Application of chitosan oligosaccharide and derivatives thereof in preparation of products for preventing and treating liver diseases

Abstract

The invention discloses application of chitosan oligosaccharide and derivatives thereof in preparation of products for preventing and treating liver diseases, and belongs to the technical field of biological medicines. The application has the beneficial effects that the invention provides the application of the chitosan oligosaccharide and the derivative thereof (such as carboxymethyl chitosan oligosaccharide) in preparation of medicines for preventing or treating liver inflammation and liver tissue fibrosis, the carboxymethyl chitosan oligosaccharide (CMCOS) reduces liver tissue fiber proliferation and fibrosis by down-regulating expression of TGF-beta1, and the HF symptom is remarkably relieved in morphology, hematology and pathology. Compared with silymarin (SLM) which is clinically applied, the CMCOS has the advantages that the biological safety is higher, and the treatment effect is better. Wide application prospects and economic values are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of chitosan oligosaccharide and its derivatives in the preparation of products for preventing and treating liver diseases. Background Art

[0002] Hepatic fibrosis (HF) can be caused by a variety of factors, including viral infections (HBV and HCV), alcohol consumption, non-alcoholic (metabolic) fatty liver disease, and congenital diseases. Alcoholic and non-alcoholic fatty liver disease are the leading causes of liver disease in Europe and the United States. In my country, chronic hepatitis B virus infection is the leading cause of liver fibrosis and even cirrhosis.

[0003] Liver fibrosis is the final result of an imbalance between damage and repair in the liver. In a normal liver, the content and spatial distribution of cells and cells, and cells and extracellular matrix (ECM) are in a delicate dynamic balance. In acute damage, fibrosis, as the body's defense response to external stimuli, is beneficial for the liver to repair itself. When the liver is attacked by various pathogenic factors (such as hepatitis B virus, hepatitis C virus, certain drugs and poisons, alcohol) for a long time, it evolves into repeated and persistent chronic liver inflammation, and a large amount of fibrous connective tissue proliferates with abnormal fiber decomposition ability, causing abnormal diffuse deposition of collagen fibers in the liver, which will eventually lead to the occurrence of fibrosis.

[0004] The development of liver fibrosis is the result of the interaction, mutual influence, and feedback of a series of cells, chemokines, and molecular signals, including hepatic stellate cells (HSCs), giant cells, endothelial cells, mesenchymal stem cells, inflammasomes, and pyroptosis. Activated HSCs become a type of hepatic myofibroblast, a major driver of liver fibrosis. Therefore, blocking the action of activated HSCs is a promising strategy for the treatment of liver fibrosis and a key focus of drug development.

[0005] Currently, there are no specific and effective anti-fibrosis treatments available clinically. Treatment of the underlying disease causing liver damage is primarily aimed at alleviating liver damage and inflammation, and preventing and treating liver fibrosis. Therefore, finding new drugs to treat interstitial hepatitis and liver fibrosis is a difficult and important task that needs to be addressed urgently.

[0006] Chitosan oligosaccharide (COS) is the only naturally occurring, positively charged, cationic alkaline amino oligosaccharide. It is an animal-derived cellulose. Also known as chito-oligosaccharide or oligochitosan, it is an oligosaccharide product with a degree of polymerization (DP) between 2 and 20, obtained by degrading chitosan using a special bio-enzyme process (chemical degradation and microwave degradation have also been reported). Its molecular weight is ≤5000 Da, making it a low-molecular-weight product with good water solubility, extensive functional effects, and high biological activity. It possesses many unique properties not found in chitosan, including high solubility, complete water solubility, and easy absorption and utilization by organisms.

[0007] Carboxymethyl chitosan (CMCOS) is an important water-soluble chitosan derivative with numerous medical benefits, such as promoting wound healing, hemostasis, scar inhibition, analgesia, and antibacterial properties. It also has extensive applications in the chemical industry, environmental protection, and health products. Carboxymethyl chitosan exhibits excellent biocompatibility and biodegradability, making it widely used in hydrogels and wound healing biomaterials, as well as in tissue engineering matrices. However, the use of chitosan oligosaccharides and carboxymethyl chitosan for the treatment of liver fibrosis has not yet been reported. Summary of the Invention

[0008] The technical problem to be solved by the present invention is how to propose a new use of chitosan oligosaccharide and its derivatives.

[0009] The present invention solves the above technical problems through the following technical means:

[0010] The first aspect of the present invention provides the use of chitosan oligosaccharides and their derivatives in the preparation of products for preventing and treating liver inflammation.

[0011] Preferably, the product includes any one or more of food, medicine, and health care products.

[0012] Preferably, chitosan oligosaccharide derivatives include but are not limited to carboxymethyl chitosan oligosaccharide and hydroxypropyl chitosan oligosaccharide.

[0013] Preferably, the molecular weight of the chitosan oligosaccharide and its derivatives is 200-5000 Da, more preferably 2000-3000 Da.

[0014] Preferably, the dosage of the chitosan oligosaccharide and its derivatives is 50-300 mg / kg (mouse), more preferably 100-200 mg / kg (mouse).

[0015] Preferably, the dosage of the carboxymethyl chitosan oligosaccharide is 150-250 mg / kg.

[0016] A second aspect of the present invention also proposes any application of carboxymethyl chitosan oligosaccharide (CMCOS) in the following:

[0017] (1) Application in the preparation of drugs for preventing and treating liver tissue fibrosis;

[0018] (2) Application in the preparation of drugs for downregulating TGF-β1;

[0019] (3) Application in the preparation of drugs for reducing excessive extracellular matrix deposition

[0020] (4) Application in the preparation of drugs for reducing the levels of ALT, AST, TBIL, and ALP.

[0021] Preferably, in (1) to (4), the drug further comprises a pharmaceutically acceptable carrier.

[0022] Preferably, the pharmaceutically acceptable carrier is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

[0023] Preferably, in (1) to (4), the drug is prepared into a pharmaceutically acceptable dosage form.

[0024] Preferably, the dosage form is tablet, pill, paste, capsule, oral solution or granule.

[0025] The third aspect of the present invention further provides a drug for preventing and treating liver-related diseases, the active ingredients of which include chitosan oligosaccharides and their derivatives.

[0026] Preferably, the liver-related diseases include liver inflammation and liver tissue fibrosis.

[0027] The beneficial effects of the present invention are:

[0028] The present invention proposes the use of chitosan oligosaccharides and their derivatives (such as carboxymethyl chitosan oligosaccharides) in the preparation of drugs for preventing or treating liver inflammation and liver fibrosis. Chitosan oligosaccharides and carboxymethyl chitosan oligosaccharides (CMCOS) reduce liver tissue fibrosis and fibrosis by downregulating TGF-β1 expression, significantly alleviating HF symptoms in morphology, hematology, and pathology. Furthermore, compared to the clinically used silymarin (SLM), CMCOS has higher biosafety and better therapeutic effects. It has broad application prospects and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure 2 is a graph showing the biological toxicity of CMCOS in Example 2 of the present invention; Figure A is a graph showing the coefficients of major organs, and Figure B is a graph showing the comparison of body weight changes;

[0030] Figure 2Figure 3 shows the effect of CMCOS and SLM on reducing liver inflammation in Example 3 of the present invention; Figure 4 shows the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and alkaline phosphatase (ALP) in serum;

[0031] Figure 3 This is a graph showing the TGF-β1 content in mice detected by Elisa in Example 4 of the present invention;

[0032] Figure 4 The H&E, Sirus red, and Masson staining images (A) and their quantification images (B) in Example 4 of the present invention are shown;

[0033] Figure 5 These are images of CMCOS reducing excessive ECM deposition in liver tissue in Example 5 of the present invention, wherein A is an IHC immunohistochemistry staining and B is an IHC quantification image.

[0034] In the figure, #P<0.05, ##P<0.01, ###P<0.001 compared with the control group; *P<0.05, **P<0.01, ***P<0.001 compared with the model group. DETAILED DESCRIPTION

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only 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 making creative efforts shall fall within the scope of protection of the present invention.

[0036] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0037] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0038] Example 1:

[0039] In order to evaluate the safety of chitosan oligosaccharides, L-02 (normal human liver cells) cells were treated with chitosan oligosaccharides at different concentrations for 24 hours. The specific operation included:

[0040] 1. Culture L-02 cells in DMEM complete medium at 37°C, 5% CO2. Replace the complete medium every two days. When cells reach 90% confluence, passage them at a 1:3 ratio.

[0041] 2. LX-2 cells were seeded in 96-well plates. On the next day, COS was added to the final concentrations of 0, 50, 100, 200, 500, 1000, 1500, 2000, 2500, and 3000 μg / ml, and the cells were treated for 24 hours.

[0042] 3. In the dark, add 20 μl of MTT solution (concentration of 5.0 mg / ml) to each well and continue incubating in the cell culture incubator for 4 hours.

[0043] 4. Discard the culture medium, add 150 μl of DMSO solution to each well, and shake on a shaker for 10 minutes.

[0044] 5. Measure the absorbance at 492 nm.

[0045] The experiment found that chitosan oligosaccharide had no significant effect on the viability of L-02 cells. Therefore, chitosan oligosaccharide has low toxicity and high safety.

[0046] Chitosan oligosaccharides inhibit the proliferation and migration of hepatic stellate cells:

[0047] Hepatic stellate cells (HSCs), a type of liver fibroblast, are a major driver of liver fibrosis. Using cell wound healing and transwell assays, we found that COS effectively inhibited the proliferation and migration of LX-2 (human hepatic stellate cells). These results suggest that COS can reduce the proliferation and migration of HSCs, thereby alleviating liver fibrosis.

[0048] Example 2:

[0049] Biosafety Determination of Carboxymethyl Chitosan Oligosaccharide (CMCOS)

[0050] To determine the safety of CMCOS, 30 Balb / c mice were randomly divided into a control group (CON) and a CMCOS group. The control group received an oral aqueous solution, while the CMCOS group received an oral 1000 mg / kg aqueous solution. The experimental period was one month, with animals receiving 0.1 mL of distilled water orally daily.

[0051] The results showed that no death or significant weight loss occurred during the administration period. Figure 1 shown)

[0052] Example 3:

[0053] Carboxymethyl chitosan oligosaccharide (CMCOS) reduces liver inflammation

[0054] Specific implementation steps:

[0055] The pharmacology of CMCOS was investigated in a carbon tetrachloride (CCl4)-induced liver fibrosis (HF) mouse model. Silymarin (SLM) treatment served as a positive control at a dose equivalent to the clinically recommended dose. Mice were randomly divided into control, model, SLM, and CMCOS groups. CCl4 in olive oil (CCl4:Olive oil mixed in a 1:1 ratio) was injected intraperitoneally twice weekly at a dose of 1 mL / kg. Mice in the blank control group underwent the same treatment, except for an equal volume of olive oil solution, for 12 weeks. After model establishment, mice in the SLM and CMCOS groups received oral administration of 100 mg / kg SLM in water and 0.5 mL CMCOS (200 mg / kg), respectively, once daily. The control and model groups received 0.5 mL of distilled water instead of oral administration. Sample collection and evaluation were performed at week 13.

[0056] The experimental results showed that carboxymethyl chitosan oligosaccharide (CMCOS) treatment reduced liver inflammation in the middle of the experiment, with significant differences compared with the control group, model group, and SLM group. This indicates that CMCOS treatment reduced the level of liver tissue inflammation and resisted CCl4-induced liver cell damage. Recovery of liver cell damage can reduce macrophage differentiation and activation of hepatic stellate cells, thereby preventing the occurrence of inflammation and fibrosis. (e.g. Figure 2 shown)

[0057] Example 4:

[0058] Carboxymethyl chitosan oligosaccharide (CMCOS) downregulates TGF-β1 and reduces liver tissue fibrosis:

[0059] Increased TGF-β1 levels were associated with increased collagen deposition and hepatic stellate cell proliferation. TGF-β1 levels in the model group remained high throughout the experiment. After 13 weeks of treatment, CMCOS effectively inhibited TGF-β1 overexpression, with a statistically significant difference compared with SLM (P < 0.01).

[0060] Specific implementation steps: 40 Balb / c mice were taken and randomly divided into 4 groups: blank (normal saline), model, SLM (100 mg / kg), and CMCOS (200 mg / kg). Intraperitoneal injection was performed twice a week, and the injection liquid was carbon tetrachloride olive oil solution (carbon tetrachloride and olive oil were mixed in a ratio of 1:1), and 1 mL / kg was injected each time. The blank control group mice were operated in the same way, but with an equal amount of olive oil solution, which lasted for 12 weeks. After modeling, the mice in the SLM group and the CMCOS group were orally administered with SLM 100 mg / kg aqueous solution and 0.5 mL CMCOS (200 mg / kg), respectively, once a day. The control group and the model group used 0.5 mL of distilled water instead of oral administration. (As Figure 3 、 4 shown)

[0061] H&E staining results showed that in the control group, hepatocytes were tightly connected, hepatic cords were arranged uniformly and orderly, and there was no obvious congestion or edema, and no obvious inflammatory cell infiltration. However, in the carbon tetrachloride model group, gaps appeared between hepatocytes, hepatic cords were loosely arranged, and hepatocyte swelling, fat droplets, and Mallory bodies were observed. Numerous inflammatory cell infiltrations were observed around the hepatic lobules and portal areas.

[0062] Sirus red and Masson staining results showed that there was no large amount of collagen deposition in the control group, while a large amount of collagen deposition appeared in the liver tissue of mice in the carbon tetrachloride model group. The amount of collagen deposition in the carboxymethyl chitosan oligosaccharide treatment group and the silymarin treatment group was less than that in the carbon tetrachloride model group, and the effect of the carboxymethyl chitosan oligosaccharide group was better than that of the silymarin group.

[0063] Example 5:

[0064] Carboxymethyl chitosan oligosaccharide (CMCOS) reduces liver fibrosis

[0065] The specific implementation steps are the same as those in Example 4.

[0066] Liver fibrosis is characterized by ECM deposition. The results showed that the liver tissue in the model group was unevenly distributed, with Mallory bodies, Collagen I, α-SMA and Fibronectin protein expressions significantly increased. Compared with SLM, CMCOS treatment avoided this situation. (e.g. Figure 5 shown)

[0067] IHC was used to detect changes in the expression of characteristic extracellular matrix proteins Collagen I, a-SMA, and Fibronectin. As shown in the figure, compared with the blank group, the liver tissue of the mice in the carbon tetrachloride model group was unevenly distributed, with Mallory bodies, and the expression of Collagen I, a-SMA, and Fibronectin proteins was significantly increased (P<0.01). Compared with the carbon tetrachloride model group, the expression levels of Collagen I, a-SMA, and Fibronectin proteins in the carboxymethyl chitosan oligosaccharide treatment group and the silymarin treatment group were decreased (P<0.05), and the effect of the carboxymethyl chitosan oligosaccharide group was better than that of the silymarin group. The results show that carboxymethyl chitosan oligosaccharide can inhibit the process of extracellular matrix deposition in the liver tissue of mice induced by CCl4, thereby alleviating liver fibrosis.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Application of chitosan oligosaccharides and their derivatives in the preparation of products for preventing and treating liver inflammation.

2. The use according to claim 1, characterized in that The products include any one or more of food, medicine, and health care products.

3. The use according to claim 1, characterized in that Chitosan oligosaccharide derivatives include carboxymethyl chitosan oligosaccharide and hydroxypropyl chitosan oligosaccharide.

4. The use according to claim 3, characterized in that The dosage of the carboxymethyl chitosan oligosaccharide is 150-250 mg / kg.

5. Any of the following applications of carboxymethyl chitosan oligosaccharide: (1) Use in the preparation of drugs for preventing and treating liver tissue and / or colon tissue fibrosis; (2) Application in the preparation of drugs for downregulating TGF-β1; (3) Application in the preparation of drugs for reducing excessive deposition of extracellular matrix; (4) Application in the preparation of drugs for reducing the levels of ALT, AST, TBIL, and ALP.

6. The use according to claim 5, characterized in that In (1) to (4), the drug further includes a pharmaceutically acceptable carrier.

7. The use according to claim 6, characterized in that The pharmaceutically acceptable carrier is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

8. The use according to claim 5, characterized in that In (1) to (4), the drug is prepared into a pharmaceutically acceptable dosage form; the dosage form is tablets, pills, ointments, capsules, oral liquids or granules.

9. A drug for preventing and treating diseases related to the liver and / or colon, characterized in that: Its active ingredients include carboxymethyl chitosan oligosaccharide.

10. The drug according to claim 9, characterized in that Liver-related diseases include liver inflammation and liver tissue fibrosis.

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