 4℃ and 12000rpm for 15min, and collect the supernatant as total protein.

[0170] (2) And take a portion as the Input for comparison;

[0171] (3) The remaining protein was added to equilibrated Protein A / G magnetic beads and incubated at 4°C for 1 hour for pre-cleaning;

[0172] (4) After centrifugation or magnetic separation, take the supernatant, add the primary antibody against the target protein and an equal amount of homologous IgG, and incubate overnight at 4°C by rotation.

[0173] (5) Then add Protein A / G magnetic beads and continue incubation for 2-4 hours;

[0174] (6) After incubation, wash the magnetic beads 3-5 times with pre-cooled IP lysis buffer, discard the supernatant and add SDS loading buffer, heat at 100℃ for 5-10 min to dissociate the protein;

[0175] (7) Finally, SDS-PAGE electrophoresis was performed and the interaction of the target proteins was detected by Western blot.

[0176] Recording of P2X7R-mediated current in BV2 cells, S2-5:

[0177] (1) To record purine energy currents, cells were transferred from the culture medium to BV2 extracellular fluid;

[0178] (2) In whole-cell patch-clamp experiments, cells are exposed to a solution with variable concentrations of divalent cations, and the composition of the electrode fluid used to record the current is recorded.

[0179] (3) The data was acquired at a sampling rate of 5 kHz, filtered at 1 kHz through a 7-pole Bessel filter, and digitized using a Digidata1440A.

[0180] The specific procedure for transfecting BV2 cells with NKAα1 siRNA using Lipofectamine™ 3000 transfection reagent is as follows:

[0181] ① Before the experiment, the ultra-clean workbench was disinfected with ultraviolet light. BV2 cells were seeded in six-well plates and cultured. When the cells were growing well and the confluence reached 30%-50%, the transfection experiment was carried out.

[0182] ② Take a 250μL centrifuge tube, add 90μL of Opti-MEM serum-free medium to each tube, add 5.0μL of NKAα1 siRNA stock solution to make the final transfection concentration of siRNA in the cells 50nM, gently pipette 3-5 times to mix, and then let stand at room temperature for 5min.

[0183] ③ In another 250μL centrifuge tube, add 95μL of Opti-MEM, then add 5.0μL of Lipofectamine™ 3000 transfection reagent, and gently pipette 3-5 times to mix and dilute the transfection reagent;

[0184] ④ Slowly add the diluted transfection reagent to the centrifuge tube containing the siRNA dilution solution, gently pipette 3-5 times to mix, and incubate at room temperature for 20 minutes to form the siRNA-Lipofectamine transfection complex.

[0185] ⑤ Gently add 200 μL of the transfection complex to a six-well plate containing BV2 cells, and gently shake the plate back and forth in a cross shape to distribute the transfection complex evenly.

[0186] ⑥ After culturing the cells in a 37℃, 5% CO2 incubator for 6 hours, replace the culture medium with fresh complete medium and continue culturing for 24 hours;

[0187] ⑦ For experimental groups that were given medication at different time points, ensure that the total transfection time is consistent for each group.

[0188] The solution with variable divalent cation concentration is a solution that does not contain magnesium chloride and contains 0.1 mM calcium chloride.

[0189] In step S3, the whole animal level experiments include the open field experiment, the new object recognition experiment, the Y-maze experiment, the Morris water maze experiment, and the conditioned fear experiment.

[0190] Example:

[0191] I. Experimental Materials

[0192] 1. Laboratory animals

[0193] This study used 4-6 month old SPF-grade PS2, PScDKO type, and their littermates' wild-type (WT) mice as experimental animals, weighing approximately 30±5g. The animals were housed at the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine during the drug administration period. All animal experimental procedures strictly adhered to relevant ethical guidelines for laboratory animals and were approved by the Experimental Animal Management and Use Committee of Shanghai University of Traditional Chinese Medicine, with ethics approval number PZSHUTCM2411130006.

[0194] The specific groupings are as follows: wild-type group (WT, n=12), WT+Clitorin group (WT+Clitorin, n=12), PScDKO group (cDKO, n=12), and PScDKO+Clitorin group (cDKO+Clitorin, n=12). Based on the research group's previous work, Clitorin was administered via intraperitoneal injection for 30 consecutive days at a dose of 10 mg / kg.

[0195] 2. Experimental cells

[0196] BV2 mouse microglia were purchased from the Cell Culture Center of the Chinese Academy of Medical Sciences. Cells were cultured in complete medium containing 10% fetal bovine serum (FBS), 90% high-glucose DMEM, and 0.1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. After resuscitation, cells were passaged three times. Cells in the logarithmic growth phase were selected after microscopic observation of good cell growth for subsequent experiments.

[0197] 3. Experimental equipment

[0198] Table 1 shows the main instruments involved in the experiment.

[0199] Table 1

[0200]

[0201]

[0202] 4. Experimental reagents

[0203] Table 2 shows the main experimental antibody reagents.

[0204] Table 2

[0205]

[0206]

[0207]

[0208] II. Experimental Methods

[0209] 1. Molecular docking verification of Ginkgo biloba extract for brain absorption with NKAα1

[0210] The 3D structures of 14 active components of Ginkgo biloba extract for brain absorption were downloaded from the PubChem database as ligands and converted to mol2 format using OpenBabel. The NKAα1 protein crystal structure was obtained from RCSB Protein Data Bank as the receptor and pretreated with PyMOL 2.6.0 for dehydration. Subsequently, energy minimization of the ligands and identification of protein activity pockets were performed in Molecular Operating Environment 2019 software, and ligand-receptor molecular docking was completed (50 operations). The results were visualized using PyMOL, and binding activity was evaluated based on binding energy.

[0211] 2. Effects of Tetramethylazazole salt colorimetric method (MTT) on the activity of BV2 cells: Detection of the effects of choleretic acid and narcissin on the activity of BV2 cells.

[0212] Take 2 × 10⁻⁶ BV2 cells in the logarithmic growth phase. 4 Cells were seeded per well in 96-well plates. After cell attachment, the culture medium was discarded, and 200 μL of medium containing 1.25, 2.5, 5, 10, 20, 40, or 80 μM litorin or narcissoside was added to each well for treatment. Six replicates were set up for each group, and a control group was included. After 24 h of treatment, 20 μL of 5 mg / mL LTT solution was added to each well for further incubation for 4 h. The culture medium was then discarded, and 150 μL of dimethylsulfoxide was added to each well. The cells were dissolved and crystallized by shaking on a shaker, and the OD value was measured at 570 nm to evaluate cell viability.

[0213] 3. qRT-PCR detection

[0214] (1) After removing the cell supernatant, add 1 mL of PBS to each well for washing, discard the PBS, add 500 μL of cell lysis buffer to each well, let stand at room temperature for 1 min, and then aspirate the lysis buffer from the well into the tube.

[0215] (2) Centrifuge at 12000 rpm for 1 min and discard the liquid inside the outer tube;

[0216] (3) Add 700 μL of wash buffer to each tube and centrifuge at 12,000 rpm for 1 min;

[0217] (4) Discard the washing solution and repeat step 3;

[0218] (5) Discard the washing solution and centrifuge at 12,000 rpm for 2 min;

[0219] (6) Transfer the inner tube to a clean 1.5 mL EP tube, open the cap and let it stand for 3 minutes to allow the alcohol in the inner tube to evaporate completely.

[0220] (7) Add 30 μL LEPC water, let stand for 2 min, centrifuge at 12000 rpm for 1 min, discard the inner tube, and detect the RNA concentration in the EP tube. As shown in Table 3, this is the target gene sequence.

[0221] Table 3

[0222]

[0223] 4. BV2 cell thermal shift assay (CETSA)

[0224] Logarithmically growing BV2 cells were treated with lysis buffer, and the supernatant protein was collected. The protein was aliquoted with 100 μC litorin or an equal volume of DMSO as a control, and incubated on ice for 2 h. The mixture was then aliquoted into 1.5 mL centrifuge tubes (80 μL per tube). Centrifuge tubes were incubated at 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, and 82 °C for 3 min, followed by incubation at room temperature for 3 min. Loading buffer was then added, and the mixture was denatured at 100 °C for 10 min for subsequent analysis.

[0225] 5. Western blot detection

[0226] Extraction of cell proteins: Prepare lysis buffer in advance and keep it on ice. After drug treatment, discard the culture medium, wash 2-3 times with PBS, add 100 μL of lysis buffer to each well, collect cells with a scraper and mix well by pipetting, transfer to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm and 4℃ for 15 min, and collect the supernatant as total cell protein.

[0227] (1) Extraction of tissue proteins: Prepare lysis buffer according to the ratio of RIPA lysis buffer: protease inhibitor: PMSF: phosphatase inhibitor = 100:1:1:1. Take about 30 mg of HPC or PFC tissue and add 100 µL of lysis buffer to a grinding tube. After adding steel balls, grind at 90 Hz for 60 s in a cryogenic grinder. Then centrifuge at 4 °C and 12000 rpm for 15 min and collect the supernatant as total tissue protein. Membrane protein extraction: Extract membrane proteins according to the instructions of the cell membrane protein and cytoplasmic protein extraction kit.

[0228] (2) BCA protein quantification and protein preparation: Prepare standard gradients of 1 mg / mL protein standard by pipetting corresponding volumes into EP tubes at concentrations of 1, 0.8, 0.5, 0.4, 0.2, 0.1, 0.05, and 0 mg / mL. Then, add 20 μL of standard or sample to a 96-well plate, with three replicates per group. Prepare BCA working solution at a ratio of A:B solution = 50:1, adding 200 μL to each well. Incubate at 37°C for 30 min, measure the OD value at 562 nm, plot a standard curve, and calculate the sample protein concentration. Based on the measured concentration, add 5× loading buffer and RIPA lysis buffer to prepare a protein sample with a final concentration of 4 μg / μL containing 1× loading buffer. Denature the sample by heating at 100°C for 10 min and store at -20°C for later use.

[0229] (3) SDS-PAGE gel preparation: An electrophoresis gel preparation device was assembled using a 1.5 mm thick glass plate. A 10% separating gel was prepared in a 50 mL centrifuge tube, mixed well, and 7.8 mL was added to the gel preparation device and sealed with anhydrous ethanol. After the separating gel had completely solidified, the ethanol was discarded and the gel was air-dried. The preparation ratio of the two SDS-PAGE gels is shown in Table 4.

[0230] Table 4

[0231]

[0232] Prepare a 4% concentrated gel according to Table 5, quickly insert it into a 10- or 15-hole comb, let it stand at room temperature for at least 30 minutes, and then transfer it to a 4°C refrigerator for later use.

[0233] Table 5

[0234]

[0235] (4) Western blot electrophoresis: Prepare the electrophoresis solution according to the ratio in Table 6, and place it in a magnetic stirrer to stir and dissolve it.

[0236] Table 6

[0237]

[0238] (5) Transfer: Prepare the transfer solution in advance according to the proportions listed in Table 7, place it in a magnetic stirrer and stir until fully dissolved, then seal and pre-cool in a refrigerator at 4°C.

[0239] Table 7

[0240]

[0241] After electrophoresis, place the sponge, filter paper, gel, NC membrane, filter paper and sponge in the order of color of the transfer clamp (black → white) and clamp them tightly. Place them in the transfer tank, add transfer buffer and put the tank in an ice box. Transfer at a constant voltage of 110V for 2 hours.

[0242] (6) Western blot blocking, incubation, and color development: After transfer, place the NC membrane in 5% BSA or 5% skim milk powder and block on a shaker (50-60 rpm) at room temperature for 60-120 min. Discard the blocking solution and rinse briefly with TBST, then add diluted primary antibody (1:3000) and incubate overnight on a shaker at 4°C. The next day, recover the primary antibody, wash the membrane three times with TBST at room temperature (100 rpm, 10 min); then add the corresponding secondary antibody (1:5000), incubate on a shaker (50-60 rpm) at room temperature for 90 min, and wash the membrane three times with TBST at room temperature (100 rpm, 10 min). Finally, add freshly prepared ECL color development solution, expose and acquire images in a gel imaging system, and analyze the gray values ​​of the target band protein using ImageJ software.

[0243] 6. Detection of BV2 cell membrane protein NKAα1 endocytosis

[0244] Cell surface proteins were labeled with Biotin-SS-NHS-Sulfo provided in the kit, followed by cell lysis. The lysed samples were then bound to Streptavidin Magnetic Beads. After washing to remove unbound components, elution was performed using DTT-containing elution buffer to obtain biotin-free cell surface proteins. These proteins were subsequently identified by Western blot analysis.

[0245] 7. NKAα1 siRNA-mediated gene silencing in BV2 cells

[0246] (1) The Atp1a1 gene sequence of mouse species Atp1a1 (ID:11928) was obtained by inputting Atp1a1 into the Gene database of the NCBI data platform (https: / / www.ncbi.nlm.nih.gov). The target gene siRNA was synthesized by Jiman Biotechnology Co., Ltd. The specific sequence is shown in Table 8.

[0247] Table 8

[0248]

[0249] (2) After centrifuging the obtained siRNA powder, dissolve it in enzyme-free water, prepare a 20 μM stock solution according to the instructions, and dispense it into 200 μL centrifuge tubes for storage at -20℃.

[0250] 8. siRNA transfection and drug administration to BV2 cells

[0251] Cells in good growth condition were seeded into six-well cell culture plates in 10% FBS antibiotic-free medium, and the cell density was controlled at 0.5~2×10⁶ cells / well. 5 / well, used for transfection after 24 hours. BV2 cells were transfected with NKAα1 siRNA using Lipofectamine™ 3000 transfection reagent. Specific steps are as follows:

[0252] (1) The ultra-clean workbench was disinfected with ultraviolet light before the experiment. BV2 cells were seeded into six-well plates and cultured. When the cells were in good growth condition and the confluence reached 30%-50%, the transfection experiment was performed.

[0253] (2) Take 250 μL centrifuge tubes, add 90 μL of Opti-MEM serum-free medium to each tube, and add 5.0 μL of NKAα1 siRNA stock solution to make the final transfection concentration of siRNA in the cells 50 nM. Gently pipette 3-5 times to mix, and then let stand at room temperature for 5 min.

[0254] (3) In another 250 μL centrifuge tube, add 95 μL of Opti-MEM and then 5.0 μL of Lipofectamine™ 3000 transfection reagent. Gently blow and aspirate 3-5 times to mix and dilute the transfection reagent.

[0255] (4) Slowly add the diluted transfection reagent to a centrifuge tube containing siRNA dilution, gently blow and aspirate 3-5 times to mix, and incubate at room temperature for 20 min to form siRNA-Lipofectamine transfection complex.

[0256] (5) Gently add 200 μL of transfection complex to a six-well plate containing BV2 cells, and gently shake the plate back and forth in a cross shape to distribute the transfection complex evenly.

[0257] (6) After culturing the cells in a 37°C, 5% CO2 incubator for 6 hours, replace the culture medium with fresh complete culture medium and continue culturing for 24 hours.

[0258] (7) For experimental groups that were given drugs at different time points, in order to ensure that the total transfection time of each group is consistent: for example, the 24h drug-giving group was given drugs immediately after changing the culture medium 6h after transfection; while the 12h drug-giving group was given drugs after changing the medium and continuing to culture for 12h before adding drugs.

[0259] 9. Immunoprecipitation

[0260] Logarithmic growth phase BV2 cells were taken, and pre-cooled IP lysis buffer (containing protease inhibitors) was added for lysis on ice. The cells were centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was collected as total protein.

[0261] A portion was used as an input control. The remaining protein was added to pre-equilibrated Protein A / G magnetic beads and incubated at 4°C for 1 hour for pre-cleaning. After centrifugation or magnetic separation, the supernatant was collected, and the primary antibody against the target protein and an equal volume of homologous IgG (negative control) were added, followed by incubation at 4°C overnight. Then, Protein A / G magnetic beads were added and incubation continued for 2-4 hours. After incubation, the magnetic beads were washed 3-5 times with pre-cooled IP lysis buffer, the supernatant was discarded, and SDS loading buffer was added. The mixture was heated at 100°C for 5-10 minutes to dissociate the protein. Finally, SDS-PAGE electrophoresis was performed, and the interaction between the target protein and protein was detected by Western blot.

[0262] 10. Recording of P2X7R-mediated current in BV2 cells

[0263] To record purine energy currents, cells were transferred from the culture medium to a bath containing the components shown in Table 9.

[0264] Table 9

[0265]

[0266] In whole-cell patch-clamp experiments, cells were exposed to a solution with variable concentrations of divalent cations (containing 0.1 mM calcium chloride but without magnesium chloride) to prevent these ions from blocking the purinergic current. The composition of the electrode fluid used to record this current is shown in Table 10.

[0267] Table 10

[0268]

[0269] Data was acquired at a 5kHz sampling rate, filtered at 1kHz using a 7-pole Bessel filter, and digitized using a Digidata 1440A (Molecular Devices, USA).

[0270] 11. Behavioral Experiments

[0271] The behavioral experiments in this section mainly include: open field experiment, novel object recognition experiment, Y-maze experiment, Morris water maze experiment, and conditioned fear experiment.

[0272] (1) Open field test: Open field test (OFT), such as Figure 1 As shown. It is mainly used to assess the spontaneous movement ability, exploratory behavior, and anxiety level of mice. The experimental setup is as follows. Figure 1As shown in Figure 1, an open-type enclosure measuring 38cm × 38cm × 25cm was used, with a camera system mounted on top for real-time recording of mouse behavior. The experimental area was divided into 16 identical squares, with the central four squares defined as the central region and the rest as peripheral regions. During the experiment, mice were gently placed in the center of the enclosure and allowed to move freely for 15 minutes. Behavioral analysis software was used to record and analyze the mice's movement behavior. Key evaluation indicators included average movement speed, total movement distance, and the proportion of time spent in the peripheral regions, to comprehensively assess the motor abilities, exploratory behavior, and anxiety levels of each group of mice.

[0273] (2) Novel object recognition experiment: The novel object recognition (NOR) experiment is used to assess the learning and memory abilities of mice. Its principle is based on the natural tendency of mice to explore novel objects. The experimental setup is the same as that of the open field experiment. Figure 2 (As shown). The experiment included a training phase and a testing phase, with the testing phase conducted 1 hour and 24 hours after training. During the training phase, two identical objects of the same size and shape were placed in a testing box at equal intervals. The mouse was then placed in the center of the box and allowed to explore freely for 8 minutes, during which the exploration time for each object was recorded. During the testing phase, one of the objects was replaced with a new object of a different shape and color. The mouse was then placed back in the testing box for 8 minutes of free exploration at 1 hour and 24 hours after training, and the exploration time for both the old and new objects was recorded. A preference index was calculated as the proportion of exploration time for the new object to the total exploration time for both objects, used to assess the mouse's memory ability.

[0274] (3) Y-maze test: The Y-maze (YM) test is used to assess the spatial recognition and memory ability of mice. Its principle is based on the mouse's exploratory behavior in a new environment. The YM consists of three arms, each 30cm long, 6cm wide, and 15cm high, namely the starting arm, the known arm (old arm), and the unknown arm (new arm). Figure 3 (As shown). The experiment consisted of a training phase and a testing phase. During the training phase, the unknown arm was closed with a barrier, leaving only the starting arm and the known arm open. The mouse was placed in the starting arm and allowed to freely explore the maze for 8 minutes. During the testing phase, the barrier was removed, leaving all three arms open. The mouse was then placed back in the starting arm and allowed to explore freely for 8 minutes. Throughout the experiment, a camera system recorded the number of times the mouse entered the unknown arm and the time spent in it, to assess its spatial recognition and memory abilities.

[0275] (4) Morris Water Maze Test: The Morris water maze (MWM) test is used to assess the spatial learning and memory abilities of mice. The experimental setup is a circular pool with a diameter of 120 cm and a height of 50 cm. Before the experiment, water is added to a depth of about 30 cm, and the water temperature is maintained at 22±2℃. Titanium dioxide is added to make the water milky white to facilitate tracking the movement trajectory of the mice. The experimental environment is isolated by a black curtain, spatial reference objects are set on the pool walls, and a camera system is installed on the top to record the behavior of the mice. Figure 4 (As shown). The pool was virtually divided into four quadrants: left, right, opposite, and target. A 10cm diameter platform was placed in the center of the target quadrant. The experiment lasted 7 days, including a training phase, a spatial exploration experiment, and a visual platform experiment. The training phase lasted 5 days. The platform was fixed in the target quadrant and placed approximately 4cm below the water surface. Mice were introduced from each of the four quadrants daily for training, and the latency to find the platform was recorded. The longest exploration time was 90 seconds. If a mouse found the platform, it stayed for 30 seconds; if it did not find it within 90 seconds, it was guided to the platform and stayed for 30 seconds. On day 6, the spatial exploration experiment was conducted. The platform was removed, and mice were introduced from the opposite quadrant into the pool. They swam freely for 90 seconds, and their swimming time and distance in the target quadrant were recorded. On day 7, the visual platform experiment was conducted. The platform was raised 2-3cm above the water surface, and mice were introduced from the opposite quadrant. The latency to reach the platform was recorded. The spatial learning and memory abilities of mice were evaluated by analyzing behavioral indicators at each stage.

[0276] (5) Conditioned Fear Test: The Fear Conditioning Test (FCT) is used to assess the situational and cue-based fear memory ability of mice, mainly reflecting the learning and memory functions related to the hippocampus and amygdala. The experiment is conducted in a transparent rectangular box (20cm×20cm×30cm), equipped with an audio modulator, an electrical stimulator, and an activity monitoring system, and placed in a soundproof and light-proof box. Figure 5 (As shown). The experiment consisted of a training phase, a situational testing phase, and a cue testing phase. In the training phase, mice were placed in an experimental box and allowed to explore freely for 3 minutes. They were then given a 75 dB sound stimulus for 28 seconds, followed by a 2-second, 0.45 mA plantar electrical stimulation at the end of the sound. The mouse's rigidity time was recorded. After 24 hours, the situational testing phase was conducted. Mice were placed in the same environment and allowed to move freely for 4 minutes. Their rigidity time was recorded under no-stimulation conditions to assess situational memory. A cue testing phase was then conducted 1 hour later. By changing the experimental environment, the baseline rigidity time was recorded first, followed by a 28-second 75 dB sound stimulus. The mouse's rigidity response was recorded. By analyzing the rigidity time of mice at different stages, their situational and cue-based fear memory abilities were evaluated.

[0277] III. Experimental Results

[0278] 1. Molecular docking of Ginkgo biloba extract with NKAα1 in the brain.

[0279] According to the molecular docking results ( Figure 6 As shown in the figure, molecular docking analysis was performed on 14 brain-entering components of ginkgo biloba extract and their target proteins. The results showed that, except for quercetin which failed to form a stable docking conformation, all other components were able to bind to the target proteins to a certain extent. It is generally believed that a molecular docking binding energy less than -5.0 kcal / mol indicates good binding activity between the ligand and receptor. In this study, the binding energies of most components were below this threshold, indicating that these compounds may have stable interactions with the target proteins. Among them, the docking results showed that Clitorin and Narcissoside had the strongest binding ability, with docking energies of -8.7 kcal / mol and -8.3 kcal / mol, respectively. The low binding energies suggest that these two compounds have a high binding affinity for the target proteins and can form stable interactions within the binding pocket. Therefore, it is speculated that Clitorin and Narcissoside may be important active components for the relevant pharmacological effects of ginkgo biloba extract.

[0280] like Figure 6 As shown, (A) is a diagram of the docking structure of various compounds with NKAα1 molecules; (B) is a diagram of the docking binding energy of various compounds with NKAα1 molecules.

[0281] 2. Effects of choleraein and narcissin on BV2 cell viability

[0282] Figure 7 The results showed that the MTT assay was used to detect the effects of Clitorin and Narcissoside on BV2 cell viability. After treatment with different drug concentrations for 24 h, the results indicated that neither compound had a significant effect on BV2 cell viability at concentrations of 20 μM and below. However, when the drug concentration was increased to 40 μM, the viability of BV2 cells decreased to below 80%, suggesting that at this concentration, both compounds had a significant impact on cell viability. Therefore, to avoid interference from the cytotoxicity of the drugs in subsequent experiments, the concentration gradient of Clitorin and Narcissoside was set within 20 μM in subsequent cell experiments.

[0283] like Figure 7 As shown, (A) shows the effect of different concentrations of Clitorin on the viability of BV2 cells; (B) shows the effect of different concentrations of Clitorin on the viability of BV2 cells.

[0284] 3. Inhibition of LPS+ATP-stimulated inflammatory factors in BV2 cells by discus extract and narcissin.

[0285] like Figure 8 As shown, to determine the optimal time for LPS+ATP stimulation of BV2 cells to induce an inflammatory response, this study first examined the expression level of il-1β mRNA at different time points. qRT-PCR results showed that within a 24-hour observation period, the highest level was reached at 4 hours, indicating that the inflammatory response in BV2 cells was most significant at this time. Therefore, subsequent experiments all selected 4 hours as the treatment time for LPS+ATP stimulation of BV2 cells. Based on this, to compare the effects of Clitorin and Narcissoside on the inflammatory response of BV2 cells, this study further examined the regulatory effects of the two compounds on il-1β mRNA expression. qRT-PCR results showed that both compounds reduced the expression level of il-1β mRNA to some extent, with Clitorin showing a more significant inhibitory effect. When the dosage concentration was 10 μM, Clitorin significantly reduced the transcription level of il-1β mRNA, suggesting that it has a good inhibitory effect on LPS+ATP-induced inflammatory responses in BV2 cells. Therefore, in subsequent experiments, Clitorin was selected as the main research object to further explore its regulatory role in the inflammatory response of BV2 cells and related molecular mechanisms.

[0286] like Figure 8 As shown, (A) is a statistical graph of il-1β mRNA in BV2 cells stimulated by LPS+ATP at different time points; (B) is a statistical graph of il-1β mRNA in BV2 cells stimulated by LPS+ATP at different concentrations of Clitorin; and (C) is a statistical graph of il-1β mRNA in BV2 cells stimulated by LPS+ATP at different concentrations of Narcissoside.

[0287] 4. Butterfly pea extract can bind directly to NKAα1 protein, enhancing its thermal stability.

[0288] The CETSA assay was used to evaluate the effect of Clitorin on the thermostability of NKAα1 protein. For example... Figure 9 Figure A shows the changes in NKAα1 protein bands with temperature between the Clitorin-treated group and the DMSO control group under a temperature gradient of 38-82℃. Overall, the intensity of the NKAα1 protein bands in both groups decreased with increasing temperature, but the attenuation was significantly reduced after Clitorin treatment. Quantitative analysis of the band gray values ​​was performed. Figure 2.4B) It was found that there was no significant difference in NKAα1 protein levels between the two groups at 46℃; when the temperature rose to 50℃, the protein content in the control group decreased significantly, while the decrease in protein levels in the Clitorin-treated group was relatively smaller. This difference further increased in subsequent higher temperature ranges, suggesting that Clitorin treatment can maintain the relative stability of NKAα1 protein under heat denaturation conditions. These results suggest that Clitorin may enhance the thermal stability of NKAα1 protein and delay the heat-induced protein denaturation process by interacting with it. This ligand-mediated protein stabilization effect may be related to its regulation of NKAα1-related signal transduction processes and the exertion of its biological effects.

[0289] like Figure 9 As shown, (A) is a band plot of NKAα1 at different heating temperatures; (B) is a statistical plot of NKAα1 bands. n=3, and the results are shown as mean±SEM. P*<0.05, P**<0.01, and P***<0.001 indicate significant differences.

[0290] 5. Butterfly pea extract inhibits the activation of the NLRP3 inflammasome in BV2 cells stimulated by LPS+ATP.

[0291] Western blot was used to detect the effect of cleitorin on NLRP3 inflammasome activation in BV2 cells. Results are as follows: Figure 10 The results showed that Clitorin treatment had no significant effect on the expression of NLRP3 inflammasome-related proteins under basal conditions. However, after LPS pretreatment followed by ATP stimulation, the expression of inflammasome-related proteins such as NLRP3, caspase-1, and IL-1β in BV2 cells was significantly increased. After Clitorin intervention, the expression levels of these inflammasome-related proteins significantly decreased. These results indicate that Clitorin can significantly inhibit LPS+ATP-induced NLRP3 inflammasome activation in BV2 cells.

[0292] like Figure 10 As shown, (A) is a band diagram of 4 groups of cellular proteins; (BD) is a statistical diagram of NLRP3, Caspase-1, and ASC protein bands. n=3, and the results are shown as mean±SEM. P***<0.001 indicates significant difference.

[0293] 6. Butterfly pea extract inhibits LPS+ATP-stimulated protein endocytosis of NKAα1 in BV2 cells.

[0294] To further explore the regulatory mechanism of Clitorin on NKAα1 membrane localization, this study used a cell surface protein biotinylation and isolation kit to label and isolate BV2 cell surface proteins, thereby detecting the distribution of NKAα1 on the cell membrane (e.g., ...). Figure 11 (As shown in the image). The results showed that, compared with the Ctrl group, the NKAα1 membrane protein level in the Ctrl+Clitorin group did not change significantly; however, under LPS+ATP stimulation, the NKAα1 protein level on the BV2 cell membrane surface decreased significantly, suggesting that inflammatory stimulation can lead to NKAα1 endocytosis from the cell membrane, reducing its membrane localization. Further observation revealed that in the LPS+ATP+Clitorin treatment group, the NKAα1 protein level on the cell membrane surface was significantly higher than that in the LPS+ATP group, indicating that Clitorin can significantly restore the distribution of NKAα1 on the cell membrane under inflammatory stimulation. These results suggest that LPS+ATP stimulation may reduce NKAα1 membrane expression by promoting NKAα1 endocytosis, while Clitorin can inhibit this process, thereby maintaining the localization of NKAα1 on the cell membrane.

[0295] like Figure 11 As shown, (A) is a band diagram of 4 groups of cellular proteins; (B) is a statistical diagram of 4 groups of cellular proteins. n=3, and the results are shown as mean±SEM. P***<0.001 indicates significant difference.

[0296] 7. Butterfly pea extract enhances the interaction between NKAα1 and P2X7 in LPS+ATP-stimulated BV2 cells.

[0297] To investigate the effect of Clitorin on the interaction between NKAα1 and P2X7R, this study used Co-IP technology to detect the binding between the two proteins in BV2 cells. Forward and reverse co-precipitation were performed using either NKAα1 or P2X7R as the immunoprecipitation antibody. The results are as follows: Figure 12 The results showed that no obvious specific bands were detected in the IgG group, indicating that the experimental system had good specificity. Compared with the Ctrl group, the binding levels of NKAα1 and P2X7R in BV2 cells were significantly reduced after LPS+ATP stimulation; however, the binding levels of both were significantly increased after Clitorin treatment compared to the LPS+ATP group. The results of forward and reverse coprecipitation were consistent, suggesting that Clitorin can enhance the interaction between NKAα1 and P2X7R under inflammatory stimulation.

[0298] like Figure 12 As shown, (A) is a protein expression band plot of NKAα1 pulled down using P2X7R; (B) is a protein expression band plot of NKAα1 pulled down using P2X7R; n=3.

[0299] 8. Butterfly pea extract inhibits P2X7R-mediated currents in BV2 cells with knocked-down NKAα1.

[0300] Whole-cell patch-clamp technique was used to detect changes in P2X7R-mediated currents in BV2 cells after siRNA knockdown of NKAα1. Results are as follows: Figure 13 The results showed that, under basal conditions, Clitorin treatment had no significant effect on BV2 cell membrane currents. However, after siRNA knockdown of NKAα1, P2X7R-mediated currents in BV2 cells were significantly enhanced, suggesting that NKAα1 deficiency can promote P2X7R channel activity. Further Clitorin treatment significantly reduced the amplitude of this enhanced current. Simultaneous detection of potassium ion concentration in the extracellular culture medium revealed that NKAα1 knockdown significantly increased extracellular K⁺ levels, while Clitorin treatment partially reduced this trend. These results indicate that NKAα1 knockdown enhances P2X7R-mediated ion channel activity and promotes potassium ion efflux in BV2 cells, while Clitorin can inhibit this process to some extent.

[0301] like Figure 13 As shown, (A) is a statistical graph of siRNA knockdown of NKAα1 mRNA; (B) is an example graph of P2X7R-mediated current; (C) is a statistical graph of the amplitude of P2X7R-mediated current (n=12); and (D) is a statistical graph of extracellular potassium ion concentration (n=8). Results are shown as mean ± SEM, and P*** < 0.001 indicates significant difference.

[0302] 9. Butterfly pea extract does not affect the motor function of PScDKO mice.

[0303] The effect of chondromycin on the voluntary movement ability of mice was assessed using OFT. Results are as follows: Figure 14 The results showed that, compared with WT mice, PScDKO mice exhibited increased total distance and average speed in the open field. Treatment with discus extract did not significantly alter the total distance or speed in either WT or PScDKO mice. Furthermore, there were no significant differences in the proportion of time spent at the edge of the open field under different treatment conditions. These results indicate that discus extract treatment does not significantly affect the spontaneous movement ability or anxiety-related behaviors of WT and PScDKO mice.

[0304] like Figure 14 As shown, (A) represents the total distance traveled by the four groups of mice in the open field experiment; (B) represents the average moving speed of the four groups of mice in the open field experiment relative to the total distance traveled; and (C) represents the percentage of time spent in the edge zone by the four groups of mice in the open field experiment. Results for n=12 mice are shown as mean±SEM, and P***<0.001 indicates significant difference.

[0305] 10. Butterfly pea extract improves recognition and memory impairment in PScDKO mice.

[0306] The effect of chondromycin on the recognition and memory abilities of PScDKO mice was assessed using the NOR assay. Results are as follows: Figure 15The results showed that, in the 1-hour test, PScDKO mice exhibited a significantly reduced proportion of time spent exploring new objects compared to WT mice, indicating impaired short-term recognition memory. Treatment with butrographis paniculata significantly increased the proportion of time spent exploring new objects in PScDKO mice and significantly improved their recognition index, while no significant changes were observed in WT mice after butrographis paniculata treatment. Further testing in the 24-hour test revealed no significant differences in the proportion of time spent exploring new objects among the different treatment conditions. These results indicate that butrographis paniculata can improve short-term recognition memory impairment in PScDKO mice, but has no significant effect on long-term recognition memory.

[0307] like Figure 15 As shown, (A) represents the percentage of time spent exploring objects at each stage among the four groups of mice; (B) represents the percentage of time spent exploring objects at each stage among the four groups of mice. Results for n=12 mice are shown as mean±SEM. P*<0.05 and P***<0.001 indicate significant differences.

[0308] 11. Butterfly pea extract improves spatial and associative memory impairment in PScDKO mice.

[0309] YM experiment results showed that WT mice exhibited more pronounced exploratory behavior in the new arm region, while PScDKO mice showed significantly reduced activity in the new arm region. Figure 16 Further investigation revealed that the frequency of PScDKO mice entering the new arm and the duration of their stay in the new arm were both reduced. Treatment with discus extract increased the exploratory behavior of PScDKO mice in the new arm region, with improvements in both the number of times they entered the new arm and the duration of their stay. Conditioned fear tests showed no significant difference in freeze time among the groups during the training phase, suggesting that the initial behavioral states of the mice were consistent. In the FCT, the freeze time of PScDKO mice was significantly shorter than that of WT mice, indicating impaired situational associative memory; however, discus extract treatment increased the freeze time of PScDKO mice. In the cue test, the freeze response of PScDKO mice to auditory stimuli was also reduced, and this response improved after discus extract treatment. These results indicate that discus extract can, to some extent, improve spatial and associative memory impairments in PScDKO mice.

[0310] like Figure 16 As shown, (A) represents the percentage of times the four groups of mice entered the new arm for exploration; (B) represents the percentage of time the four groups of mice spent exploring the new arm; and (C) represents the percentage of time the mice remained still during each stage of the conditioned fear test. Results for n=12 mice are shown as mean±SEM. P*<0.05, P**<0.01, and P***<0.001 indicate significant differences.

[0311] 12. Butterfly pea extract improves spatial reference memory impairment in PScDKO mice.

[0312] To assess the effects of discus extract on spatial learning and memory abilities in PScDKO mice, the Morris water maze (MWM) test was used. Figure 17 The results showed that during the training phase, the escape latency of all groups of mice gradually shortened with increasing training time, indicating the gradual formation of spatial learning. Compared with WT mice, PScDKO mice showed a slower decline in latency in the later stages of training, with a significantly lagging learning curve, suggesting impaired spatial learning ability. Treatment with discus extract improved the learning curve and shortened the escape latency of PScDKO mice. In the spatial exploration test, WT mice mainly searched within the quadrant where the original platform was located, while the swimming trajectories of PScDKO mice were more dispersed. After discus extract treatment, the search behavior of PScDKO mice in the target quadrant significantly increased. Further analysis revealed that PScDKO mice had a reduced swimming distance and dwell time in the target quadrant, and fewer instances of crossing the original platform location; all of these indicators improved after discus extract treatment. In the visual platform test, there were no significant differences in swimming speed and time to reach the platform among the groups of mice, suggesting that PScDKO and discus extract treatments did not significantly affect the visual function or basic motor abilities of the mice. The above results indicate that butterfly bean extract can improve spatial learning and memory impairment in PScDKO mice.

[0313] like Figure 17 As shown, (A) is a statistical graph of the escape latency of mice in each group during the training phase; (B) is the average movement speed of mice in each group during the visualization phase; (C) is the total number of times mice crossed the target platform; (D) is the percentage of distance traveled across the target quadrant; (E) is the percentage of time taken to cross the target quadrant; and (F) is the time taken for mice in each group to reach the platform during the visualization phase. Results for n=12 mice are shown as mean ± SEM. P*<0.05, P**<0.01, and P***<0.001 indicate significant differences.

[0314] 13. Butterfly pea extract enhances the interaction between NKAα1 and P2X7 in PScDKO mice.

[0315] To investigate the effect of chondroidin on the interaction between NKAα1 and P2X7R, a Co-IP experiment was used. The results are as follows: Figure 18 The results showed that NKAα1 and P2X7R interacted to some extent in WT mice, while the binding level of the two was significantly reduced in PScDKO mice. Treatment with discus extract significantly enhanced the interaction between NKAα1 and P2X7R in PScDKO mice. No obvious nonspecific bands were detected in the IgG group. These results indicate that discus extract can promote the interaction between NKAα1 and P2X7R in the brain tissue of PScDKO mice.

[0316] like Figure 18 As shown, (A) is a protein expression band of NKAα1 pulled down using P2X7R; (B) is a protein expression band of NKAα1 pulled down using P2X7R; n=3 mice.

[0317] 14. Butterfly pea extract inhibits the activation of the NLRP3 inflammasome in PScDKO mice.

[0318] To evaluate the effect of chondromycin on NLRP3 inflammasome activation in the brains of PScDKO mice, the expression levels of NLRP3 inflammasome-related proteins NLRP3, ASC, and Caspase-1 were measured. Results are as follows: Figure 19 The results showed that, compared with WT mice, the expression of NLRP3, ASC, and Caspase-1 was significantly increased in PScDKO mice, indicating that the NLRP3 inflammasome was activated. Treatment with discus extract significantly reduced the expression levels of these proteins. These results indicate that discus extract can inhibit the activation of the NLRP3 inflammasome in the brain of PScDKO mice, thereby alleviating the inflammatory response.

[0319] like Figure 19 As shown, (A) is a band diagram of NLRP3, Caspase-1, and ASC protein expression in the HPC of the four groups of mice; (BD) is a statistical graph of the relative expression levels of NLRP3, Caspase-1, and ASC proteins in the PFC of the four groups of mice; (E) is a band diagram of NLRP3, Caspase-1, and ASC protein expression in the PFC of the four groups of mice; (FH) is a statistical graph of the relative expression levels of NLRP3, Caspase-1, and ASC proteins in the PFC of the four groups of mice. n=3 mice, and the results are shown as mean±SEM. P**<0.01 and P***<0.001 indicate significant differences.

[0320] IV. Conclusion

[0321] Experimental studies have found that:

[0322] (1) Molecular docking results showed that the brain-entering components of ginkgo biloba extract, discoidin and narcissin, had a good binding ability with NKAα1.

[0323] (2) Butterfly pea extract and narcissin had no significant effect on the viability of BV2 cells within the experimental concentration range, and could inhibit the production of LPS+ATP-induced inflammatory factors.

[0324] (3) Butterfly pea extract can directly bind to NKAα1 protein and enhance its thermal stability, while inhibiting LPS+ATP-induced NLRP3 inflammasome activation.

[0325] (4) Butterfly pea extract can inhibit the internalization of NKAα1 protein under inflammatory stimulation and enhance the interaction between NKAα1 and P2X7R;

[0326] (5) Knockdown of NKAα1 can enhance P2X7R-mediated current and promote potassium ion efflux in BV2 cells, while chocolate can significantly inhibit this change.

[0327] (6) Butterfly pea extract improves learning and memory dysfunction in PScDKO mice;

[0328] (7) Butterfly pea extract enhances the interaction between NKAα1 and P2X7R;

[0329] (8) Butterfly pea extract inhibits the activation of the NLRP3 inflammasome.

Claims

1. The application of discus extract in ginkgo biloba ester in improving neuroinflammatory cognitive impairment, characterized in that, The following steps are used: S1. First, we screened the active monomers of ginkgo biloba extract that may act on NKAα1 through molecular docking, and verified their direct binding ability to NKAα1 through CETSA. S2, then in an LPS+ATP-induced microglial inflammation model, the regulatory effects of this monomer on NKAα1 endocytosis, NKAα1-P2X7R interaction, P2X7R-mediated current and K⁺ efflux, and NLRP3 inflammasome activation were systematically evaluated, and its anti-inflammatory molecular mechanism was elucidated. S3. Finally, through whole-animal level experiments, we further explored the role of discus extract in improving cognitive impairment in PScDKO mice and its regulatory effect on the NKAα1-P2X7-NLRP3 signaling axis.

2. The application of discus extract in ginkgo biloba ester according to claim 1 in improving neuroinflammatory cognitive impairment, characterized in that, The specific process of step S1 is as follows: S1-1, Molecular docking verification of Ginkgo biloba extract for brain absorption with NKAα1: (1) The 3D structures of 14 active ingredients of ginkgo biloba extract entering the brain were downloaded from the PubChem database as ligands and converted into mol2 format using OpenBabel. (2) The crystal structure of NKAα1 protein was obtained from RCSB Protein Data Bank as the receptor and pretreated with PyMOL 2.6.0 for dehydration. (3) Subsequently, the ligand was minimized and the protein activity pocket was identified in Molecular Operating Environment 2019 software to complete the ligand-receptor molecular docking. The results were visualized using PyMOL and the binding activity was evaluated based on the binding energy. S1-2, Tetramethylazozid salt colorimetric method for detecting the effects of chondruvicin and narcisin on BV2 cell viability: (1) Take BV2 cells in the logarithmic growth phase and seed them in 96-well plates. After the cells adhere to the plate, discard the culture medium and add culture medium containing 1.25, 2.5, 5, 10, 20, 40, and 80 μM litorin or narcissoside to treat the cells. 200 μL per well, 6 replicates per group, and a control group is also set up. (2) After 24 hours of treatment, add 20 μL of 5 mg / mL MTT solution to each well and continue incubation for 4 hours; (3) Then discard the culture medium, add 150 μL of dimethylsulfoxide to each well, shake on a shaker to dissolve and crystallize, and measure the OD value at a wavelength of 570 nm to evaluate cell viability; S1-3, qRT-PCR detection: (1) After removing the cell supernatant, add 1 mL of PBS to each well for washing, discard the PBS, add 500 μL of cell lysis buffer to each well, let stand at room temperature for 1 min, and then aspirate the lysis buffer from the well into the tube. (2) Centrifuge at 12000 rpm for 1 min and discard the liquid inside the outer tube; (3) Add 700 μL of wash buffer to each tube and centrifuge at 12,000 rpm for 1 min; (4) Discard the washing solution and repeat step (3) once, that is, add 700 μL of wash buffer to each tube, centrifuge at 12000 rpm for 1 min; (5) Discard the washing solution and centrifuge at 12,000 rpm for 2 min; (6) Transfer the inner tube to a clean 1.5 mL EP tube, open the cap and let it stand for 3 minutes to allow the alcohol in the inner tube to evaporate completely. (7) Add 30 μL LEPC water, let stand for 2 min, centrifuge at 12000 rpm for 1 min, discard the inner tube, and detect the RNA concentration in the EP tube; S1-4, BV2 cell heat transfer experiment: (1) Take BV2 cells in the logarithmic growth phase, treat them with lysis buffer and collect the supernatant protein; (2) Add 100 μM litorin or an equal volume of DMSO control to the protein and incubate on ice for 2 h; (3) Dispense into 1.5mL centrifuge tubes, 80μL per tube, set 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82℃, react for 3min, place at room temperature for 3min, add loading buffer and denature in 100℃ water bath for 10min for subsequent analysis; S1-5, Western blot analysis: (1) Extraction of cell proteins: Prepare lysis buffer in advance and keep it on ice; (2) After the drug treatment is completed, discard the culture medium, wash with PBS 2-3 times, add 100μL of lysis buffer to each well, collect the cells with a scraper and blow them evenly, transfer them to a 1.5mL centrifuge tube, centrifuge at 12000rpm and 4℃ for 15min, and collect the supernatant as the total cell protein.

3. The application of discus extract in ginkgo biloba ester according to claim 2 in improving neuroinflammatory cognitive impairment, characterized in that: The ligand-receptor molecular docking was performed 50 times.

4. The application of discus extract in ginkgo biloba ester according to claim 2 in improving neuroinflammatory cognitive impairment, characterized in that: The specific procedure for Western blot testing is as follows: (1) Extraction of cell proteins: ① Prepare the lysis buffer according to the ratio of RIPA lysis buffer, protease inhibitor: PMSF: phosphatase inhibitor = 100:1:1:1; ② Take about 30 mg of HPC or PFC tissue and add 100 µL of lysis buffer into a grinding tube. After adding steel balls, grind the tissue at 90 Hz for 60 s in a cryogenic grinder. ③Then centrifuge at 4℃ and 12000rpm for 15min, and collect the supernatant as the total tissue protein; extract membrane proteins according to the instructions of the cell membrane protein and cytoplasmic protein extraction kit; (2) BCA protein quantification and protein preparation: ① Prepare standard gradients of 1 mg / mL protein standard by pipetting the corresponding volumes into EP tubes, with concentrations of 1, 0.8, 0.5, 0.4, 0.2, 0.1, 0.05, and 0 mg / mL respectively; ②Then add 20 μL of standard or sample to a 96-well plate, with 3 replicates per group; ③ Prepare BCA working solution according to solution A: solution B = 50:1, add 200 μL to each well, incubate at 37℃ for 30 min, measure the OD value at 562 nm, plot the standard curve and calculate the protein concentration of the sample; ④ Based on the measured concentration, add 5× loading buffer and RIPA lysis buffer to prepare a protein sample with a final concentration of 4 μg / μL containing 1× loading buffer. Denature the sample by heating at 100℃ for 10 min and store at -20℃ for later use. (3) Preparation of SDS-PAGE gel: ① The experiment used a 1.5mm thick glass plate to assemble the electrophoresis gel preparation device. 10% separating gel was prepared in a 50mL centrifuge tube. After mixing, 7.8mL was added to the gel preparation device and sealed with anhydrous ethanol. ② After the separating gel has completely solidified, discard the ethanol and allow it to dry. (4) Western blot electrophoresis: Prepare the electrophoresis solution according to the required ratio, and place it in a magnetic stirrer to stir and dissolve it evenly; (5) Transfer: Prepare the transfer solution in advance according to the required ratio, place it in a magnetic stirrer and stir until fully dissolved, then seal and pre-cool in a refrigerator at 4°C; (6) Western blot blocking, incubation and color development: ① After the transfer is complete, place the NC membrane in 5% BSA or 5% skim milk powder and seal it in a shaker at room temperature for 60-120 minutes; ② Discard the blocking solution and rinse briefly with TBST, then add diluted primary antibody and incubate overnight at 4°C on a shaker at low speed; ③ The primary antibody was recovered the next day, and the membrane was washed three times at room temperature using TBST. ④Then add the corresponding secondary antibody, incubate on a shaker at room temperature for 90 minutes, and then wash the membrane 3 times with TBST at room temperature; ⑤ Finally, add the freshly prepared ECL colorimetric solution, expose and acquire images in a gel imaging system, and use ImageJ software to analyze the gray values ​​of the target band protein.

5. The application of discus extract in ginkgo biloba ester according to claim 1 in improving neuroinflammatory cognitive impairment, characterized in that, The specific process of step S2 is as follows: Detection of S2-1, BV2 cell membrane protein NKAα1 endocytosis: (1) Cells were lysed after labeling the proteins on the cell surface with Biotin-SS-NHS-Sulfo provided in the kit. The lysed samples were then bound to Streptavidin Magnetic Beads. (2) After washing to remove unbound components, elute with DTT-containing eluent to obtain cell surface proteins without biotin labeling. The cell surface proteins can then be identified by Western blot analysis. S2-2, NKAα1 siRNA-mediated gene silencing in BV2 cells: (1) Input Atp1a1 into the Gene database in the NCBI data platform to obtain the gene sequence of Atp1a1 of mouse species, and synthesize the siRNA of the target gene; (2) After centrifuging the obtained siRNA powder, dissolve it in enzyme-free water to prepare a 20 μM stock solution and dispense it into 200 μL centrifuge tubes for storage at -20℃ for later use. S2-3, siRNA transfection and drug administration in BV2 cells: (1) Take cells in good growth condition and seed them in a six-well cell culture plate with 10% FBS antibiotic-free medium, and control the cell density to 0.5~2×105 / well for transfection after 24h; (2) BV2 cells were transfected with NKAα1 siRNA using Lipofectamine™ 3000 transfection reagent; S2-4, Immunoprecipitation: (1) Take BV2 cells in the logarithmic growth phase, add pre-cooled IP lysis buffer and lyse on ice, centrifuge at 4℃ and 12000rpm for 15min, and collect the supernatant as total protein. (2) And take a portion as the Input for comparison; (3) The remaining protein was added to equilibrated Protein A / G magnetic beads and incubated at 4°C for 1 hour for pre-cleaning; (4) After centrifugation or magnetic separation, take the supernatant, add the primary antibody against the target protein and an equal amount of homologous IgG, and incubate overnight at 4°C by rotation. (5) Then add Protein A / G magnetic beads and continue incubation for 2-4 hours; (6) After incubation, wash the magnetic beads 3-5 times with pre-cooled IP lysis buffer, discard the supernatant and add SDS loading buffer, heat at 100℃ for 5-10 min to dissociate the protein; (7) Finally, SDS-PAGE electrophoresis was performed and the interaction of the target proteins was detected by Western blot. Recording of P2X7R-mediated current in BV2 cells, S2-5: (1) To record purine energy currents, cells were transferred from the culture medium to BV2 extracellular fluid; (2) In whole-cell patch-clamp experiments, cells are exposed to a solution with variable concentrations of divalent cations, and the composition of the electrode fluid used to record the current is recorded. (3) The data was acquired at a sampling rate of 5 kHz, filtered at 1 kHz through a 7-pole Bessel filter, and digitized using a Digidata1440A.

6. The application of discus extract in ginkgo biloba ester according to claim 5 in improving neuroinflammatory cognitive impairment, characterized in that: The specific procedure for transfecting BV2 cells with NKAα1 siRNA using Lipofectamine™ 3000 transfection reagent is as follows: ① Before the experiment, the ultra-clean workbench was disinfected with ultraviolet light. BV2 cells were seeded in six-well plates and cultured. When the cells were growing well and the confluence reached 30%-50%, the transfection experiment was carried out. ② Take a 250μL centrifuge tube, add 90μL of Opti-MEM serum-free medium to each tube, add 5.0μL of NKAα1 siRNA stock solution to make the final transfection concentration of siRNA in the cells 50nM, gently pipette 3-5 times to mix, and then let stand at room temperature for 5min. ③ In another 250μL centrifuge tube, add 95μL of Opti-MEM, then add 5.0μL of Lipofectamine™ 3000 transfection reagent, and gently pipette 3-5 times to mix and dilute the transfection reagent; ④ Slowly add the diluted transfection reagent to the centrifuge tube containing the siRNA dilution solution, gently pipette 3-5 times to mix, and incubate at room temperature for 20 minutes to form the siRNA-Lipofectamine transfection complex. ⑤ Gently add 200 μL of the transfection complex to a six-well plate containing BV2 cells, and gently shake the plate back and forth in a cross shape to distribute the transfection complex evenly. ⑥ After culturing the cells in a 37℃, 5% CO2 incubator for 6 hours, replace the culture medium with fresh complete medium and continue culturing for 24 hours; ⑦ For experimental groups that were given medication at different time points, ensure that the total transfection time is consistent for each group.

7. The application of discus extract in ginkgo biloba ester according to claim 5 in improving neuroinflammatory cognitive impairment, characterized in that: The solution with variable divalent cation concentration is a solution that does not contain magnesium chloride and contains 0.1 mM calcium chloride.

8. The application of cholerae extract in ginkgo biloba ester according to claim 1 in improving neuroinflammatory cognitive impairment, characterized in that: In step S3, the whole animal level experiments include the open field experiment, the new object recognition experiment, the Y-maze experiment, the Morris water maze experiment, and the conditioned fear experiment.

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