Tobacco exosome as well as preparation method and application thereof

Abstract

The invention relates to a tobacco exosome as well as a preparation method and application thereof. The tobacco exosome comprises nicotine, lipid composition, a small molecule compound and protein, and in the tobacco exosome, the concentration of the nicotine is higher than 1 ng / mL. The tobacco exosome prepared by the invention can effectively inhibit inflammatory activity induced by microglial cells LPS, inhibit secretion of inflammatory factors PGE2 and transcriptional expression of iNOS, IL-1beta and IL-6. In a mouse neuroinflammation model experiment, the tobacco exosome prepared by the invention can improve behavioral changes of a mouse neuroinflammation model, and the improvement effect is better than that of other plant exosomes, such as potato exosomes.

Description

Technical Field

[0001] The present invention relates to plant exosomes and a preparation method and application thereof, and in particular to tobacco exosomes and a preparation method and application thereof. Background Art

[0002] Neuroinflammation is an immune response characterized by excessive activation of central nervous system glia. Microglia are the first immune effectors to respond to external stimuli to mediate neuroinflammation. When sensing damage-associated molecular patterns or pathogen-associated molecular patterns, microglia transform into a proinflammatory state, manifested by proliferation, migration to the site of injury, and release of proinflammatory factors, including TNF-α, interleukin-1β, interleukin-6, and prostaglandin E2. Mild neuroinflammation is beneficial to the repair and maintenance of neurons, while persistent uncontrolled glial activation and microenvironmental disruption exacerbate neuronal damage and death in aging and neurodegenerative diseases. Neuroinflammatory responses are closely related to neurodegenerative diseases, leading to synaptic degeneration, neuronal cell death, and cognitive dysfunction.

[0003] Tobacco contains a variety of anti-inflammatory active ingredients, such as nicotine, solanesol, chlorogenic acid, rutin, etc., among which nicotine is the main neuroactive ingredient of tobacco. Nicotine is a nicotinic acetylcholine receptor agonist, which regulates the release of neurotransmitters, affects multiple pathways in the central nervous system and peripheral nervous system, produces neuroprotective and neuro-immunomodulatory effects, and can be used as a potential drug for neurological diseases. However, direct use of tobacco will bring potential toxicity or side effects. Factors such as its growth conditions and harvest time will affect the content of its active ingredients, making quality control and standardization difficult. Although naturally extracted active ingredients are popular due to their good pharmacological effects and minimal side effects. However, recent studies have pointed out that it has disadvantages such as poor solubility, poor permeability and bioavailability, and instability in the body, which limits its permeability through the blood-brain barrier and clinical evaluation applications.

[0004] Plant-derived exosomes are a type of nanoparticle double-layer membrane vesicles isolated from fresh plant juice, containing lipids, polysaccharides, nucleic acids and small molecules. Plant exosomes have diverse functions and are widely used in drug delivery. They not only have high drug delivery efficiency, but also can achieve targeted delivery and better exert drug efficacy. However, not all plant exosomes can exert drug efficacy, which is related to many factors. So far, there have been no reports on the isolation and functional research of tobacco exosomes. Summary of the invention

[0005] In order to fill the gap in the prior art, the present invention provides a tobacco exosome and a preparation method and application thereof. It has been verified that the tobacco exosomes prepared by the present invention can effectively inhibit the inflammatory activity induced by LPS in microglia, inhibit the secretion of inflammatory factor PGE2, and the transcriptional expression of iNOS, IL-1β, and IL-6. In the mouse neuroinflammation model experiment: the tobacco exosomes prepared by the present invention can improve the behavioral changes of the mouse neuroinflammation model, and the improvement effect is better than other plant exosomes, such as potato exosomes.

[0006] The present invention is achieved by adopting the following technical solutions.

[0007] On the one hand, the present invention provides a tobacco exosome, wherein the tobacco exosome comprises nicotine, a lipid composition, a small molecule compound and a protein, wherein, in the tobacco exosome, the concentration of the nicotine is higher than 1 ng / mL.

[0008] Preferably, in the tobacco exosomes, the concentration of nicotine is 1.76 ng / mL.

[0009] Preferably, in the lipid composition of the tobacco exosomes, phosphatidylethanolamine accounts for 4.53 w / w%, phosphatidylcholine accounts for 5.15 w / w%, and ceramide accounts for 3.04 w / w%.

[0010] Preferably, in the lipid composition of the tobacco exosomes, sphingolipid lipids (SPH) account for 40.33w / w%, triglycerides (TG) account for 19.00w / w%, and bis(monoacylglycerol) phosphate (BisMePA) accounts for 6.95w / w%.

[0011] Preferably, the lipid composition in the tobacco exosomes is as follows Figure 2 shown.

[0012] Preferably, the diameter of the tobacco exosomes is 100-150 nm, preferably 125-135 nm.

[0013] In another aspect, the present invention provides a method for preparing the above tobacco exosomes, the method comprising the following steps:

[0014] Step 1, preparation of tobacco leaf juice: taking 18-22 parts by weight of tobacco leaves, preferably 20 parts by weight, removing surface stains, washing the leaves with pure water for 3-5 times, adding PBS buffer solution about 10 times the weight of the tobacco leaves, crushing and squeezing the juice to obtain tobacco leaf juice;

[0015] Step 2, centrifuging the tobacco leaf juice obtained in step 1 at 3000 rpm for 15 min, discarding the precipitate, taking the supernatant, and centrifuging the supernatant again at 10000 rpm for 15 min, discarding the precipitate, and taking the supernatant;

[0016] Step 3, taking the supernatant prepared in step 2 and filtering it with a 0.22 μm filter to obtain a filtrate, taking 100 mL of the filtrate, diluting it 5 times with 0.9% saline, concentrating it 10 times with a tangential flow device, and collecting a first concentrated solution;

[0017] Step 4, diluting the first concentrated solution obtained in step 3 by 5 times with 0.9% physiological saline (first dilution), concentrating it by 10 times with a tangential flow device, and collecting a second concentrated solution;

[0018] Step 5, diluting the second concentrated solution prepared in step 4 by 5 times with 0.9% saline (second dilution), concentrating it by 10 times with a tangential flow device, and collecting a third concentrated solution;

[0019] Step 6, diluting the third concentrated solution prepared in step 5 by 5 times with 0.9% physiological saline (third dilution), concentrating by 10 times with a tangential flow device, and collecting a fourth concentrated solution;

[0020] Step 7, dilute the fourth concentrated solution prepared in step 6 5 times with 0.9% saline (fourth dilution), concentrate it 10 times with a tangential flow device, and collect the fifth concentrated solution, which is the exosomes.

[0021] In step 2-7, the molecular weight cut-off of the tangential flow device is 300 kDa.

[0022] In a specific embodiment, the present invention provides a method for preparing the above tobacco exosomes comprising the following steps:

[0023] Step 1, preparation of tobacco leaf juice: taking 18-22 g, preferably 20 g, of tobacco leaves, removing surface stains, washing the leaves with pure water for 3-5 times, adding 180-220 mL, preferably 200 mL, of PBS buffer, crushing and squeezing the leaves to obtain tobacco leaf juice;

[0024] Step 2, centrifuging the tobacco leaf juice obtained in step 1 at 3000 rpm for 15 min, discarding the precipitate, taking the supernatant, and centrifuging the supernatant again at 10000 rpm for 15 min, discarding the precipitate, and taking the supernatant;

[0025] Step 3, taking the supernatant prepared in step 2 and filtering it with a 0.22 μm filter to obtain a filtrate, taking 100 mL of the filtrate, diluting it 5 times with 0.9% saline, concentrating it 10 times with a tangential flow device, and collecting a first concentrated solution;

[0026] Step 4, diluting the first concentrated solution obtained in step 3 by 5 times with 0.9% physiological saline (first dilution), concentrating it by 10 times with a tangential flow device, and collecting a second concentrated solution;

[0027] Step 5, diluting the second concentrated solution prepared in step 4 by 5 times with 0.9% saline (second dilution), concentrating it by 10 times with a tangential flow device, and collecting a third concentrated solution;

[0028] Step 6, diluting the third concentrated solution prepared in step 5 by 5 times with 0.9% physiological saline (third dilution), concentrating by 10 times with a tangential flow device, and collecting a fourth concentrated solution;

[0029] Step 7, dilute the fourth concentrated solution prepared in step 6 5 times with 0.9% saline (fourth dilution), concentrate it 10 times with a tangential flow device, and collect the fifth concentrated solution, which is the exosomes.

[0030] In the present invention, the TFF method (tangential flow filtration) is adopted. For the TFF method, the selection of the molecular weight cut-off parameter will affect the molecular size of the retained substance. Common choices are 5kDa, 10kDa, 30kDa, 50kDa, 100kDa, 300kDa, and 500kDa. Among them, 5kDa is suitable for the separation and concentration of small molecules and the removal of salts and ions, 10kDa and 30kDa are suitable for the removal of small to medium molecular impurities, 50kDa is suitable for the separation and concentration of large molecules and the removal of medium to large molecular impurities, 100kDa is suitable for the separation and concentration of very large molecules and the removal of large molecular impurities, and 300kDa and 500kDa can be used to retain nano-scale products. In the present application, 300kDa was used, and the size of the exosomes captured was more uniform than that of 500kDa. Under the condition of 500kDa, the particle size was distributed at 223.0nm and 361.1nm with 42.9% and 7% respectively, while under the condition of 300kDa, the particle size was mainly concentrated in 100nm-150nm, with 10.9% distribution at 204.3nm.

[0031] The preparation method of the present invention can obtain exosomes with a high final particle number.

[0032] In another aspect, the present invention provides a use of the above tobacco exosomes in preparing a drug for treating neuroinflammation.

[0033] The tobacco exosomes prepared by the present invention have a lipid bilayer structure, a uniform particle size distribution, and are rich in lipids, proteins, small molecule active substances and nicotine, and can be used to treat neuroinflammation. The tobacco exosomes have a short preparation cycle, excellent biocompatibility, no obvious toxicity to cells of healthy tissues, a small immune response caused in the body, and a reduced risk of rejection. Cell experiments have shown that the tobacco exosomes prepared by the present invention can effectively inhibit the inflammatory activity induced by LPS in microglia, inhibit the secretion of inflammatory factor PGE2, and the transcriptional expression of iNOS, IL-1β, and IL-6. Tobacco exosomes can effectively cross the blood-brain barrier and improve the efficiency of the delivery of effective ingredients in tobacco exosomes to the brain. In the mouse neuroinflammation model experiment: the tobacco exosomes prepared by the present invention can improve the behavioral changes in the mouse neuroinflammation model, and the improvement effect is better than potato exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 (A) Electron micrograph of tobacco exosomes prepared in Example 1 of the present invention; (B) Particle size distribution diagram of tobacco exosomes prepared in Example 1 of the present invention; (C) Electron micrograph of potato exosomes prepared in Comparative Example 1; (D) Particle size distribution diagram of potato exosomes prepared in Comparative Example 1; (E) Nicotine concentration in tobacco exosomes prepared in Example 1 of the present invention and potato exosomes prepared in Comparative Example 1. In Figures (A) and (C), 200 μm represents the size scale.

[0035] Figure 2 Lipid composition of tobacco exosomes prepared in Example 1 of the present invention.

[0036] Figure 3 The molecular weight distribution range of the protein in the tobacco exosomes prepared in Example 1 was obtained by gel protein electrophoresis analysis.

[0037] Figure 4 Tobacco exosomes inhibit microglial inflammatory activity: (A) PGE2 secretion concentration of BV2 cells stimulated by LPS after tobacco exosome treatment; (B) iNOS transcription level of BV2 cells stimulated by LPS after tobacco exosome treatment; (C) IL-1β transcription level of BV2 cells stimulated by LPS after tobacco exosome treatment; (D) IL-6 transcription level of BV2 cells stimulated by LPS after tobacco exosome treatment, PBS refers to phosphate buffered saline, LPS refers to lipopolysaccharide. ns indicates no difference, # indicates the difference between LPS group and experimental group, * the difference between blank control group and LPS group. # indicates p<0.05, ## indicates p<0.01, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001, the smaller the p, the greater the difference.

[0038] Figure 5 Tobacco exosomes are superior to potato exosomes in improving the behavioral study of the mouse neuroinflammation model: (A) Analysis of the time mice stayed in the rotarod test after treatment; (B) Mouse climbing test, PBS refers to phosphate buffered saline, LPS refers to lipopolysaccharide (Lipopolysaccharide). ns means no difference, # means the difference between the LPS group and the experimental group, * the difference between the blank control group and the LPS group. # means p<0.05, ## means p<0.01, ** means p<0.01, *** means p<0.001, **** means p<0.0001, the smaller the p, the greater the difference. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0040] Example 1 Preparation of tobacco exosomes of the present invention

[0041] The preparation of tobacco exosomes of the present invention specifically comprises the following steps:

[0042] Step 1, preparation of tobacco leaf juice: taking 20 g of tobacco leaves, removing surface stains, washing the leaves with purified water for 3-5 times, then washing with distilled water for 3-5 times, drying, adding 200 mL of PBS buffer solution, crushing and squeezing the leaves to obtain tobacco leaf juice;

[0043] Step 2: Centrifuge the tobacco leaf juice obtained in step 1 at 3000 rpm for 15 min, discard the precipitate and take the supernatant. Centrifuge the supernatant at 10000 rpm for 15 min, discard the precipitate and take the supernatant.

[0044] Step 3, take 100 mL of the supernatant prepared in step 2 filtered through a 0.22 μm filter, dilute it 5 times with 0.9% saline, concentrate it 10 times with a tangential flow device (300 kda), and collect the first concentrated solution.

[0045] Step 4, dilute the first concentrated solution obtained in step 3 5 times with 0.9% physiological saline (first dilution), concentrate it 10 times with a tangential flow device (300 kda), and collect the second concentrated solution.

[0046] Step 5, dilute the second concentrated solution prepared in step 4 5 times with 0.9% physiological saline (second dilution), concentrate it 10 times with a tangential flow device (300 kda), and collect the third concentrated solution.

[0047] Step 6, dilute the third concentrated solution prepared in step 5 5 times with 0.9% physiological saline (third dilution), concentrate 10 times with a tangential flow device (300 kda), and collect the fourth concentrated solution.

[0048] Step 7, dilute the fourth concentrated solution prepared in step 6 5 times with 0.9% saline (fourth dilution), concentrate it 10 times with a tangential flow device (300 kda), and collect the fifth concentrated solution, which is tobacco exosomes.

[0049] Example 2: Experimental study on the ratio of tobacco leaf to PBS buffer solution

[0050] The same preparation method as in Example 1 was used, except that the masses of tobacco leaves and PBS buffer solution in step 1 were as follows:

[0051] A: 23.7 g tobacco leaves; 150 mL PBS buffer solution;

[0052] B: 41.7 g tobacco leaves; 150 mL PBS buffer solution;

[0053] C: 18 g tobacco leaves; 180 mL PBS buffer solution;

[0054] D: 22 g tobacco leaves; 220 mL PBS buffer solution;

[0055] E: 20 g tobacco leaves; 200 mL PBS buffer solution (i.e., Example 1);

[0056] The total number of tobacco exosome particles in the above samples was detected by NTA (Nanoparticle Tracking Analysis) method, and the results are shown in Table 1.

[0057] Table 1 Total number of tobacco exosome particles in the test samples

[0058] sample Extract total particle number Unit particle number Sample A <![CDATA[0.96×10 10 ]]> <![CDATA[0.4×10 9 pcs / g]]> Sample B <![CDATA[1.1×10 10 ]]> <![CDATA[0.264×10 9 pcs / g]]> Sample C <![CDATA[2.1×10 10 ]]> <![CDATA[1.167×10 9 pcs / g]]> Sample D <![CDATA[2.6×10 10 ]]> <![CDATA[1.182×10 9 pcs / g]]> Sample E <![CDATA[2.5×10 10 ]]> <![CDATA[1.25×10 9 pcs / g]]>

[0059] Example 3: Experiments using different molecular weight cut-offs

[0060] The same preparation method as in Example 1 was used, except that in steps 2-7, the molecular weight cutoff of the tangential flow device was 500 kDa.

[0061] Comparative Example 1: Preparation of potato exosomes

[0062] Studies have shown that the risk of Parkinson's disease is negatively correlated with the consumption of Solanaceae plants, that is, the higher the consumption of Solanaceae plants, the lower the risk of disease. Moreover, this trend is strengthened by weighting Solanaceae plants according to nicotine concentration. In addition to tobacco plants, Solanaceae plants also include peppers, potatoes, and tomatoes. Combining existing literature and experimental conditions, we chose potatoes and extracted potato exosomes for comparison.

[0063] Potato exosomes were prepared using a method similar to that in Example 1. The specific preparation steps are as follows:

[0064] Step 1, preparation of potato juice: taking 20 g of potatoes, removing surface stains, removing potato skins, washing with purified water for 3-5 times, then washing with distilled water for 3-5 times, crushing and juicing to obtain potato juice;

[0065] Step 2: Centrifuge the potato juice obtained in step 1 at 3000 rpm for 15 min, discard the precipitate and take the supernatant. Centrifuge the supernatant at 10000 rpm for 15 min, discard the precipitate and take the supernatant.

[0066] Step 3, take 100 ml of the supernatant prepared in step 2 filtered through a 0.22 μm filter, dilute it 5 times with 0.9% saline, concentrate it 10 times with a tangential flow device (300 kda), and collect the first concentrated solution.

[0067] Step 4, dilute the first concentrated solution obtained in step 3 5 times with 0.9% physiological saline (first dilution), concentrate it 10 times with a tangential flow device (300 kda), and collect the second concentrated solution.

[0068] Step 5, dilute the second concentrated solution prepared in step 4 5 times with 0.9% physiological saline (second dilution), concentrate it 10 times with a tangential flow device (300 kda), and collect the third concentrated solution.

[0069] Step 6, dilute the third concentrated solution prepared in step 5 5 times with 0.9% physiological saline (third dilution), concentrate 10 times with a tangential flow device (300 kda), and collect the fourth concentrated solution.

[0070] Step 7, dilute the fourth concentrated solution prepared in step 6 5 times with 0.9% saline (fourth dilution), concentrate it 10 times with a tangential flow device (300 kda), and collect the fifth concentrated solution, which is potato exosomes.

[0071] Experimental Example 1

[0072] The tobacco exosomes prepared in Example 1 were observed by transmission electron microscopy, and the results are shown in Figure 1 A. The particle size of the tobacco exosomes obtained in Example 1 was measured by a particle size nanometer particle size analyzer, and the results are shown in Figure 1 As shown in B.

[0073] The results showed that the tobacco exosomes prepared by the present invention had a diameter of about 100 to 150 nm and a uniform size distribution.

[0074] The tobacco exosomes obtained in steps C and D in Example 2 have different total particle numbers, but no obvious difference in morphology under electron microscopy, and the measured particle size distribution is basically the same.

[0075] The size of exosomes captured by 300kDa is more uniform than that of 500kDa. Under 500kDa conditions, the particle size is distributed at 223.0nm and 361.1nm with 42.9% and 7% respectively, while under 300kDa conditions, the particle size is mainly concentrated in 100nm-150nm, with 10.9% distributed at 204.3nm.

[0076] Table 2 Particle size distribution ratio at different molecular weight cut-off

[0077]

[0078] The potato exosomes prepared in Comparative Example 1 were observed by transmission electron microscopy, and the results are shown in Figure 1 C. The particle size of the tobacco exosomes obtained in Comparative Example 1 was measured by a particle size nanometer particle size analyzer, and the result is shown in Figure 1 As shown in D.

[0079] The results showed that the potato exosomes prepared in Comparative Example 1 had a diameter of approximately 135 to 225 nm, and a uniform size distribution.

[0080] Experimental Example 2

[0081] Using a high performance liquid chromatography-mass spectrometer, this experimental example detected the nicotine content in the exosomes prepared in Example 1, samples C, D and Comparative Example 1.

[0082] The nicotine contents in the exosomes prepared in Example 1, Samples C and D, and Comparative Example 1 were 1.76 ng / mL, 1.64 ng / mL, 1.67 ng / mL, and 0.665 ng / mL, respectively. Figure 1 E shows the nicotine content in the exosomes prepared in Example 1 and Comparative Example 1.

[0083] The results showed that the concentration of nicotine in the tobacco exosomes prepared by the present invention was significantly higher than that in the potato exosomes.

[0084] Experimental Example 3

[0085] Using liquid chromatography-mass spectrometry, this experimental example detected the lipid composition of the exosomes prepared in Example 1.

[0086] The results are as follows Figure 2 shown.

[0087] Through lipidomics analysis, it can be seen that the tobacco exosomes prepared by the present invention include SPH (sphingolipids, 40.33%), TG (triglycerides, 19.00%), BisMePA (bis(monoacylglycerol) phosphate, 6.95%), PC (phosphatidylcholine, 5.15%), phosphatidylethanolamine, 4.53%, and ceramide accounts for 3.04w / w%.

[0088] Experimental Example 4

[0089] Using liquid chromatography-mass spectrometry, this experimental example detected the small molecule compounds in the exosomes prepared in Example 1.

[0090] Through metabolomics analysis, 779 small molecule compounds were detected, including a variety of bioactive ingredients such as isoflavones, coumarin derivatives, and flavonoids, which have good antioxidant and anti-inflammatory biological activities.

[0091] Coumarin and its derivatives, flavonoids and isoflavones are shown in Tables 3-5 below.

[0092] Through gel protein electrophoresis analysis, the molecular weight distribution range of proteins in tobacco exosomes is mainly 10kDa-180kDa ( Figure 3 ).

[0093] Table 3 Coumarin and its derivatives

[0094]

[0095]

[0096] Table 4 Flavonoid compounds

[0097]

[0098]

[0099] Table 5 Isoflavone compounds

[0100]

[0101] Experimental Example 5 :Experiment on the inhibition of microglial inflammatory activity by tobacco exosomes

[0102] 1. Materials

[0103] Tobacco exosome group: exosomes prepared in Example 1.

[0104] Potato exosome group: exosomes prepared in Comparative Example 1.

[0105] Blank control group: PBS buffer

[0106] LPS group: Add PBS buffer containing LPS (final concentration 1 μg / mL)

[0107] Tobacco exosomes + LPS group: exosomes prepared in Example 1, added with PBS buffer containing LPS (final concentration of 1 μg / mL)

[0108] Potato exosomes + LPS group: exosomes prepared in comparative example 1, added with PBS buffer containing LPS (final concentration of 1 μg / mL)

[0109] 2. Experimental process

[0110] BV2 cells were cultured at 10 6 The cells were inoculated at a density of 10 / ml in a 6-well plate and cultured in a carbon dioxide incubator for 24 hours. A blank control group, LPS group, tobacco exosome + LPS group, potato exosome + LPS group, and tobacco exosome group were set up; the final concentration of tobacco exosomes and potato exosomes after addition was 10 6 After tobacco exosomes and potato exosomes were added to cells and incubated for 4 hours, LPS was added for 2 hours. The supernatant of the cell culture fluid was collected, and the concentration of PGE2 in the supernatant was detected according to the instructions of the ELISA kit.

[0111] PGE2 is a type of prostaglandin. Neuroinflammation is related to Parkinson's disease. Its increase will aggravate neuroinflammation. Figure 4 As shown in A, tobacco exosomes can effectively inhibit the production of inflammatory factor PGE2 in BV2 cells induced by LPS and inhibit the inflammatory activity of microglia.

[0112] Collect BV2 cells after the experiment, extract RNA according to the centrifugal column method of RNA extraction kit, and the whole experiment process is carried out on ice. After measuring the concentration and OD260 / OD280 ratio, reverse transcription cDNA synthesis is performed. Prepare primers and dilute cDNA. According to the 20μL reaction system, add template, forward primer, reverse primer, SYBR, and enzyme-free water. After adding the sample, implement fluorescent quantitative detection, pre-denaturation at 95℃ for 2min, denaturation at 95℃ for 15s, return / extension at 60℃ for 15-30s, and 40 cycles. Collect CT values ​​and calculate the transcription levels of iNOS, IL-1β, and IL-6.

[0113] iNOS is mainly expressed by microglia and astrocytes in neuroinflammation. Overexpression of iNOS is associated with neuronal damage and apoptosis. IL-1β is a key proinflammatory cytokine in neuroinflammation. It triggers inflammatory responses through the IL-1R1 signaling pathway and promotes the release of other inflammatory factors. IL-6 also plays an important role in neuroinflammation. IL-6 is involved in the production of β-amyloid protein, further exacerbating the process of neuroinflammation. The experimental results are as follows Figure 4 As shown in B, C, and D, tobacco exosomes can effectively inhibit the transcriptional expression of LPS-induced BV2 cell inflammation-related proteins iNOS, IL-1β, and IL-6. Comparing the tobacco exosomes + LPS group and the potato exosomes + LPS group, tobacco exosomes are better than potato exosomes in inhibiting inflammatory factors. Therefore, tobacco exosomes have anti-inflammatory regulatory effects and are better than potato exosomes.

[0114] Experimental Example 6

[0115] 1. Materials:

[0116] Tobacco exosome group: exosomes prepared in Example 1.

[0117] Potato exosome group: exosomes prepared in Comparative Example 1.

[0118] Blank control group: PBS buffer.

[0119] LPS group: PBS buffer containing LPS was injected into the substantia nigra of the mouse brain

[0120] Tobacco exosomes + LPS group: Exosomes prepared in Example 1, PBS buffer containing LPS was injected into the substantia nigra

[0121] Potato exosomes + LPS group: Exosomes prepared in comparative example 1, PBS buffer containing LPS was injected into the substantia nigra of the brain

[0122] 2. Experimental process

[0123] 25 male C57BL / 6 mice were purchased from Vital River, 6-8 weeks old, SPF grade, and weighing 18-20 g. The mice were randomly divided into groups and housed in standardized independent ventilation cages (temperature 22±2°C, relative humidity 45-65%, and 12-hour light / dark cycle).

[0124] To investigate the effect of tobacco exosomes on LPS-induced neuroinflammation, 25 mice were randomly divided into five groups: blank control group, LPS group, tobacco exosomes + LPS group, potato exosomes + LPS group, and tobacco exosomes group. Exosomes were injected intraperitoneally for three consecutive days (10 830 minutes after the last intraperitoneal injection, LPS (2 μg) or PBS was injected into the substantia nigra. The drug was administered continuously for three days and the behavioral changes of the mice were analyzed.

[0125] For the rotarod test, on the training day, each mouse was placed on the rotarod for training until it could stay on the rotarod for more than 2 minutes at a speed of 4 r / min. On the test day, the speed of the rotarod was accelerated from 4 r / min to 40 r / min within 5 minutes, and each mouse could walk freely on the rotarod, and the time spent on the rotarod was recorded. This experiment was repeated three times for each mouse.

[0126] The experimental results are as follows Figure 5 As shown in A, tobacco exosomes and potato exosomes can effectively inhibit LPS-induced neuroinflammation in mice and prolong the time mice stay on the rotating rod. The improvement effect of tobacco exosomes is better than that of potato exosomes. The time of staying on the rotating rod in the LPS group is 67.4s, the average time of staying on the rotating rod in the tobacco exosome group is prolonged to 155.5s, and the average time of staying on the rotating rod in the potato exosome group is prolonged to 124.9s.

[0127] For the pole climbing test, a 50 cm high, 1 cm diameter, rough-surfaced wooden pole was used. On the training day, mice were placed on the pole and asked to crawl from the top down to the cage three times. On the test day, each mouse was placed head down on the top of the pole and the time required to climb back to the cage was recorded. The experiment was repeated three times for each mouse and the average time was analyzed.

[0128] The experimental results are as follows Figure 5 As shown in B, tobacco exosomes can effectively inhibit LPS-induced neuroinflammation in mice and shorten the climbing time of mice. The improvement effect of tobacco exosomes is better than that of potato exosomes. The climbing time of the LPS group was 13.8s, the climbing time of the tobacco exosome group was shortened to 8.0s, and the climbing time of the potato exosome group was shortened to 10.5s.

Claims

1. A tobacco exosome, comprising nicotine, lipid composition, small molecule compounds and protein, wherein: In the tobacco exosomes, the concentration of nicotine is higher than 1 ng / mL.

2. The tobacco exosomes according to claim 1, wherein In the tobacco exosomes, the concentration of nicotine is 1.76 ng / mL.

3. The tobacco exosomes according to claim 1 or 2, wherein In the lipid composition of the tobacco exosomes, phosphatidylethanolamine accounted for 4.53 w / w%, phosphatidylcholine accounted for 5.15 w / w%, and ceramide accounted for 3.04 w / w%.

4. The tobacco exosome according to any one of claims 1 to 3, wherein In the lipid composition of the tobacco exosomes, sphingolipid lipids (SPH) accounted for 40.33 w / w%, triglycerides (TG) accounted for 19.00 w / w%, and bis(monoacylglycerol) phosphate (BisMePA) accounted for 6.95 w / w%.

5. The tobacco exosomes according to any one of claims 1 to 4, wherein The lipid composition in the tobacco exosomes is shown in FIG2 .

6. The tobacco exosome according to any one of claims 1 to 5, wherein The diameter of the tobacco exosomes is 100-150 nm, preferably 125-135 nm.

7. A method for preparing tobacco exosomes according to any one of claims 1 to 6, the method comprising the following steps: Step 1, preparation of tobacco leaf juice: taking 18-22 parts by weight of tobacco leaves, preferably 20 parts by weight, removing surface stains, washing the leaves with purified water for 3-5 times, and then washing with distilled water for 3-5 times, adding PBS buffer solution in an amount of about 10 times the weight of the tobacco leaves, crushing and squeezing the leaves to obtain tobacco leaf juice; Step 2, centrifuging the tobacco leaf juice obtained in step 1 at 3000 rpm for 15 min, discarding the precipitate, taking the supernatant, and centrifuging the supernatant again at 10000 rpm for 15 min, discarding the precipitate, and taking the supernatant; Step 3, taking the supernatant prepared in step 2 and filtering it with a 0.22 μm filter to obtain a filtrate, taking 100 mL of the filtrate, diluting it 5 times with 0.9% saline, concentrating it 10 times with a tangential flow device, and collecting a first concentrated solution; Step 4, diluting the first concentrated solution obtained in step 3 by 5 times with 0.9% physiological saline (first dilution), concentrating it by 10 times with a tangential flow device, and collecting a second concentrated solution; Step 5, diluting the second concentrated solution prepared in step 4 by 5 times with 0.9% physiological saline (second dilution), concentrating it by 10 times with a tangential flow device, and collecting a third concentrated solution; Step 6, diluting the third concentrated solution prepared in step 5 by 5 times with 0.9% physiological saline (third dilution), concentrating by 10 times with a tangential flow device, and collecting a fourth concentrated solution; Step 7, dilute the fourth concentrated solution prepared in step 6 5 times with 0.9% saline (fourth dilution), concentrate it 10 times with a tangential flow device, and collect the fifth concentrated solution, which is the exosomes.

8. The preparation method according to claim 7, wherein: In step 2-7, the molecular weight cut-off of the tangential flow device is 300 kDa.

9. Use of the tobacco exosomes according to any one of claims 1 to 6 in the preparation of a medicament for treating neuroinflammation.

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