Application of ackermania kwangsiensis in preparation of medicine for preventing and / or treating liver injury

Abstract

The invention provides application of ackermania kwangsiensis in preparation of a medicine for preventing and / or treating liver injury, and belongs to the technical field of biological medicine manufacturing. The ackermania kwangsiensis viable bacteria or pasteurization inactivated bacteria provided by the invention can effectively reduce the activity of alanine aminotransferase (ALT) and asparaginic acid aminotransferase (AST) in mouse serum, improve the activity of superoxide dismutase (SOD) in the liver, reduce the content of malondialdehyde (MDA), and improve the activity of ackermania kwangsiensis. Meanwhile, hepatic disorder, hepatic cell degeneration, liver inflammatory cell infiltration and small vacuoles in cytoplasm of mice can be effectively improved. Therefore, the ackermania kwangsiensis can be used as the unique effective component to prepare the medicine for preventing and / or treating the liver injury, and has a good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biological drug manufacturing, and specifically relates to the application of Akkermansia guangxiensis in preparing drugs for preventing and / or treating liver damage. Background Art

[0002] D-galactose is a naturally occurring monosaccharide, and its main metabolic site is the liver. However, excessive D-galactose will be converted into aldose and hydrogen peroxide during metabolism, and will produce a large number of oxygen free radicals, leading to oxidative stress in the body, accelerating the release of alanine transaminase (ALT) and aspartate aminotransferase (AST) in the body, accelerating the apoptosis and necrosis of liver cells, and ultimately leading to liver damage. Currently known treatments are mainly drug therapy, but drug therapy has stability and some side effects.

[0003] Probiotics have beneficial functions such as improving intestinal flora, preventing cardiovascular diseases, and enhancing immunity. In liver diseases, their auxiliary treatment also has significant effects. Akkermansia is a "star" probiotic, and its application has received more and more attention. The applicant developed and screened a new type of Guangxi Akkermansia (Akkermansia guangxiensis) strain N21116 in the early stage. It is a functional microbial strain isolated and screened from the intestines of centenarians, and has certain anti-aging, antioxidant, weight loss and lipid-lowering effects (ZL202310332284.3). There is no report on the use of Akkermansia for the treatment of liver damage. Summary of the invention

[0004] In view of this, the object of the present invention is to provide the use of Akkermansia guangxiensis in the preparation of a medicament for preventing and / or treating liver damage.

[0005] The present invention provides application of Akkermansia Guangxi or a probiotic containing Akkermansia Guangxi in preparing a medicine for preventing and / or treating liver damage.

[0006] Preferably, the Akkermansia Guangxi includes the Akkermansia Guangxi strain N21116;

[0007] The deposit number of the Guangxi Akkermansia strain N21116 is GDMCC No: 62888.

[0008] Preferably, the Akkermansia guangxiensis includes live bacteria or inactivated bacteria.

[0009] Preferably, the inactivated bacteria are inactivated bacteria obtained by pasteurization of Guangxi Akkermansia strain N21116.

[0010] Preferably, the liver damage includes a decrease in biochemical indices and / or oxidative stress indices and pathological changes in liver tissue.

[0011] Preferably, the biochemical indexes include alanine aminotransferase and / or aspartate aminotransferase;

[0012] The oxidative stress indicators include superoxide dismutase and / or malondialdehyde;

[0013] The pathological changes of liver tissue include disorder of hepatic cords, degeneration of hepatocytes, small non-colored vacuoles in the cytoplasm and infiltration of inflammatory cells.

[0014] Preferably, the liver damage is induced by D-galactose.

[0015] Preferably, the dosage form of the probiotic containing Akkermansia guangxi includes powder or aqueous solution.

[0016] Preferably, the bacterial concentration of Akkermansia guangxi is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0017] Preferably, the dosage form of the drug includes at least one of the following: compressed tablets, oral liquid, powder, granules, capsules and drops.

[0018] The present invention provides the use of Guangxi Akkermansia or a probiotic containing the Guangxi Akkermansia in the preparation of a drug for preventing and / or treating liver damage. The present invention uses a D-galactose-induced liver injury mouse model as an experimental subject to verify the anti-liver injury efficacy of Guangxi Akkermansia. Experiments have shown that Guangxi Akkermansia, whether live or pasteurized, can significantly improve the symptoms of liver damage caused by oxidative stress. The results show that compared with the model group, the Guangxi Akkermansia intervention group of live or pasteurized bacteria can significantly reduce the ALT and AST activities in mouse serum; at the same time, significantly increase the SOD activity in the mouse liver and reduce the MDA content; and can also significantly improve the mouse liver cord disorder, hepatocyte degeneration, and liver inflammatory cell infiltration. It can be seen that the Guangxi Akkermansia has a good liver damage repair effect and can achieve liver protection. The scheme of the present invention not only enriches the types of liver damage treatment drugs but also expands the application of Guangxi Akkermansia, further improving its industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of Guangxi Akkermansia strain N21116 on the ALT activity in the serum of mice with liver injury induced by D-galactose;

[0020] Figure 2The effect of Guangxi Akkermansia strain N21116 on AST activity in the serum of mice with liver damage induced by D-galactose;

[0021] Figure 3 The effect of Guangxi Akkermansia strain N21116 on the SOD activity in liver tissue of mice with liver damage induced by D-galactose;

[0022] Figure 4 The effect of Guangxi Akkermansia strain N21116 on the MDA content in the tissues of mice with liver damage induced by D-galactose;

[0023] Figure 5 The figure shows the effect of Guangxi Akkermansia strain N21116 on the pathological morphology of mice with liver damage induced by D-galactose, where A is PBS+H2O; B is D-Gal+H2O; C is D-Gal+N21116 (live); and D is D-Gal+N21116 (pasteurized). DETAILED DESCRIPTION

[0024] The present invention provides application of Akkermansia Guangxi or a probiotic containing Akkermansia Guangxi in preparing a medicine for preventing and / or treating liver damage.

[0025] In the present invention, the Guangxi Akkermansia preferably includes Guangxi Akkermansia strain N21116. The deposit number of the Guangxi Akkermansia strain N21116 is GDMCC No: 62888. The strain N21116 is disclosed and deposited in a patent with a publication number of 2023118474389 and a patent name of the use of Guangxi Akkermansia strain or its probiotic agent in the preparation of products for prolonging life, anti-aging, and delaying Parkinson's disease.

[0026] In the present invention, the Guangxi Akkermansia preferably includes live bacteria or inactivated bacteria. The preparation method of the live bacteria of the Guangxi Akkermansia preferably inoculates the activated Guangxi Akkermansia into a composite culture medium for culture, obtains a culture solution, separates the bacteria, and obtains live bacteria. The inoculation amount of the activated Guangxi Akkermansia during the culture is preferably 5% to 10%, which can be 8%. The culture is preferably anaerobic culture at 37°C for 24 to 72h, which can be anaerobic culture for 48h. The method for isolating the bacteria is preferably centrifugation. The speed of the centrifugation is preferably 6000 to 10000rpm, which can be 8000rpm. The time of the centrifugation is preferably 5 to 10min, which can be 8min. The temperature of the centrifugation is preferably 3 to 5°C, which can be 4°C. The inactivated bacteria are preferably inactivated bacteria obtained by pasteurization of Guangxi Akkermansia strain N21116. The method of pasteurization treatment is preferably inactivated at 65°C for 30min.

[0027] In the present invention, the dosage form of the probiotic containing Guangxi Akkermansia preferably includes powder or aqueous solution. The bacterial concentration of Guangxi Akkermansia is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g, can be 5×10 8 CFU / mL, 10×10 8 CFU / mL, 20×10 8 CFU / mL, 30×10 8 CFU / mL, 40×10 8 CFU / mL, 50×10 8 CFU / mL or 5×10 8 CFU / g, 10×10 8 CFU / g, 20×10 8 CFU / g, 30×10 8 CFU / g, 40×10 8 CFU / g, 50×10 8 CFU / g. In an embodiment of the present invention, the preparation method of a probiotic is described by taking the preparation of a powder as an example. The preparation method of the powder is preferably to mix the live bacteria prepared above with a freeze-drying protective agent, and vacuum freeze-dry to obtain a bacterial powder. The mass ratio of the live bacteria or inactivated bacteria to the freeze-drying protective agent is preferably 1:2 to 1:2, more preferably 1:1. The freeze-drying protective agent preferably includes 10% skim milk, 10% trehalose, 1% sorbitol and 5% vitamins.

[0028] In the present invention, the liver injury is preferably chemically induced liver injury. In an embodiment of the present invention, the liver injury is preferably induced by D-galactose. In an embodiment of the present invention, the single dose of D-galactose is 200 mg / kg.bw. The duration of D-galactose injection is 8 weeks.

[0029] In the present invention, the liver damage preferably includes a decrease in biochemical indicators and / or oxidative stress indicators and pathological changes in liver tissue. The biochemical indicators preferably include alanine aminotransferase and / or aspartate aminotransferase. The oxidative stress indicators preferably include superoxide dismutase and / or malondialdehyde. The pathological changes in liver tissue preferably include disorder of hepatic cords, the occurrence of hepatocyte degeneration, the presence of uncolored small vacuoles in the cytoplasm, and inflammatory cell infiltration.

[0030] In the present invention, the Guangxi Akkermansia strain N21116, whether live or pasteurized, can significantly improve liver damage induced by D-galactose. Specifically, the strain N21116, as the only effective ingredient, can significantly reduce ALT and AST in mouse serum, and can significantly increase SOD activity in mouse liver tissue, significantly reduce MDA content, and the strain N21116 can also significantly improve mouse liver cord disorder, hepatocyte degeneration, liver inflammatory cell infiltration, and small vacuoles in the cytoplasm. In summary, the Guangxi Akkermansia, whether live or inactivated, can improve liver damage induced by D-galactose.

[0031] In the present invention, the dosage form of the drug preferably includes at least one of the following: tablets, oral liquids, powders, granules, capsules and drops. The present invention has no particular limitation on the specific preparation of the drug, and the preparation method of the drug preparation known in the art can be used.

[0032] The application of Akkermansia guangxiensis provided by the present invention in the preparation of a medicament for preventing and / or treating liver damage is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Sources of materials in the examples:

[0034] 1. Sources of materials and reagents

[0035] D-galactose was purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.;

[0036] Alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute.

[0037] 2. Animal source description

[0038] Six-week-old SPF female C57BL / 6J mice weighing 15-18 g were housed in separate cages and allowed to eat and drink freely. The experimental environment temperature was 22°C ± 2°C and the relative humidity was 50% ± 10%.

[0039] 3. Composite culture medium formula: glucose 7.2 g / L, yeast extract powder 9.0 g / L, soy peptone 6.0 g / L, L-cysteine ​​0.5 g / L, sodium nitrate 0.425 g / L, N-acetylglucosamine 2.0 g / L, sodium chloride 4.5 g / L, disodium hydrogen phosphate 5.0 g / L and threonine 8.0 g / L.

[0040] Example 1

[0041] Preparation method of freeze-dried bacterial powder of Guangxi Akkermansia strain N21116

[0042] The Guangxi Akkermansia strain N21116 stored in a glycerol tube frozen at -80°C was inoculated on a solid composite culture medium for streak culture, and a pure single colony was picked and inoculated into a liquid composite culture medium, and the culture was gradually expanded to 5L according to a 10% inoculation amount, and anaerobically cultured at 37°C for 48h, and the bacterial liquid was collected, centrifuged at 4°C and 8000rpm for 8min, the supernatant was discarded, and the bacterial body was washed with physiological saline and centrifuged again to obtain bacterial mud. The whole process was operated under anaerobic conditions.

[0043] The freeze-drying protective agent (formula is 10% skim milk, 10% trehalose, 1% sorbitol, 5% vitamin C) and the above-mentioned bacterial mud in a mass ratio of 1:1 are mixed, poured into a freeze-drying plate and placed in a vacuum freeze-drying equipment for freeze-drying. The specific freeze-drying procedure is as follows: the pre-freezing temperature is 50°C, the time is maintained for 4 hours, the primary drying temperature is 30°C, the time is 25 hours, the secondary drying temperature is 35°C, the time is 16 hours, and finally the freeze-dried bacterial powder is obtained.

[0044] The freeze-dried bacterial powder was resuspended in physiological saline and counted using the plate counting method. The concentration of live bacteria was adjusted to 1×10 8 CFU / mL, recorded as Guangxi Akkermansia strain N21116 live bacteria (N21116lp), for later use.

[0045] The viable bacteria of the Guangxi Akkermansia strain N21116 prepared above were placed in an oven at 65° C. and inactivated for 30 min, and the obtained bacterial solution was recorded as pasteurized bacteria of the Guangxi Akkermansia strain N21116 (N21116ps).

[0046] Example 2

[0047] Construction method of liver injury model and efficacy verification of Guangxi Akkermansia strain N21116

[0048] 1. Establishment of liver injury model and grouping of experimental animals

[0049] A total of 40 SPF-grade 6-8-week-old female C57BL / 6J mice were randomly divided into 4 groups after 1 week of adaptive feeding, with 10 mice in each group. During the feeding period, a 12h / 12h light-dark cycle was maintained, the ambient temperature was 22-25℃, and the relative humidity was 40%-60%. The specific groups are as follows:

[0050] Normal group: basal feed and daily intragastric administration of normal saline;

[0051] Model group: basal diet, daily subcutaneous injection of D-galactose (200 mg / kg) and daily intragastric administration of an equal amount of normal saline;

[0052] The N21116 live bacteria intervention group was fed with basic feed, subcutaneous injection of D-galactose (200 mg / kg) and oral administration of 200 μL of live bacteria N21116 (1×10 8 CFU / mL);

[0053] N21116 pasteurized bacteria intervention group: basal diet, daily subcutaneous injection of D-galactose (200 mg / kg) and daily intragastric administration of 200 μL N21116 pasteurized bacteria (1×10 8 CFU / mL);

[0054] The experiment lasted for 8 weeks. After the last oral gavage, the mice were fasted for 24 hours, anesthetized with 1% sodium pentobarbital, and blood was taken from the heart and centrifuged for 8 minutes. The serum was packaged and stored at -80°C for the determination of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The mice were killed by cervical dislocation, and the liver was quickly removed. The right lobe of the liver was fixed with formalin for pathological histological examination; the left lobe of the liver was reserved for the determination of superoxide dismutase (SOD) and malondialdehyde (MDA) oxidative stress indicators, and the rest of the liver was frozen at -80°C for future use.

[0055] 2. Index detection

[0056] 2.1 Analysis of mouse serum biochemical indicators

[0057] Mouse serum was obtained, and the AST and ALT levels in the serum were determined according to the operating procedures in the kit instructions.

[0058] Results Figure 1 and Figure 2 Compared with the blank group, the ALT and AST activities in the serum of the mice in the model group were significantly increased; compared with the model group, after N21116 intervention, ALT and AST in the serum of the mice were significantly decreased regardless of whether they were live bacteria or pasteurized bacteria.

[0059] 2.2 Analysis of oxidative stress indicators in mouse liver tissue

[0060] The left lobe of mouse liver was taken, PBS solution was added and placed in a grinder to prepare 10% liver homogenate, and the SOD and MDA content in the liver tissue was determined according to the operating steps in the kit instructions to evaluate the antioxidant capacity and liver damage degree of mice.

[0061] Results Figure 3 and Figure 4 Compared with the blank group, the superoxide dismutase (SOD) content in the model group was significantly decreased, and the malondialdehyde (MDA) content was significantly increased; compared with the model group, after N21116 intervention, the SOD activity was significantly increased, and the MDA content was significantly decreased, regardless of live bacteria or pasteurized bacteria.

[0062] 2.3 Liver histopathological analysis

[0063] The right lobe of mouse liver tissue was fixed with 10% formalin solution, dehydrated, embedded, and sliced ​​to a thickness of 3 to 5 μm. It was then stained with hematoxylin-eosin to prepare stained sections, which were then placed under an optical microscope to observe their structure and morphology.

[0064] Results Figure 5 .Depend on Figure 5 It can be seen that the model group ( Figure 5 Middle B) presents pathological morphology: disordered hepatic cords, a large number of hepatocyte degeneration, unpigmented small vacuoles in the cytoplasm, and inflammatory cell infiltration. Guangxi Akkermansia N21116, regardless of live bacteria ( Figure 5 C) or pasteurized bacteria ( Figure 5 D) all showed certain improvement, among which the live bacteria group was closer to the blank group, reflecting the protective effect on the liver.

[0065] 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 Akkermansia guangxiensis or a probiotic containing the Akkermansia guangxiensis in the preparation of a medicament for preventing and / or treating liver damage.

2. The use according to claim 1, characterized in that: The Guangxi Akkermansia includes the Guangxi Akkermansia strain N21116; The deposit number of the Guangxi Akkermansia strain N21116 is GDMCC No: 62888.

3. The application according to claim 1, characterized in that: The Akkermansia guangxiensis includes live bacteria or inactivated bacteria.

4. The use according to claim 3, characterized in that: The inactivated bacteria are inactivated bacteria obtained by pasteurization of Guangxi Akkermansia strain N21116.

5. The use according to claim 1, characterized in that: The liver damage includes a decrease in biochemical indices and / or oxidative stress indices and pathological changes in liver tissue.

6. The use according to claim 5, characterized in that: The biochemical indexes include alanine aminotransferase and / or aspartate aminotransferase; The oxidative stress indicators include superoxide dismutase and / or malondialdehyde; The pathological changes of liver tissue include disorder of hepatic cords, degeneration of hepatocytes, small non-colored vacuoles in the cytoplasm and infiltration of inflammatory cells.

7. The use according to claim 1, characterized in that: The liver damage was induced by D-galactose.

8. The use according to claim 1, characterized in that: The dosage form of the probiotic containing Akkermansia guangxiensis includes powder or aqueous solution.

9. The use according to claim 1, characterized in that: The bacterial concentration of Guangxi Akkermansia is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

10. The use according to any one of claims 1 to 9, characterized in that: The dosage form of the drug includes at least one of the following dosage forms: compressed tablets, oral liquid, powder, granules, capsules and drops.

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