Application of tobacco extracellular vesicles in Alzheimer's disease

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of tobacco extracellular vesicles in Alzheimer's disease. It is found that the tobacco extracellular vesicles can significantly activate a PI3K-AKT signal channel in primary astrocytes, the expression quantity of P-PI3K protein and / or P-AKT protein in the PI3K-AKT signal channel in the astrocytes is increased, degradation of A beta is promoted, the learning and memory ability is improved, cognitive impairment is relieved, and then the Alzheimer's disease is prevented or treated. The application provides a new idea for the treatment of Alzheimer's disease.

Description

Technical Field

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

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

[0003] According to statistics, more than 50 million people are affected by Alzheimer's disease worldwide. The main pathological hallmarks of Alzheimer's disease include abnormal aggregation of amyloid plaques (Aβ, also known as β-amyloid protein) in the brain and entanglement of hyperphosphorylated tau protein (a microtubule-associated protein) in the fibril structure. At present, the treatment of Alzheimer's disease focuses on reducing the content of Aβ in the patient's brain. In recent years, the U.S. Food and Drug Administration (FDA) has approved new drugs such as aducanumab and licanumab for reducing the deposition of Aβ in the brain of AD patients. However, the long-term efficacy and safety of these drugs still need further verification. Therefore, exploring strategies to reduce the deposition of Aβ 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 of the invention

[0004] In view of this, the present invention provides the use of tobacco extracellular vesicles in Alzheimer's disease.

[0005] To achieve the above solution, the technical solution of the present invention is as follows:

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

[0007] Optionally, the nerve comprises a cranial nerve.

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

[0009] Optionally, the drug includes an active ingredient, the active ingredient includes a PI3K-AKT signaling pathway activator, the PI3K-AKT signaling pathway activator can activate the PI3K-AKT signaling pathway in astrocytes, and the PI3K-AKT signaling pathway activator includes the tobacco extracellular vesicles.

[0010] Optionally, the drug is a drug that can increase the expression level of P-PI3K protein and / or P-AKT protein in the PI3K-AKT signaling pathway in astrocytes.

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

[0012] Optionally, the tobacco extracellular vesicles are extracellular vesicles prepared using tobacco cells as raw materials.

[0013] Optionally, the tobacco extracellular vesicles are in a cup-shaped structure.

[0014] Optionally, the average particle size of the tobacco extracellular vesicles is 185-190 nm.

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

[0016] The tobacco cells are cultured, and the culture fluid is subjected to differential centrifugation and ultracentrifugation in sequence to obtain the tobacco extracellular vesicles.

[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 carried out under dark conditions.

[0020] Optionally, 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.

[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 PBS buffer.

[0023] Optionally, the drug includes an active ingredient, the active ingredient includes a PI3K-AKT signaling pathway activator, the PI3K-AKT signaling pathway activator can activate the PI3K-AKT signaling pathway in astrocytes, and the PI3K-AKT signaling pathway activator includes the tobacco extracellular vesicles.

[0024] Optionally, the drug further comprises a pharmaceutically acceptable excipient.

[0025] Optionally, the pharmaceutically acceptable excipients include at least one of solvents, cosolvents, excipients, emulsifiers, dispersants, wetting agents, binders, film formers, disintegrants, thickeners, defoamers, stabilizers, preservatives and fillers.

[0026] It should be noted that, in the present application, solvents include but are not limited to: benzene, toluene, xylene, acetone, dimethylformamide, dimethyl sulfoxide, chloroform, dichloromethane, cyclohexane, cyclohexanone, N-methylpyrrolidone, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, cyclohexanol, methyl glycol ether, ethyl glycol ether, petroleum ether, acetonitrile, paraffin, tetralin, isophorone, soybean oil, cottonseed oil and other substances.

[0027] In the present application, the co-solvent includes but is not limited to: methanol, ethanol, isopropanol, n-butanol and the like.

[0028] In the present application, excipients include but are not limited to: polyvinyl chloride, oleic acid and other substances.

[0029] In the present application, the emulsifier includes but is not limited to: sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, calcium dodecylbenzene sulfonate, nonylphenol polyoxyethylene ether phosphate amine salt, castor oil polyoxyethylene ether phosphate amine salt, triphenyl ethyl phenol polyoxyethylene ether phosphate amine salt, polyoxyethylene fatty amide, alkyl pyridinium salt, alkyl isoquinolinium salt, dialkyl morpholinium salt, oleamide sulfonate, diisopropyl naphthalene sulfonate, lignin sulfonate, naphthalene sulfonate formaldehyde condensate, styrylphenyl polyoxyethylene ether, phenethylphenol polyoxyethylene polyoxypropylene ether, nonylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene block polymer, alkylphenol formaldehyde resin polyoxyethylene ether, polyoxyethylene sorbitan fatty acid ester, sorbitan monostearate, sorbitan monostearate polyoxyethylene ether, polyoxypropylene glycol monofatty acid ester and the like.

[0030] 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, naphthalenesulfonic acid formaldehyde condensate sodium salt, dodecyltrimethylammonium salt, alkyldimethylbenzylammonium salt, dipolyoxyethylene dodecyl 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, alkyl alcohol amide and polyoxyethylene alkylamine and the like.

[0031] In the present application, the wetting agent includes but is not limited to: sodium dodecyl sulfate, calcium dodecylbenzene sulfonate, sodium isooctyl 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, triphenyl phenol polyoxyethylene ether, castor oil polyoxyethylene ether, fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene polyether formaldehyde condensate, alkylphenol polyoxyethylene ether phosphate, polyoxyethylene fatty acid esters, higher fatty acid glycerides, sorbitan fatty acid esters and the like.

[0032] In the present application, the adhesive includes but is not limited to: starch, polyvinyl pyrrolidone, carboxymethyl cellulose, carboxyethyl cellulose, gelatin, gum arabic, xanthan gum and the like.

[0033] 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.

[0034] In the present application, disintegrants include but are not limited to: urea, sodium sulfate, calcium sulfate, sodium bicarbonate, ammonium sulfate, magnesium chloride, sodium chloride and the like.

[0035] In the present application, thickeners include but are not limited to: gum arabic, xanthan gum, gelatin, polyvinyl alcohol, magnesium aluminum silicate, sodium carboxymethyl cellulose and the like.

[0036] In the present application, the defoaming agent includes but is not limited to: butyl phosphate, isobutyl phosphate, n-octanol, silicone and other substances.

[0037] In the present application, the stabilizer includes but is not limited to: triphenyl phosphite, N-soybean oil-based trimethylene diamine, dialkyl succinate sulfonate, epichlorohydrin, butyl glycidyl ether and the like.

[0038] In the present application, preservatives include but are not limited to: benzoic acid, sodium benzoate and other substances.

[0039] In the present application, fillers include but are not limited to: attapulgite, diatomaceous earth, kaolin, white carbon black, bentonite, montmorillonite, calcium carbonate, zeolite, acid clay, white clay, pyrophyllite, talc, calcite and other substances.

[0040] In a second aspect, the present application also provides the use of tobacco extracellular vesicles as a PI3K-AKT signaling pathway activator, wherein the PI3K-AKT signaling pathway activator can activate the PI3K-AKT signaling pathway in astrocytes.

[0041] As described above, the use of tobacco extracellular vesicles of the present invention in Alzheimer's disease has the following beneficial effects:

[0042] The present application found that tobacco extracellular vesicles can significantly activate the PI3K-AKT signaling pathway in primary astrocytes, promote the degradation of Aβ, improve learning and memory abilities, alleviate cognitive dysfunction, and thereby prevent or treat Alzheimer's disease.

[0043] This application provides a new idea for the treatment of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a process flow chart of the preparation of tobacco extracellular vesicles of the present application, where centrifugation means centrifugation;

[0045] Figure 2 This is a transmission electron micrograph of tobacco extracellular vesicles;

[0046] Figure 3 This is the particle size distribution diagram of tobacco extracellular vesicles, Diameter represents the diameter, and Particles represents the number of particles;

[0047] Figure 4 This is the escape latency diagram of mice in the water maze experiment, Escape latency means escape latency, and Day means days;

[0048] Figure 5 Provide a road map for mice to find the platform in a water maze experiment;

[0049] Figure 6 This is a graph of the number of times mice crossed the target platform in the water maze experiment. Number of target crossing indicates the number of times the target platform was crossed;

[0050] Figure 7 This is a graph of the time that mice stayed in the target quadrant in the water maze experiment. Target Quadrant Occupancy represents the target quadrant occupancy rate;

[0051] Figure 8This is a graph of the total distance traveled by mice in the water maze experiment. Total traveled distance represents the total distance traveled;

[0052] Fig. 9 This is the immunofluorescence image of Aβ in the mouse hippocampus. Hippocampus represents hippocampus.

[0053] Fig.10 This is a statistical diagram of the Aβ plaque area in the mouse hippocampus. Aβplaque area represents the Aβ plaque area.

[0054] Fig.11 This is a statistical diagram of the number of Aβ plaques in the hippocampus of mice. Number of Aβplaque indicates the number of Aβ plaques;

[0055] Fig.12 The figure is the change of total Aβ content in the hippocampus of mice. Aβ42ng / ml of total protein indicates the content of amyloid β protein 42 relative to total protein, in ng / ml, the same below;

[0056] Fig.13 The graph shows the changes of Aβ content in the culture medium of primary astrocytes. Control means negative control group, the same below.

[0057] Fig.14 This is the result of Western Blot expression analysis of PI3K / AKT pathway proteins in primary astrocytes. DETAILED DESCRIPTION

[0058] The present invention is further described below through specific examples, but it should be pointed out that the specific material ratios, process conditions and results described in the embodiments of the present invention are only used to illustrate the present invention and cannot be used to limit the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

[0059] The present invention is described in detail below by specific examples. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0060] (I) Preparation of tobacco extracellular vesicles

[0061] according to Figure 1 The process shown is to prepare tobacco extracellular vesicles, and the specific steps are:

[0062] Tobacco BY-2 cells provided by the College of Life Sciences of Hunan Agricultural University were suspended and cultured at 25°C, 120 rpm and in the dark for 7 days to obtain a cell culture solution;

[0063] The cell culture medium was subjected to differential centrifugation, specifically: first centrifuged at 4°C and 2000×g for 20 min, then centrifuged at 4°C and 10000×g for 30 min;

[0064] The supernatant obtained by differential centrifugation was subjected to ultracentrifugation, specifically: first centrifuged at 4°C and 100,000×g for 2 h, resuspended the obtained precipitate in 500 μL sterile PBS buffer to obtain a suspension, and the suspension was centrifuged again at 4°C and 120,000×g for 2 h, and the obtained precipitate (i.e., tobacco extracellular vesicles) was resuspended in 500 μL sterile PBS buffer (same as above) to obtain a secondary suspension, which was stored in a -80°C refrigerator for later use.

[0065] (II) Detection of the particle size and morphology of grass extracellular vesicles

[0066] The morphology of the tobacco extracellular vesicles prepared in (I) was observed using a transmission microscope (TEM). The specific steps were as follows: 5 μL 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 its morphological characteristics were observed using a transmission microscope (TEM). The results are shown in FIG. Figure 2 shown.

[0067] Depend on Figure 2 It can be seen that the tobacco extracellular vesicles prepared in the present application present a clear cup-shaped structure.

[0068] The size distribution and particle concentration of the tobacco extracellular vesicles prepared in (a) were observed and analyzed using a nanoparticle tracking analyzer (ZetaView, ParticLe Metrix, Germany). The specific steps were as follows: the sample pool was cleaned with deionized water, the instrument was calibrated using polystyrene microspheres, and the sample pool was cleaned with PBS buffer (same as above). The secondary suspension was diluted 50 times with PBS buffer (same as above) and then loaded for detection. The results are shown in Figure 2. Figure 3 shown.

[0069] Depend on Figure 3 It can be seen that the average particle size of the tobacco extracellular vesicles prepared in this application is 188.10±6.06nm, and the content is 4.73±0.02×10 9 Particles / mL.

[0070] (II) Application in Alzheimer’s disease

[0071] 2.1 Research Materials

[0072] 2.1.1 Animals: Clean grade 5xFAD mice and C57BL / 6J mice, 3 months old, were housed in the animal housing unit of Chongqing Medical University under controlled conditions (12 / 12 h light / dark cycle, humidity 60% ± 5%, temperature 22 ± 3 °C) with free access to food and water. All animals were handled in accordance with the Guide for the Care and Experimental Animals. All experiments were approved and performed in accordance with the guidelines of the Ethics Committee of Chongqing Medical University.

[0073] 2.1.2 Reagents: tobacco extracellular vesicles (secondary suspension prepared in (I)), PBS buffer, primary astrocytes, fetal bovine serum, F12 medium, 1% penicillin / streptomycin;

[0074] 2.1.3 Instruments: water maze apparatus, pipette, disposable pipette tip, centrifuge, transmission electron microscope, nanoparticle tracking analyzer, 6-well plate culture flask, T75 culture flask.

[0075] 2.2 Test methods

[0076] 2.2.1 Establishment of Alzheimer's disease animal model and grouping and drug treatment

[0077] 5xFAD (Tg6799, Jackson Laboratory) male mice were mated with wild-type C57BL / 6J female mice, and heterozygous 5xFAD mice were screened by PCR. Three-month-old female 5xFAD and female C57BL / 6J mice 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. The mice in the experimental group (5xFAD+tEVs) were given tobacco extracellular vesicles (the secondary suspension prepared in (I), with a dose of 0.125 mg / kg) through the nasal cavity, and the mice in the negative control group (5xFAD+PBS) were given an equal amount of PBS buffer. In the wild-type control group, 6 C57BL / 6J mice (WT+PBS) were given an equal amount of PBS buffer. The administration cycle was one month.

[0078] 2.2.2 Mouse water maze behavior test

[0079] One month after administration, the wild-type control group and two groups of 5xFAD mice were subjected to water maze behavioral tests. The water maze trough (120 cm in diameter) was filled with water and a transparent platform was used. This transparent platform was invisible in the water, and four different visual cues were placed in each quadrant.

[0080] Before each day's experiment, the water temperature was set to 22 ± 1 °C. 5xFAD mice were treated with drugs 1 h before the start of the test. Specifically, the experimental group mice (5xFAD + tEVs) were intranasally administered with tobacco extracellular vesicles (the secondary suspension prepared in (I), with a dose of 0.125 mg / kg), and the negative control group mice (5xFAD + PBS) and the wild-type control group (WT + PBS) were administered with an equal amount of PBS buffer;

[0081] The mice were placed in the behavioral testing room for at least 15 min before behavioral testing. Four training experiments were conducted every day for 5 consecutive days (60 s per group, with an interval of 1 h). In each training trial, each mouse was allowed to stay on the platform for 15 s after finding it, and then was removed from the maze and returned to its home cage. If the mouse did not find the platform within 60 s, it was guided to the platform and returned to its home cage after 15 s. The results are shown in the table below. Figure 4 and Figure 5 As shown, Figure 4 This is the escape latency diagram of mice in the water maze experiment. Figure 5 Provide a road map for mice to find the platform in a water maze experiment;

[0082] On the 6th day, a spatial exploration test was conducted and the swimming trajectory was recorded and analyzed using a video tracking system. Figures 6 to 8 As shown, Figure 6 This is a graph showing the number of times mice crossed the target platform in the water maze experiment. Figure 7 This is a graph of the time that mice stay in the target quadrant in the water maze experiment. Figure 8 This is a graph showing the total distance traveled by mice in the water maze experiment.

[0083] like Figure 4 and Figure 5 As shown, in the 5-day training experiment in the water maze, the time taken by the mice in the negative control group to find the platform was significantly reduced compared with the wild-type group mice. In addition, the time taken by the mice in the experimental group to find the platform was significantly shortened compared with the negative control group ( Figure 4 ), preferring to swim in the target quadrant and move on the platform ( Figure 5 ).

[0084] like Figure 6-8 As shown, in the spatial exploration experiment, when the total travel distance was basically the same, the time the negative control group spent in the target quadrant and the number of times the mice crossed the platform were significantly reduced compared with the wild-type control group, and compared with the negative control group, the time the mice in the experimental group spent in the target quadrant and the number of times they crossed the platform were significantly increased.

[0085] comprehensive Figures 4 to 8 It can be seen that tobacco extracellular vesicles have the effect of improving cognitive impairment in 5xFAD mice.

[0086] 2.2.3 Detection of Aβ plaque content in the hippocampus

[0087] In order to explore the mechanism by which tobacco extracellular vesicles improve cognitive impairment in 5xFAD mice, the present application detected the Aβ plaque content in the hippocampus of each group of mice. Specifically, the mice were killed after the mouse water maze behavioral test in 2.2.3, and paraffin sections of brain tissues of each group of mice were prepared, and then immunofluorescence staining of brain tissue sections was performed. The specific steps were as follows:

[0088] First, treat with xylene I for 10 min, then treat with xylene II for 10 min, then treat with anhydrous ethanol for 5 min, then treat with 95% ethanol solution (volume concentration) for 5 min, then treat with 85% ethanol solution for 5 min, then treat with 75% ethanol solution for 5 min, and then treat with pure water for 5 min;

[0089] The tissue sections were placed in a repair box containing Tris-EDTA antigen repair buffer (pH = 9, biosharp #BL617A) in a microwave oven for antigen repair, with medium heat for 8 minutes until boiling, then turned off the heat for 8 minutes to keep warm and then turned to medium-low heat for 7 minutes;

[0090] After cooling naturally, the slides were placed in PBS and washed on a decolorizing shaker for 3 times, 5 minutes each time. After the sections were slightly dried, a circle was drawn around the tissue with a histochemical pen (to prevent the antibody from flowing away), and 5% goat serum was dripped into the circle to evenly cover the tissue, and the tissue was blocked at room temperature for 30 minutes.

[0091] Then the brain sections were incubated with the primary antibody (mouse-anti-β-amyLoid, 1:100, BioLegend #803004) at 4°C overnight, and after the incubation, the sections were washed three times with PBS buffer and then treated with the secondary antibody (Goat anti-mouse Cy3, 1:200, Thermo Fisher #A10521) in blocking buffer in the dark for 1 h;

[0092] Then, the brain slices were washed again with PBS buffer for 3 times, and stained with Hoechst 33342 (10 μg / mL) diluted with PBS (1:2500) for 10 min in the dark. Finally, the slices were washed again with PBS buffer for 3 times, and blocked with anti-fluorescence quenching mounting medium (Biyuntian #P0126). The slices were photographed using a confocal microscope. Representative image results are shown in Figure 2. Fig. 9 As shown;

[0093] ImageJ (Fiji) was used to analyze the number and area of ​​Aβ(6E10) plaques. The stained images were opened with imageJ, and the dentate gyrus and cortical areas were specifically selected in each image using the "freehand selection" tool. After selecting the analysis area, the image was made 8-bit, and then the fluorescence-positive area was observed using the "threshold" tool, and quantification was performed using the "analyze particles" tool. The results are shown in Fig.10 and Fig.11 shown.

[0094] like Fig. 9 As shown, compared with the negative control group, the number of Aβ plaques in the hippocampus and cortex regions of the brain of the experimental group mice was significantly reduced.

[0095] like Fig.10 and Fig.11 As shown, compared with the negative control group, the size and number of Aβ plaques in the hippocampus of the experimental group mice were significantly reduced.

[0096] 2.2.4 ELISA detection of Aβ overall level in mouse hippocampus tissue

[0097] In order to further explore the mechanism of tobacco extracellular vesicles in improving cognitive impairment, the present application conducted an ELISA test on the overall level of Aβ in the hippocampus tissue of each group of mice. Specifically, the overall level of Aβ in the hippocampus of each group of mice after the mouse water maze behavioral test in 2.2.3 was detected. The specific steps were as follows:

[0098] The protein in the mouse hippocampus was extracted using a commercial kit, and the protein content was detected by the BCA method after the extraction. After the determination, the samples were stored at -80°C for future use. Specifically, the Aβ42 ELISA commercial kit (Invitrogen #KHB3441) was used to analyze the Aβ level in the mouse hippocampus. The Aβ42 peptide synthesized in the kit was used to establish a standard curve, and the BCA assay was used to calibrate the total protein amount in each sample. The results are shown in Fig.12 shown.

[0099] like Fig.12 As shown, compared with the negative control group, the overall level of Aβ42 in the hippocampus of the experimental group mice was significantly reduced.

[0100] 2.2.5 Detection of Aβ content in primary astrocyte culture medium

[0101] In order to further explore the mechanism of tobacco extracellular vesicles in improving cognitive impairment, this application conducted a test on the Aβ content in the culture medium of primary astrocytes. Specifically:

[0102] Mouse primary astrocytes were cultured and purified from postnatal day 3 C57 mouse pups as follows:

[0103] The cortex was dissected in ice-cold Hanks balanced salt solution (HBSS) without calcium and magnesium to remove the meninges, and the brain tissue was washed with HBSS and digested with 0.25% trypsin-EDTA at 37°C for 10 min to obtain a mixture;

[0104] The mixture was filtered using a 70 μm cell strainer, and then centrifuged at 1000 × g for 5 min. The resulting precipitate was resuspended in a complete medium containing DMEM-F12, 10% fetal bovine serum and 1% penicillin / streptomycin, and the cells were then seeded on a poly-L-lysine-coated T75 culture flask. After 3 h, all the culture medium was replaced to remove non-adherent cells. After the initial replacement, the culture medium was replaced every 3 days. After the cells reached confluence, they were shaken at 250 rpm for 6 h, and the culture medium was aspirated to remove less adherent microglia. Astrocytes were washed with PBS buffer, and astrocytes were separated from the T75 culture flask using 0.05% trypsin-EDTA. After the cells were digested, an equal amount of trypsin was added to terminate the digestion, and the supernatant was discarded after centrifugation at 1000 × g for 5 min. The resulting precipitate was the primary astrocyte.

[0105] Aβ oligomers were constructed, specifically in the following steps: freeze-dried Aβ peptide (invitrogen #03-111) was dissolved in HFIP (Ron #R003996, hexafluoroisopropanol) to obtain a solution with a concentration of 1 mM of Aβ oligomers, the solution was divided, HFIP was evaporated to obtain an Aβ peptide film, the Aβ peptide film was stored at -80°C, 24 h before use, the Aβ peptide film was dissolved in DMSO (Biosharp #BL165A) under sterile conditions and ultrasonically treated, the DMSO solution containing the Aβ peptide film was added to DMEM-F12 culture medium to make its final concentration 1 μM, and the Aβ oligomer solution was obtained by incubating at 4°C for 24 h;

[0106] Take a 6-well plate and culture primary astrocytes at 1×10 5 / well were inoculated in a 6-well plate, and the culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture. The culture medium was removed the next day, and the cultured astrocytes were exposed to tobacco extracellular vesicles (5μM, 10μM, experimental group) and cultured for 24h. The tobacco extracellular vesicle solution was removed after culture, and the Aβ oligomer solution (1μM, i.e., the final concentration of Aβ oligomers in the system was 1μM) was continued to be co-incubated with astrocytes at 37°C and 5% CO2 for 24h. After the incubation, 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; and then a commercially available Aβ42 ELISA kit was used to analyze the Aβ content level. The results are shown in Fig.13 shown.

[0107] like Fig.13 As shown, compared with the negative control group, the Aβ content level in the astrocyte culture medium of the experimental group was significantly decreased, and there was a clear dose-dependency between the Aβ content level in the astrocyte culture medium and the concentration of tobacco extracellular vesicles.

[0108] 2.2.6 Tobacco extracellular vesicles activate the expression of PI3K / AKT pathway proteins in astrocytes

[0109] In order to further explore the mechanism of tobacco extracellular vesicles in improving cognitive impairment, this application conducted a detection of PI3K / AKT pathway proteins in astrocytes activated by tobacco extracellular vesicles. The specific steps are as follows:

[0110] Take a 6-well plate and culture primary astrocytes at 1×10 5 / well were inoculated in a 6-well plate, and the culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture. The culture medium was removed the next day, and the cultured astrocytes were exposed to tobacco extracellular vesicles (5μM, 10μM, experimental group) and cultured for 24h. After the tobacco extracellular vesicle solution was removed, the cell lysate 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;

[0111] Then, Western Blot experiments were used to perform qualitative and quantitative analysis on cells in different groups, and Image J image processing software was used to analyze the optical density values ​​of the bands to determine the effect of tobacco extracellular vesicles on the expression of proteins in the PI3K-AKT signaling pathway of primary astrocytes. The results are shown in Fig.14 shown.

[0112] like Fig.14 As shown in the figure, compared with the negative control group, the expression of key phosphorylated proteins P-PI3K and P-AKT in the PI3K-AKT signaling pathway in astrocytes in the experimental group increased significantly, indicating that the pathway was activated. This result shows that tobacco extracellular vesicles can promote the degradation of Aβ by activating the PI3K-AKT signaling pathway, thereby preventing and treating Alzheimer's disease.

[0113] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Application of tobacco extracellular vesicles in the preparation of drugs for preventing or treating diseases caused by β-amyloid protein-induced nerve damage.

2. The use according to claim 1, characterized in that The nerves include cranial nerves.

3. The use according to claim 2, characterized in that Diseases caused by the damage of β-amyloid protein to nerves include cognitive dysfunction, behavioral impairment or Alzheimer's disease.

4. The use according to claim 3, characterized in that The medicine comprises an active ingredient, wherein the active ingredient comprises a PI3K-AKT signaling pathway activator, wherein the PI3K-AKT signaling pathway activator can activate the PI3K-AKT signaling pathway in astrocytes, and the PI3K-AKT signaling pathway activator comprises the tobacco extracellular vesicles.

5. The use according to claim 4, characterized in that The drug is a drug that can increase the expression of P-PI3K protein and / or P-AKT protein in the PI3K-AKT signaling pathway in astrocytes.

6. The use according to claim 4, characterized in that The drug is a drug capable of promoting the degradation of β-amyloid protein.

7. The use according to claim 4, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that The pharmaceutically acceptable excipients include at least one of solvents, cosolvents, excipients, emulsifiers, dispersants, wetting agents, adhesives, film formers, disintegrants, thickeners, defoamers, stabilizers, preservatives and fillers.

9. The use according to claim 8, characterized in that The solvent includes at least one of benzene, toluene, xylene, acetone, dimethylformamide, dimethyl sulfoxide, chloroform, dichloromethane, cyclohexane, cyclohexanone, N-methylpyrrolidone, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, cyclohexanol, methyl glycol ether, ethyl glycol ether, petroleum ether, acetonitrile, paraffin, tetralin, isophorone, soybean oil and cottonseed oil.

10. Use of tobacco extracellular vesicles as PI3K-AKT signaling pathway activators, characterized in that: The PI3K-AKT signaling pathway activator can activate the PI3K-AKT signaling pathway in astrocytes.

Images