Application of a kind of Zanthoxylum bungeanum exosome in the preparation of a drug for preventing and / or treating hepatic diseases

Abstract

The present invention discloses the application of Zanthoxylum bungeanum exosomes in the preparation of a drug for preventing and / or treating hepatic diseases, and specifically relates to the field of biomedical technologies. The application of Zanthoxylum bungeanum exosomes in the preparation of a drug for preventing and / or treating hepatic diseases is based on the principle of inhibiting the proliferation, migration and fibrosis indicators of hepatic stellate cells. For the alcohol-induced chemical injury liver fibrosis model in mice, intervention with Zanthoxylum bungeanum exosomes can significantly improve the pathological changes of liver tissue, inhibit collagen deposition, reduce the number of lipid droplets in liver tissue, and decrease the activities of serum AST and ALT, with significantly better effects than the non-drug-administered model group. The present invention provides a drug containing Zanthoxylum bungeanum exosomes or related biological materials. Compared with traditional therapeutic drugs, this drug has the advantages of being easy to separate, easy to detect, rich in sources, high in safety, good in stability, and can also be used as a drug delivery system to load targeted drugs, etc., thereby improving the treatment efficiency of liver fibrosis diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to the application of Zanthoxylum bungeanum exosomes in the preparation of a drug for preventing and / or treating liver diseases. Background Art

[0002] Liver fibrosis is a disease caused by various chronic liver injuries, such as viral hepatitis, alcoholic liver disease, fatty liver, autoimmune hepatitis, etc. The excessive proliferation and abnormal deposition of extracellular matrix components in liver tissue cause a large amount of fibrous connective tissue to proliferate and form scars, and the fibrous scars will damage the liver structure, leading to hepatocyte necrosis and dysfunction of normal liver functions, and aggravating liver injury.

[0003] The activation of hepatic stellate cells is a key factor in the occurrence of liver fibrosis. Research shows that after hepatocyte injury, a variety of cytokines will be secreted, and these cytokines dynamically regulate the process of liver fibrosis lesions through pathways such as the TGF-β1 / Smad signaling pathway, the p53 pathway, and ferroptosis. Among them, hepatic stellate cells are transformed into fibroblasts after being activated by cytokines, and ultimately cause excessive accumulation of extracellular matrix in the liver. Fibrocytes, bone marrow-derived cells, and hepatocytes can also be transformed into fibroblasts through different stimulatory factors to trigger liver fibrosis.

[0004] At present, there is no anti-fibrosis drug with definite curative effect in Western medicine. However, some traditional Chinese medicine components such as flavonoids, phenolic acids, alkaloids, and polysaccharides have been found to have significant anti-fibrosis effects in clinical treatment. In recent years, among the single traditional Chinese medicines for anti-liver fibrosis, Paeonia lactiflora, Salvia miltiorrhiza, Astragalus membranaceus, and Trionyx sinensis have the highest usage frequency. Among the compound traditional Chinese medicines, clearing heat and removing dampness, promoting blood circulation to remove blood stasis, soothing the liver and strengthening the spleen, nourishing the liver and kidney, etc. are the main ones. The traditional Chinese medicine compound capsules mainly include Fuzheng Huayu Capsule, Biejia Ruangan Capsule, Jigucao Capsule, etc. However, research shows that treating liver fibrosis with a large dose of Scutellaria baicalensis will aggravate the degree of liver fibrosis. In addition, some traditional Chinese medicines such as Tripterygium wilfordii and Polygonum multiflorum have been reported to have liver damage effects. Therefore, how to better deliver the active ingredients in plants into hepatocytes, affect the behavior of hepatocytes, and clarify the molecular mechanism of plant active ingredients regulating hepatocytes remains to be further studied in depth.

[0005] Plant extracellular vesicles are membranous vesicles secreted by plant cells, with a lipid bilayer as the basic skeleton and encapsulating various active substances such as proteins and nucleic acids, and play an important role in processes such as plant growth and development, tissue repair, and self-defense.

[0006] It has been found that plant-derived vesicles have the potential to treat alcoholic liver damage. After healthy mice were orally administered ginger-derived vesicles, the vesicles were found to accumulate in large quantities in the liver and mesenteric lymph nodes, and inhibit the production of reactive oxygen species in hepatocytes. For mice with alcoholic liver damage, vesicle treatment can significantly reduce liver weight and the number of lipid droplets, and reduce triglyceride levels, which is significantly better than the untreated model group, indicating that ginger vesicles have the potential to prevent and treat alcoholic liver damage.

[0007] Zanthoxylum bungeanum refers to the fruit of various species of Zanthoxylum bungeanum in the Rutaceae family, which is widely used in Chinese kitchens for its spicy flavor and medicinal properties. The essential oil content of fresh Zanthoxylum bungeanum is very high, up to 11%, and is described as having a fresh, spicy, floral, cool and green aroma. Modern pharmacological studies have shown that Zanthoxylum bungeanum has analgesic and anti-inflammatory, liver-protecting, antioxidant, mosquito-repellent and insect-killing, anti-tumor, hypoglycemic, and anti-Alzheimer's effects. It is still unclear what the effect and mechanism of Zanthoxylum bungeanum on liver fibrosis is. Summary of the invention

[0008] To this end, the present invention provides an application of Zanthoxylum bungeanum exosomes in the preparation of a medicament for preventing and / or treating liver diseases, so as to fill the gap in the existing research on Zanthoxylum bungeanum for liver fibrosis.

[0009] The present invention has discovered for the first time that Zanthoxylum bungeanum exosomes and related biomaterials can be used to prevent and / or treat liver fibrosis, and can achieve the effect of preventing and / or treating liver fibrosis by inhibiting the proliferation, migration and fibrosis indicators of liver stellate cells, and can be used as a drug for preventing and / or treating liver fibrosis. The present invention found that for the alcohol-induced mouse chemical injury liver fibrosis model, Zanthoxylum bungeanum exosome intervention can significantly improve liver tissue pathological changes, inhibit collagen deposition, reduce the number of lipid droplets in liver tissue, and reduce serum AST and ALT activity, and the effect is significantly better than that of the unmedicated model group. The present invention provides a drug containing Zanthoxylum bungeanum exosomes or related biomaterials. Compared with traditional therapeutic drugs, the drug has the advantages of easy separation, easy detection, abundant sources, high safety, good stability, and can also be used as a drug delivery system to load targeted drugs, thereby improving the efficiency of liver fibrosis disease treatment.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] According to one aspect of the present invention, a use of Zanthoxylum bungeanum exosomes in the preparation of a medicament for preventing and / or treating liver diseases is provided.

[0012] Furthermore, the liver diseases include cirrhosis, hepatitis, liver fibrosis and fatty liver.

[0013] The liver fibrosis includes alcoholic liver disease and non-alcoholic liver disease.

[0014] In one embodiment, the liver fibrosis treatment drug has at least one of the following functions:

[0015] (1) Inhibiting the activation, proliferation and migration of hepatic stellate cells;

[0016] (2) Improving the histopathological changes of the liver tissue;

[0017] (3) Reducing the expression level of liver fibrosis marker genes.

[0018] The liver fibrosis marker genes of the present invention include α-SMA and COL1.

[0019] Furthermore, the liver diseases include chemically induced liver fibrosis, virus-induced liver fibrosis, fatty hepatitis, alcoholic cirrhosis and fatty liver cirrhosis.

[0020] Furthermore, the chemically induced liver fibrosis includes carbon tetrachloride-induced liver fibrosis.

[0021] Furthermore, the preparation method of the Zanthoxylum bungeanum exosomes includes: grinding fresh Zanthoxylum bungeanum into a homogenate in PBS, centrifuging multiple times, filtering the supernatant and then centrifuging again, and resuspending the precipitate with PBS to obtain Zanthoxylum bungeanum exosomes.

[0022] Furthermore, the method of multiple centrifugation is centrifugation at 1000g for 10 min, 4000g for 20 min, and 10000g for 30 min; the re-centrifugation is centrifugation at 100000g for 60 min.

[0023] Furthermore, the Zanthoxylum bungeanum includes young stems and leaves, immature green Zanthoxylum bungeanum and mature red Zanthoxylum bungeanum.

[0024] The Zanthoxylum bungeanum exosomes have a double-membrane structure under a transmission electron microscope, mostly in a disc shape, and the particle size of the Zanthoxylum bungeanum exosomes is 50-200 nm.

[0025] According to another aspect of the present invention, a drug for treating liver diseases, the drug contains Zanthoxylum bungeanum exosomes or vesicles and a pharmaceutically acceptable carrier.

[0026] Furthermore, the dosage form of the drug is tablets, capsules, pills, powders, granules, suspensions, oral solutions, injections.

[0027] The above various dosage forms can be prepared according to the conventional processes in the field of pharmaceutical preparations.

[0028] In the uses described above, for the administration time, administration frequency, and administration times of various active ingredients, etc., it needs to be determined according to the actual situation of the subject, which is within the technical scope mastered by those skilled in the art. For example, when applying the treatment plan for mice to humans, the effective dose of all active ingredients for humans can be converted from their effective doses for mice, which is also easily achievable for those of ordinary skill in the art.

[0029] The present invention provides the use of Zanthoxylum bungeanum exosomes in the preparation of drugs for preventing and / or treating liver fibrosis. Zanthoxylum bungeanum exosomes can inhibit the progression of liver fibrosis at both the cellular level and the tissue level, thereby inhibiting the development of liver fibrosis.

[0030] The present invention has the following advantages:

[0031] The present invention discovers for the first time that Zanthoxylum bungeanum exosomes can reverse liver fibrosis by inhibiting the proliferation and migration ability of hepatic stellate cells, and can be used as a drug for preventing and / or treating liver fibrosis.

[0032] The present invention discovers that for an alcohol-induced mouse liver fibrosis model, intervention with Zanthoxylum bungeanum exosomes can significantly reduce the expression levels of alanine aminotransferase and aspartate aminotransferase in mice, improve the histopathological changes of the liver tissue, inhibit collagen deposition, and reduce the number of lipid droplets in the liver tissue.

[0033] The present invention provides a drug containing Zanthoxylum bungeanum exosomes or related biological materials. Compared with traditional therapeutic drugs, this drug has good biosafety and therapeutic effects, can be absorbed orally, and can also be used as a drug delivery system to load targeted drugs, etc., thereby improving the efficiency of liver fibrosis treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained based on the provided drawings.

[0035] The structures, ratios, sizes, etc. illustrated in this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the limited conditions for the implementation of the present invention. Therefore, they do not have technical substance significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the effects that the present invention can produce and the purposes that can be achieved, should still fall within the scope that can be covered by the technical content disclosed in the present invention.

[0036] Figure 1Electron microscope and nano-particle size statistical charts of exosomes isolated from young tender stems and leaves of Zanthoxylum bungeanum (abbreviated as "stem") provided in Example 1 of the present invention, where A is the electron microscope image of the stem; B is the nano-particle size statistical chart of the stem.

[0037] Figure 2 Electron microscope and nano-particle size statistical charts of exosomes isolated from immature fruits of green Zanthoxylum bungeanum (abbreviated as "green") provided in Example 1 of the present invention, where A is the electron microscope image of the green; B is the nano-particle size statistical chart of the green.

[0038] Figure 3 Electron microscope and nano-particle size statistical charts of exosomes isolated from immature fruits of red Zanthoxylum bungeanum (abbreviated as "red") provided in Example 1 of the present invention, where A is the electron microscope image of the red; B is the nano-particle size statistical chart of the red.

[0039] Figure 4 Absorption of Zanthoxylum bungeanum exosomes in hepatic stellate cells (LX-2) provided in Example 2 of the present invention.

[0040] Figure 5 Cytotoxicity analysis of Zanthoxylum bungeanum exosomes on non-activated LX-2 provided in Example 3 of the present invention.

[0041] Figure 6 Analysis of the effect of Zanthoxylum bungeanum exosomes on the cell proliferation activity of activated LX-2 provided in Example 3 of the present invention.

[0042] Figure 7 Analysis of the effect of Zanthoxylum bungeanum exosomes on the expression of fibrosis marker genes in activated LX-2 provided in Example 3 of the present invention, where A is the expression of α-SMA (alpha smooth muscle actin); B is the expression of COL1A1 (type I collagen).

[0043] Figure 8 Analysis of the effect of Zanthoxylum bungeanum exosomes on the protein expression of fibrosis marker genes in activated LX-2 provided in Example 3 of the present invention, where A is the detection of the expression of α-SMA and COL1 by immunoblotting; B is the relative statistical situation of the expression abundance of α-SMA in Figure A; C is the relative statistical situation of the expression abundance of COL1 in Figure A.

[0044] Figure 9 Analysis of the effect of Zanthoxylum bungeanum exosomes on the cell migration of non-activated LX-2 provided in Example 4 of the present invention.

[0045] Figure 10 Flow chart representation of the animal (C57BL / 6J mice) experiment provided in Example 5 of the present invention.

[0046] Figure 11 This is an analysis of the effect of Zanthoxylum bungeanum exosomes on changes in mouse liver function provided in Example 5 of the present invention, wherein A is the change in alanine aminotransferase (ALT) in the blood; B is the change in aspartate aminotransferase (AST).

[0047] Figure 12 This is an analysis of the effect of Zanthoxylum bungeanum exosomes provided in Example 5 of the present invention on the degree of liver fibrosis in mice. DETAILED DESCRIPTION

[0048] 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.

[0049] Example 1

[0050] This embodiment provides a kind of Zanthoxylum bungeanum exosomes:

[0051] 1. Experimental purpose: Isolation and characterization of Zanthoxylum bungeanum exosomes

[0052] 2. Experimental methods:

[0053] The ultracentrifuge (L-100XP) used in the experiment was produced by Beckman Company of the United States, the transmission electron microscope was JEM-1400 of Japan, and the nanoparticle tracking analyzer (Particle Metrix) was produced by Meerbusch Company of Germany.

[0054] Freshly picked young stems and leaves, immature and mature fruits of Sichuan Hanyuan Zanthoxylum bungeanum were used to separate Zanthoxylum bungeanum exosomes (referred to as stem, green and red, respectively) by differential centrifugation. The specific steps were as follows: fresh Zanthoxylum bungeanum was ground into a homogenate in 10 times the volume of PBS, centrifuged at 1000g for 10min, 4000g for 20min, and 10000g for 30min, the supernatant was filtered through a 0.2-0.5μm filter membrane and centrifuged at 100000g for 60min, and the precipitate was resuspended in PBS to obtain Zanthoxylum bungeanum exosomes. The particle size of the exosomes was determined by nanoparticle tracking analysis (NTA), and the morphology and structure of the exosomes were observed using transmission electron microscopy (TEM).

[0055] 3. Experimental results: Electron microscopy showed that the exosomes of Zanthoxylum bungeanum contained a typical lipid bilayer membrane structure, which was round and about 100 nm in size, which was consistent with the characteristics of plant exosomes ( Figure 1 A. Figure 2 A. Figure 3A). The particle sizes of the stem, green, and red exosomes were measured by NTA, and it was found that the particle sizes of the three types of exosomes were mainly distributed in the range of 50 - 200 nm, which was consistent with the results observed by TEM ( Figure 1 B, Figure 2 B, Figure 3 B). This indicates that plant exosomes with a vesicle structure can be isolated from Zanthoxylum bungeanum by the above method.

[0056] Example 2

[0057] 1. Experimental purpose: This example provides the absorption of Zanthoxylum bungeanum exosomes by human hepatic stellate cells (LX-2);

[0058] 2. Experimental method:

[0059] The hepatic stellate cells used in the experiment were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. The 24-well cell slides (WHB-24-CS-10) were purchased from Shanghai Wohong Biotechnology Co., Ltd. The reagent DiI (CAS 41085-99-8) was purchased from AATBioquest Inc., USA. DAPI (BL105A) was purchased from biosharp. The fluorescence microscope (Leica Microsystems) was produced by Wetzlar, Germany.

[0060] 2.1 Labeling exosomes with DiI

[0061] The DiI powder was dissolved in DMSO to prepare a solution with a working concentration of 1 mM, filtered through a 0.22 μm filter membrane, and stored in the dark. Exosomes with a concentration of 1×10 11 per mL were co-incubated with 1 μM DiI in the dark for 30 min and resuspended with PBS. Centrifuged at 100,000 g for 2 h to remove the excess DiI dye.

[0062] 2.2 Co-culturing DiI-labeled exosomes with cells

[0063] The LX-2 cells were seeded onto the cell slides at a seeding concentration of 3×10 4 per well. After 24 h, the DiI-labeled exosomes were added to the culture medium at a concentration of 1×10 10 per mL. After 24 h, fluorescence staining was performed.

[0064] 2.3 Fluorescence staining and confocal microscopy observation

[0065] The cell slides were fixed with 4% paraformaldehyde for 30 min, then washed 4 times with PBS for 5 min each time to wash away the residual paraformaldehyde. The cell nuclei were stained with 1 mg / mL DAPI and incubated in the dark for 15 min. After washing with PBS, the slides were mounted with a mounting medium containing an anti-fluorescence quencher. Observed and images were collected under a confocal microscope.

[0066] 3. Experimental results:

[0067] Previous studies have shown that plant vesicles derived from ginger have the potential to prevent and treat alcoholic liver injury. To clarify whether Zanthoxylum exosomes have a regulatory effect on liver fibrosis, the present invention selects hepatic stellate cells, which are widely used in fibrosis research, as model cells.

[0068] The results of confocal microscopy observations on the localization of three types of DiI-labeled exosomes, namely, green, red, and blue, in recipient cells showed that a large amount of red fluorescence, i.e., DiI-labeled exosomes, could be observed in LX-2 cells, and the three types of Zanthoxylum-derived exosomes were mainly localized in the cytoplasmic region of LX-2 cells, indicating that the three types of Zanthoxylum exosomes isolated in the present invention could be taken up by LX-2 cells( Figure 4 ).

[0069] Example 3

[0070] 1. Experimental purpose: This example examines the effect of Zanthoxylum exosomes on the fibrosis of human hepatic stellate cells;

[0071] 2. Experimental method:

[0072] The experimental drug TGF-β1 was purchased from RD Company in the United States, and the CCK8 cell proliferation detection kit (C0037) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The 95% alcohol used in the experiment was purchased from Sichuan Yijieshi Medical Technology Co., Ltd. The total RNA extraction kit (MiPure Cell / Tissue miRNAKit, RC201), reverse transcription kit( III RTSuperMix for qPCR, R323-01) and qPCR kit (ChamQ Universal SYBR qPCR Master Mix, Q711-02 / 03) were purchased from Nanjing Novozymes Biotechnology Co., Ltd. The α-SMA antibody (114395-1-AP), COL1 antibody (14695-1-AP) and GAPDH antibody (60004-1-lg) were all purchased from proteintech company.

[0073] The LX-2 cells were seeded at a density of 2×10 5Cells were seeded at a density of [[number of cells]] per well in 96-well or 6-well plates. After the cells adhered to the plate (for more than 6 hours), the experimental groups were set according to the requirements: a control group (without any treatment), a positive group (treated with 100 mmol / L alcohol), a stem group (treated with stem exosomes after 12 hours of treatment with 100 mmol / L alcohol), a green group (treated with green exosomes after 12 hours of treatment with 100 mmol / L alcohol), and a red group (treated with red exosomes after 12 hours of treatment with 100 mmol / L alcohol). After 48 hours, the CCK8 kit was used to detect cell proliferation. Total RNA was extracted from the cells in the 6-well plates and reverse-transcribed according to the instructions. The expression levels of the fibrosis marker genes α-SMA and COL1A1 in LX-2 cells were analyzed by fluorescence quantitative PCR. The reverse transcription primers were designed and synthesized by Shanghai Sangon Biotech Co., Ltd. (the specific sequences are shown in Table 1).

[0074] Table 1 Primer sequences related to gene expression detection in Example 3

[0075]

[0076]

[0077] 3. Experimental results:

[0078] 3.1 Effects of Zanthoxylum exosomes on cell proliferation

[0079] To clarify whether Zanthoxylum exosomes have a regulatory effect on liver fibrosis after being taken up by LX-2 cells, this example analyzed the effects of Zanthoxylum exosomes on the proliferation of LX-2 cells. As Figure 5 shown, compared with the control group, the three types of Zanthoxylum exosomes had no significant effect on the proliferation of LX-2 cells and had no toxic effect on LX-2 cells.

[0080] Next, the present invention activated LX-2 from a quiescent state to a fibrotic state with 100 mmol / L alcohol to simulate the process of liver fibrosis. As Figure 6 shown, after treating LX-2 cells with 100 mmol / L alcohol, it could significantly induce their proliferation. After adding the three types of Zanthoxylum exosomes, namely stem, green, and red, the proliferation degree of LX-2 cells was significantly weakened (P<0.01). This indicates that Zanthoxylum exosomes can partially counteract the fibrotic process of LX-2 cells induced by alcohol.

[0081] 3.2 Effects of Zanthoxylum exosomes on the expression of fibrosis marker genes in LX-2 cells

[0082] To detect whether Zanthoxylum exosomes have certain anti-fibrotic activity, the present invention detected the expression levels of fibrosis marker genes in LX-2 cells. As Figure 7As shown in the figure, after treating LX-2 cells with 100 mmol / L alcohol, the expression of the marker genes α-SMA and COL1A1 was significantly up-regulated (the former was up-regulated by about 2.5 times, and the latter was about 1.5 times). In the presence of Zanthoxylum exosomes, the expression of both marker genes was significantly decreased (P<0.01). In addition, in this example, the protein expression levels of α-SMA and COL1 in LX-2 cells were analyzed by immunoblotting, and it was found that treatment with Zanthoxylum exosomes could significantly reduce the protein expression levels (P<0.05 and P<0.01, Figure 8 ). This indicates that Zanthoxylum exosomes can inhibit the expression of fibrosis marker genes at both the gene level and the protein level, thus indicating that Zanthoxylum exosomes have anti-fibrotic activity.

[0083] Example 4

[0084] 1. Experimental purpose: This example examines the effect of Zanthoxylum exosomes on the migration of LX-2 cells;

[0085] 2. Experimental method:

[0086] When culturing LX-2 cells in serum-free medium until they were basically confluent in a 6-well plate, a scratch was made in the middle of the culture well with a pipette tip. After changing the medium, three kinds of Zanthoxylum exosomes, namely stem, green, and red, were added respectively. After 24 hours, the effect of Zanthoxylum exosomes on cell migration was analyzed by measuring the scratch area.

[0087] 3. Experimental results:

[0088] The enhancement of the migration ability of LX-2 cells is also closely related to its fibrosis process. To further clarify the anti-fibrotic activity of Zanthoxylum exosomes, this example used a scratch test to evaluate the migration ability of LX-2. As Figure 9 shown, the three kinds of Zanthoxylum exosomes, namely stem, green, and red, significantly reduced the migration degree of LX-2 cells (P<0.01). Compared with the control, the migration rates were reduced by about 52%, 53%, and 49% respectively. Therefore, the present invention shows that Zanthoxylum exosomes have anti-fibrotic activity that inhibits the migration of LX-2 cells.

[0089] Example 5

[0090] 1. Experimental purpose: This example examines the effect of Zanthoxylum exosomes on alcohol-induced liver fibrosis;

[0091] 2. Experimental method

[0092] Experimental animals: The experimental animals used in the present invention are C57BL / 6 mice, 4 weeks old, weighing 20-24 g, male, provided by Chengdu Dashuo Experimental Animal Co., Ltd. The 95% alcohol used in the experiment was purchased from Sichuan Yijieshi Medical Technology Co., Ltd., and the alcohol liquid feed was purchased from Nantong Trolphy Feed Technology Co., Ltd.

[0093] Thirty mice were randomly divided into a normal control group, a model group, a stem exosome treatment group (stem group), a green exosome treatment group (green group), and an infrared exosome treatment group (red group), with 6 mice in each group. The modeling method was as follows Figure 10 : The model group and the treatment groups were fed a 5% alcohol liquid diet for 8 weeks (during which they were gavaged with 31.5% alcohol, twice a week). From the 4th week, the control group and the model group were given an equal volume of PBS solution, and the zanthoxylum exosome treatment groups were respectively given intragastric treatment with three kinds of exosomes, namely stem, green, and red, at a dose of 1×10 10 individuals / Kg, dissolved in PBS solution, once every two days, for a total of 4 weeks of intervention. After 8 weeks, all the mice were anesthetized and sacrificed, and serum and liver tissues were collected to detect serum liver function and perform histological (H&E) analysis of the liver tissue.

[0094] 3. Experimental results:

[0095] 3.1 Effects of zanthoxylum exosomes on serological indicators

[0096] Compared with the control group, the activities of serum AST and ALT in the model group were significantly increased (P<0.01); compared with the model group, the activities of serum AST and ALT in the zanthoxylum exosome treatment groups were significantly decreased (P<0.01), as shown in Figure 11 . Therefore, the present invention shows that the treatment with zanthoxylum exosomes improves the liver function of mice with liver fibrosis.

[0097] 3.2 Effects of zanthoxylum exosomes on histological changes in the liver

[0098] H&E staining showed that the hepatocytes of mice in the normal group had normal morphology, clear hepatic lobule structure, and no collagen deposition; in the model group, a large number of hepatocytes were necrotic, part of the normal hepatic lobule structure disappeared, collagen deposition was relatively severe, and the number of lipid droplets was relatively abundant; compared with the model group, the hepatocyte necrosis and inflammatory cell infiltration in the zanthoxylum exosome treatment groups were significantly reduced, and the number of lipid droplets was less ( Figure 12 ). Therefore, the present invention shows that the treatment with zanthoxylum exosomes restores the hepatic tissue morphology of mice with liver fibrosis.

[0099] Through the above experiments, it can be shown that zanthoxylum exosomes have a good therapeutic effect on liver fibrosis and can be used to prepare drugs for treating or preventing liver diseases such as anti-liver fibrosis.

[0100] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A use of Zanthoxylum bungeanum exosomes in the preparation of a medicament for preventing and / or treating liver fibrosis, characterized in that: The method for preparing the exosomes of Zanthoxylum bungeanum comprises: grinding fresh Zanthoxylum bungeanum into a homogenate in PBS, centrifuging the mixture multiple times, filtering the supernatant, centrifuging the mixture again, and resuspending the precipitate in PBS to obtain the exosomes of Zanthoxylum bungeanum.

2. The use according to claim 1, characterized in that: The method of multiple centrifugation is centrifugation at 1000g for 10 minutes, centrifugation at 4000g for 20 minutes, and centrifugation at 10000g for 30 minutes; and the next centrifugation is centrifugation at 100000g for 60 minutes.

3. The use according to claim 2, characterized in that: The pepper comprises young stems and leaves, immature green pepper and mature red pepper.

4. A drug for treating liver fibrosis, characterized in that: The medicine comprises the Zanthoxylum bungeanum exosomes as claimed in claim 1 and a pharmaceutically acceptable carrier.

5. The drug according to claim 4, characterized in that The dosage form of the medicine is tablet, capsule, pill, powder, granule, suspension and oral solution.

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