Preparation method of taxus chinensis exosome and application of taxus chinensis exosome in medicine for preventing and treating hepatic diseases

Abstract

The invention discloses a preparation method of taxus chinensis exosomes and application of the taxus chinensis exosomes in medicines for preventing and treating hepatic diseases, and belongs to the technical field of biological medicines. The method comprises the following steps: grinding fresh taxus chinensis into homogenate in PBS (Phosphate Buffer Solution) of which the volume is 5-20 times that of the fresh taxus chinensis, carrying out centrifugal separation, filtering supernate through a filter membrane, then carrying out centrifugal separation, and carrying out PBS resuspension precipitation, so as to obtain the taxus chinensis exosome. The taxus chinensis exosome can inhibit proliferation and migration of hepatic stellate cells, so that the taxus chinensis exosome is a medicine for reversing fatty liver and preventing and / or treating fatty liver. The medicine has the advantages of being easy to separate and detect, rich in source, high in safety, good in stability, good in treatment effect, capable of being absorbed through oral administration, capable of serving as a medicine delivery system to load targeted medicine and the like, and therefore the fatty liver treatment efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing yew exosomes as a yew exosome and an application of the same in drugs for preventing and treating liver diseases. Background Art

[0002] Fatty liver is a disease characterized by excessive fat storage and fatty degeneration in liver cells during liver fat metabolism. Many factors, such as alcoholism and obesity, can lead to the occurrence of this disease. Patients are often asymptomatic, but a few may experience fatigue, mild discomfort in the right upper abdomen, dull pain in the liver area, or distension and pain in the upper abdomen.

[0003] Fatification of hepatocytes is a key factor in the development of fatty liver. In non-alcoholic fatty liver, more than 30% of hepatocytes in the liver lobule undergo fatty degeneration, mainly macrovesicular fatty degeneration. It is not accompanied by inflammation, necrosis and fibrosis of hepatocytes. In patients with alcoholic fatty liver, ethanol-induced liver damage first manifests as fatty degeneration of hepatocytes. In mild cases, single hepatocytes or small pieces of hepatocytes are affected, mainly in the central area of ​​the lobule, and further develop into diffuse distribution. There is no inflammation or necrosis of hepatocytes, and the lobule structure is intact.

[0004] At present, there is no effective drug for the treatment of fatty liver in Western medicine. In clinical practice, the treatment drugs for liver damage currently include ornithine aspartate, polyene phosphatidylcholine, diammonium glycyrrhizinate, silymarin, bicyclol, reduced glutathione and other preparations. Some drugs have certain efficacy in drug-induced liver injury and cholestatic liver disease, but the therapeutic effect and biosafety of these drugs on fatty hepatitis and liver fibrosis still need further clinical trials to confirm. However, clinical treatment has found that some Chinese herbal medicine ingredients such as flavonoids, alkaloids and polysaccharides have significant effects on reversing liver damage. However, studies have shown that Chinese herbal medicine ingredients have certain liver damage effects when used, and even aggravate the degree of fatty liver. Therefore, how to better deliver the active ingredients in plants to hepatocytes and affect the behavior of hepatocytes, as well as clarify the molecular mechanism of plant active ingredients regulating hepatocytes, requires further in-depth research.

[0005] Plant exosomes are membranous vesicles secreted by plant cells, with a phospholipid bilayer structure, which contain proteins and microRNAs. Protein analysis shows that plant exosomes contain proteins involved in signal transduction (such as immune response), as well as proteins involved in oxidative stress response and reactive oxygen signal transduction and various membrane transport proteins. Plant exosomes contain relatively rich microRNAs, which can enter cells or bacteria under the mediation of exosomes to regulate cross-kingdom gene expression.

[0006] It has been found that plant-derived exosomes have the potential to treat alcoholic liver damage. Studies have shown that after oral administration, ginger-derived exosomes can accumulate in large quantities in the liver of mice and inhibit the production of reactive oxygen species in hepatocytes, thereby reducing liver damage. For mice with alcoholic liver damage, exosome treatment can significantly reduce liver weight and the number of lipid droplets, and reduce triglyceride levels. Therefore, ginger exosomes have the potential to prevent and treat alcoholic liver damage.

[0007] Taxus chinensis is an endangered plant with economic value, which is listed as a first-class protected plant in my country. Its bark contains a terpenoid compound, paclitaxel, which is a world-famous anti-tumor drug and is widely used to treat various solid tumors, such as breast, ovarian and lung cancer. Therefore, the rational use of Taxus chinensis to produce paclitaxel has been a hot topic of research in my country in recent years. Since the content of paclitaxel in its bark is very low, direct extraction, chemical synthesis, fermentation and other methods have certain disadvantages. Therefore, it is of great practical significance to develop more bioactive molecules in Taxus chinensis. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for preparing Taxus chinensis exosomes and application thereof in drugs for preventing and or treating liver diseases.

[0009] A method for preparing yew exosomes comprises: grinding fresh yew into a homogenate in 5-20 times volume of PBS, centrifuging, filtering the supernatant through a 0.2-0.5 μm filter membrane and then centrifuging again, resuspending the precipitate in PBS, and obtaining yew exosomes.

[0010] Furthermore, the preparation method of yew exosomes includes: grinding fresh yew into a homogenate in 10 times the volume of PBS, centrifuging at 1000g for 10 minutes, 4000g for 20 minutes, and 12000g for 40 minutes, filtering the supernatant with a 0.2-0.5μm filter membrane, centrifuging at 100000g for 90 minutes, resuspending the precipitate in PBS, and the precipitate is yew exosomes. Under a transmission electron microscope, yew exosomes are double-membrane structures, mostly in the shape of round cakes, and the particle size of yew exosomes is 50-200nm.

[0011] Furthermore, the fresh yew comprises yew leaves and stems.

[0012] The present invention also claims to protect the use of Taxus exosomes in preparing a medicine for treating liver diseases.

[0013] Furthermore, the liver diseases include fatty liver, cirrhosis and hepatitis.

[0014] Furthermore, the fatty liver includes chemically damaged fatty liver and virally damaged fatty liver; the cirrhosis includes alcoholic cirrhosis and fatty cirrhosis; and the hepatitis includes fatty hepatitis.

[0015] The present invention also claims a drug for treating fatty liver, which comprises Taxus exosomes or vesicles and a pharmaceutically acceptable carrier.

[0016] Furthermore, the dosage form of the drug is tablets, capsules, pills, powders, granules, suspensions, oral solutions, and injections.

[0017] Furthermore, the drug for treating fatty liver has at least one of the following functions:

[0018] (1) Inhibit the proliferation and migration of hepatic stellate cells;

[0019] (2) Improvement of pathological changes in liver tissue;

[0020] (3) Improve liver function and reduce the expression levels of alanine aminotransferase and aspartate aminotransferase.

[0021] The use of the yew exosomes of the present invention in the preparation of drugs for treating liver diseases has the following beneficial effects:

[0022] (1) The Taxus chinensis exosomes of the present invention can inhibit the proliferation and migration ability of hepatic stellate cells, thereby achieving the effect of reversing fatty liver, and can be used as a drug for preventing and / or treating fatty liver.

[0023] (2) The Taxus chinensis exosomes of the present invention intervened in the alcohol-induced fatty liver model in mice, and significantly reduced the expression levels of alanine aminotransferase and aspartate aminotransferase in mice, improved the pathological changes of liver tissue, inhibited collagen deposition, and reduced the number of lipid droplets in liver tissue.

[0024] (3) Compared with traditional therapeutic drugs, the drug containing yew exosomes or related biomaterials provided by the present invention has the advantages of being easy to separate, easy to detect, abundant in source, highly safe, stable, and having a good therapeutic effect, can be absorbed orally, and can also be used as a drug delivery system to load targeted drugs, thereby improving the efficiency of fatty liver treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the implementation mode of the present invention or the technical solution in the prior art, the drawings required for the implementation mode or the prior art description are briefly introduced below. Obviously, the drawings described below are only exemplary. For ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work. The structure, proportion, size, etc. illustrated in this specification are only used to match the content disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions that the present invention can be implemented, so it has no technical substantive significance. Any structural modification, change in proportional relationship or adjustment of size, without affecting the effect that the present invention can produce and the purpose that can be achieved, should still fall within the scope that the technical content disclosed by the present invention can cover.

[0026] Figure 1 This is an electron microscope image of exosomes isolated from Taxus leaves (referred to as leaves) provided in Example 1 of the present invention;

[0027] Figure 2 This is an electron microscope image of exosomes isolated from the stems and branches of Taxus chinensis (referred to as stems) provided in Example 1 of the present invention;

[0028] Figure 3 This is a statistical diagram of the nanoparticle size of exosomes isolated from the leaves of Taxus chinensis (referred to as leaves) provided in Example 1 of the present invention;

[0029] Figure 4 A statistical graph of the nanoparticle size of exosomes isolated from the stems and branches of Taxus chinensis (referred to as stems) provided in Example 1 of the present invention;

[0030] Figure 5 The absorption of Taxus chinensis exosomes in hepatic stellate cells (LX-2) provided in Example 2 of the present invention;

[0031] Figure 6 A statistical graph of the relative fluorescence intensity of the yew exosomes provided in Example 2 of the present invention after being absorbed by hepatic stellate cells;

[0032] Figure 7 This is an analysis of the effect of Taxus chinensis exosomes on the cell proliferation activity of activated LX-2 provided in Example 3 of the present invention, wherein A is the effect of leaves on the cell proliferation activity of LX-2; B is the effect of stems on the cell proliferation activity of LX-2;

[0033] Figure 8 Analysis of the effect of Taxus chinensis exosomes on the cell migration of activated LX-2 cells provided in Example 3 of the present invention;

[0034] Fig. 9This is a statistical graph of the cell migration of Taxus chinensis exosomes to activated LX-2 cells provided in Example 3 of the present invention;

[0035] Fig.10 A flowchart of an animal experiment provided in Example 4 of the present invention is shown;

[0036] Fig.11 The changes in liver function of mice after treatment with Taxus chinensis exosomes provided in Example 4 of the present invention, wherein A is the change in alanine aminotransferase (ALT) in the blood; B is the change in aspartate aminotransferase (AST);

[0037] Fig.12 Analysis of the effect of Taxus chinensis exosomes treatment on the degree of fatty liver in mice provided in Example 4 of the present invention (HE staining);

[0038] Fig.13 Analysis of the effect of Taxus exosomes on the degree of fatty liver in mice after treatment with Taxus exosomes provided in Example 4 of the present invention (Oil Red O staining). DETAILED DESCRIPTION

[0039] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The present invention provides the use of yew exosomes in the preparation of a drug for preventing and / or treating fatty liver. The yew exosomes can inhibit the progression of fatty liver at both the cellular level and the tissue level, thereby inhibiting the development of fatty liver.

[0041] Example 1

[0042] A method for preparing yew exosomes, comprising the following steps:

[0043] Freshly picked yew leaves and stems were used to separate yew exosomes (referred to as leaf exosomes and stem exosomes, respectively) by differential centrifugation. The specific steps were as follows: fresh yew was ground into a homogenate in 10 times the volume of PBS, and the mixture was centrifuged at 1000g for 10 min, 4000g for 20 min, and 12000g for 40 min. The supernatant was filtered through a 0.2-0.5 μm filter membrane and then centrifuged at 100000g for 90 min. The precipitate was resuspended in PBS, and the precipitate was the yew exosomes.

[0044] The yew exosomes were analyzed using a transmission electron microscope (Japan JEM-1400) and a nanoparticle tracking analyzer (Particle Metrix). Electron microscopy revealed that the yew exosomes contained a typical lipid bilayer membrane structure, which was round and about 100 nm in size, consistent with the characteristics of plant exosomes ( Figure 1 and Figure 2 ).

[0045] The particle size of Taxus exosomes was determined by nanoparticle tracking analysis (e.g. Figure 3 and Figure 4 ), the average particle sizes of Taxus exosomes were 152.4nm and 167.7nm, respectively, and were mainly distributed in the range of 100-300nm, which was consistent with the results of electron microscopy observations.

[0046] This indicates that the Taxus exosomes separated from Taxus by the above method have a vesicle structure.

[0047] Example 2

[0048] Taxus exosomes are taken up by human hepatic stellate cells (LX-2)

[0049] 1. Experimental instruments and consumables:

[0050] Hepatic stellate cells were provided by Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. DiI dye (CAS

[0051] 41085-99-8) was purchased from AAT Bioquest, USA, 24-well cell slide (WHB-24-CS-10) was purchased from Shanghai Wohong Biotechnology Co., Ltd., DAPI (BL105A) was purchased from biosharp, and the fluorescence microscope (Leica Microsystems) was a German Wetzlar brand.

[0052] 2. Experimental Procedure

[0053] In order to clarify whether Taxus exosomes have a regulatory effect on fatty liver, the present invention selected hepatic stellate cells, which are widely used in fibrosis research, as model cells.

[0054] DiI labeled exosomes: DiI dye (CAS 41085-99-8) was prepared with DMSO to a working concentration of 1 mM solution, filtered through a 0.22 μm filter membrane, and stored in the dark. The exosomes obtained in Example 1 with a concentration of 1 mg / mL were incubated with 1 μM DiI dye in the dark for 30 min, and resuspended in PBS. Centrifuged at 100000 g for 2 h to remove excess DiI dye.

[0055] DiI-labeled exosomes were co-cultured with cells: LX-2 cells were inoculated onto cell slides at a cell inoculation concentration of 3×10 4 After 24 hours, DiI-labeled exosomes were added to the culture medium at a concentration of 0.1 mg / mL. After 24 hours, fluorescence staining was performed.

[0056] Fluorescence staining and confocal microscopy observation: The cell slides were fixed with 4% paraformaldehyde for 30 minutes, and then washed with PBS for 4 times, 5 minutes each time, to wash away the residual paraformaldehyde. The cell nuclei were stained with 1 mg / mL DAPI, incubated for 15 minutes in a dark environment, washed with PBS, and sealed with a sealing agent containing an anti-fluorescence quencher. The images were observed and collected under a confocal microscope. The results are shown in the figure. Figure 5 and Figure 6 shown.

[0057] 3. Experimental results

[0058] The results of confocal microscopy observation of the localization of DiI-labeled exosomes of Taxus chinensis in recipient cells showed that a large amount of red fluorescence, i.e., DiI-labeled exosomes, could be observed in LX-2 cells, and both types of exosomes derived from Taxus chinensis were localized in the cytoplasmic region of LX-2 cells, indicating that both types of Taxus chinensis exosomes isolated in the present invention could be taken up by LX-2 cells and had the potential to treat alcoholic liver injury.

[0059] Example 3

[0060] Effects of Taxus chinensis exosomes on adipogenesis of human hepatic stellate cells

[0061] Experimental instruments and consumables: The experimental drug TGF-β1 was purchased from RD Company in the United States, 96-well plates and 6-well plates were purchased from Corning (Shanghai) Co., Ltd., and CCK8 cell proliferation detection kit (C0037) was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd. 95% alcohol was purchased from Sichuan Yijieshi Medical Technology Co., Ltd.

[0062] Experimental procedure: LX-2 cells were cultured at 2×10 5 Cells / well were seeded in 96-well or 6-well plates. After the cells adhered (more than 6 hours), LX-2 cells were activated by treating with 100mmol / L alcohol for 12 hours, and then treated with different concentrations of Taxus exosomes (2μg / mL, 10μg / mL, 30μg / mL). The proliferation of hepatic stellate cells was detected using the CCK8 kit for 6 consecutive days.

[0063] When LX-2 cells were cultured in serum-free conditions until they were almost contaminated in a 6-well plate, a scratch was made in the middle of the culture well with a pipette tip. After replacing the culture medium, Taxus exosomes were added. After 48 hours, the scratch area was measured to analyze the effect of Taxus exosomes on cell migration. The results are shown in the figure. Figure 7 , Figure 8 and Fig. 9 shown.

[0064] In this example, 100mmol / L alcohol was used to activate LX-2 from a static state to a fatty state to simulate the fatty liver process. After the addition of yew exosomes, the proliferation of LX-2 cells was significantly weakened from the second day (P<0.01, P<0.05). This indicates that yew exosomes can partially offset the alcohol-induced LX-2 cell proliferation process, and yew exosomes have a regulatory effect on fatty liver after being taken up by LX-2 cells.

[0065] This example uses a scratch test to evaluate the migration ability of LX-2. Fig. 9 , Taxus exosomes significantly reduced the migration degree of LX-2 cells (P<0.01). Compared with the control, the migration rates were reduced by about 43% and 33%, respectively. This indicates that the Taxus exosomes of the present invention have anti-lipidation activity that inhibits the migration of LX-2 cells.

[0066] Example 4

[0067] Effects of Taxus chinensis exosomes on alcohol-induced fatty liver

[0068] Experimental grouping: The experimental animals used in the present invention were C57BL / 6 mice, 4 weeks old, weighing 20-24g, male, provided by Chengdu Dashuo Experimental Animal Co., Ltd., and alcohol liquid feed was purchased from Nantong Trofi Feed Technology Co., Ltd. 24 mice were randomly divided into a normal control group, a model group, a stem exosome treatment group (stem group) and a leaf exosome treatment group (leaf group), with 6 mice in each group. The process of mouse fatty liver modeling and yew exosome treatment is as follows Fig.10 shown.

[0069] The model group and the treatment group were fed with 5% alcohol liquid feed for 4 weeks. From the 4th week, the control group and the model group were given an equal volume of PBS solution, and the yew exosome treatment group was given two kinds of exosomes from stems and leaves by gavage, with a dose of 1 mg / Kg, dissolved in PBS solution, once every two days, for a total of 2 weeks. After the 6th week, all mice were anesthetized and killed, and serum and liver tissue were collected to detect serum liver function and perform liver tissue pathology (H&E) analysis. The results obtained are as follows Fig.11 shown.

[0070] Experimental results:

[0071] Effects of Taxus exosomes on serological parameters

[0072] Compared with the control group, the serum AST and ALT activities in the model group were significantly increased (P<0.01); compared with the model group, the serum AST and ALT activities in the Taxus exosome treatment group were significantly decreased (P<0.01). Therefore, the present invention shows that Taxus exosome treatment significantly improves the liver function of mice with fatty liver.

[0073] Effects of Taxus chinensis exosomes on liver histological changes

[0074] The results of staining liver tissue are as follows Fig.12 and Fig.13 As shown;

[0075] HE staining showed that the hepatocyte morphology of mice in the normal group was relatively normal, the hepatic lobule structure was clear, and there was no collagen deposition; the hepatocytes of mice in the model group were massively necrotic, the normal hepatic lobule structure partially disappeared, the collagen deposition was relatively serious, and the number of white lipid droplets was relatively abundant; compared with the model group, the hepatocyte necrosis and inflammatory cell infiltration in the Taxus chinensis exosome treatment group were significantly reduced, and the number of lipid droplets was relatively small.

[0076] Oil red O staining was used to detect the presence and number of fat droplets in liver tissue to reflect the degree of fatty liver development. After treatment with Taxus exosomes, the number of red fat droplets visible in the liver was significantly reduced.

[0077] The present invention shows that the yew exosome treatment restored the liver tissue morphology of mice with fatty liver and alleviated the development of fatty liver.

[0078] The above experiments indicate that yew exosomes have a good therapeutic effect on fatty liver and can be used to prepare drugs for the treatment or prevention of liver diseases such as fatty liver.

[0079] In the above-mentioned uses, the application time, number of applications, and frequency of application of various active ingredients need to be determined according to the actual conditions of the subject, which is within the technical scope of those skilled in the art. For example, when the treatment plan for mice is applied to humans, the effective doses of all active ingredients for humans can be converted by their effective doses for mice, which is also easy to achieve for those of ordinary skill in the art.

[0080] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for preparing Taxus exosomes, characterized in that: The method for preparing the yew exosomes comprises: grinding fresh yew into a homogenate in 5-20 times the volume of PBS, centrifuging, filtering the supernatant through a 0.2-0.5 μm filter membrane, centrifuging again, and resuspending the precipitate in PBS to obtain the yew exosomes.

2. The preparation method according to claim 1, characterized in that: The fresh yew comprises yew leaves and stems.

3. Application of yew exosomes in the preparation of drugs for treating liver diseases.

4. The use according to claim 3, characterized in that: The liver diseases include fatty liver, cirrhosis and hepatitis.

5. The use according to claim 4, characterized in that: The fatty liver includes chemically damaged fatty liver and virally damaged fatty liver; the cirrhosis includes alcoholic cirrhosis and fatty cirrhosis; and the hepatitis includes fatty hepatitis.

6. A drug for treating fatty liver, characterized in that: The drug comprises the yew exosomes according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier.

7. The drug according to claim 6, characterized in that The dosage form of the medicine is tablet, capsule, pill, powder, granule, suspension, oral solution and injection.

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