Application of bacteroides multiforme in preparation of medicine for treating and / or preventing hepatic fibrosis

Abstract

The invention discloses an application of bacteroides multiforme in preparation of a medicine for treating and / or preventing hepatic fibrosis. In-depth study on the potential treatment effect of the bacteroides multiforme on hepatic fibrosis discovers that the bacteroides multiforme as a unique active component can significantly reduce the content of serum liver function indexes AST and ALT in hepatic fibrosis diseases caused by carbon tetrachloride; the liver tissue lesion degree, the inflammation infiltration condition, the liver collagenous fiber area and the colon tissue damage degree in the liver fibrosis disease caused by carbon tetrachloride can be obviously reduced; therefore, the bacteroides multiforme can effectively improve liver function impairment, hepatitis, hepatic histopathologic change and intestinal injury caused by hepatic fibrosis and improve hepatic fibrosis, a new thought is provided for clinical treatment of hepatic fibrosis, a new treatment choice is provided for patients, and prognosis and life quality of the patients are improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of Bacteroides polymorpha in the preparation of drugs for the treatment and / or prevention of liver fibrosis. Background Technology

[0002] The liver is one of the most important organs in the human body, serving as the hub of metabolism and performing vital functions such as nutrient metabolism and bile secretion. Liver fibrosis is a disease caused by abnormal proliferation of connective tissue in the liver due to various pathogenic factors. It is generally caused by liver damage, which further leads to liver fibrosis, cirrhosis, and eventually liver cancer. However, liver disease is a reversible process; timely intervention in the early stages can effectively prevent the disease from worsening. Liver fibrosis is a key stage in the development of various chronic liver diseases into cirrhosis and even liver cancer, and its main characteristic is the excessive deposition of extracellular matrix (ECM) proteins.

[0003] Bacteroides polymorpha ( Bacteroides thetaiotaomicron Bacteroides (Bt) is a Gram-negative bacterium belonging to the genus Bacteroides. It is one of the most abundant commensal bacteria in the human gut and plays an important role in regulating host metabolism and immune function. In recent years, Bt bacteria have been found to have good effects in treating alcohol-related liver disease (Recovery of Bacteroides thetaiotaomicron ameliorates hepatic steatosis in experimental alcohol-related liver disease), non-alcoholic fatty liver disease (AL201858), and liver function disorders (CN112280720A, milk-induced liver metabolic dysfunction). Currently, there are no reports on the use of Bt bacteria for the treatment and / or prevention of liver fibrosis and diseases caused by fibrosis. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an application of Bacteroides polymorpha as the sole active ingredient in the preparation of drugs for treating and / or preventing liver fibrosis.

[0005] A second objective of this invention is to provide the use of Bacteroides polymorpha as the sole active ingredient in the preparation of a medicament for the treatment and / or prevention of diseases caused by liver fibrosis.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: Both CCl4-induced liver fibrosis and clinical liver fibrosis (such as that caused by viral or alcoholic hepatitis) involve hepatic stellate cells (HSCs) as the core pathological component. Activated HSCs secrete large amounts of collagen fibers (such as type I and type III collagen), leading to abnormal deposition of extracellular matrix (ECM) in the liver and the formation of fibrous septa. Therefore, a carbon tetrachloride-induced liver fibrosis model can be used to evaluate the therapeutic effect of Bacteroides polymorpha on liver fibrosis.

[0007] This invention aims to provide a novel method for improving the treatment of liver fibrosis-related diseases using Bacteroides thetaiotaomicron (Bt). Studies have found that administering an effective amount of Bacteroides thetaiotaomicron to mice treated with carbon tetrachloride significantly reduced serum AST and ALT levels in carbon tetrachloride-induced liver fibrosis compared to the carbon tetrachloride-only treatment group. This effectively improved liver function, reduced the severity of liver tissue lesions in mice, improved hepatocyte structure, reduced inflammatory cell infiltration, effectively alleviated collagen deposition levels, reduced the area of ​​collagen fiber formation in the liver, improved the intestinal barrier and intestinal damage, and effectively reduced the number of goblet cells in the colon. In contrast, the inactivated Bacteroides thetaiotaomicron group showed no significant improvement compared to the carbon tetrachloride-treated group. In short, Bacteroides thetaiotaomicron can significantly improve liver function impairment, liver inflammation, liver histopathological changes, and intestinal damage caused by liver fibrosis, thus providing a new approach for the clinical treatment of liver fibrosis.

[0008] Therefore, this invention provides the use of Bacteroides polymorpha as the sole active ingredient in the preparation of medicaments for the treatment and / or prevention of liver fibrosis.

[0009] This invention also provides the use of Bacteroides polymorpha as the sole active ingredient in the preparation of medicaments for the treatment and / or prevention of diseases caused by liver fibrosis.

[0010] Furthermore, the liver fibrosis is caused by factors including, but not limited to, viral hepatitis, alcoholic hepatitis, fatty liver, autoimmune diseases, or carbon tetrachloride.

[0011] Furthermore, the drug achieves its therapeutic effect by reducing the degree of liver tissue lesions, inflammatory cell infiltration, collagen fiber production, and / or intestinal damage.

[0012] Furthermore, the reduction of liver tissue lesions refers to the improvement of liver lobule structure damage.

[0013] Furthermore, the drug achieves its therapeutic effect by lowering the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0014] Furthermore, the *Bacteroides polymorpha* is a live *Bacteroides polymorpha*.

[0015] Furthermore, the number of Bacteroides polymorpha in the drug is 1 × 10⁻⁶. 7 ~1×10 10 CFU.

[0016] Furthermore, the number of Bacteroides polymorpha in the drug is 1 × 10⁻⁶. 9 CFU.

[0017] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0018] Furthermore, the excipients are one or more of the following: carrier, excipient, diluent, lubricant, wetting agent, emulsifier, suspension stabilizer, and preservative.

[0019] Furthermore, the dosage form of the drug is any one of injection, oral dosage, tablet, capsule, or emulsion.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of *Bacteroides polymorpha* as the sole active ingredient in the preparation of drugs for the treatment and / or prevention of liver fibrosis. Through in-depth research into the potential therapeutic effects of *Bacteroides polymorpha* on liver fibrosis, this invention has discovered that *Bacteroides polymorpha*, as the sole active ingredient, can significantly reduce the levels of serum liver function indicators AST and ALT in carbon tetrachloride-induced liver fibrosis. It can also significantly reduce the degree of liver tissue lesions and inflammatory infiltration, liver collagen fiber area, and colon tissue damage in carbon tetrachloride-induced liver fibrosis. Therefore, *Bacteroides polymorpha* can effectively improve liver function impairment, liver inflammation, liver histopathological changes, and intestinal damage caused by liver fibrosis, thereby improving liver fibrosis. This invention provides a new approach for the clinical treatment of liver fibrosis, offering patients new treatment options and improving their prognosis and quality of life. Attached Figure Description

[0021] Figure 1 Comparison of liver function indicators AST and ALT in the serum of mice from different groups of Bacteroides polymorpha.

[0022] Figure 2 H&E staining pathological images of mouse liver tissue from different groups of Bacteroides polymorpha.

[0023] Figure 3 Masson staining pathological images, Sirius Red staining pathological images, and collagen fiber statistical diagrams of mouse liver tissue from different groups of Bacteroides polymorpha.

[0024] Figure 4 H&E staining pathological images of mouse colon tissue from different groups of Bacteroides polymorpha.

[0025] Figure 5 H&E staining pathological images of mouse liver tissue from different groups of Bacteroides acidogeneticus.

[0026] Figure 6 Masson staining and Sirius Red staining pathological images of mouse liver tissue from different groups of Bacteroides acetamipridae.

[0027] Figure 7 H&E staining pathological images of mouse colon tissue from different groups of Bacteroides acidogeneticus. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] The following description illustrates exemplary embodiments of the present invention, including various details of the embodiments to aid understanding; these should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0031] The *Bacteroides polymorpha* used in the embodiments of this invention ( Bacteroides thetaiotaomicron Purchased from Beina Biotechnology - Henan Provincial Industrial Microbial Strains Engineering Technology Research Center, strain identification project number BNCCJD22062204.

[0032] Example 1: Construction of the Experimental Model 1. Experimental grouping and treatment Forty SPF-grade male C57BL / 6 mice were used in this experiment. After one week of acclimatization, four antibiotics (0.5 g / L vancomycin + 1 g / L ampicillin + 1 g / L metronidazole + 1 g / L neomycin sulfate) were administered in combination. The mice were given 10 μL / g of the antibiotic combination via gavage for one week to create a pseudo-sterile environment (ABX). The animals were randomly divided into four groups of 10 mice each, and the treatments for each group were as follows: ① Control group (administered by gavage with an equal volume of PBS); ② Carbon tetrachloride (CCl4) model group (intraperitoneal injection of 10% CCl4 5 mL / kg); ③ Carbon tetrachloride (CCl4) + Bacteroides polymorpha group (gavage bacterial count 1×10⁻⁶) 9 CFU / each); ④ Carbon tetrachloride (CCl4) + inactivated Bacteroides polymorpha group (inactivated bacterial load by gavage was 1×10⁻⁶) 9 CFU / animal, inactivated by pasteurization, inactivation conditions: 70℃ water bath for 30 min).

[0033] The above four treatments were administered every other day for 8 consecutive weeks. After the second administration at the end of the 8th week, the animals were weighed, anesthetized with pentobarbital, enucleated to collect blood, euthanized, and their tissues were collected for subsequent pharmacodynamic evaluation.

[0034] Example 2: Bacteroides polymorpha has an ameliorative effect on carbon tetrachloride-induced liver fibrosis in animals. I. Experimental Methods 1. Serum biochemical index determination Take an appropriate amount of mouse serum into a centrifuge tube and detect the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of each group of mice according to the kit.

[0035] 2. Hematoxylin-eosin staining (H&E staining) Mouse liver and colon tissues were taken from the same location, fixed in paraformaldehyde for 24 hours, dehydrated, cleared, impregnated with paraffin, and embedded to prepare paraffin sections. The paraffin sections were baked in a 65℃ oven for 4 hours to melt the paraffin, and then successively immersed in xylene and ethanol solutions of different concentrations to dewax and rehydrate. They were then stained with hematoxylin and eosin, and finally mounted with neutral resin. The sections were stored under dry conditions at room temperature, and images were acquired by randomly selecting fields of view at room temperature.

[0036] 3. Masson staining Mouse liver tissue was collected from the same location, fixed with paraformaldehyde for 24 h, dehydrated, cleared, impregnated with paraffin, and embedded to prepare paraffin sections. After melting, dewaxing, and rehydration, the paraffin sections were fixed in 3% mercuric chloride saturated picric acid solution, treated with 0.5% glacial acetic acid, and then stained with a 1:2 mixture of 1% acid fuchsin solution and 1% ponceau red solution. After treatment with 0.5% glacial acetic acid and 1% phosphomolybdic acid, the sections were counterstained with 1% brilliant green solution. After dehydration and clearing, the sections were mounted with neutral resin and stored under dry conditions at room temperature. Images were acquired by randomly selecting fields of view at room temperature.

[0037] 4. Sirius Red staining. Mouse liver tissue was collected from the same location, fixed with paraformaldehyde for 24 h, dehydrated, cleared, impregnated with paraffin, and embedded to prepare paraffin sections. After melting, dewaxing, and rehydration, the paraffin sections were stained sequentially with 1% Sirius red-saturated picric acid solution and hematoxylin solution. After dehydration and clearing, they were mounted with neutral resin, dried and stored at room temperature, and images were acquired by randomly selecting fields of view at room temperature.

[0038] II. Experimental Results 1. Bacteroides polymorpha has an ameliorative effect on carbon tetrachloride-induced liver fibrosis in animals. like Figure 1 As shown, compared with the control group, the serum ALT and AST levels of mice in the model group were significantly increased. Compared with the model group, the serum ALT and AST levels of mice in the Bacteroides polymorpha group were significantly decreased. However, the inactivated Bacteroides polymorpha group could not reduce serum ALT and AST levels in mice with liver fibrosis, suggesting that Bacteroides polymorpha has an ameliorative effect on carbon tetrachloride-induced liver injury in mice.

[0039] 2. Bacteroides polymorpha can improve liver pathological damage in mice with liver fibrosis. H&E staining results of mouse liver tissue are as follows Figure 2 As shown, compared with the control group, the model group mice exhibited inflammatory cell infiltration, ballooning degeneration, and destruction of liver lobules in their hepatocytes. After intervention with Bacteroides polymorpha, the liver fibrosis mice showed milder liver tissue lesions, reduced inflammatory cell infiltration, and a more normal hepatocyte structure, while inactivated Bacteroides polymorpha showed no significant changes. Masson staining and Sirius Red staining results are shown below. Figure 3 As shown, compared with the control group, the normal structure of liver tissue in the model group mice was destroyed, a large amount of collagen fibers were generated, fibrous septa were formed and liver lobule structure was destroyed, forming pseudolobules. After intervention with Bacteroides polymorpha, the degree of liver tissue lesions in the liver fibrosis mice was milder than that in the model group, and the area of ​​collagen fiber formation was reduced. However, there was no significant change after intervention with inactivated Bacteroides polymorpha, indicating that Bacteroides polymorpha can have an effective therapeutic effect on liver fibrosis and the liver diseases it causes.

[0040] 3. Bacteroides polymorpha can improve intestinal damage in mice with liver fibrosis. H&E staining results of mouse colon tissue are as follows Figure 4 As shown, compared with the control group, the model group mice had disordered arrangement of glands in the lamina propria, some of which were necrotic or disappeared, and the number of goblet cells in the intestinal epithelium was significantly reduced. After intervention with Bacteroides polymorpha, the above intestinal lesions were improved, while there was no significant change after intervention with inactivated Bacteroides polymorpha.

[0041] In summary, Bacteroides polymorpha can significantly reduce the levels of serum liver function indicators AST and ALT in carbon tetrachloride-induced liver fibrosis, and can significantly reduce the degree of liver tissue lesions and inflammatory infiltration, liver collagen fiber area and colon tissue damage in carbon tetrachloride-induced liver fibrosis. Therefore, Bacteroides polymorpha can effectively improve liver function impairment, liver inflammation, liver tissue pathological changes and intestinal damage caused by liver fibrosis, and improve liver fibrosis.

[0042] Comparative Example 1: Effects of Bacteroides acidogeneticus (Ba) on mice with liver fibrosis I. Experimental grouping and treatment of Bacteroides acidogeneticus (Ba) Same as Example 1.

[0043] The processing for each group is as follows: ① Control group (administered by gavage with an equal volume of PBS) ② Carbon tetrachloride (CCl4) model group (intraperitoneal injection of 10% CCl4 5 mL / kg) ③ Carbon tetrachloride (CCl4) + Bacteroides acidogenic group (gavage bacterial count 1×10⁻⁶) 9 CFU / each ④ Carbon tetrachloride (CCl4) + inactivated Bacteroides acid-producing group (inactivated bacteria amount by gavage: 1×10⁻⁶) 9 CFU / each II. Experimental Methods Same as Example 2.

[0044] III. Experimental Results Compared with Bt bacteria, the liver pathological damage in mice with liver fibrosis after treatment with Ba bacteria in the control group was as follows: Figure 5-6 As shown, H&E staining, Masson staining, and Sirius Red staining results indicated increased liver cell volume, increased lipid droplet vacuoles, dense collagen fibers, and severe liver fibrosis; intestinal damage, such as... Figure 7 As shown, H&E staining results indicated an increased number of goblet cells in the colonic epithelium, shallower crypt depth, and no significant improvement in intestinal barrier function. Therefore, *Bacillus subtilis* does not have a therapeutic effect on liver fibrosis and its related diseases.

[0045] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. The use of Bacteroides polymorpha as the sole active ingredient in the preparation of drugs for the treatment and / or prevention of liver fibrosis.

2. The use of Bacteroides polymorpha as the sole active ingredient in the preparation of drugs for the treatment and / or prevention of diseases caused by liver fibrosis.

3. The application according to claim 1 or 2, characterized in that, The liver fibrosis is caused by viral hepatitis, alcoholic hepatitis, fatty liver, autoimmune diseases, or carbon tetrachloride.

4. The application according to claim 1 or 2, characterized in that, The drug achieves its therapeutic effect by reducing the degree of liver tissue lesions, inflammatory cell infiltration, collagen fiber production, and / or intestinal damage.

5. The application according to claim 4, characterized in that, The reduction of liver tissue lesions is achieved by improving the destruction of liver lobule structure.

6. The application according to claim 1 or 2, characterized in that, The drug achieves its therapeutic effect by lowering the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

7. The application according to claim 1 or 2, characterized in that, The number of Bacteroides polymorpha in the drug is 1×10⁻⁶. 7 ~1×10 10 CFU.

8. The application according to claim 7, characterized in that, The number of Bacteroides polymorpha in the drug is 1×10⁻⁶. 9 CFU.

9. The application according to claim 1 or 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

10. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is any one of injection, oral dosage, tablet, capsule, or emulsion.

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