Use of tobacco extracellular vesicles in alzheimer's disease

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to application of tobacco extracellular vesicles in Alzheimer's disease. It is found that the tobacco extracellular vesicles can significantly activate the PI3K-AKT signal pathway in primary astrocytes, improve the expression amount of P-PI3K protein and / or P-AKT protein in the PI3K-AKT signal pathway in astrocytes, promote degradation of A beta, improve learning and memory ability, reduce cognitive dysfunction, and thus prevent or treat Alzheimer's disease. The application provides a new idea for treatment of Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to application of tobacco extracellular vesicles in Alzheimer's disease. BACKGROUND

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system, mainly occurring in the elderly or pre-elderly. Alzheimer's disease is irreversible, and its main features include progressive cognitive impairment and behavioral impairment. The clinical symptoms include memory impairment, aphasia, apraxia, agnosia, and impairment of visual spatial ability. In addition, the patient's abstract thinking and calculation ability are also impaired, often accompanied by changes in personality and behavior.

[0003] According to statistics, more than 50 million people worldwide are affected by Alzheimer's disease. The main pathological markers of Alzheimer's disease include abnormal aggregation of amyloid plaques (Aβ, also known as β-amyloid protein) in the brain and tangles of hyperphosphorylated tau protein (a microtubule-associated protein) in the fibril structure. At present, the treatment of Alzheimer's disease mainly focuses on reducing the content of Aβ in the brain of patients. In recent years, the U.S. Food and Drug Administration (FDA) has approved new drugs such as aducanumab and lecanemab for use in reducing the deposition of Aβ in the brain of AD patients. However, the long-term efficacy and safety of these drugs still need to be further verified. Therefore, exploring strategies to reduce Aβ deposition in AD patients is of great significance for improving the quality of life of AD patients, reducing the economic burden of society, and promoting the development of related medical fields. SUMMARY

[0004] Therefore, the present application provides application of tobacco extracellular vesicles in Alzheimer's disease.

[0005] To achieve the above-mentioned scheme, the technical solutions of the present application are as follows:

[0006] In a first aspect, the present application provides application of tobacco extracellular vesicles in the preparation of a drug for preventing or treating a disease caused by β-amyloid protein damage to nerves.

[0007] Optionally, the nerve includes a brain nerve.

[0008] Optionally, the disease caused by β-amyloid protein damage to nerves includes cognitive impairment, behavioral impairment, or Alzheimer's disease.

[0009] Optionally, the medicine comprises an active ingredient, the active ingredient comprises a PI3K-AKT signaling pathway activator, the PI3K-AKT signaling pathway activator is capable of activating a PI3K-AKT signaling pathway in a star-shaped glial cell, and the PI3K-AKT signaling pathway activator comprises the tobacco extracellular vesicle.

[0010] Optionally, the medicine is a medicine capable of increasing the expression amount of P-PI3K protein and / or P-AKT protein in a PI3K-AKT signaling pathway in a star-shaped glial cell.

[0011] Optionally, the medicine is a medicine capable of promoting the degradation of beta-amyloid protein.

[0012] Optionally, the tobacco extracellular vesicle is an extracellular vesicle prepared by using a tobacco cell as a raw material.

[0013] Optionally, the tobacco extracellular vesicle has a cup shape.

[0014] Optionally, the tobacco extracellular vesicle has an average particle size of 185-190 nm.

[0015] Optionally, the tobacco extracellular vesicle is prepared by a method comprising the following steps:

[0016] The tobacco cell is cultured, and the culture solution is sequentially subjected to differential centrifugation and ultracentrifugation to obtain the tobacco extracellular vesicle.

[0017] Optionally, the culture temperature is 20-30°C, preferably 22-30°C.

[0018] Optionally, during the culture process, the rotation speed is 100-150 rpm, preferably 110-150 rpm.

[0019] Optionally, the culture is performed in a dark condition.

[0020] Optionally, the differential centrifugation comprises: first centrifuging at 3-5°C and 1500-2500 g for 15-25 min, and then centrifuging at 3-5°C and 8000-12000 g for 25-35 min.

[0021] Optionally, the ultracentrifugation comprises: first centrifuging at 3-5°C and 80000-120000 g for 1-3 h, resuspending the obtained precipitate in a buffer, and then centrifuging at 3-5°C and 100000-150000 g for 1-3 h.

[0022] Optionally, the buffer is selected from a PBS buffer.

[0023] Optionally, the medicine comprises an active ingredient, and the active ingredient comprises a PI3K-AKT signaling pathway activator capable of activating a PI3K-AKT signaling pathway in a star-shaped glial cell, and the PI3K-AKT signaling pathway activator comprises the tobacco extracellular vesicle.

[0024] Optionally, the medicine further comprises a pharmaceutically acceptable adjuvant.

[0025] Optionally, the pharmaceutically acceptable adjuvant comprises at least one of a solvent, a cosolvent, an excipient, an emulsifier, a dispersant, a wetting agent, a binder, a film-forming agent, a disintegrant, a thickening agent, an antifoaming agent, a stabilizer, a preservative, and a filler.

[0026] In the present application, the cosolvent includes but is not limited to methanol, ethanol, isopropanol, n-butanol, and the like.

[0027] In the present application, the excipient includes but is not limited to polyvinyl chloride, oleic acid, and the like.

[0028] In the present application, the emulsifier includes but is not limited to sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, calcium dodecylbenzenesulfonate, nonylphenol polyoxyethylene ether phosphate amine salt, castor oil polyoxyethylene ether phosphate amine salt, triphenylethyl phenol polyoxyethylene ether phosphate amine salt, polyoxyethylene fatty amide, alkylpyridinium salt, alkylisoquinolinium salt, dialkylmorpholinium salt, oleamide sulfonate, diisopropyl naphthalene sulfonate, lignin sulfonate, naphthalene sulfonate formaldehyde condensate, styrylphenyl polyoxyethylene ether, phenylethyl phenol polyoxyethylene polyoxypropylene ether, nonylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene block polymer, alkylphenol formaldehyde resin polyoxyethylene ether, polyoxyethylene sorbitol fatty acid ester, sorbitan monostearate, sorbitan monostearate polyoxyethylene ether, polyoxypropylene glycol monofatty acid ester, and the like.

[0029] In the present application, the dispersant includes but is not limited to lignin sulfonate, sodium p-hydroxyphenyl lignin sulfonate, fatty alcohol polyoxyethylene ether sulfate, alkyl naphthalene sulfonate, naphthalene sulfonate formaldehyde condensate sodium salt, dodecyltrimethylammonium salt, alkyl dimethyl benzyl ammonium salt, diphthalene glycol ether phosphate, styrene-maleate copolymer, fatty alcohol polyoxyethylene ether phosphate, nonylphenol polyoxyethylene ether, glycerol fatty acid polyoxyethylene ether, polyoxyethylene alkyl aryl ether, polyoxyethylene lanolin alcohol, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glyceryl monofatty acid ester, alkylolamide, and polyoxyethylene alkylamine, and the like.

[0030] In the present application, the wetting agent includes, but is not limited to, sodium dodecyl sulfate, calcium dodecylbenzenesulfonate, sodium isooctanol succinate sulfonate, sodium oleyl methyl taurate, sodium polyoxyethylene lauryl ether carboxylate, sodium lauroyl sarcosinate, sodium N-lauroyl glutamate, dioctyl sulfosuccinate, polyoxyethylene alkyl aryl ether phosphate, trisiloxane polyoxyethylene ether, triphenylethyl phenol polyoxyethylene ether, castor oil polyoxyethylene ether, fatty alcohol polyoxyethylene ether, alkyl phenol polyoxyethylene polyether formaldehyde condensate, alkyl phenol polyoxyethylene ether phosphate, polyoxyethylene fatty acid ester, higher fatty acid glyceride, sorbitan fatty acid ester and the like.

[0031] In the present application, the binder includes, but is not limited to, starch, polyvinylpyrrolidone, carboxymethyl cellulose, carboxyethyl cellulose, gelatin, gum arabic, xanthan gum and the like.

[0032] In the present application, the film-forming agent includes, but is not limited to, polyvinyl alcohol, polyvinyl acetate, polyacrylamide, carboxymethyl cellulose, hydroxypropyl cellulose, gum arabic, gelatin, xanthan gum, starch and the like.

[0033] In the present application, the disintegrant includes, but is not limited to, urea, sodium sulfate, calcium sulfate, sodium bicarbonate, ammonium sulfate, magnesium chloride, sodium chloride and the like.

[0034] In the present application, the thickening agent includes, but is not limited to, gum arabic, xanthan gum, gelatin, polyvinyl alcohol, magnesium aluminum silicate, sodium carboxymethyl cellulose and the like.

[0035] In the present application, the antifoaming agent includes, but is not limited to, butyl phosphate, isobutyl phosphate, n-octanol, silicone and the like.

[0036] In the present application, the stabilizer includes, but is not limited to, triphenyl phosphite, N-soybean oil-based triethylene diamine, dialkyl succinate sulfonate, epichlorohydrin, butyl glycidyl ether and the like.

[0037] In the present application, the preservative includes, but is not limited to, benzoic acid, sodium benzoate and the like.

[0038] In the present application, the filler includes, but is not limited to, attapulgite, diatomite, kaolin, white carbon black, bentonite, montmorillonite, calcium carbonate, zeolite, acid clay, white clay, pyrophyllite, talc, calcite and the like.

[0039] In a second aspect, the present application also provides the use of the tobacco extracellular vesicle as a PI3K-AKT signaling pathway activator, which can activate the PI3K-AKT signaling pathway in astrocytes.

[0040] As described above, the use of the tobacco extracellular vesicle in Alzheimer's disease has the following beneficial effects:

[0041] The present application finds that tobacco extracellular vesicles can significantly activate the PI3K-AKT signaling pathway in primary astrocytes, promote the degradation of Aβ, improve learning and memory ability, reduce cognitive dysfunction, and thus prevent or treat Alzheimer's disease.

[0042] The present application provides a new idea for the treatment of Alzheimer's disease. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The flow chart for the preparation process of the tobacco extracellular vesicles of the present application, centrifugation represents centrifugation;

[0044] Figure 2 The transmission electron micrograph of the tobacco extracellular vesicles;

[0045] Figure 3 The particle size distribution graph of the tobacco extracellular vesicles, Diameter represents diameter, and Particles represents particle number;

[0046] Figure 4 The escape latency graph of mice in the water maze experiment, Escape latency represents escape latency, and Day represents day;

[0047] Figure 5 The route graph of mice searching for the platform in the water maze experiment;

[0048] Figure 6 The number of target crossing graph of mice in the water maze experiment, Number of target crossing represents the number of target crossing;

[0049] Figure 7 The target quadrant occupancy graph of mice in the water maze experiment, Target Quadrant Occupancy represents target quadrant occupancy;

[0050] Figure 8 The total traveled distance graph of mice in the water maze experiment, Total traveled distance represents total traveled distance;

[0051] Figure 9 The Aβ immunofluorescence image of the hippocampus region of mice, Hippocampus represents hippocampus;

[0052] Figure 10 The Aβ plaque area statistical graph of the hippocampus region of mice, Aβ plaque area represents Aβ plaque area;

[0053] Figure 11Figure 4 is a graph showing the number of Aβ plaques in the hippocampus of mice, wherein Number of Aβ plaque represents the number of Aβ plaques;

[0054] Figure 12 Figure 5 is a graph showing the total Aβ content in the hippocampus of mice, wherein Aβ42 ng / ml of total protein represents the content of amyloid β protein 42 relative to the total protein, and the unit is ng / ml, and the same below;

[0055] Figure 13 Figure 6 is a graph showing the Aβ content in the culture medium of primary astrocytes, wherein Control represents the negative control group, and the same below;

[0056] Figure 14 Figure 7 is a Western Blot analysis result of the PI3K / AKT pathway protein in primary astrocytes. DETAILED DESCRIPTION

[0057] The present application is further described below through specific examples, but it should be noted that the specific material ratio, process conditions and results described in the embodiments of the present application are only used to illustrate the present application, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the spirit and principle of the present application should be covered within the protection scope of the present application.

[0058] The present application is further described below through specific examples, but it should be noted that the specific material ratio, process conditions and results described in the embodiments of the present application are only used to illustrate the present application, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the spirit and principle of the present application should be covered within the protection scope of the present application.

[0059] (I) Preparation of tobacco extracellular vesicles

[0060] According to the process shown in Figure 1, the specific steps are as follows: Figure 1

[0061] The tobacco BY-2 cells provided by the School of Life Sciences of Hunan Agricultural University were suspended and cultured at 25°C, 120rpm and in the dark for 7 days to obtain a cell culture solution;

[0062] The cell culture solution was subjected to differential centrifugation, specifically: first centrifuged at 4°C and 2000xg for 20min, and then centrifuged at 4°C and 10000xg for 30min;

[0063] ​The supernatant obtained by differential centrifugation was subjected to ultracentrifugation, specifically: first centrifuged at 4°C and 100000xg for 2h, the obtained precipitate was resuspended in 500μL sterile PBS buffer to obtain a suspension, the suspension was centrifuged again at 4°C and 120000xg for 2h, the obtained precipitate (i.e. tobacco extracellular vesicles) was resuspended in 500μL sterile PBS buffer (as above) to obtain a secondary suspension, which was stored in a -80°C refrigerator for standby.

[0064] (B) Detection of the particle size and morphology of the grass extracellular vesicles

[0065] The morphology of the tobacco extracellular vesicles prepared in (A) was observed by transmission electron microscopy (TEM), and the specific steps were as follows: 5μL of the secondary suspension was fixed on a copper grid coated with a continuous carbon film, and after sedimentation and drying, it was negatively stained with uranyl acetate (1%, w / v), and then the morphology characteristics were observed by transmission electron microscopy (TEM), and the results are shown in Figure 2 .

[0066] As can be seen from Figure 2 , the tobacco extracellular vesicles prepared in the present application have a clear cup-shaped structure.

[0067] The size distribution and particle concentration of the tobacco extracellular vesicles prepared in (A) were observed and analyzed by nanoparticle tracking analyzer (ZetaView, ParticLe Metrix, Germany), and the specific steps were as follows: the sample pool was cleaned with deionized water, the instrument was calibrated with polystyrene microspheres, and the sample pool was cleaned with PBS buffer (as above), the secondary suspension was diluted 50 times with PBS buffer (as above) and then loaded for detection, and the results are shown in Figure 3 .

[0068] As can be seen from Figure 3 , the average particle size of the tobacco extracellular vesicles prepared in the present application is 188.10±6.06nm, and the content is 4.73±0.02x10 9 Particles / mL.

[0069] (B) Application in Alzheimer's disease

[0070] 2.1 Research materials

[0071] 2.1.1 Animals: clean grade 5xFAD mice and C57BL / 6J mice, 3 months old, all mice were raised in the animal feeding unit of Chongqing Medical University, under controlled conditions (12 / 12h light / dark cycle, humidity 60%±5%, temperature 22±3°C), food and water were freely available, all animals were treated in accordance with the "Guide for Care and Use of Laboratory Animals", all experiments were approved and performed in accordance with the guidelines of the Chongqing Medical University Ethics Committee,

[0072] 2.1.2 Reagents: Tobacco extracellular vesicles (secondary suspension prepared in (I)), PBS buffer, primary astrocytes, fetal bovine serum, F12 medium, 1% penicillin / streptomycin;

[0073] 2.1.3 Instruments: Water maze instrument, pipette, disposable tip, centrifuge, transmission electron microscope, nanoparticle tracking analyzer, 6-well plate culture bottle, T75 culture bottle.

[0074] 2.2 Test method

[0075] 2.2.1 Establishment and grouping of Alzheimer's disease AD animal models and drug treatment

[0076] 5xFAD (Tg6799, Jackson Laboratory) male mice were mated with wild-type C57BL / 6J female mice, and hybrid 5xFAD mice were screened by PCR; 3-month-old female 5xFAD and female C57BL / 6J of the same age were selected for drug treatment, specifically: 12 5xFAD mice were randomly divided into an experimental group and a negative control group, and the experimental group mice (5xFAD+tEVs) were given tobacco extracellular vesicles (secondary suspension prepared in (I)) through the nose at a dose of 0.125 mg / kg, and the negative control group mice (5xFAD+PBS) were given an equal amount of PBS buffer; the wild-type control group of 6 C57BL / 6J mice (WT+PBS) was given an equal amount of PBS buffer; the drug administration period was one month.

[0077] 2.2.2 Behavioral detection of mice in water maze

[0078] After one month of drug administration, the wild-type control group and the two groups of 5xFAD mice were subjected to water maze behavioral detection, the water maze tank (120 cm in diameter) was filled with water, and a transparent platform was used, which was invisible in water, and four different visual cues were placed in each quadrant;

[0079] Before each day's test, the water temperature was set to 22±1℃, and the 5xFAD mice were treated with drugs 1h before starting the test, specifically: the experimental group mice (5xFAD+tEVs) were given tobacco extracellular vesicles (secondary suspension prepared in (I)) through the nose at a dose of 0.125 mg / kg, and the negative control group mice (5xFAD+PBS) and the wild-type control group (WT+PBS) were given an equal amount of PBS buffer;

[0080] The mice were placed in the behavior test room for at least 15 min before the behavior test, and the behavior test was performed for 5 consecutive days, 4 training experiments per day (60 s per group, 1 h interval), in each training experiment, each mouse was allowed to stay on the platform for 15 s after finding the platform, and then was removed from the maze and returned to the home cage, if the mouse did not find the platform within 60 s, it was guided to the platform, and after 15 s, it returned to its home cage, and the results are shown in Figure 4 and Figure 5 , wherein, Figure 4 is a plot of the escape latency of the mice in the water maze experiment, Figure 5 is a plot of the platform search route of the mice in the water maze experiment;

[0081] A spatial exploration experiment was performed on the 6th day, and the swimming trajectory was recorded and analyzed using a video tracking system, and the results are shown in Figures 6 to 8 , wherein, Figure 6 is a plot of the number of times the mice crossed the target platform in the water maze experiment, Figure 7 is a plot of the time the mice spent in the target quadrant in the water maze experiment, Figure 8 is a plot of the total distance traveled by the mice in the water maze experiment.

[0082] As shown in Figure 4 and Figure 5 , compared with the wild type group of mice, the negative control group of mice found the platform significantly reduced the time in the water maze in the 5-day training experiment, in addition, compared with the negative control group, the experimental group of mice found the platform significantly shortened the time ( Figure 4 ), and was more inclined to swim in the target quadrant and move on the platform ( Figure 5 ).

[0083] As shown in Figures 6-8 , in the spatial exploration experiment, the time spent in the target quadrant and the number of times crossing the platform were significantly reduced in the negative control group compared with the wild type control group, and the time spent in the target quadrant and the number of times crossing the platform were significantly improved in the experimental group of mice compared with the negative control group, under the condition that the total distance traveled was basically the same.

[0084] In summary Figures 4 to 8 , it can be seen that tobacco extracellular vesicles have the effect of improving the cognitive impairment of 5xFAD mice.

[0085] 2.2.3 Detection of Aβ plaque content in hippocampal region

[0086] To explore the mechanism of tobacco extracellular vesicles improving the cognitive impairment of 5xFAD mice, the present application detects the Aβ plaque content in the hippocampal region of each group of mice, specifically, after the mice are killed after the behavior test in the water maze in 2.2.3, the brain tissue paraffin sections of each group of mice are prepared, and then the brain tissue section immunofluorescence staining is performed, and the specific steps are as follows:

[0087] Firstly, the brain slices were treated with xylene I for 10 min, then xylene II for 10 min, followed by absolute ethanol for 5 min, then 95% ethanol solution (volume concentration) for 5 min, followed by 85% ethanol solution for 5 min, then 75% ethanol solution for 5 min, and finally pure water for 5 min;

[0088] The tissue sections were placed in a repair box of Tris-EDTA antigen repair buffer (pH = 9, biosharp #BL617A) in a microwave oven for antigen repair, medium heat for 8 min to boiling, turn off the heat for 8 min, and then turn to low heat for 7 min;

[0089] After natural cooling, the slides were placed in PBS and shaken on a decolorizing shaker for 3 times, 5 min each time. After the slices were slightly dried, a circle was drawn around the tissue with a histological pen (to prevent the antibody from flowing away), 5% goat serum was added evenly to cover the tissue in the circle, and the room temperature was blocked for 30 min;

[0090] Then the brain slices were incubated with the primary antibody (mouse-anti-β-amyloid, 1:100, BioLegend #803004) at 4°C overnight. After incubation, the slices were washed with PBS buffer three times, and then incubated with the secondary antibody (Goat anti-mouse Cy3, 1:200, Thermo Fisher #A10521) in blocking buffer for 1 h in the dark;

[0091] Then the brain slices were washed with PBS buffer three times, and then stained with Hoechst 33342 (10 μg / mL) diluted with PBS (1:2500) in the dark for 10 min. Finally, the slices were washed with PBS buffer three times, and then blocked with an anti-fluorescence quenching mounting medium (Bi Yun Tian #P0126). The representative image results are shown in Figure 9 ;

[0092] The number and area of Aβ (6E10) plaques were analyzed using ImageJ (Fiji). The staining image was opened using ImageJ, and the dentate gyrus and cortical regions were specifically selected in each image using the "freehand selection" tool. After selecting the analysis region, the image was made into 8-bit, and then the fluorescent positive region was observed using the "threshold" tool. The "particle analysis" tool was used for quantification, and the results are shown in Figure 10 and Figure 11 .

[0093] As shown in Figure 9 , compared with the negative control group, the number of Aβ plaques in the hippocampal and cortical regions of the experimental group mice was significantly reduced.

[0094] As shown in Figure 10 and Figure 11 Compared with the negative control group, the size and number of Aβ plaques in the hippocampal region of the experimental group mice were significantly reduced.

[0095] 2.2.4 ELISA detection of Aβ whole level in mouse hippocampal tissue

[0096] To further explore the mechanism of tobacco extracellular vesicles in improving cognitive impairment, the present application carries out ELISA detection of the Aβ whole level in the hippocampal tissue of each group of mice. Specifically, the Aβ whole level in the hippocampus of each group of mice after the mouse water maze behavior detection in 2.2.3 is detected, and the specific steps are as follows:

[0097] The protein in the mouse hippocampus is extracted by using a commercially available kit, and the protein content is detected by BCA method after extraction. After the determination is completed, it is stored at -80℃ for standby, specifically, the Aβ level in the mouse hippocampus is analyzed by using Aβ42 ELISA commercially available kit (Invitrogen # KHB3441), the synthesized Aβ42 peptide in the kit is used to establish a standard curve, and the total protein mass in each sample is corrected by using BCA determination method, and the results are shown in Figure 12 .

[0098] As shown in Figure 12 Compared with the negative control group, the whole content level of Aβ42 in the hippocampus of the experimental group mice was significantly reduced.

[0099] 2.2.5 Detection of Aβ content in primary astrocyte culture medium

[0100] To further explore the mechanism of tobacco extracellular vesicles in improving cognitive impairment, the present application carries out detection of Aβ content in primary astrocyte culture medium, specifically:

[0101] Mouse primary astrocytes are cultured from C57 mouse pups at 3 days after birth and purified, and the specific steps are as follows:

[0102] The cortex is dissected in ice-cold calcium and magnesium-free Hanks balanced salt solution (HBSS) to remove the dura mater, the brain tissue is washed with HBSS, and the mixture is obtained by digesting with 0.25% trypsin-EDTA at 37℃ for 10 min.

[0103] The mixture was filtered using a 70 μm cell strainer, then centrifuged at 1000xg for 5 min, the obtained precipitate was resuspended in complete medium containing DMEM-F12, 10% fetal bovine serum and 1% penicillin / streptomycin, then the cells were inoculated on poly-L-lysine coated T75 flasks, 3 h later the complete medium was replaced to remove non-adherent cells, and the medium was replaced every 3 days after the first replacement, after the cells reached confluence, they were shaken at 250 rpm for 6 h, and the medium was removed to remove less adherent microglial cells, the astrocytes were washed with PBS buffer, and the astrocytes were separated from the T75 flasks using 0.05% trypsin-EDTA, after the cells were digested, an equal amount of trypsin was added to terminate digestion, centrifuged at 1000xg for 5 min, the supernatant was discarded, and the obtained precipitate was the primary astrocytes;

[0104] The Aβ oligomers were constructed as follows: lyophilized Aβ peptide (invitrogen #03-111) was dissolved in HFIP (Rohen #R003996, hexafluoroisopropanol) to obtain a solution of Aβ oligomers at a concentration of 1 mM, the solution was aliquoted, the HFIP was evaporated, and Aβ peptide films were obtained, which were stored at -80°C, 24 h before use, the Aβ peptide films were dissolved in DMSO (Biosharp #BL165A) under sterile conditions and ultrasonicated, the DMSO solution containing Aβ peptide films was added to DMEM-F12 medium to a final concentration of 1 μM, and the Aβ oligomer solution was obtained by incubating at 4°C for 24 h;

[0105] The 6-well plates were taken, and the primary astrocytes were inoculated in the 6-well plates at a concentration of 1×10 5 cells / well, and the plates were incubated in a cell incubator at 37°C and 5% CO2 overnight, the next day the medium was removed, and the cultured astrocytes were exposed to tobacco extracellular vesicles (5 μM, 10 μM, experimental group) for 24 h, then the tobacco extracellular vesicle solution was removed, the Aβ oligomer solution (1 μM, i.e. the final concentration of Aβ oligomers in the system was 1 μM) was added to the astrocytes, and the co-incubation was continued at 37°C and 5% CO2 for 24 h, after the incubation was completed, the cell culture medium was collected and stored at -80°C, and a negative control group was set up, the difference between the negative control group and the experimental group was that an equal volume of PBS buffer was used instead of tobacco extracellular vesicles, then the Aβ content level was analyzed using a commercially available Aβ42 ELISA kit, and the results are shown in Figure 13 .

[0106] As shown in Figure 13 , compared with the negative control group, the Aβ content level in the astrocyte culture medium of the experimental group was significantly reduced, and there was a clear dose-dependent relationship between the Aβ content level in the astrocyte culture medium and the concentration of tobacco extracellular vesicles.

[0107] 2.2.6 Tobacco extracellular vesicles activate the expression of PI3K / AKT pathway proteins in astrocytes

[0108] To further explore the mechanism of tobacco extracellular vesicles improving cognitive impairment, the present application detects the activation of PI3K / AKT pathway proteins in astrocytes by tobacco extracellular vesicles, and the specific steps are as follows:

[0109] Take 6-hole plates, and inoculate the primary astrocytes in the 6-hole plates at a number of 1×10 5 / well, and place the culture plates in a cell incubator at 37°C and 5% CO2 for overnight culture. The next day, after removing the culture medium, expose the cultured astrocytes to tobacco extracellular vesicles (5 μM, 10 μM, experimental group) for 24 h, remove the tobacco extracellular vesicle solution after the incubation is completed, collect the cell lysate, and store it at -80°C. Set up a negative control group, and the difference between the negative control group and the experimental group is that an equal volume of PBS buffer is used instead of tobacco extracellular vesicles.

[0110] Then, use Western Blot experiment to qualitatively and quantitatively analyze the cells in different groups, use Image J image processing software to analyze the optical density value of the band, to determine the effect of tobacco extracellular vesicles on the expression of PI3K-AKT signaling pathway proteins in primary astrocytes, and the results are shown in Figure 14 .

[0111] As shown in Figure 14 , compared with the negative control group, the expression amount of key phosphoprotein P-PI3K and P-AKT in the PI3K-AKT signaling pathway in the experimental group astrocytes increased significantly, indicating that the pathway was activated. The results show that tobacco extracellular vesicles can activate the PI3K-AKT signaling pathway, promote the degradation of Aβ, and achieve the effect of preventing and treating Alzheimer's disease.

[0112] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. Use of a tobacco extracellular vesicle in the preparation of a medicament for the treatment of Alzheimer's disease, characterized in that, The tobacco extracellular vesicles are prepared by a method comprising the following steps: The tobacco cells are cultured in dark conditions, and the culture solution is subjected to differential centrifugation and ultracentrifugation in sequence to obtain the tobacco extracellular vesicles; The temperature of the culture is 20-30°C, and the rotation speed during the culture is 100-150 rpm; The differential centrifugation comprises: first centrifugation at 3-5°C and 1500-2500g for 15-25 min, and then centrifugation at 3-5°C and 8000-12000g for 25-35 min; The ultracentrifugation comprises: first centrifugation at 3-5°C and 80000-120000g for 1-3h, resuspension of the obtained precipitate in PBS buffer, and then centrifugation at 3-5°C and 100000-150000g for 1-3h.

2. Use according to claim 1, wherein The medicine further comprises a pharmaceutically acceptable excipient.

3. Use according to claim 2, wherein the compound is ###0002### The pharmaceutically acceptable excipient comprises at least one of a solvent, a cosolvent, an excipient, an emulsifier, a dispersant, a wetting agent, a binder, a film former, a disintegrant, a thickening agent, an antifoaming agent, a stabilizer, a preservative, and a filler.

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