Application of domoic acid in preparation of medicine for removing beta-amyloid protein

Abstract

The invention discloses application of domoic acid in preparation of a medicine for removing beta-amyloid protein, and belongs to the technical field of biology. The concentration of the domoic acid is 100 to 500 nM; the domoic acid is used for promoting microglial cells to phagocytize beta-amyloid protein; experiments prove that 100-500nM of DA can promote the microglial cells to phagocytize the beta-amyloid protein, and does not influence the activity of the microglial cells. The discovery provides a new choice and technical support for promoting the microglial cells to phagocytize the beta-amyloid protein or clearing the beta-amyloid protein, and has important research and clinical application values.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of domoic acid in the preparation of a drug for clearing β-amyloid protein. Background Art

[0002] As the core effector cells for clearing Aβ in the central nervous system, the dynamic balance of the chemotactic migration ability and phagocytic function of microglia is a key link in regulating the pathological development. Existing studies have shown that microglia specifically recognize and phagocytose Aβ deposits through surface receptors, and then degrade and clear plaques through lysosomal enzymes. This process can significantly reduce the Aβ pathological burden and delay neuronal damage.

[0003] Currently, the treatment strategies for Aβ clearance mainly include monoclonal antibody therapy and microglia activators, but both have significant limitations. Monoclonal antibodies (such as Aducanumab) have a low blood-brain barrier penetration rate and require high-dose and high-frequency administration to reach the therapeutic concentration. AL002, as a microglia activator, can target and enhance the function of the TREM2 protein, but did not show effective therapeutic effects in phase II clinical trials.

[0004] The literature "Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia" discloses that short-term in vitro exposure to 10 μM - 1 mM domoic acid may lead to the activation of rat neonatal microglia, but is accompanied by the release of the pro-inflammatory mediators tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9). Currently, the prior art does not disclose whether domoic acid at higher or lower concentrations will have an impact on microglia. Summary of the Invention

[0005] The object of the present invention is to provide the application of domoic acid in the preparation of a drug for clearing β-amyloid protein, so as to solve the problems existing in the above prior art. 100 - 500 nM DA can promote microglia to phagocytose β-amyloid protein without affecting the activity of microglia.

[0006] To achieve the above object, the present invention provides the following solution:

[0007] The present invention provides the use of domoic acid in the preparation of a drug for clearing β-amyloid protein, and the concentration of the domoic acid is 100-500 nM.

[0008] Optionally, the domoic acid promotes microglia to phagocytose β-amyloid protein.

[0009] The present invention also provides the use of domoic acid in the preparation of a preparation for promoting microglia to phagocytose β-amyloid protein.

[0010] The present invention also provides a drug for clearing β-amyloid protein, and the active ingredient is domoic acid;

[0011] The concentration of the domoic acid is 100-500 nM.

[0012] Optionally, the domoic acid promotes microglia to phagocytose β-amyloid protein.

[0013] The present invention also provides a preparation for promoting microglia to phagocytose β-amyloid protein, and the active ingredient is domoic acid; the concentration of the domoic acid is 100-500 nM.

[0014] The present invention discloses the following technical effects:

[0015] Verified by experiments, DA at 100-500 nM can activate microglia to promote microglia to phagocytose β-amyloid protein, and has no effect on the activity of microglia. This discovery of the present invention provides a new option and technical support for promoting microglia to phagocytose β-amyloid protein or clearing β-amyloid protein, and has important research and clinical application values. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1 For the effect of DA on the Aβ phagocytosis efficiency of HMC3 cells detected by flow cytometry; A: Representative flow cytometry result graph; B: The proportion of FITC-positive HMC3 cells in the total cell number; C: Statistical analysis of the average fluorescence intensity of FITC in FITC-positive HMC3 cells;

[0018] Figure 2Effect of DA on the phagocytosis efficiency of Aβ by primary microglia in mice; A: Representative immunofluorescence results; B: Statistical analysis of the proportion of FITC-positive microglia in the total number of cells; C: Fluorescence intensity of FITC in FITC-positive microglia.

[0019] Figure 3 To detect the effect of DA treatment on the viability of HMC3 cells by MTT assay. Detailed implementation manners

[0020] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0021] It should be understood that the terms used in the present invention are only for describing particular implementation manners and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0023] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0024] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0025] The domoic acid (DA) used in the present invention has the molecular formula C 15 H 21 NO6, and is purchased from Dalian Tomson Yibai Biotechnology Co., Ltd., batch number: GBW(E)100782, purity > 99%.

[0026] Effect of DA on the phagocytic function of HMC3 cells in Example 1

[0027] To evaluate the effect of DA on the phagocytic function of microglia, in this example, HMC3 cells were treated with 100 nM and 500 nM of DA for 12 hours respectively, and then co-cultured with fibrillar FITC-labeled amyloid-β 1-42 (Aβ1-42) for 12 hours, and its phagocytosis was evaluated by flow cytometry.

[0028] 1. Seeding of HMC3 cells and drug treatment

[0029] (1) Cell seeding: HMC3 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 1×10 6 cells / well, and 2 mL of DMEM / F12 medium containing 10% fetal bovine serum (FBS) was added to each well;

[0030] (2) Plate treatment: The culture plate was shaken 5 times by the horizontal cross method to ensure uniform cell distribution, and then incubated in an incubator at 37°C and 5% CO2 for 24 hours until the cells were completely adherent;

[0031] (3) Drug pretreatment: The original medium was discarded, and the three experimental groups were respectively added with complete medium containing 100 nM DA and 500 nM DA, and the blank control group was not treated, and the cells were treated for 12 h under the culture conditions.

[0032] 2. Evaluation of the phagocytic function of cells on Aβ peptides by flow cytometry

[0033] The FITC-labeled Aβ1-42 peptide (product number: M212900, Beijing Myriad Genetics Co., Ltd.) was dissolved in DMSO to prepare a 200 μM stock solution, which was aliquoted and stored at -80°C for later use.

[0034] (1) Twelve hours before use, the FITC-Aβ1-42 stock solution was diluted to 100 μM with PBS and incubated in a 37°C constant temperature incubator for 12 hours to promote Aβ fiber cross-linking;

[0035] (2) After the cells were treated with the drug for 12 hours, the cell culture medium was replaced with serum-free medium (containing the drug) and fibrillar FITC-Aβ1-42 with a final concentration of 0.5 μM was added, and the cells were cultured for another 12 h;

[0036] (3) After 24 hours, the cells were digested and stained with 5 μg / mL of DAPI to label dead cells;

[0037] (4) Detect HMC3 cells by flow cytometry. DAPI-negative cells are living cells. According to the FITC fluorescence signal intensity in living cells, quantitatively analyze the effect of DA on the phagocytosis level of FITC-Aβ1-42 by HMC3 cells.

[0038] 6. Experimental Results

[0039] 6.1 DA increases the phagocytosis of Aβ by HMC3 cells

[0040] The results are as Figure 1 shown. In the blank group, the proportion of FITC-positive cells in HMC3 cells was 43%. After treatment with 100 nM DA, the proportion of FITC-positive cells was 54%. At the same time, in HMC3 cells treated with 100 nM DA, the amount of Aβ phagocytosed by phagocytosis-positive cells (expressed as the mean fluorescence intensity in FITC-positive cells) increased by 1.19 times compared with the blank control group, with statistical significance (**, p < 0.001). After treatment with 500 nM DA, the proportion of FITC-positive HMC3 cells was 51%, and at the same time, the amount of Aβ phagocytosed by phagocytosis-positive cells also increased by 1.11 times. Therefore, it is considered that 100 nM and 500 nM DA significantly promoted the phagocytosis of Aβ by HMC3 cells.

[0041] At the same time, this example further found that treatment with 100 - 500 nM DA did not lead to an increase in the level of inflammatory factors. This finding indicates that 100 - 500 nM DA does not induce a strong inflammatory response while promoting the phagocytosis of β-amyloid by HMC3.

[0042] Example 2. Effect of DA on the phagocytic function of primary mouse microglia

[0043] 1. Isolation and culture of primary mouse microglia

[0044] (1) Pretreatment of culture flask: 18 hours before the experiment, add 0.5 mg / mL polylysine solution to a T25 culture flask and coat it overnight at 37°C;

[0045] (2) Preparation of single-cell suspension: Take the midbrain tissue of P1 (one day after birth) mice, treat it with a Neural Tissue Dissociation Kit (130 - 094 - 802, Miltenyi Biotec), filter it through a 70 μm filter, and resuspend it in glial cell medium (DMEM, 10% FBS, 1% double antibody) after centrifugation at 300 × g for 10 min;

[0046] (3) Culture of mixed glial cells: Inoculate the cells into a pre-coated T25 flask and culture them at 37°C and 5% CO2 for 7 - 8 days;

[0047] (4) Microglia isolation: Incubate on a shaker at 37°C at 180 rpm for 1 h, collect the suspension cell culture medium, centrifuge at 300×g for 10 min, resuspend and seed into pre-coated 24-well plates.

[0048] 2. Immunofluorescence evaluation of Aβ peptide phagocytosis

[0049] (1) Drug pretreatment: After inoculating microglia for 24 h, discard the original culture medium. Add complete culture medium containing 100 nM DA and 500 nM DA to the three experimental groups respectively, and do not treat the blank control group. Incubate for 12 h under culture conditions.

[0050] (2) Take the FITC-Aβ1-42 stock solution, dilute it to 100 μM with PBS, and incubate in a 37°C incubator for 12 h to promote Aβ fiber cross-linking.

[0051] (3) After treating the cells with drugs for 12 h, replace the cell culture medium with serum-free culture medium (containing drugs) and add fibrillar FITC-Aβ1-42 at a final concentration of 0.5 μM, and continue to culture for 12 h.

[0052] (4) Immunofluorescence staining after 12 h of culture: Fixation: Treat with 4% PFA at room temperature for 15 min; Permeabilization: Treat with 0.2% Triton X-100 / PBS at room temperature for 10 min (wash with PBS before and after); Blocking: Block with 10% goat serum + 2% BSA / PBST at room temperature for 1 h; Primary antibody: Anti-Iba1 at 4°C overnight; Secondary antibody: Alexa Fluor Cy3-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) at room temperature for 1 h; Nuclear staining: Hoechst 33342 at room temperature for 10 min; Mount the slides and image.

[0053] (6) Microscopic imaging: Use a 10× objective of an Olympus FV3000 confocal microscope to randomly select 5 fields of view / sample for each sample for image acquisition, with a z-axis step size of 0.7 μm.

[0054] (7) Image processing: Export the images using FV31S-SW Viewer (2.5), perform cell segmentation using the online software cellpose (version 2.0), then use ImageJ to calculate the number of FITC-positive microglia, and analyze the average fluorescence intensity of FITC-Aβ1-42 in FITC-positive cells.

[0055] 3. Results

[0056] The results are as Figure 2As shown, in the microglia of the blank control group, the number of FITC-positive cells was 70%, in the microglia treated with 100 nM, the number of FITC-positive cells was 69.6%, and in the microglia treated with 500 nM, the number of FITC-positive cells was 69.8%. There was no statistical difference among the three groups. However, when the average fluorescence intensity of FITC in the FITC-positive cells was statistically analyzed, it was found that in the microglia treated with 100 nM and 500 nM DA, the fluorescence intensity of FITC was significantly higher than that of the blank control group (**, p<0.01; *, p<0.05). Therefore, it can be seen that the treatment with DA significantly enhanced the phagocytosis efficiency of primary microglia against Aβ and improved the phagocytic function of primary microglia.

[0057] Example 3 Effect of DA on the Activity of Microglia

[0058] To further verify whether DA at concentrations of 100 nM and 500 nM affects cell survival, the present invention also detected the effect of DA on the viability of HMC3 cells by the MTT method.

[0059] Cell viability detection method:

[0060] (1) Cell culture and seeding: Seed HMC3 cells in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and culture them in a 37°C, 5% CO2 incubator until the logarithmic growth phase;

[0061] (2) Plate treatment: Seed at a density of 5×10 3 cells / well into a 96-well plate and add complete medium to a final volume of 100 μL per well;

[0062] (3) Pretreatment and drug administration: After pre-culturing for 24 hours after seeding, add 100 nM and 500 nM DA for treatment, and the blank control group is not treated;

[0063] (4) MTT detection: After 24 hours of drug treatment, add 10 μL of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) with a concentration of 5 mg / mL to each well. After incubating at 37°C for 4 hours, discard the supernatant, add 100 μL of dimethyl sulfoxide (DMSO) to each well, and shake for 10 minutes to fully dissolve the formazan crystals;

[0064] (5) Data collection and analysis: Use an enzyme-linked immunosorbent assay reader to measure the absorbance (OD value) at a wavelength of 570 nm. Cell viability calculation formula: Cell viability (%) = [(OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)]×100.

[0065] The results are as Figure 3As shown, it can be seen that under the treatment of DA at a concentration of 100 nM, the survival rate of HMC3 cells was 97.7% compared with the control group. Under the treatment of DA at a concentration of 500 nM, the survival rate of HMC3 cells was 99.3% compared with the control group. There was no significant difference compared with the control group, indicating that the treatment of HMC3 cells with 100 nM and 500 nM of DA for 24 hours did not show cytotoxicity and did not affect cell viability.

[0066] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of domoic acid in the preparation of a drug for clearing β-amyloid protein, characterized in that, The concentration of the domoic acid is 100 - 500 nM.

2. The application according to claim 1, wherein The domoic acid promotes microglia to phagocytize β-amyloid.

3. Application of domoic acid in the preparation of a preparation for promoting microglia to phagocytize β-amyloid.

4. A drug for clearing β-amyloid protein, characterized in that, The active ingredient is domoic acid; The concentration of the domoic acid is 100 - 500 nM.

5. The drug according to claim 4, wherein The domoic acid promotes microglia to phagocytize β-amyloid.

6. A preparation for promoting the phagocytosis of β-amyloid protein by microglia, characterized in that, The active ingredient is domoic acid; The concentration of the domoic acid is 100 - 500 nM.

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