Application of broccoli exosome in preparation of medicine for treating Alzheimer's disease

Abstract

The invention provides an application of broccoli exosome in preparation of a medicine for treating Alzheimer's disease, and relates to the technical field of disease treatment medicines. The broccoli exosome can improve the permeability of the intestinal tract, balance microbial flora in the intestinal tract and prevent intestinal inflammatory factors from entering the brain, so that neuroinflammation is relieved. According to the application, the treatment of the Alzheimer's disease by using the broccoli-derived exosome is proposed for the first time. Oral administration is carried out on a mouse with Alzheimer's disease, and after administration lasts for one month, the result proves that behavioral defects of the mouse can be remarkably improved, and pathological protein A beta deposition of the brain is also remarkably reduced; the broccoli source exosome has the advantages of being low in raw material cost, high in biological safety, edible, good in effect, simple in extraction process and capable of being produced on a large scale, and powerful support is provided for clinical application.

Description

Technical Field

[0001] The present application relates to the technical field of disease treatment drugs, and more specifically, to the use of broccoli exosomes in the preparation of drugs for treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease and one of the common causes of death in the elderly. According to statistics from Alzheimer's Disease International (ADI), there are currently about 48 million patients with Alzheimer's disease in the world, and China is the country with the largest number of patients, with about 10 million Alzheimer's patients. According to ADI statistics, the cost of caring for AD patients worldwide exceeds 600 billion US dollars, accounting for about 1% of the global GDP. As China's aging population becomes increasingly serious, the prevalence of AD will also increase, and the social and economic problems it brings are becoming increasingly serious. It has become a high-burden disease for society. However, there is currently no effective drug to stop or reverse the pathological process of AD. Therefore, the development of effective drugs to treat AD has become an urgent problem that needs to be solved.

[0003] In recent years, the role of exosomes in the pathogenesis of AD has received increasing attention. Recent studies have shown that mesenchymal cells-derived exosomes (MSCs-Exos) can effectively improve cognitive impairment in AD mice. In addition, clinical results have shown that after continuous nasal administration of MSCs-Exos to AD patients for 12 weeks, their cognitive impairment was significantly improved. However, the therapeutic mechanism of MSCs-Exos for AD is not clear, and longer-term exploration and research are needed. More importantly, MSCs-Exos are derived from human / animal cells, and potential toxicity, ethical restrictions, high costs, and large-scale production issues during the preparation process are also challenges faced in its further clinical application. Summary of the invention

[0004] The purpose of this application is to provide the use of broccoli exosomes in the preparation of drugs for the treatment of Alzheimer's disease, which has the advantages of low cost, high biosafety, edibility, good effect, simple extraction process and large-scale production, providing strong support for clinical application.

[0005] In order to solve the above technical problems, the technical solution adopted in this application is:

[0006] The present application provides the use of broccoli exosomes in the preparation of a drug for treating Alzheimer's disease, wherein the broccoli exosomes are prepared by the following method:

[0007] S1. Take the corolla of broccoli, mix it with PBS, homogenize it at high speed for 1 minute, stop and place it on ice to cool down, repeat the above operation 3 times; then homogenize it at high speed for 30 seconds, stop and place it on ice to cool down, repeat the above operation 4 times, let it stand for 30-60 minutes, and take the supernatant;

[0008] S2. Perform differential centrifugation on the homogenized broccoli suspension: centrifuge at 1000-2000g at 4°C for 10-20min, take the supernatant; centrifuge at 3000-5000g at 4°C for 20-30min, take the supernatant; centrifuge at 10000g at 4°C for 40min, take the supernatant; centrifuge at 100000-150000g at 4°C for 60-90min, take the precipitate, dissolve it with PBS, and obtain a mixed solution;

[0009] S3. The mixed solution is subjected to microfiltration and ultrafiltration to obtain broccoli exosome concentrate.

[0010] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0011] 1. The preliminary data of this laboratory showed that broccoli exosomes are rich in sulforaphane (SFN). SFN has broad-spectrum antioxidant and anti-nonspecific inflammatory effects, and shows great prospects in the treatment of neurodegenerative diseases (such as AD and PD). In addition, the applicant also proposed for the first time that broccoli exosomes can improve dementia through the "brain-gut axis": the "brain-gut axis" is the key pathway for transmitting harmful signals in the intestinal cavity into the brain, that is, through the mediation of the immune system, peripheral circulatory system, and enteric nervous system, harmful factors in the intestine are delivered to the brain, over-activating microglia in the brain, and ultimately causing brain inflammation and neuronal apoptosis. Broccoli exosomes can improve intestinal permeability, balance the microbial flora in the intestine, and prevent intestinal inflammatory factors from entering the brain, thereby reducing neuroinflammation. Therefore, in the present invention, we proposed for the first time to use broccoli-derived exosomes to treat Alzheimer's disease. The drug was administered orally to Alzheimer's mice for one month, and the results showed that it could significantly improve their behavioral defects and significantly reduce the deposition of pathological protein Aβ in the brain, providing strong support for its clinical application.

[0012] 2. Compared with MSCs-Exos derived from animal cells, the exosomes derived from broccoli in the present invention have the advantages of low raw material cost, high biosafety, edibility, good effect, simple extraction process and large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 This is a particle size characterization diagram of broccoli exosomes in this application;

[0015] Figure 2 This is the electron microscopic characterization image of broccoli exosomes in this application;

[0016] Figure 3 The correct rate of choice of mice in the Y maze experiment of this application;

[0017] Figure 4 The time it takes for mice to reach the platform in the water maze experiment of this application;

[0018] Figure 5 The number of times mice crossed the platform, the time they stayed in the target quadrant, and the total distance they swam in the water maze experiment of this application;

[0019] Figure 6 This is a comparison chart of Aβ immunohistochemical staining of mouse brain tissue in the experimental example of this application. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0021] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0022] Application of broccoli exosomes in the preparation of drugs for the treatment of Alzheimer's disease.

[0023] In some embodiments of the present application, the dosage forms of the above-mentioned drugs include injections and oral preparations.

[0024] In some embodiments of the present application, the above-mentioned drug also includes a pharmaceutically acceptable excipient.

[0025] In some embodiments of the present application, the particle size of the broccoli exosomes is 114.7 nm, and they are specifically products extracted from broccoli corollas.

[0026] In some embodiments of the present application, the above extraction method specifically comprises the following steps:

[0027] S1. Take the corolla of broccoli, mix it with PBS, homogenize it at high speed for 1 minute, stop and place it on ice to cool down, repeat the above operation 3 times; then homogenize it at high speed for 30 seconds, stop and place it on ice to cool down, repeat the above operation 4 times, let it stand for 30-60 minutes, and take the supernatant;

[0028] S2. Perform differential centrifugation on the homogenized broccoli suspension: centrifuge at 1000-2000g at 4°C for 10-20min, take the supernatant; centrifuge at 3000-5000g at 4°C for 20-30min, take the supernatant; centrifuge at 10000g at 4°C for 40min, take the supernatant; centrifuge at 100000-150000g at 4°C for 60-90min, take the precipitate, dissolve it with PBS, and obtain a mixed solution;

[0029] S3. The mixed solution is subjected to microfiltration and ultrafiltration to obtain broccoli exosome concentrate.

[0030] In some embodiments of the present application, in the above step S1, the mass ratio of broccoli corolla to PBS is 1:(2-4); and the high-speed rotation speed is 25000-30000 rpm.

[0031] In some embodiments of the present application, the volume ratio of the precipitate to PBS in the above step S2 is 1:9.

[0032] In some embodiments of the present application, in the above step S3, the solution is filtered by microfiltration using a 0.22 μm filter membrane; and the solution is filtered and concentrated by ultrafiltration using a 50 kDa ultrafiltration tube.

[0033] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0034] Example 1

[0035] The extraction of broccoli exosomes includes the following steps:

[0036] S1. Homogenization: Take fresh broccoli corolla, mix with PBS at a mass ratio of 1:2, and place in a homogenizer. In the first step, homogenize at 28000rpm for 1min, stop and place on ice to cool down, and repeat the above operation 3 times. In the second step, homogenize at 30000rpm for 30s, stop and place on ice to cool down, and repeat the above operation 4 times. Let it stand for 50min, take the supernatant, and get the broccoli suspension.

[0037] S2. Perform differential centrifugation on the homogenized broccoli suspension: centrifuge at 1000g for 10 min at 4°C, take the supernatant. Centrifuge at 3000g for 20 min at 4°C, take the supernatant. Centrifuge at 10000g for 40 min at 4°C, take the supernatant. Centrifuge at 120000g for 90 min at 4°C, take the precipitate, and dissolve it with 9 times the volume of PBS.

[0038] S3. First filter the broccoli solution with a 0.22 μm filter membrane, and then filter and concentrate it with a 50 kDa ultrafiltration tube to obtain the final broccoli exosome solution, which is stored in a -80°C refrigerator.

[0039] Example 2

[0040] The extraction of broccoli exosomes includes the following steps:

[0041] S1. Homogenization: Take fresh broccoli corolla, mix with PBS at a mass ratio of 1:3, and place in a homogenizer. In the first step, homogenize at 25000rpm for 1min, stop and place on ice to cool down, and repeat the above operation 3 times. In the second step, homogenize at 25000rpm for 30s, stop and place on ice to cool down, and repeat the above operation 4 times. Let it stand for 30min, take the supernatant, and get the broccoli suspension.

[0042] S2. Perform differential centrifugation on the homogenized broccoli suspension: centrifuge at 1000g for 20 minutes at 4℃, take the supernatant. Centrifuge at 3000g for 30 minutes at 4℃, take the supernatant. Centrifuge at 10000g for 40 minutes at 4℃, take the supernatant. Centrifuge at 100000g for 90 minutes at 4℃, take the precipitate, and dissolve it with 9 times the volume of PBS.

[0043] S3. First, filter the broccoli solution with a 0.22 μm filter membrane, and then filter and concentrate it with a 50 kDa ultrafiltration tube to obtain the final broccoli exosome solution, which is stored in a -80°C refrigerator.

[0044] Example 3

[0045] The extraction of broccoli exosomes includes the following steps:

[0046] S1. Homogenization: Take fresh broccoli corolla, mix with PBS at a mass ratio of 1:4, and place in a homogenizer. In the first step, homogenize at 30,000 rpm for 1 minute, stop and place on ice to cool down, and repeat the above operation 3 times. In the second step, homogenize at 28,000 rpm for 30 seconds, stop and place on ice to cool down, and repeat the above operation 4 times. Let it stand for 60 minutes, take the supernatant, and get the broccoli suspension.

[0047] S2. Perform differential centrifugation on the homogenized broccoli suspension: centrifuge at 2000g for 10 min at 4°C, take the supernatant. Centrifuge at 5000g for 20 min at 4°C, take the supernatant. Centrifuge at 10000g for 40 min at 4°C, take the supernatant. Centrifuge at 150000g for 60 min at 4°C, take the precipitate, and dissolve it with 9 times the volume of PBS.

[0048] S3. First filter the broccoli solution with a 0.22 μm filter membrane, and then filter and concentrate it with a 50 kDa ultrafiltration tube to obtain the final broccoli exosome solution, which is stored in a -80°C refrigerator.

[0049] Experimental example

[0050] 1. Characterization of the particle size of broccoli exosomes: The broccoli exosome solution prepared in Example 1 was diluted to 1 μg / mL, and the particle size of the nanomicelles was measured using a Particle Metrix nanoparticle tracking analyzer. Figure 1 As shown, the particle size of broccoli exosomes is 114.7 nm, confirming that the product prepared in the present application is broccoli exosomes.

[0051] 2. Electron microscopic characterization of broccoli exosomes: 20 μL of broccoli exosomes prepared in Example 1 were adsorbed on a copper mesh, stained with saturated uranyl acetate for 30 seconds, and then rinsed with PBS and observed on a microscope. Figure 2 As shown, observation of the exosome morphology by transmission electron microscopy showed that broccoli exosomes had a typical double-layer membrane structure with a smooth surface and a spherical shape.

[0052] 3. APP / PS1 mouse drug administration experiment: 5-month-old APP / PS1 mice were grouped for drug administration. Broccoli exosomes were administered by gavage every other day for two months. The grouping is shown in Table 1, followed by behavioral experiments.

[0053] Table 1. Grouping of mice for drug administration

[0054]

[0055]

[0056] 1. APP / PS1 mouse behavioral experiment-Y maze

[0057] (1) 5-7 days before the experiment, the mice were stroked thoroughly for 1-2 minutes every day to allow the animals to fully adapt to the experimenter and the environment and to have no fear.

[0058] (2) The mice were restricted in their food intake for 3 days before the experiment and fasted for 6 h each day to reduce their body weight to 80%-90% of their normal food intake, thereby increasing their motivation to forage for food.

[0059] (3) Two days before the training experiment, the gates of the two arms of the Y-maze were opened, food was placed in both arms, and the mouse was placed in the starting arm of the Y-maze to explore freely for 5 min. After the mouse was taken out, it was cleaned with alcohol to remove the odor and the next mouse was placed in. This was repeated four times per day for each mouse.

[0060] (4) Training experiment: Each experiment consists of two parts, a forced choice wheel and a test wheel. Forced choice wheel: select an arm of any Y-maze, close its gate, and place the mouse in the starting arm. The mouse can only choose the other arm to obtain food. After eating, take it out immediately and wash it with alcohol to remove the odor. Test wheel: open the gates of both arms and place the mouse in the starting arm. If the mouse chooses to enter an arm it has never entered, it is considered a correct choice and is rewarded with food; if it repeatedly enters the same arm, it is considered an incorrect choice and is not given food, and is confined to the arm for 10 seconds. Each mouse undergoes 6 training experiments per day, with an interval of 15 minutes between each experiment, and the mouse's correct choice rate is calculated every day. The training experiment ends when the mouse's correct choice rate is higher than 85% for 2 consecutive days.

[0061] Figure 3 The figure shows the statistics of the correct selection rate of mice in the Y-maze. Compared with the control mice, the correct selection rate of APP / PS1 mice increased significantly after Exosome administration, which means that Exosome administration alleviated the decline of working memory in APP / PS1 mice.

[0062] 2. APP / PS1 mouse behavioral experiment - water maze

[0063] (1) First, determine the position of the outer cylinder and make four marks in the southeast, northwest, and northeast directions, so that the east-west line is perpendicular to the north-south line, and ensure that the position will not change during the seven-day experiment. Place visible markers 10 cm above the southeast, northwest, and northeast marks, using the following: square, triangle, circle, or five-pointed star. Determine the position of the platform in the southwest.

[0064] (2) On the first day, water was placed 1 cm below the platform, white pigment was added, and the water temperature was adjusted to 21.5±2℃. Mice were placed in the four quadrants in random order and the video was turned on. During each training session, the mouse had a maximum of 1 min to find the platform. If the platform was found, the mouse was allowed to stay on the platform for 30 s and then taken back. If the platform was not found after 1 min, the guide guided the mouse to the platform, where it stayed on the platform for 30 s, was taken back, its hair was dried with a towel, and the next mouse was prepared. Each mouse was required to enter each of the four quadrants once a day. The time taken for the mouse to find the platform was recorded.

[0065] (3) From the second to the fifth day, add water to 1 cm above the platform and proceed with the rest of the process as in step (2).

[0066] (4) On the sixth day, a platform-removal exploration experiment was conducted. The platform was removed in advance and the mouse was placed in the northeast quadrant of the bucket. The mouse was allowed to search in the bucket without the platform. The video was turned on and the mouse was fished out after 60 seconds.

[0067] The time it took for mice to reach the platform during the water maze training period was Figure 4 As shown, the time to reach the platform in the Exosome-administered group during the six-day training was significantly different from that in the model group, suggesting that Exosome administration improved the learning ability of APP / PS1 mice.

[0068] In the platform exploration experiment, the number of times the mouse crossed the platform, the time the mouse stayed in the quadrant where the platform was located, and the total distance the mouse swam were Figure 5 As shown. Compared with the model group, the number of shuttles across the platform in the Exosome-administered group increased significantly, and the search time in the quadrant where the platform was located in the administration group increased significantly, indicating that Exosomes can improve the memory ability of APP / PS1 mice. In addition, the total swimming distance of the mice in the Exosome-administered group increased significantly compared with the model group, indicating that Exosomes can improve the impaired motor ability of APP / PS1 mice.

[0069] 3. Aβ immunofluorescence staining

[0070] (1) Sample preparation: After the mice were killed, brain tissue samples were obtained and fixed in 4% paraformaldehyde. After 24 hours, the samples were dehydrated in 30% sucrose solution. Three days later, the samples were placed in an embedding box, embedded in OCT, and frozen in a refrigerator at -80°C. The samples were cut into 5 μm thick frozen sections and stored at -80°C.

[0071] (2) Place the frozen sections at room temperature for 30 min and wash three times with 1× PBS.

[0072] (3) Add peroxidase inhibitor, incubate at room temperature for 10 min, and wash three times with 1× PBS.

[0073] (4) Place in citrate buffer solution, microwave for 5 min, cool to room temperature, and wash three times with 1× PBS.

[0074] (5) Place in 0.5% Triton X-100 for 10 min and wash three times with 1× PBS.

[0075] (6) Add 5% BSA and incubate at 37°C for 30 min.

[0076] (7) Add primary antibody β-Amyloid (1:1000 dilution) and incubate at 4°C overnight.

[0077] (8) Wash three times with 1× PBS.

[0078] (9) Add secondary antibody Anti-Rabbit IgG (Alexa Flour 555) at a dilution of 1:1000 and incubate at 37°C for 1 h in the dark.

[0079] (10) Wash three times with 1× PBS.

[0080] (11) DAPI (1:5000 dilution) for 5 min. Wash three times with 1× PBS.

[0081] (12) Seal the slides with anti-fluorescence quenching sealing solution.

[0082] The results are as follows Figure 6 As shown, compared with the model group mice, Exosome administration significantly reduced the deposition of Aβ in the mouse brain tissue.

[0083] In summary, the use of broccoli exosomes in the embodiments of the present application in the preparation of drugs for treating Alzheimer's disease has the following advantages:

[0084] 1. The preliminary data of this laboratory showed that broccoli exosomes are rich in sulforaphane (SFN). SFN has broad-spectrum antioxidant and anti-nonspecific inflammatory effects, and shows great prospects in the treatment of neurodegenerative diseases (such as AD and PD). In addition, the applicant also proposed for the first time that broccoli exosomes can improve dementia through the "brain-gut axis": the "brain-gut axis" is the key pathway for transmitting harmful signals in the intestinal cavity into the brain, that is, through the mediation of the immune system, peripheral circulatory system, and enteric nervous system, harmful factors in the intestine are delivered to the brain, over-activating microglia in the brain, and ultimately causing brain inflammation and neuronal apoptosis. Broccoli exosomes can improve intestinal permeability, balance the microbial flora in the intestine, and prevent intestinal inflammatory factors from entering the brain, thereby reducing neuroinflammation. Therefore, in the present invention, we proposed for the first time to use broccoli-derived exosomes to treat Alzheimer's disease. The drug was administered orally to Alzheimer's mice for one month, and the results showed that it could significantly improve their behavioral defects and significantly reduce the deposition of pathological protein Aβ in the brain, providing strong support for its clinical application.

[0085] 2. Compared with MSCs-Exos derived from animal cells, the exosomes derived from broccoli in the present invention have the advantages of low cost, high biosafety, edibility, good effect, simple extraction process and large-scale production.

[0086] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present application.

Claims

1. Application of broccoli exosomes in the preparation of drugs for the treatment of Alzheimer's disease.

2. The use of broccoli exosomes according to claim 1 in preparing a drug for treating Alzheimer's disease, characterized in that: The dosage forms of the drug include injections and oral preparations.

3. The use of broccoli exosomes according to claim 2 in the preparation of a drug for treating Alzheimer's disease, characterized in that: The drug also includes pharmaceutically acceptable excipients.

4. The use of broccoli exosomes according to claim 1 in preparing a drug for treating Alzheimer's disease, characterized in that: The particle size of the broccoli exosomes is 100-120 nm, and the broccoli exosomes are specifically extracted from the corolla of broccoli.

5. The use of broccoli exosomes according to claim 4 in preparing a drug for treating Alzheimer's disease, characterized in that: The extraction method specifically comprises the following steps: S1. Take the corolla of broccoli, mix it with PBS, homogenize it at high speed for 1 minute, stop and place it on ice to cool down, repeat the above operation 3 times; then homogenize it at high speed for 30 seconds, stop and place it on ice to cool down, repeat the above operation 4 times, let it stand for 30-60 minutes, and take the supernatant; S2. Perform differential centrifugation on the homogenized broccoli suspension: centrifuge at 1000-2000g at 4°C for 10-20min, take the supernatant; centrifuge at 3000-5000g at 4°C for 20-30min, take the supernatant; centrifuge at 10000g at 4°C for 40min, take the supernatant; centrifuge at 100000-150000g at 4°C for 60-90min, take the precipitate, dissolve it with PBS, and obtain a mixed solution; S3. The mixed solution is subjected to microfiltration and ultrafiltration to obtain broccoli exosome concentrate.

6. The use of broccoli exosomes according to claim 5 in preparing a drug for treating Alzheimer's disease, characterized in that: In the step S1, the mass ratio of broccoli corolla to PBS is 1:(2-4); the high speed rotation speed is 25000-30000 rpm.

7. The use of broccoli exosomes according to claim 5 in preparing a drug for treating Alzheimer's disease, characterized in that: The volume ratio of the precipitate to PBS in step S2 is 1:

9.

8. The use of broccoli exosomes according to claim 5 in preparing a drug for treating Alzheimer's disease, characterized in that: In the step S3, the solution is filtered by microfiltration using a 0.22 μm filter membrane; and the solution is filtered and concentrated by ultrafiltration using a 50 kDa ultrafiltration tube.

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