Use of Akkermansia muciniphila in preparing products for preventing, treating and / or assisting in treating male reproductive damage

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to an application of Akkermansia muciniphila in preparing a product for preventing, treating and / or assisting in treating male reproductive damage. The present invention provides an application of Akkermansia muciniphila in preparing a product for preventing, treating and / or assisting in treating male reproductive damage, wherein the Akkermansia muciniphila is deposited in CGMCC with a deposit number of CGMCC No. 20955. The Akkermansia muciniphila can effectively improve the total number of sperm, viability, vitality and related motor parameter levels to reduce reproductive damage induced by enteritis.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to an application of Akkermansia muciniphila in preparing a product for preventing, treating and / or assisting in treating male reproductive damage. Background Art

[0002] Inflammatory bowel disease is a chronic inflammatory disease of the gastrointestinal tract. Crohn's disease and ulcerative colitis are the two main forms of inflammatory bowel disease. Inflammatory bowel disease is relatively common worldwide. The incidence of sexual dysfunction in male patients with inflammatory bowel disease is 15%-25%, which is higher than the 5% incidence of sexual dysfunction in men in the general population (Park YE, Kim T O. Sexual dysfunction and fertility problems in men with inflammatory bowel disease[J]. The World Journal of Men's Health, 2020, 38(3): 285.). Wdowiak et al. (2021) systematically analyzed the semen of 50 patients with ulcerative colitis and 50 healthy men and found that the sperm count, sperm motility, sperm motility and related motility parameters of patients with colitis were significantly lower than those in healthy people (Wdowiak, A.; Gujski, M.; Bojar, I.; Raczkiewicz, D.; Bartosińska, J.; Wdowiak-Filip, A.; Filip, R. Chronic Inflammation Impairs Male Fertility—A Case-Control Study in Ulcerative Colitis Patients. J. Clin. Med. 2021, 10, 1460.).

[0003] The intestinal flora is very important for host health, and there is increasing evidence that the intestinal microbiota communicates with the host's distal organs, including the brain, liver, muscle, and testis, through various complex mechanisms. Recently, the intestinal microbiota has been found to be a major regulator of androgen production and metabolism, and can even cross the blood-testis barrier to regulate spermatogenesis, that is, the intestinal microbiota can regulate male reproductive activity through the microbial-gut-testis axis, thereby regulating the host's physiology and health (Li X, Cheng W, Shang H, et al. The interplay between androgen and gutmicrobiota: is there a microbiota-gut-testis axis [J]. Reproductive Sciences, 2022, 29 (6): 1674-1684.). The latest study found that single Lactobacillus rhamnosus PB01 intervention can significantly improve sperm motility and kinematic parameters (Dardmeh F, Alipour H, Gazerani P, et al. Lactobacillus rhamnosus PB01 (DSM 14870) supplementation affects markers of sperm kinematic parameters in a diet-induced obesity mice model [J]. PloS One, 2017, 12 (10): e0185964.).

[0004] Chinese patent application 202111408137.7 discloses a probiotic preparation for improving sperm motility, and the use of the probiotic preparation, wherein the probiotic preparation for improving sperm motility includes Christensenella minuta. The application provides the use of the probiotic preparation for preparing a health food for improving sperm motility, and also provides the use of the probiotic preparation for preparing a medicine for improving sperm motility.

[0005] Chinese patent application 202211665868.4 discloses a composite probiotic fermentation composition and application for improving male sperm motility. The composition includes the following components: Bifidobacterium lactis, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium longum, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus acidophilus; the animal bifidobacterium is a mixture of animal bifidobacterium BB-12, animal bifidobacterium B94 and animal bifidobacterium ZK-77 with a mass ratio of 1:1-3:6-10. The composite bacteria of the present invention are used for the fermentation of the Chinese medicine composition of the present invention to obtain a fermentation liquid, which can effectively improve male sperm motility, and the prepared Chinese medicine liquid can be directly used to prepare downstream products for improving male sperm motility.

[0006] Currently, there is a need in the art for more microorganisms that can be used to improve male sperm motility, and prevent and / or treat male reproductive damage. Summary of the invention

[0007] The inventor creatively isolated a strain of Akkermansia muciniphila from a healthy human body, which has been deposited in CGMCC with the deposit number CGMCC No.20955. The inventor also identified and characterized its morphological taxonomy and biochemical genetic functions, and explored its applications in different fields (other patent applications have been submitted).

[0008] Biodeposit Information:

[0009] Biomaterial: AKK PROBIO

[0010] Taxonomic nomenclature: Akkermansia muciniphila

[0011] Storage time: October 26, 2020

[0012] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0013] Deposit number: CGMCC No.20955.

[0014] The inventors were surprised to find that AKK PROBIO has achieved unexpected positive effects in many fields such as anti-tumor, cognitive disease, metabolic disease, inflammatory disease, bone and joint disease, cardiovascular disease, etc. However, considering the practice of Chinese patent law, we now apply for protection for each indication that is not unique.

[0015] This application is directed to the use of AKK PROBIO in the preparation of products for preventing, treating and / or assisting in the treatment of male reproductive damage.

[0016] The applicant only recorded relevant experimental data such as microbial morphological taxonomic identification and biochemical genetic performance characterization in the submitted microbial patent application. This application does not record any information related to the microorganisms, nor does it protect the microorganisms themselves.

[0017] In order to facilitate the review of the present invention and quickly understand the inventor's previous work, the relevant information of AKK PROBIO is now briefly disclosed in the invention content of this application.

[0018] 1. AKK PROBIO

[0019] The conclusive information in this section is for the convenience of the review of the present invention, but is not intended to limit the scope of protection of the present invention. The specific experimental process and experimental results are recorded in the microbiological patent filed on the same day as the present invention.

[0020] 1. Akkermansia muciniphila CGMCC No.20955 was screened and isolated by the inventors;

[0021] 2. The deposit information of Akkermansia muciniphila CGMCC No.20955;

[0022] 3. 16srRNA of Akkermansia muciniphila CGMCC No.20955;

[0023] 4. Physiological and biochemical properties of Akkermansia muciniphila CGMCC No.20955:

[0024] (1) Hydrophobicity: increases with time, reaching more than 30% at 60 min;

[0025] (2) Self-aggregation: It increases with time and stabilizes at around 52% after 20 hours.

[0026] (3) Gastrointestinal fluid tolerance:

[0027] The survival rate in gastric juice showed a decreasing trend with the extension of time, and the survival rate was about 85% at 240 minutes;

[0028] The survival rate in intestinal fluid showed an overall downward trend, and at 240 min the survival rate was above 80%;

[0029] (4) Biofilm formation ability: weak;

[0030] (5) Acute toxicity test:

[0031] The results of bacterial reverse mutation test showed that the strain had no mutagenicity;

[0032] Mice were acutely gavaged with high, medium, and low doses of Akkermansia muciniphila CGMCC No.20955, and no mortality occurred;

[0033] There was no significant difference in the daily body weight changes between the test group mice and the control saline group;

[0034] During the acute gavage period, there was no significant difference in the daily food intake of the test mice and the control saline group mice;

[0035] During the acute gavage period, there was no significant difference in the daily changes in blood glucose between the test mice and the control saline group mice;

[0036] There were no significant differences in the blood parameters between the acute gavage group and the control saline group;

[0037] In the liver, the triglyceride levels in the test mice were significantly lower than those in the control saline group mice;

[0038] There was no significant difference in serum triglyceride levels between the test mice and the control mice;

[0039] There was no significant difference in cholesterol levels in serum and liver between the test mice and the control mice;

[0040] There was no significant difference in the bile acid levels in serum and liver between the test mice and the control mice;

[0041] There was no significant difference in glucose levels in serum and liver between the test mice and the control mice;

[0042] There was no significant difference in the total protein levels in serum and liver between the test mice and the control mice;

[0043] There was no significant difference in the activity of alanine aminotransferase in serum and liver between the test mice and the control mice.

[0044] There was no significant difference in the activity of aspartate aminotransferase in serum and liver between the test mice and the control mice;

[0045] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0046] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0047] There was basically no significant difference in the weight of the heart, liver, spleen, kidney, thymus, brain, testis, lung, stomach and intestine of the test mice and the control mice;

[0048] There was no significant pathological damage in the liver and kidneys of the test mice and the control group mice.

[0049] (6) Subchronic toxicity test:

[0050] Mice were gavaged with high, medium, and low doses of AKK PROBIO for 90 consecutive days without mortality.

[0051] There was no significant difference in the weekly body weight change between the test group mice and the control saline group;

[0052] During the continuous gavage period, there was no significant difference in the weekly changes in food intake between the test mice and the control saline group mice;

[0053] During the continuous gavage period, the changes in blood glucose in the mice given a medium dose of oral administration were significantly different from those in the control saline group, but there were no significant differences between the other groups and the control group.

[0054] There were no significant differences in the blood parameters between the mice in the gavage group and the control saline group;

[0055] There was no significant difference in triglyceride levels in the liver and serum between the test mice and the control mice;

[0056] There was no significant difference in cholesterol levels in the liver and serum between the test mice and the control mice;

[0057] The bile acid levels in the liver of the mice given the medium dose and the control group were significantly different, while there were no significant differences in the serum and liver between the other groups and the control group.

[0058] There was no significant difference in glucose levels in the liver and serum between the test mice and the control mice;

[0059] There was no significant difference in the total protein content in the liver and serum between the test mice and the control mice;

[0060] There was no significant difference in the activity of alanine aminotransferase in serum and liver between the test mice and the control mice.

[0061] There was no significant difference in the activity of aspartate aminotransferase in serum and liver between the test mice and the control mice;

[0062] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0063] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0064] There was basically no significant difference in the weight of the heart, liver, spleen, kidney, thymus, brain, testis, lung, pancreas, stomach and intestine between the test mice and the control mice;

[0065] There was no significant pathological damage in the liver and kidneys of the test mice and the control mice;

[0066] There was no significant difference in blood sugar regulation ability between the test mice and the control group mice.

[0067] (7) Drug sensitivity analysis:

[0068] AKK PROBIO was found to be sensitive to ampicillin, ceftriaxone, ceftriaxone, meropenem, tetracycline, moxifloxacin and chloramphenicol.

[0069] Akkermansia muciniphila (A. muciniphila) belongs to the Verrucomicrobia phylum and is widely found in the intestines of humans and other animals. A. muciniphila was first isolated from a fecal sample of a healthy Caucasian woman in 2004. 16S rRNA gene sequence analysis showed that the isolate belonged to a species of the Verrucomicrobia phylum (Derrien M, Vaughan EE, Plugge CM, et al. Akkermansia muciniphila gen. nov., sp. Nov., a human intestinal mucin-degrading bacterium [J]. International journal of systematic and evolutionary microbiology, 2004, 54 (5): 1469-1476.).

[0070] Most studies have shown that A. muciniphila and its membrane protein Amuc-1100 play an important probiotic role in a variety of metabolic diseases such as diabetes, obesity, alcoholic liver disease, etc. Although the beneficial role of A. muciniphila in metabolic diseases has been recognized, its role in reproductive damage, especially reproductive damage caused by inflammatory bowel disease (IBD), has not been reported.

[0071] Therefore, the present invention chooses to explore the role of AKK PROBIO in the prevention, treatment and / or auxiliary treatment of male reproductive damage.

[0072] The technical solution proposed by the present invention is as follows:

[0073] In one aspect, the present invention provides the use of Akkermansia muciniphila in preparing a product for preventing, treating and / or assisting in treating male reproductive damage, wherein the Akkermansia muciniphila is deposited in CGMCC with a deposit number of CGMCC No.20955.

[0074] In some embodiments, the male reproductive damage includes, but is not limited to, low sperm count, decreased sperm motility, decreased sperm motility, testicular tissue damage, decreased sperm head lateral swing amplitude, decreased sperm linearity, decreased whipping frequency, decreased average angular displacement, decreased forward motion, slower linear motion rate, slower average path rate or slower curve rate, etc.

[0075] In some embodiments, the male reproductive damage is induced by intestinal inflammation.

[0076] In some preferred embodiments, the enteritis may be ulcerative colitis.

[0077] In some embodiments, the product comprises a living or dead form of the Akkermansia muciniphila, and isolates and / or secretions thereof.

[0078] In some embodiments, the living form of Akkermansia muciniphila can be any biologically active Akkermansia muciniphila, such as live bacteria in fermentation broth, live bacteria in solid culture medium, live bacteria in fermentation broth precipitate, live bacteria in fermentation broth supernatant, live bacteria naturally existing in nature, live bacteria existing in the intestines of humans or animals, etc.

[0079] In some embodiments, the dead form of Akkermansia muciniphila can be any Akkermansia muciniphila without biological activity, such as dead bacteria in fermentation broth, dead bacteria in solid culture medium, dead bacteria in fermentation broth precipitate, dead bacteria in fermentation broth supernatant, dead bacteria naturally existing in nature, dead bacteria existing in the intestines of humans or animals, etc.

[0080] In some embodiments, the product comprises a drug or a drug raw material.

[0081] In another aspect, the present invention provides a probiotic preparation, comprising an effective amount of a living form or a dead form of Akkermansia muciniphila, and an isolate and / or secretion thereof. The Akkermansia muciniphila has a deposit number of CGMCC No.20955.

[0082] In some embodiments, the probiotic preparation can be used as a health product.

[0083] In some preferred embodiments, the probiotic preparation further includes food-acceptable excipients, including, but not limited to, erythritol, D-mannitol, fumaric acid, glycerol, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L(+)-tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glyceride, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, beet red, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, magnesium stearate, α-cyclodextrin, γ-cyclodextrin, dextrin, and the like.

[0084] In some preferred embodiments, the probiotic preparation further includes food-acceptable compounds, or food-acceptable proteins and / or polypeptides, such as iron sulfate, succinic acid, zinc sulfate, zinc gluconate, β-carotene, polyphenol antioxidants, dietary fiber, soy protein isolate, meat protein, cereal protein, milk protein, plant polypeptides, animal polypeptides, etc.

[0085] In some embodiments, the dosage form of the probiotic preparation includes a liquid preparation, a solid preparation, a semi-solid preparation or a gaseous preparation.

[0086] In some embodiments, each unit of the probiotic preparation contains at least about 10 8 CFU / g or CFU / mL.

[0087] The unit may refer to each tablet, each dose, each pill, each drop pill, each capsule and the like. Those skilled in the art should know that the unit depends on the dosage form / carrier form of the preparation and does not constitute a limitation on the scope of protection of the present invention.

[0088] In some embodiments, each unit of the probiotic preparation contains about 10 8 -10 11 CFU / g or CFU / mL.

[0089] In some preferred embodiments, each unit of the probiotic preparation may contain about 1.0×10 8 , 2.0×10 8 , 3.0×10 8 4.0×10 8, 5.0×10 8 , 6.0×10 8 , 7.0×10 8 8.0×10 8 9.0×10 8 , 1.0×10 9 , 2.0×10 9 , 3.0×10 9 4.0×10 9 , 5.0×10 9 , 6.0×10 9 , 7.0×10 9 8.0×10 9 9.0×10 9 , 1.0×10 10 , 2.0×10 10 , 3.0×10 10 4.0×10 10 , 5.0×10 10 , 6.0×10 10 , 7.0×10 10 8.0×10 10 9.0×10 10 , 1.0×10 11 .

[0090] In some preferred embodiments, each unit of the probiotic preparation may contain about 10 9 -10 11 CFU / g or CFU / mL.

[0091] In some preferred embodiments, each unit of the probiotic preparation may contain about 10 9 -10 10 CFU / g or CFU / mL.

[0092] In some preferred embodiments, each unit of the probiotic preparation may contain about 1.0×10 9 , 1.1×10 9 , 1.2×10 9 , 1.3×10 9 , 1.4×10 9 , 1.5×10 9 , 1.6×10 9 , 1.7×10 9 , 1.8×10 9 , 1.9×10 9、2.0×10 9 、2.1×10 9 、2.2×10 9 、2.3×10 9 、2.4×10 9 、2.5×10 9 、2.6×10 9 、2.7×10 9 、2.8×10 9 、2.9×10 9 、3.0×10 9 、3.1×10 9 、3.2×10 9 、3.3×10 9 、3.4×10 9 、3.5×10 9 、3.6×10 9 、3.7×10 9 、3.8×10 9 、3.9×10 9 、4.0×10 9 、4.1×10 9 、4.2×10 9 、4.3×10 9 、4.4×10 9 、4.5×10 9 、4.6×10 9 、4.7×10 9 、4.8×10 9 、4.9×10 9 、5.0×10 9 、5.1×10 9 、5.2×10 9 、5.3×10 9 、5.4×10 9 、5.5×10 9 、5.6×10 9 、5.7×10 9 、5.8×10 9 、5.9×10 9 、6.0×10 9 、6.1×10 9 、6.2×10 9 、6.3×10 9 、6.4×10 9 、6.5×10 9 、6.6×10 9 、6.7×10 9 、6.8×10 9 、6.9×10 9, 7.0×10 9 , 7.1×10 9 , 7.2×10 9 , 7.3×10 9 , 7.4×10 9 , 7.5×10 9 , 7.6×10 9 , 7.7×10 9 , 7.8×10 9 ,7.9×10 9 8.0×10 9 , 8.1×10 9 , 8.2×10 9 , 8.3×10 9 , 8.4×10 9 , 8.5×10 9 , 8.6×10 9 , 8.7×10 9 , 8.8×10 9 , 8.9×10 9 9.0×10 9 , 9.1×10 9 , 9.2×10 9 , 9.3×10 9 , 9.4×10 9 , 9.5×10 9 , 9.6×10 9 , 9.7×10 9 , 9.8×10 9 , 9.9×10 9 , 1.0×10 10 CFU / g or CFU / mL.

[0093] In still another aspect, the present invention provides a medicament for preventing and / or treating male reproductive damage, comprising an effective amount of a living form or a dead form of Akkermansia muciniphila, and an isolate and / or secretion thereof, wherein the deposit number of Akkermansia muciniphila is CGMCC No.20955.

[0094] The male reproductive damage includes, but is not limited to, low sperm count, decreased sperm motility, decreased sperm viability, testicular tissue damage, decreased sperm head lateral swing amplitude, decreased sperm linearity, decreased whipping frequency, decreased average angular displacement, decreased forward directionality, slower linear motion rate, lower average path rate, slower curve rate, etc.

[0095] In some embodiments, the male reproductive damage is induced by intestinal inflammation.

[0096] In some preferred embodiments, the enteritis may be ulcerative colitis.

[0097] In some embodiments, the living form of Akkermansia muciniphila can be any biologically active Akkermansia muciniphila, such as live bacteria in fermentation broth, live bacteria in solid culture medium, live bacteria in fermentation broth precipitate, live bacteria in fermentation broth supernatant, live bacteria naturally existing in nature, live bacteria existing in the intestines of humans or animals, live bacteria in freeze-dried powder, etc.

[0098] In some embodiments, the dead form of Akkermansia muciniphila can be any Akkermansia muciniphila without biological activity, such as dead bacteria in fermentation broth, dead bacteria in solid culture medium, dead bacteria in fermentation broth precipitate, dead bacteria in fermentation broth supernatant, dead bacteria naturally existing in nature, dead bacteria existing in the intestines of humans or animals, dead bacteria in freeze-dried powder, etc.

[0099] In some embodiments, the drug further includes pharmaceutically acceptable excipients, which include, but are not limited to solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavors, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0100] In some embodiments, the dosage form of the drug includes, but is not limited to, powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, powder mist, aerosol, suppository, drops or pills.

[0101] In some embodiments, each unit of the drug contains at least about 10 8 CFU / g or CFU / mL.

[0102] In some preferred embodiments, each unit of the drug may contain about 10 8 -10 11 CFU / g or CFU / mL.

[0103] In some preferred embodiments, each unit of the drug may contain about 1.0×10 8 , 2.0×10 8 , 3.0×10 84.0×10 8 , 5.0×10 8 , 6.0×10 8 , 7.0×10 8 8.0×10 8 9.0×10 8 , 1.0×10 9 , 2.0×10 9 , 3.0×10 9 4.0×10 9 , 5.0×10 9 , 6.0×10 9 , 7.0×10 9 8.0×10 9 9.0×10 9 , 1.0×10 10 , 2.0×10 10 , 3.0×10 10 4.0×10 10 , 5.0×10 10 , 6.0×10 10 , 7.0×10 10 8.0×10 10 9.0×10 10 , 1.0×10 11 .

[0104] In some preferred embodiments, each unit of the drug may contain about 10 9 -10 11 CFU / g or CFU / mL.

[0105] In some preferred embodiments, each unit of the drug may contain about 10 9 -10 10 CFU / g or CFU / mL.

[0106] In some preferred embodiments, each unit of the drug may contain about 1.0×10 9 , 1.1×10 9 , 1.2×10 9 , 1.3×10 9 , 1.4×10 9 , 1.5×10 9 , 1.6×10 9 , 1.7×10 9 , 1.8×10 9 , 1.9×109 、2.0×10 9 、2.1×10 9 、2.2×10 9 、2.3×10 9 、2.4×10 9 、2.5×10 9 、2.6×10 9 、2.7×10 9 、2.8×10 9 、2.9×10 9 、3.0×10 9 、3.1×10 9 、3.2×10 9 、3.3×10 9 、3.4×10 9 、3.5×10 9 、3.6×10 9 、3.7×10 9 、3.8×10 9 、3.9×10 9 、4.0×10 9 、4.1×10 9 、4.2×10 9 、4.3×10 9 、4.4×10 9 、4.5×10 9 、4.6×10 9 、4.7×10 9 、4.8×10 9 、4.9×10 9 、5.0×10 9 、5.1×10 9 、5.2×10 9 、5.3×10 9 、5.4×10 9 、5.5×10 9 、5.6×10 9 、5.7×10 9 、5.8×10 9 、5.9×10 9 、6.0×10 9 、6.1×10 9 、6.2×10 9 、6.3×10 9 、6.4×10 9 、6.5×10 9 、6.6×10 9 、6.7×10 9 、6.8×10 9 、6.9×109 , 7.0×10 9 , 7.1×10 9 , 7.2×10 9 , 7.3×10 9 , 7.4×10 9 , 7.5×10 9 , 7.6×10 9 , 7.7×10 9 , 7.8×10 9 ,7.9×10 9 8.0×10 9 , 8.1×10 9 , 8.2×10 9 , 8.3×10 9 , 8.4×10 9 , 8.5×10 9 , 8.6×10 9 , 8.7×10 9 , 8.8×10 9 , 8.9×10 9 9.0×10 9 , 9.1×10 9 , 9.2×10 9 , 9.3×10 9 , 9.4×10 9 , 9.5×10 9 , 9.6×10 9 , 9.7×10 9 , 9.8×10 9 , 9.9×10 9 , 1.0×10 10 CFU / g or CFU / mL.

[0107] The unit may refer to each tablet, each dose, each pill, each pill drop, each capsule and the like. Those skilled in the art should know that the unit depends on the dosage form / carrier form of the drug and does not constitute a limitation on the scope of protection of the present invention.

[0108] In yet another aspect, the present invention provides a method for preventing, treating and / or assisting in the treatment of male reproductive damage, comprising administering to the subject an effective amount of a living form or a dead form of Akkermansia muciniphila, an isolate and / or secretion thereof, the probiotic preparation or the medicament;

[0109] The deposit number of the Akkermansia muciniphila is CGMCC No.20955.

[0110] In some embodiments, the male reproductive damage includes, but is not limited to at least one of low sperm count, decreased sperm motility, decreased sperm motility, testicular tissue damage, decreased sperm head lateral swing amplitude, decreased sperm linearity, decreased whipping frequency, reduced average angular displacement, reduced forward motion, slower linear motion rate, slower average path rate, and slower curve rate.

[0111] In some embodiments, the male reproductive damage is induced by intestinal inflammation.

[0112] In some preferred embodiments, the enteritis may be ulcerative colitis.

[0113] The present invention establishes a DSS colitis model, prepares a DSS solution with pure water and gives it to animals to drink freely to establish the model, evaluates the effect of Akkermansia muciniphila in treating and / or preventing male reproductive damage induced by enteritis, and finds that Akkermansia muciniphila can be used as a protective component for improving male sperm motility and improving male reproductive damage. The present invention takes the intestine-testis axis as the entry point, not only providing a new idea for the exploration of related mechanisms, but also providing an effective method for the subsequent prevention and / or treatment of male reproductive damage caused by enteritis-related inducements using Akkermansia muciniphila.

[0114] The Akkermansia muciniphila, namely AKK PROBIO, provided by the present invention can significantly increase the density of sperm count, and obviously improve the viability, vitality and related movement parameter levels, so as to alleviate male reproductive damage induced by enteritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 The effect of Akkermansia muciniphila on sperm morphology, density and number in enteritis. (A) Sperm morphology and density; (B) Sperm number. Different lowercase letters indicate significant differences between groups (p<0.05), and the same lowercase letters indicate no significant differences between groups (p>0.05).

[0116] Figure 2The effects of Akkermansia muciniphila intervention on sperm motility, motility rate and related parameter levels in sperm damage caused by enteritis. (A) Sperm motility; (B) Sperm motility; (C) Amplitude of lateral head displacement (ALH); (D) Sperm linearity (LIN); (E) Beat cross frequency (BCF); (F) Mean angular displacement (MAD). Different lowercase letters indicate significant differences between groups (p<0.05), and the same lowercase letters indicate no significant differences between groups (p>0.05).

[0117] Figure 3 The effects of Akkermansia muciniphila intervention on sperm and sperm-related motility parameters in sperm damage caused by enteritis. (A) Straightness coefficient (STR); (B) Straight-line velocity (VSL); (C) Average path velocity (VAP); (D) Curvilinear velocity (VCL). Different lowercase letters indicate significant differences between groups (p<0.05), and the same lowercase letters indicate no significant differences between groups (p>0.05). DETAILED DESCRIPTION

[0118] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0119] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0120] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0121] Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0122] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" as used throughout the application, especially in the claims, may be replaced by the term "consisting of".

[0123] The terms "optional", "either", "any", or "any" as used herein mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. As used herein, "a" and "an" are used herein to refer to one or more than one of the grammatical objects.

[0124] The term "and / or" used herein should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0125] The terms "Akkermansia muciniphila", "AKK" and "AKK PROBIO" used herein refer to Akkermansia muciniphila deposited in CGMCC with the deposit number CGMCC No.20955.

[0126] The term "viable form of Akkermansia muciniphila" as used herein refers to Akkermansia muciniphila having at least one of the following properties: culturability, transcribability, metabolic activity, and cell membrane integrity. Culturability refers to the ability of bacteria to form colonies on a culture medium; transcribability refers to the ability of bacterial cells to produce mRNA; metabolic activity refers to the ability of bacteria to exhibit life phenomena related to their metabolism, such as redox ability, respiration, active enzymes, and substrate uptake ability; and cell membrane integrity refers to the fact that the bacterial cell membrane is not damaged.

[0127] Otherwise, it is the "death form of Akkermansia muciniphila".

[0128] The term "isolate" as used herein refers to a microbial substance in which a specific microorganism is separated from a population or a mixed microbial population and exists in a certain medium, wherein the medium includes fermentation broth supernatant, fermentation broth precipitate, bacterial suspension, freeze-dried powder, etc.

[0129] The term "secretions" as used herein refers to products secreted by specific microbial individuals during growth or metabolism, including metabolites such as methanol, ethanol, propanol, isopropanol, n-butanol, succinic acid, tartaric acid, etc.; extracellular products such as proteins, fats, starch, cellulose, antibiotics, etc.

[0130] The term "drug raw material" or "pharmaceutical raw material" used in this article refers to the raw material drug containing the active ingredient of the drug and the biological and chemical substances that serve as its source.

[0131] The term "probiotic preparation" used in this article refers to a preparation prepared according to the principles of microecology using normal beneficial microorganisms or their promoting substances that are beneficial to the host. It has the function of maintaining or adjusting the balance of microecology, preventing and treating diseases and promoting the health of the host.

[0132] The term "subject" as used herein refers to a person who participates in a clinical trial and serves as a recipient of the trial drug, including patients and healthy subjects. Preferably, the subject is a patient.

[0133] The term "adjuvant therapy" as used herein refers to products that are not directly used to prevent or combat disease, but are used to reduce the side effects of treatment, improve patient tolerance, and enhance the effectiveness of treatment. Products used for adjuvant therapy generally refer to products that help promote the absorption of the main drug, prevent or reduce adverse reactions, and enhance body functions.

[0134] To make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in many publications, such as "Molecular Cloning Experiment Guide (Fourth Edition)" (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience.

[0135] The experimental materials involved in the present invention are:

[0136] Dextran sulfate sodium (DSS), MP Biomedicals LLC, USA

[0137] Experimental animals and feeding:

[0138] C57BL / 6JNifdc male mice were purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd. (License No.: SCXK (Yue) 2022-0063). They were adaptively raised in the Experimental Animal Center of Jinan University with a 12-hour light cycle, a temperature of 20-25°C, and a relative humidity of 50±5%, and were fed freely. All experimental operations were carried out in accordance with the relevant requirements of the "Jinan University Experimental Animal Ethics Committee".

[0139] Example 1 Establishment of animal model and experimental grouping

[0140] 1.1 Animal model building

[0141] C57BL / 6JNifdc male mice were adaptively fed with ordinary feed, and 32 mice were randomly divided into 4 groups by weight stratification method, with 8 mice in each group, namely: Control group, DSS group, DSS+AL group (AL was low dose (10 8 CFU / Akkermansia muciniphila), DSS+AH group (AH was high dose (10 9CFU / animal) of Akkermansia muciniphila). DSS colitis model was established. Except for the Control group, animals in other groups were given 3% DSS solution prepared with pure water to drink freely for 8 days, and then given normal drinking water to drink freely for 2 days to establish the model.

[0142] The results showed that the present invention successfully established a DSS colitis model. After the animals were given DSS solution to drink freely to establish the model, the feces of the mice showed characteristic phenotypes such as soft feces, rotten feces, and bloody feces compared with the Control group.

[0143] 1.2 Intervention

[0144] All groups of mice were intervened on the first day of modeling and continued until the end of the experiment: the Control group and the DSS group were gavaged with the corresponding volume of water, while the DSS+AL group and the DSS+AH group were gavaged with the corresponding volume of Akkermansiamuciniphila intervention.

[0145] Example 2 Determination of the effect of Akkermansia muciniphila on sperm morphology, density and number

[0146] Sperm parameter determination:

[0147] · Turn on the sperm analyzer half an hour in advance and preheat the sperm counting plate to 37°C. At the same time, turn on the water bath half an hour in advance to preheat the physiological saline to 37°C.

[0148] ·Put the right epididymal tail in 1.0mL preheated physiological saline and cut it into pieces quickly. After 5 minutes of release at 37℃, take 10.0μL of the well-mixed sperm suspension and add it to the sperm counting plate. Use the Computer-aided Semen Analysis System (CASA) to take continuous frame dynamic pictures of the sperm to determine sperm motility and movement-related parameters. Then, when the sperm is still, take a single frame static picture of the sperm for sperm quantity and morphology analysis.

[0149] Based on the pictures taken, the CASA system is used to perform dynamic and static analysis of sperm.

[0150] The computer-assisted semen analysis system was used to determine the effects of Akkermansia muciniphila on sperm morphology, density and number. The results showed that compared with the control group, the DSS-treated group showed significant reproductive damage: sperm count and density were significantly reduced ( Figure 1). However, the Akkermansia muciniphila intervention groups (DSS+AL group and DSS+AH group) significantly improved reproductive damage.

[0151] Example 3 Determination of the effect of Akkermansia muciniphila on sperm motility and motility rate

[0152] The sperm parameters were determined as in Example 2.

[0153] Computer-assisted semen analysis system was used to determine the effects of Akkermansia muciniphila on sperm motility and viability.

[0154] The results showed that compared with the control group, the sperm motility, motility rate, sperm head lateral swing amplitude (ALH), whipping frequency (BCF) and mean angular displacement (MAD) of the DSS-treated group were significantly decreased, while the intervention of Akkermansia muciniphila rescued this trend ( Figure 2 ).

[0155] Example 4 Determination of the effect of Akkermansia muciniphila on sperm motility-related parameters

[0156] The sperm parameters were determined as in Example 2.

[0157] Computer-assisted semen analysis system was used to determine the effects of Akkermansia muciniphila on sperm motility-related parameters.

[0158] The results showed that compared with the control group, the sperm linear velocity (VSL), average path velocity (VAP), and curvilinear velocity (VCL) in the DSS-treated group were significantly decreased, while the intervention of Akkermansia muciniphila rescued this trend ( Figure 3 ).

[0159] From the above results, it can be seen that Akkermansia muciniphila is effective in treating and / or preventing male reproductive damage caused by enteritis, and can serve as a protective component for improving male sperm motility and improving male reproductive damage.

[0160] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Akkermansia muciniphila Akkermansia muciniphila ) for use in the preparation of a product for treating and / or assisting in the treatment of male reproductive damage, characterized in that: Said Akkermansia muciniphila Deposited in CGMCC, the deposit number is CGMCC No. 20955; The product is a medicine or a medicine raw material; the male reproductive damage is induced by enteritis; the male reproductive damage includes low sperm count, decreased sperm motility, decreased sperm viability, testicular tissue damage, decreased sperm head lateral swing amplitude, decreased whipping frequency, reduced average angular displacement, slowed linear motion rate, slowed average path rate or slowed curve rate.

2. The use according to claim 1, characterized in that: The product includes the Akkermansia muciniphila At least 10 8 CFU / g or CFU / mL.

3. The use according to claim 2, characterized in that: The product includes the Akkermansia muciniphila 10 8 -10 11 CFU / g or CFU / mL.

4. The use according to claim 3, characterized in that The product includes the Akkermansia muciniphila 10 8 -10 9 CFU / g or CFU / mL.

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