Extraction method of sea-buckthorn exosome and application of sea-buckthorn exosome in relieving ulcerative bowel disease

Abstract

The invention belongs to the technical field of exosome preparation, and provides an extraction method of sea-buckthorn exosomes and application of the sea-buckthorn exosomes in relieving ulcerative bowel disease, fresh sea-buckthorn berries are cleaned and crushed, residues are filtered out, and juice is collected; carrying out primary centrifugal treatment on the juice, discarding precipitate and collecting supernatant; carrying out centrifugal treatment on the obtained supernatant again, discarding the precipitate and collecting the supernatant; carrying out third centrifugal treatment on the obtained supernatant, discarding the precipitate and collecting the supernatant; carrying out centrifugal treatment on the obtained supernate in an ultra-high-speed centrifuge to obtain exosome precipitates, and resuspending the exosome precipitates in a proper amount of precooled PBS (Phosphate Buffer Solution); according to the method, the high-purity hippophae rhamnoides exosome can be obtained through multi-stage centrifugation and sucrose gradient centrifugation, and the hippophae rhamnoides exosome has potential anti-inflammatory and anti-oxidation effects, can be used for treating inflammatory bowel diseases, can also be expanded to the field of treatment of other inflammatory diseases, and has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome preparation, and specifically relates to a method for extracting seabuckthorn exosomes and an application thereof in alleviating ulcerative enteropathies. Background Art

[0002] Surveys show that more than 90% of the population in my country suffer from intestinal diseases. As an important digestive tract and immune organ, the importance of the intestine to the human body is self-evident. In recent years, with the improvement of people's living standards, people's eating habits and dietary structure have undergone tremendous changes, resulting in a sharp increase in the incidence of intestinal diseases, of which colitis accounts for a large proportion. Colitis is a nonspecific inflammatory lesion of the colon and rectal mucosa. Genetic, mental factors and autoimmune reactions are the main factors causing colitis; ulcerative colitis (UC) and Crohn's disease (CD) are two manifestations of inflammatory bowel disease, of which ulcerative colitis is mainly manifested as diffuse intestinal damage, usually throughout the rectum and colon, resulting in abdominal pain, diarrhea and bloody stools in individuals. Because of its repeated course and lifelong disease, it is mainly treated with drugs in clinical practice. Due to its long treatment cycle, the risk of intestinal cancer increases. Therefore, there is an urgent need to develop a drug with mild properties and good therapeutic effects.

[0003] Exosomes are membrane-enclosed nanoscale vesicle structures that can be secreted by both plant and animal cells in nature. They contain some active molecules, such as DNA, microRNA (miRNA) and proteins, and play an important role in intercellular communication. Studies have shown that plant-derived exosomes not only play an important role in their own metabolism and immune defense, but can also regulate cross-border gene expression, such as anti-inflammatory, antiviral, and anti-tumor. Therefore, they can be used as new bioactive ingredients in clinical drug development.

[0004] Seabuckthorn is a plant that is both a medicine and an food. Its nutritional ingredients and bioactive ingredients are the highest among fruits and teas. Modern pharmacological studies have shown that seabuckthorn fruit extracts are rich in flavonoids, triterpenes and polyphenols, and have anti-inflammatory, antioxidant and anti-tumor pharmacological effects. To this end, those skilled in the art have proposed a method for extracting seabuckthorn exosomes and its application in alleviating ulcerative enteropathy to solve the problems raised by the background technology. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for extracting seabuckthorn exosomes and an application thereof in alleviating ulcerative enteropathies, so as to solve the problems existing in the prior art.

[0006] The method for extracting seabuckthorn exosomes and the application thereof in alleviating ulcerative bowel disease comprises the following steps:

[0007] S1. Wash and crush fresh seabuckthorn berries, filter to remove residues, and collect juice;

[0008] S2, subjecting the juice to a first centrifugal treatment, discarding the precipitate and collecting the supernatant;

[0009] S3, centrifuging the supernatant obtained in step S2 again, discarding the precipitate and collecting the supernatant;

[0010] S4, centrifuging the supernatant obtained in step S3 for the third time, discarding the precipitate and collecting the supernatant;

[0011] S5, centrifuging the supernatant obtained in step S4 in an ultra-high speed centrifuge to obtain an exosome precipitate, and resuspending the exosome precipitate in an appropriate amount of pre-cooled PBS buffer;

[0012] S6. The resuspended solution was slowly added to discontinuous sucrose media of different mass fractions (w / w) (8%, 30%, 45% and 60%), and then ultracentrifuged at 4°C and 150,000 g for 120 min to collect the bands between the 30% and 45% sucrose gradient layers;

[0013] S7, diluting the band obtained in step S6 with a buffer solution, then performing ultracentrifugation to wash away the sucrose solution, and resuspending the final precipitate in a pre-cooled buffer solution;

[0014] S8, filtering the exosome-containing buffer through a 0.22 μm filter to obtain high-purity seabuckthorn exosomes (H-ELNs);

[0015] S9, BCA to measure the protein concentration, and store at -80°C until use (if it is to be used further, store at 4°C to prevent damage to the exosome membrane cavity during repeated freezing and thawing).

[0016] Preferably, the sea buckthorn berries are sourced from Luliang, Shanxi.

[0017] Preferably, in step S2, the centrifugation refers to centrifugation at 4°C and 1000g for 10 minutes.

[0018] Preferably, in step S3, the re-centrifugation treatment refers to centrifugation at 4°C and 3000g for 20 minutes.

[0019] Preferably, in step S4, the third centrifugation treatment refers to centrifugation at 4°C and 10,000 g for 30 minutes.

[0020] Preferably, in step S5, the centrifugation refers to centrifugation at 4°C and 150,000 g for 120 minutes.

[0021] Preferably, in step S7, the ultracentrifugation treatment is ultracentrifugation again at 4°C and 150,000 g for 120 minutes.

[0022] Through the technical scheme, seabuckthorn exosome particles can be obtained, and the obtained product exhibits nano-scale inward-saucer-shaped particles; and the surface of the particles has a negative charge; cell experiments show that the nanoparticles can be taken up by macrophages RAW264.7 and down-regulate the expression of related inflammatory factors, and animal experiments show that seabuckthorn exosomes have a good improvement effect on inflammatory colitis and have no obvious toxic effects. In summary, seabuckthorn exosomes have the effect of alleviating colitis and can be prepared into related pharmaceutical products.

[0023] Use of seabuckthorn exosomes prepared by the above method in preparing a medicine for treating inflammatory colitis.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention can obtain high-purity seabuckthorn exosomes through multi-stage centrifugation and sucrose gradient centrifugation;

[0026] 2. The cell experiments of the present invention show that the seabuckthorn exosomes can be taken up by macrophages RAW264.7 and down-regulate the expression of related inflammatory factors; animal experiments further confirm that the seabuckthorn exosomes have a good improvement effect on inflammatory colon disease and have no obvious toxic effects;

[0027] 3. The present invention uses seabuckthorn, a plant with both medicinal and edible properties, as a raw material to develop a new bioactive ingredient, seabuckthorn exosomes, which provides new ideas and means for the treatment of inflammatory bowel diseases such as ulcerative colitis;

[0028] 4. The seabuckthorn exosomes of the present invention have potential anti-inflammatory and antioxidant effects, and can not only be used to treat inflammatory bowel disease, but can also be expanded to the treatment of other inflammatory diseases, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart of the method for preparing seabuckthorn exosomes of the present invention;

[0030] Figure 2 This is a schematic diagram of the seabuckthorn exosome sample of the present invention;

[0031] Figure 3 It is a schematic diagram of the detection results of the particle size and potential of the seabuckthorn exosome particles of the present invention;

[0032] Figure 4 This is a schematic diagram of the uptake of the mouse macrophage RAW 264.7 of the present invention;

[0033] Figure 5 This is a schematic diagram of the expression results of inflammatory factors in mouse macrophage RAW 264.7 of the present invention;

[0034] Figure 6 This is a schematic diagram of the adaptive feeding state of mice of the present invention;

[0035] Figure 7 Schematic diagram of weight changes of mice in various groups of the present invention;

[0036] Figure 8 It is a schematic diagram of the length of the colon of each group of mice of the present invention;

[0037] Fig. 9 This is a schematic diagram of indicators of severity of intestinal inflammation of the present invention;

[0038] Fig.10 This is a schematic diagram of intestinal HE staining of different treatment groups of the present invention; DETAILED DESCRIPTION

[0039] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0040] Embodiment: The present invention provides a method for extracting seabuckthorn exosomes, such as Figure 1 As shown, the following steps are included:

[0041] S1. Wash and crush fresh seabuckthorn berries, filter to remove residues, and collect juice;

[0042] S2, centrifuging the juice at 4°C and 1000g for 10 minutes, discarding the precipitate and collecting the supernatant;

[0043] S3, centrifuging the supernatant obtained in step S2 at 4°C and 3000g for 20 minutes, discarding the precipitate and collecting the supernatant;

[0044] S4, centrifuging the supernatant obtained in step S3 at 4°C and 10000g for 30 minutes, discarding the precipitate and collecting the supernatant;

[0045] S5, centrifuging the supernatant obtained in step S4 in an ultracentrifuge at 4°C and 150,000 g for 120 minutes to obtain an exosome precipitate, and resuspending the exosome precipitate in an appropriate amount of pre-cooled PBS buffer;

[0046] S6. Slowly add the resuspended solution into discontinuous sucrose media of different mass fractions (w / w) (8%, 30%, 45% and 60%), ultracentrifuge at 4°C and 150,000 g for 120 min, and collect the bands between the 30% and 45% sucrose gradient layers;

[0047] S7, diluting the band obtained in step S6 with a buffer solution, ultracentrifuging again at 4°C and 150,000 g for 120 minutes, washing away the sucrose solution, and resuspending the final precipitate in a pre-cooled buffer solution;

[0048] S8, filtering the exosome-containing buffer through a 0.22 μm filter to obtain high-purity seabuckthorn exosomes (H-ELNs);

[0049] S9, BCA to measure the protein concentration, and store at -80°C until use (if it is to be used further, store at 4°C to prevent damage to the exosome membrane cavity during repeated freezing and thawing).

[0050] As can be seen from the above, the present invention has the following effects:

[0051] 1) Efficient extraction: The present invention provides a detailed and efficient method for extracting seabuckthorn exosomes, which can obtain high-purity seabuckthorn exosomes through multi-stage centrifugation and sucrose gradient centrifugation;

[0052] 2) Therapeutic application: Cell experiments showed that the seabuckthorn exosomes can be taken up by macrophages RAW264.7 and down-regulate the expression of related inflammatory factors; animal experiments further confirmed that seabuckthorn exosomes have a good improvement effect on inflammatory colon disease and have no obvious toxic effects;

[0053] 3) Innovation: The present invention uses seabuckthorn, a plant with both medicinal and edible properties, as raw material to develop a new bioactive ingredient, seabuckthorn exosomes, which provides new ideas and means for the treatment of inflammatory bowel diseases such as ulcerative colitis;

[0054] 4) Application prospects: Seabuckthorn exosomes have potential anti-inflammatory and antioxidant effects. They can not only be used to treat inflammatory bowel disease, but can also be expanded to the treatment of other inflammatory diseases, and have broad application prospects.

[0055] The above embodiments are further described below in conjunction with the accompanying drawings.

[0056] Application example 1: Transmission electron microscopy observation of seabuckthorn exosome samples

[0057] The obtained seabuckthorn exosome suspension was pipetted with a pipette to 20 μL onto the carbon film copper mesh and placed for 3-5 minutes. The excess liquid was removed by filter paper. 2% phosphotungstic acid was dropped onto the carbon support film copper mesh and placed for 1-2 minutes. The excess liquid was removed by filter paper and dried at room temperature. The results were observed under a transmission electron microscope. Figure 2 As shown, inward-looking saucer-shaped particles with a size between 100-200 nm can be observed under a transmission electron microscope.

[0058] Application Example 2: Particle Size and Potential Analysis of Seabuckthorn Exosomes

[0059] After rinsing the sample pool of the nanoparticle analyzer with ultrapure water, the instrument was calibrated with polystyrene microspheres (100 nm), and the high-purity seabuckthorn exosome suspension was diluted to an appropriate concentration range with PBS buffer and tested on the machine. The results were repeated three times. Figure 3 As shown, nanoparticle tracking analysis (NTA) and Zeta potential results showed that the particle size of seabuckthorn exosomes was about 150nm, the charge on the particle surface was negative and the Zeta potential result was -15.92±0.63mV.

[0060] Experimental Example 1: Cell experiment: Mouse macrophage RAW 264.7 cell uptake

[0061] Specifically, Figure 4 Shown include:

[0062] (1) RAW 264.7 cells in the logarithmic growth phase were washed with pre-cooled PBS buffer, collected by pipetting in a Pasteur tube, and centrifuged at 1000 rpm for 5 min;

[0063] (2) Discard the supernatant and add an appropriate amount of complete culture medium to resuspend the cells at 1x10 5 The cells / well were seeded in a 12-well plate (with a processed glass slide of appropriate size placed in the well in advance) and cultured for 24 h;

[0064] (3) After the DiD storage solution was prepared into a working solution with a concentration of 5 μM using an appropriate amount of PBS, it was thoroughly mixed with H-ELNs and incubated in the dark for 30 min;

[0065] (4) After the incubation, centrifuge at 150,000 g for 120 min, discard the supernatant, dilute and resuspend the exosome precipitate at the bottom with a certain amount of PBS, and add it to the 12-well plate seeded with cells in advance;

[0066] (5) After 24 h, the 12-well plate was removed from the cell culture incubator, the culture medium was discarded, and the plate was washed twice with PBS for 5 min each time.

[0067] (6) After washing, discard PBS and add 500 μL of 4% paraformaldehyde solution to each well for fixation at room temperature for 15 min;

[0068] (7) After fixation, aspirate and discard PBS and wash twice, each time for 5 minutes; add 500 μL 0.2% Triton X-100 for permeabilization for 5 minutes, aspirate and discard, wash 3 times with PBS, each time for 5 minutes; add DAPI dye and incubate at room temperature for 15 minutes. After incubation, wash 3 times with PBS, each time for 5 minutes;

[0069] (8) After preparation, fluorescence microscopy was used to observe the uptake of exosomes by RAW 264.7 cells.

[0070] Test Example 2: Figure 5 As shown, mouse macrophage RAW 264.7 cells expressed inflammatory factors, including:

[0071] ① In advance, uniformly inoculate the cells in a 12-well plate at a density of 5 × 10 5 RAW 264.7 cells were cultured overnight, and then 1 mL of H-ELNs with a final concentration of 60 μg / mL and 120 μg / mL were added for pretreatment for 3 h, and then LPS was added to a final concentration of 1 μg / mL for treatment for 24 h; only 1 mL of culture medium was added to the blank control group;

[0072] ②After 24 hours, discard the culture medium and gently rinse twice with pre-cooled PBS to collect the cells;

[0073] ③ The total RNA in the cells was extracted by Trizol method, and the RNA concentration was measured by NanoDrop;

[0074] ④ According to the instructions of the reverse transcription kit, calculate the volume V required for reverse transcription of 1 μg of RNA, as shown in Table 1, prepare the RNA reverse transcription reaction system in a PCR tube, pipette and mix after the preparation, set the reverse transcription program in the PCR instrument according to Table 2, and perform reverse transcription;

[0075] ⑤After reverse transcription is completed, take out the cDNA sample, add 80 μL ddH2O, and store at -20℃;

[0076] ⑥ Prepare the qRT-PCR reaction system as shown in Table 3, gently pipette to mix and centrifuge. The primers used are shown in Table 4;

[0077] ⑦Use a fluorescent quantitative PCR instrument to set the qRT-PCR reaction program as shown in Table 5 and perform qRT-PCR;

[0078] Table 1 Reverse transcription system

[0079]

[0080] Table 2 Reverse transcription procedures

[0081]

[0082] Table 3 qRT-PCR reaction system

[0083]

[0084]

[0085] Table 4 Primers used for qRT-PCR

[0086]

[0087] Table 5 qRT-PCR reaction procedure

[0088]

[0089] Note: The denaturation and annealing / extension steps were performed for 40 cycles, with β-actin as the internal reference. ΔΔ The relative expression level of the target gene was calculated by the CT method.

[0090] The following is a further description of the embodiments in conjunction with the accompanying drawings and animal experiments.

[0091] like Figure 6 As shown, experimental animals: C57BL / 6 mice, SPF grade, 6-8 weeks old, body weight 18-20g, ambient temperature: (25±1)℃, humidity: 55%±5%, given 12h light / dark cycle.

[0092] After one week of adaptive feeding, the mice were randomly divided into a control group (Control), an H-ELNs treatment group (Prevent and Treat) and a model group (Mode l). The mice in the control group had normal diet and water, while the mice in the model group (Mode l) and the H-ELNs treatment group (including the Prevent group and the Treat group) had normal water on days 0-14 of the experiment and were given 3% (w / v) DSS on days 14-21 of the experiment. In addition, the Prevent group in the H-ELNs treatment group was treated with 100 mg / kg / mouse by gavage every day for 21 consecutive days, and the Treat group was treated with 200 mg / kg / mouse by gavage every day starting from day 14 of the experiment for seven consecutive days.

[0093] Furthermore, weight changes: During the modeling period, the weight of mice in each group was weighed daily, and the difference between the daily weight and the previous day and the ratio of the weight on the day before the modeling was the degree of weight loss; the results are shown in Figure 7 As shown, from the fourth day of modeling, the body weight of mice in all groups except the control group began to decrease significantly, indicating that H-ELNs can alleviate the body weight fluctuation caused by DSS-induced colitis.

[0094] Furthermore, Figure 8 As shown, after the experiment, the mice were killed and the colons of each group of mice were collected and their lengths were measured. It can be seen that H-ELNs can significantly improve the shortening of the colon caused by DSS.

[0095] Furthermore, the stool consistency and blood in the stool of the mice were observed and recorded daily. The Disease Activity Index (DAI) is an important indicator of the severity of intestinal inflammation, which is evaluated based on the degree of weight loss, stool consistency and blood in the stool, and is the sum of the three evaluation scores. Fig. 9 As shown, H-ELNs can reduce the severity of intestinal diseases. The specific evaluation criteria are shown in Table 6:

[0096] Table 6 Disease activity index evaluation criteria

[0097]

[0098] Furthermore, after the experiment, the mice were killed and the intestines of the mice were taken for HE staining. Fig.10 As shown, H-ELNs can improve the intestinal damage caused by DSS-induced colitis.

[0099] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for extracting seabuckthorn exosomes, characterized in that: The following steps are involved: S1. Wash and crush fresh seabuckthorn berries, filter to remove residues, and collect juice; S2, subjecting the juice to a first centrifugal treatment, discarding the precipitate and collecting the supernatant; S3, centrifuging the supernatant obtained in step S2 again, discarding the precipitate and collecting the supernatant; S4, centrifuging the supernatant obtained in step S3 for the third time, discarding the precipitate and collecting the supernatant; S5, centrifuging the supernatant obtained in step S4 in an ultra-high speed centrifuge to obtain an exosome precipitate, and resuspending the exosome precipitate in an appropriate amount of pre-cooled PBS buffer; S6. The resuspended solution was slowly added to discontinuous sucrose media of different mass fractions (w / w) (8%, 30%, 45% and 60%), and then ultracentrifuged at 4°C and 150,000 g for 120 min to collect the bands between the 30% and 45% sucrose gradient layers; S7, diluting the band obtained in step S6 with a buffer solution and then performing ultracentrifugation to wash away the sucrose solution, and resuspending the final precipitate in a pre-cooled buffer solution; S8. Filter the exosome-containing buffer through a 0.22 μm filter to obtain high-purity seabuckthorn exosomes (H-ELNs).

2. The method for extracting seabuckthorn exosomes according to claim 1, characterized in that: In step S2, the centrifugation treatment refers to centrifugation at 4°C and 1000 g for 10 minutes.

3. The method for extracting seabuckthorn exosomes according to claim 1, characterized in that: In step S3, the re-centrifugation treatment refers to centrifugation at 4°C and 3000 g for 20 minutes.

4. The method for extracting seabuckthorn exosomes according to claim 1, characterized in that: In step S4, the third centrifugation treatment refers to centrifugation at 4°C and 10,000 g for 30 minutes.

5. The method for extracting seabuckthorn exosomes according to claim 1, characterized in that: In step S5, the centrifugation treatment refers to centrifugation at 4°C and 150,000 g for 120 minutes.

6. The method for extracting seabuckthorn exosomes according to claim 1, characterized in that: In step S7, the ultracentrifugation treatment is performed again at 4°C and 150,000 g for 120 minutes.

7. Use of seabuckthorn exosomes prepared by the method according to any one of claims 1 to 6 in the preparation of a medicament for treating inflammatory colitis.

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