Application of Glucosamine or pharmaceutically acceptable salt thereof in preparation of medicine for preventing and / or treating alcoholic hepatitis

Abstract

The invention provides an application of Glucosamine or a pharmaceutically acceptable salt thereof in preparation of a medicine for preventing and / or treating alcoholic hepatitis, and belongs to the technical field of biological medicines. The pharmaceutically acceptable salt of the Glucosamine disclosed by the invention is selected from at least one of hydrochloride and sulfate of the Glucosamine. The inventor accidentally finds that Glucosamine can inhibit polarization of M1 type macrophages and reduce release of pro-inflammatory cytokines, so that the Glucosamine has a protective effect on alcoholic hepatitis caused by long-term drinking and can obviously inhibit liver inflammation and reduce the death rate, experimental mice do not show abnormal reaction, and the Glucosamine can be used for preparing a medicine for treating alcoholic hepatitis caused by long-term drinking. Therefore, the Glucosamine can be applied to preparation of the medicine for preventing and / or treating alcoholic hepatitis caused by long-term and large-amount drinking.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of glucosamine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating alcoholic hepatitis. Background Art

[0002] In recent years, with the improvement of people's living standards and the popularization of drinking culture, the incidence of alcoholic liver damage has been increasing year by year and has become a global public health problem. According to the severity of alcohol damage to the liver, it can be divided into three stages: alcoholic fatty liver, alcoholic hepatitis and alcoholic cirrhosis. Alcoholic hepatitis is an intermediate stage of alcoholic liver damage. On the basis of alcoholic fatty liver, liver cell damage activates liver macrophages (Kupffer cells) and recruits more macrophages in the blood to enter the liver and polarize, which further induces liver immune cell infiltration and leads to the occurrence of hepatitis. Clinically, symptoms such as jaundice, nausea, vomiting, and loss of appetite will appear. The short-term mortality rate of alcoholic hepatitis is as high as 20-50%. In addition, 70% of patients with alcoholic hepatitis will progress to alcoholic cirrhosis. Although the medical level is constantly improving, the treatment measures for alcoholic hepatitis are not ideal.

[0003] Macrophages are an important cell in the innate immune system, playing an important role in maintaining the body's immune response and treating chronic inflammatory and autoimmune diseases. Macrophages can be divided into two subtypes according to their functions and activation: classically activated M1 macrophages and alternatively activated M2 macrophages. M1 macrophages are induced by Th1 cytokines (TNFα, IFN-γ) and bacterial components (lipopolysaccharide, etc.). Activated M1 macrophages release high levels of proinflammatory cytokines, such as TNFα, IL-6, and IL-1β, thereby promoting Th1 responses, and induce adaptive immune responses through antigen presentation, amplifying inflammatory responses, and promoting the body's clearance of pathogens. M2 macrophages mainly secrete anti-inflammatory cytokines such as Arginase-I, IL-10, and TGF-β, which have the functions of reducing inflammation and immunosuppression, and play an important role in wound healing and tissue repair.

[0004] Under the induction of alcohol, liver damage increases, and M1 macrophages become abnormally polarized and dominant, which is the main cause of alcoholic hepatitis by amplifying the inflammatory response to enhance the clearance of damaged cells. At the same time, under the stimulation of inflammatory factors such as LPS and IFN-γ, the monocytes infiltrating in alcoholic fatty liver hepatitis activate transcription factors such as NF-κB and STAT1, and then polarize to form M1 macrophages, further promoting inflammation and liver damage. It can be seen that limiting the function of M1 macrophages can help relieve and treat alcoholic liver inflammation. Therefore, it is necessary to provide a drug that can relieve and / or treat alcoholic liver inflammation by limiting the function of M1 macrophages, providing a new way to treat alcoholic liver inflammation. Summary of the invention

[0005] The purpose of the present invention is to provide the use of glucosamine or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating alcoholic hepatitis, in order to discover a new drug for treating liver inflammation caused by long-term heavy drinking.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of glucosamine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating alcoholic hepatitis. The chemical formula of the glucosamine is:

[0008]

[0009] Preferably, the pharmaceutically acceptable salt of glucosamine includes at least one selected from the group consisting of hydrochloride and sulfate of glucosamine.

[0010] Preferably, the alcoholic hepatitis is alcoholic hepatitis caused by chronic drinking.

[0011] Preferably, the dosage form of the drug is an oral dosage form.

[0012] Preferably, the oral dosage forms include capsules and tablets.

[0013] Preferably, the medicine also includes pharmaceutical excipients.

[0014] Preferably, the pharmaceutical excipients include one or more of an emulsifier, a flavoring agent, a colorant, a solubilizer and a wetting agent.

[0015] Preferably, the application is that glucosamine or a pharmaceutically acceptable salt thereof prevents and / or treats alcoholic hepatitis by inhibiting M1 macrophage polarization.

[0016] Preferably, the application is that glucosamine or a pharmaceutically acceptable salt thereof prevents and / or treats alcoholic hepatitis by inhibiting the expression of M1 type proinflammatory factors IL-1β, IL-6 and TNFα.

[0017] Glucosamine is a natural amino monosaccharide, which is currently mainly used to improve joint movement and relieve pain. The inventor unexpectedly discovered that Glucosamine can inhibit the polarization of M1 macrophages and reduce the release of proinflammatory cytokines, thereby protecting against alcoholic hepatitis caused by long-term heavy drinking. It can significantly inhibit liver inflammation and reduce mortality, and the experimental mice did not show abnormal reactions, indicating that Glucosamine can be used to prepare drugs for preventing and / or treating alcoholic hepatitis caused by long-term heavy drinking. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 The survival status of each group of mice in Example 1;

[0020] Figure 2 The results of the measurement of serum liver function biochemical indexes ALT and AST levels in each group of mice in Example 1;

[0021] Figure 3 The H&E staining results of liver tissues of mice in each group in Example 1 and the results of liver triglyceride level determination, the scale bar in the figure is 100 μm;

[0022] Figure 4 The results of immunohistochemical detection of F4 / 80 positive cells (macrophages) and MPO positive cells (neutrophils) in the liver of each group of mice in Example 1, the scale bars in the figure are 100 μm (F4 / 80) and 50 μm (MPO), respectively;

[0023] Figure 5 The results of measuring the gene expression levels of pro-inflammatory factors in the liver of each group of mice in Example 1;

[0024] Figure 6 The survival status of each group of mice in Example 2;

[0025] Figure 7 The gene expression of pro-inflammatory factors IL-1β and TNFα in the liver of each group of mice in Example 2;

[0026] Figure 8 The results of immunohistochemical detection of F4 / 80 positive cells (macrophages) and MPO positive cells (neutrophils) in the liver of each group of mice in Example 2, the scale bar in the figure is 100 μm;

[0027] Fig. 9 These are the results of measuring the expression of macrophage M1 proinflammatory factors IL-1β, IL-6 and M2 marker genes CD206 and CD163 in Example 3. DETAILED DESCRIPTION

[0028] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1

[0030] The purpose of this example is to study the protective effect of glucosamine (glucosamine, GlcN) on alcohol-induced liver inflammation. Wherein, the CAS number of glucosamine is 3416-24-8, and the chemical formula is:

[0031]

[0032] Grouping and administration: 8-week-old C57BL / 6J mice were divided into three groups: normal control group (Ctrl), alcohol group (EtOH) and drug administration group (EtOH+GlcN), with 6 to 8 mice in each group. Among them, the alcohol group was fed with 5% alcohol liquid feed (Lieber-DeCarli feed); the drug administration group was given 5% alcohol liquid feed containing 1mg / mL GlcN, and the GlcN intake was about 500-600mg / kg / day; the normal control group was fed with control liquid feed containing the same calories. The feed was prepared daily and replaced at 5 pm. The mice were free to eat and continued to be fed for 40 days. The composition of Lieber-DeCarli liquid feed is 26% protein, 18% carbohydrates, and 56% fat in terms of energy composition. The amount of liquid feed consumed by each group of mice per day was about 10-15mL.

[0033] During the feeding period, the survival of mice in each group was recorded. After the feeding, the mice were killed, and the serum and liver tissues of the mice were collected, and the liver tissue samples were preserved and fixed. The liver triglyceride content of mice in each group was detected by chloroform-methanol extraction method; the levels of serum liver function biochemical indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of mice in each group were detected by ultraviolet colorimetry; liver tissue sections were stained with H&E for pathological analysis; immunohistochemistry F4 / 80 was used to detect the infiltration of liver macrophages and immunohistochemistry MPO was used to detect the infiltration of neutrophils; real-time fluorescence quantitative PCR (qPCR) was used to detect the gene expression of liver proinflammatory factors (IL-1β, IL-6, TNFα).

[0034] The survival of mice in each group was Figure 1 The results of ALT and AST level determination are shown in Figure 2 The results of H&E staining and liver triglyceride level determination are shown in Figure 3 The results of liver F4 / 80 and MPO immunohistochemistry are shown in Figure 4 The mRNA expression levels of each pro-inflammatory factor are shown in Figure 5 shown.

[0035] Depend on Figure 1 to Figure 5 It can be seen that there is obvious fatty degeneration and inflammatory infiltration in the liver tissue of the mice in the alcohol group, and the serum ALT and AST levels are significantly increased, indicating that the chronic alcoholic hepatitis model was successfully established; the drug administration group (EtOH+GlcN) can significantly prolong the survival of mice, reduce the serum ALT and AST levels of mice, reduce liver fatty degeneration and liver triglyceride levels, reduce the number of F4 / 80-positive and MPO-positive cells in the mouse liver, reduce the infiltration of macrophages and neutrophils, and inhibit the transcriptional expression of proinflammatory factors IL-1β, IL-6 and TNFα genes. It can be seen that GlcN can improve the survival rate of chronic alcohol model mice, reduce liver fat accumulation, alleviate liver damage and significantly inhibit liver inflammation, and has a good protective effect on alcoholic hepatitis.

[0036] Example 2

[0037] The purpose of this example is to study the protective effect of glucosamine (GlcN) on liver inflammation induced by the classic Gao-Binge alcoholic liver disease model (10 days of alcohol feeding plus a one-time high-dose alcohol gavage).

[0038] Grouping and dosing (the control liquid feed and 5% alcohol liquid feed used below are the same as those in Example 1): 8-week-old C57BL / 6J mice were divided into three groups: a control group (GB-Ctrl), an alcohol group (GB-EtOH) and a drug-treated group (GB-EtOH+GlcN). Each group of mice was given a control liquid feed for 5 days for adaptive feeding; during the formal feeding period, the alcohol group was given a 5% alcohol liquid feed, the drug-treated group was given a 5% alcohol liquid feed containing 1 mg / mL GlcN, and the control group was given a control liquid feed containing the same calories; the amount of liquid feed consumed by each group of mice per day was about 10 to 15 mL. The feed was prepared daily and replaced at 5 pm. The mice were free to eat and fed continuously for 10 days. The daily GlcN intake of each mouse was about 500 to 600 mg / kg / day. At 8:00 a.m. on the 11th day, the mice were gavaged at a dose of 20 mL / kg. The control group was gavaged with 45% maltodextrin, and the alcohol group and the drug group were gavaged with 31.5% alcohol. The volume of the gavage was 20 times (μL) of the body weight (g), for example, a 20g mouse was gavaged with 400 μL. The mice were killed 9 hours after gavage, and blood and liver tissue were collected for experiments.

[0039] The survival status of mice in each group was recorded during the feeding period. After the mice were killed, the liver tissues were embedded and sliced, and the number of macrophages and neutrophils was detected by immunohistochemistry F4 / 80 and MPO. The RNA of mouse liver tissue was extracted to detect the gene expression of proinflammatory factors (IL-1β, TNFα).

[0040] The survival of mice in each group was Figure 6 As shown in Figure 2, the gene expressions of pro-inflammatory factors IL-1β and TNFα were Figure 7 As shown in the figure, the results of immunohistochemistry F4 / 80 and MPO detection of macrophages and neutrophils are as follows Figure 8 As shown. It can be seen that the drug administration group (GB-EtOH+GlcN) can reduce the mortality of mice compared with the alcohol group, inhibit the transcriptional expression of pro-inflammatory factors IL-1β and TNFα genes, and significantly reduce the infiltration of macrophages and neutrophils. This shows that GlcN can improve the survival rate of mice in the classic Gao-Binge alcoholic hepatitis model and alleviate the aggravation of alcohol-induced liver inflammation.

[0041] Example 3

[0042] The purpose of this example is to study the effect of Glucosamine (GlcN) on the polarization of M1 and M2 types of bone marrow-derived macrophages (BMDM).

[0043] Methods: Primary bone marrow macrophages (BMDM) were isolated from C57BL / 6J WT mice. BMDM were cultured at 2×10 5The cells were seeded in a 12-well plate at a concentration of 1.54 cells / well and treated with M-CSF (40 ng / mL) for 7 days. They were then treated with lipopolysaccharide LPS (100 ng / mL) and IL-4 (10 ng / mL) for 24 hours to induce M1 and M2 polarization, respectively. GlcN (100 nM) was then added for intervention for 6 hours. Total RNA was then extracted and reverse transcribed into cDNA. qPCR was used to detect the expression of M1 proinflammatory factors IL-1β and IL-6 and M2 marker genes CD206 and CD163.

[0044] Test results such as Fig. 9 As shown in the figure, it can be seen that after mouse bone marrow-derived macrophages (BMDM) were treated with LPS, the mRNA levels of macrophage M1 polarization markers IL-1β and IL-6 were significantly increased, while GlcN treatment significantly reduced the expression levels of IL-1β and IL-6 ( Fig. 9 Meanwhile, GlcN could significantly enhance the expression of IL4-induced macrophage M2 polarization markers CD206 and CD163 ( Fig. 9 C and D), indicating that GlcN can inhibit LPS-induced M1 polarization and promote IL-4-induced M2 polarization. Therefore, after administration, GlcN can inhibit macrophage M1 polarization and thus exert an anti-inflammatory effect, have a significant protective effect on alcohol-induced liver damage, and greatly improve the survival rate of mice.

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of glucosamine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating alcoholic hepatitis, characterized in that: The chemical formula of the Glucosamine is:

2. The use according to claim 1, characterized in that The pharmaceutically acceptable salt of glucosamine includes at least one selected from the group consisting of hydrochloride and sulfate of glucosamine.

3. The use according to claim 2, characterized in that The alcoholic hepatitis is alcoholic hepatitis caused by chronic drinking.

4. The use according to claim 3, characterized in that The dosage form of the drug is an oral dosage form.

5. The use according to claim 4, characterized in that The oral dosage forms include capsules and tablets.

6. The use according to claim 5, characterized in that The medicine also includes pharmaceutical excipients.

7. The use according to claim 6, characterized in that The pharmaceutical excipients include one or more of an emulsifier, a flavoring agent, a colorant, a cosolvent and a wetting agent.

8. The use according to claim 7, characterized in that The application is that glucosamine or a pharmaceutically acceptable salt thereof prevents and / or treats alcoholic hepatitis by inhibiting M1 macrophage polarization.

9. The use according to claim 8, characterized in that The application is that glucosamine or a pharmaceutically acceptable salt thereof prevents and / or treats alcoholic hepatitis by inhibiting the expression of M1 type proinflammatory factors IL-1β, IL-6 and TNFα.

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