Use of Akkermansia muciniphila in preparing products for preventing, treating and / or assisting in the treatment of pain

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to the use of Akkermansia muciniphila in the preparation of products for preventing, treating and / or assisting in the treatment of pain. The present invention provides Akkermansia muciniphila, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 26, 2020, with a deposit number of CGMCC No. 20955. The Akkermansia muciniphila has a good analgesic effect, and its effect is equivalent to that of Fenbid Capsules.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of Akkermansia muciniphila in preparing products for preventing, treating and / or assisting in treating pain. Background Art

[0002] What modern medicine calls pain is a complex physiological and psychological activity and one of the most common clinical symptoms. It includes the pain sensation caused by harmful stimuli acting on the body, as well as the body's pain response to harmful stimuli (somatic motor response and / or visceral autonomic response, often accompanied by strong emotional colors). Pain can serve as a warning that the body is hurt, causing a series of defensive protective responses in the body. But on the other hand, pain as an alarm also has its limitations (for example, when pain occurs in cancer, it is too late). And some long-term severe pain has become an unbearable torture to the body. Therefore, analgesia is an important task facing medical workers.

[0003] Painkillers are generally divided into several categories, such as non-steroidal anti-inflammatory drugs, narcotic analgesics, antispasmodics, and central analgesics. Different painkillers have different effects and side effects. Most painkillers may have side effects such as dizziness, drowsiness, and nausea, and painkillers will pass through the liver and kidneys during metabolism, which will also have certain harm to liver and kidney function; in addition, narcotic analgesics such as morphine and pethidine have strong analgesic effects, which can cause addiction and dependence, and also have certain sedative and respiratory depression effects; non-steroidal anti-inflammatory drugs are commonly used analgesics, such as aspirin, ibuprofen, celecoxib, etc., but the analgesic effect is weak, and it may also induce gastric ulcers, bleeding, liver damage, and even allergic asthma and other side effects. Although these analgesic methods can achieve good clinical results in a short period of time in clinical practice, they will cause serious harm to the patient's body and mind, and even make the patient addicted.

[0004] Bacteria of the genus Akkermansia, in particular those of the species Akkermansia muciniphila, are thought to play an important role in the prevention and / or treatment of metabolic disorders, such as those associated with obesity (WO2014 / 075745; WO2014 / 077246). Oral administration of Akkermansia muciniphila to mice on a restricted diet or a high-fat diet was found to normalize diet-induced metabolic endotoxemia, obesity, and adipose tissue CD11c markers without any change in food intake (WO2014 / 075745). In addition, Akkermansia muciniphila treatment reduced body weight and improved body composition (i.e., fat / lean mass ratio). It was found that under a high-fat diet, Akkermansia muciniphila treatment increased mRNA expression of markers of adipocyte differentiation and lipid oxidation without affecting lipogenesis. It was also found that colonization with Akkermansia muciniphila completely reversed diet-induced fasting hyperglycemia and similarly reduced the insulin resistance index after treatment. Akkermansia is present in the human gut since early life, albeit at very low levels. The presence of Akkermansia is associated with intestinal integrity, and its relative abundance and absolute number are known to increase rapidly with age. Animal experiments have confirmed the effects of Akkermansia on intestinal barrier function (Jreunanen et al., 2015, ApplenVironMicrobiol.81:3655-62), and have demonstrated that its administration can prevent diet-induced obesity. However, there are no reports on the use of Akkermansia for pain relief.

[0005] The present invention is the development of downstream application technology carried out by the inventor for the independently developed microbial resources: Akkermansia muciniphila with the deposit number of CGMCC No.20955 (other patents have been submitted on the same day).

[0006] After obtaining Akkermansia muciniphila, the inventors conducted application experiments in multiple fields including analgesia, cardiovascular disease, anti-tumor, cognitive disease, metabolic disease, inflammatory disease, bone and joint disease, and obtained positive experimental results in some fields.

[0007] Taking into account the relevant provisions of the Patent Law on unity, protection is requested separately for each indication that does not have unity.

[0008] The present invention is directed to the protection of analgesic application technology.

[0009] Since relevant evidence such as microbial identification and performance testing was published in the microbial patent submitted on the same day, in order to facilitate the review of the present invention and quickly understand the inventor's previous work, the relevant information of Akkermansiamuciniphila is briefly disclosed in the content of the present invention.

[0010] Akkermansia muciniphila:

[0011] The conclusive information in this section is for the convenience of reviewing 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.

[0012] 1. Akkermansia muciniphila was obtained by separation and screening by the present inventors.

[0013] 2. The deposit information of Akkermansia muciniphila: The deposit number is: CGMCC No.20955.

[0014] 3. 16srRNA of Akkermansia muciniphila: SEQ ID NO.1.

[0015] 4. Physiological and biochemical properties of Akkermansia muciniphila:

[0016] (1) Hydrophobicity: It increases with time and reaches more than 30% at 60 min.

[0017] (2) Self-aggregation: It increases with time and tends to be stable after 20 hours, maintaining at around 52%.

[0018] (3) Gastrointestinal fluid tolerance:

[0019] 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;

[0020] The survival rate in intestinal fluid showed a downward trend as a whole, and the survival rate was above 80% at 240 minutes.

[0021] (4) Biofilm formation ability: weak.

[0022] (5) Acute toxicity test:

[0023] The results of bacterial reverse mutation test showed that the strain had no mutagenicity;

[0024] Mice were acutely gavaged with high, medium, and low doses of Akkermansia muciniphila, and no mortality occurred;

[0025] There was no significant difference in the daily body weight changes between the test group mice and the control saline group;

[0026] 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;

[0027] 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;

[0028] There were no significant differences in the blood parameters between the acute gavage group and the control saline group;

[0029] In the liver, the triglyceride levels in the test mice were significantly lower than those in the control saline group mice;

[0030] There was no significant difference in serum triglyceride levels between the test mice and the control mice;

[0031] There was no significant difference in cholesterol levels in serum and liver between the test mice and the control mice;

[0032] There was no significant difference in the bile acid levels in serum and liver between the test mice and the control mice;

[0033] There was no significant difference in glucose levels in serum and liver between the test mice and the control mice;

[0034] There was no significant difference in the total protein levels in serum and liver between the test mice and the control mice;

[0035] There was no significant difference in the activity of alanine aminotransferase in serum and liver between the test mice and the control mice.

[0036] There was no significant difference in the activity of aspartate aminotransferase in serum and liver between the test mice and the control mice;

[0037] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0038] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0039] 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;

[0040] There was no significant pathological damage in the liver and kidneys of the test mice and the control group mice.

[0041] (6) Subchronic toxicity test:

[0042] Mice were gavaged with high, medium, and low doses of AKK PROBIO for 90 consecutive days without mortality.

[0043] There was no significant difference in the weekly body weight change between the test group mice and the control saline group;

[0044] 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;

[0045] 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.

[0046] There were no significant differences in the blood parameters between the mice in the gavage group and the control saline group;

[0047] There was no significant difference in triglyceride levels in the liver and serum between the test mice and the control mice;

[0048] There was no significant difference in cholesterol levels in the liver and serum between the test mice and the control mice;

[0049] 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.

[0050] There was no significant difference in glucose levels in the liver and serum between the test mice and the control mice;

[0051] There was no significant difference in the total protein content in the liver and serum between the test mice and the control mice;

[0052] There was no significant difference in the activity of alanine aminotransferase in serum and liver between the test mice and the control mice.

[0053] There was no significant difference in the activity of aspartate aminotransferase in serum and liver between the test mice and the control mice;

[0054] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0055] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0056] 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;

[0057] There was no significant pathological damage in the liver and kidneys of the test mice and the control mice;

[0058] There was no significant difference in blood sugar regulation ability between the test mice and the control group mice.

[0059] (7) Drug sensitivity analysis of Akkermansia muciniphila:

[0060] Akkermansia muciniphila was found to be sensitive to ampicillin, ceftriaxone, ceftriaxone, meropenem, tetracycline, moxifloxacin and chloramphenicol. Summary of the invention

[0061] In order to solve the above problems, the present invention provides Akkermansia muciniphila, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 26, 2020, with a deposit number of CGMCC No. 20955. The Akkermansia muciniphila has a good analgesic effect.

[0062] The present invention is directed to the downstream application technology of Akkermansia muciniphila for protection.

[0063] Specifically, the use of Akkermansia muciniphila in analgesia.

[0064] Specifically, in terms of application areas, The aforementioned applications include:

[0065] 1. Use Akkermansia muciniphila for the prevention, treatment, auxiliary prevention and auxiliary treatment of pain in therapeutic and pharmaceutical purposes;

[0066] 2. Akkermansia muciniphila is used for the prevention, treatment, auxiliary prevention and auxiliary treatment of pain by using raw material preparation.

[0067] The present invention uses model mice as an example to verify the application of Akkermansia muciniphila in analgesia. The scope of application of the present invention should not be limited to therapeutic methods or pharmaceutical uses based on the commonness of animal experiments in the medical field.

[0068] Specifically, in terms of the type of pain, the aforementioned applications include, but are not limited to: nociceptive pain, inflammatory pain, neuropathic pain, cancer pain and / or psychiatric pain.

[0069] Based on the fact that all kinds of pain are caused by certain stimulation of receptors, which are converted into pain signals through nerve endings, those skilled in the art can reasonably conclude or infer that Akkermansia muciniphila can be equally applied to or at least has the potential for application in other pains based on experiments on Akkermansia muciniphila in a specific pain (burn).

[0070] More specifically, the nociceptive pain is pain caused by damage to the skin, muscles, ligaments, fascia, and bones.

[0071] More specifically, the noxious pain may be a burn.

[0072] More specifically, the inflammatory pain is pain caused by biological inflammation or chemical inflammation.

[0073] More specifically, the inflammatory pain may be rheumatoid arthritis, rheumatoid arthritis and / or ankylosing spondylitis.

[0074] More specifically, the neuropathic pain is pain directly caused by damage or disease of the somatic sensory nervous system.

[0075] More specifically, the neuropathic pain may be postherpetic neuralgia and / or diabetic neuropathy.

[0076] More specifically, the cancer pain is the pain caused by the compression and infiltration of surrounding organs and nerves by the tumor.

[0077] More specifically, the cancer pain may be pain caused by liver cancer, gastric cancer, pancreatic cancer, bile duct cancer and / or bone metastasis of malignant tumors.

[0078] Specifically, from Pharmaceutical field In particular, the drug comprises:

[0079] (1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila;

[0080] (2) Pharmaceutically acceptable excipients.

[0081] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0082] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0083] The dosage form of the medicine is tablet, capsule, granule or injection.

[0084] More specifically, the medicine comprises an analgesic medicine.

[0085] Specifically, in terms of the active dose in the above fields, the viable count of Akkermansia muciniphila is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

[0086] Preferably, the viable count of Akkermansia muciniphila is 1×10 8 -1×10 12 CFU / g or 1×10 8 -1×10 12 CFU / mL.

[0087] More preferably, the viable count of Akkermansia muciniphila is 1×10 9 -1×10 12 CFU / g or 1×10 9 -1×10 12 CFU / mL, such as 1×10 9 CFU / g (CFU / mL), 2×10 9 CFU / g (CFU / mL), 3×10 9 CFU / g (CFU / mL), 4×10 9 CFU / g (CFU / mL), 5×10 9 CFU / g (CFU / mL), 6×10 9 CFU / g (CFU / mL), 7×10 9 CFU / g (CFU / mL), 8×10 9 CFU / g (CFU / mL), 9×10 9 CFU / g (CFU / mL), 10×10 9 CFU / g, etc. Other point values ​​within this numerical range can be selected.

[0088] Specifically, from Raw material form For the purposes of the present invention, the aforementioned application includes the use of fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria and / or any ingredient, component, composition, metabolite directly or indirectly derived from Akkermansia muciniphila; and may or may not contain current or future excipients permitted in a specific application field.

[0089] More specifically, the fermentation liquid refers to the liquid obtained by inoculating bacteria into a culture medium and culturing for a period of time.

[0090] More specifically, the fermentation broth supernatant refers to the clarified liquid on the upper layer of the fermentation broth after centrifugation; it contains abundant metabolites of bacterial growth and reproduction and some bacterial fragments. The acidic substances and bacteriocins secreted by bacteria have antagonistic and killing effects on harmful bacteria; the amino acids and synthesized vitamins after bacteria decompose food are all in the culture medium, and also include enzymes secreted by bacteria that are useful to the human body; and some bacterial components also have an immune-promoting effect on the human body.

[0091] More specifically, the fermentation liquid precipitate refers to the liquid precipitate obtained by centrifugation, including free protein, residual bacteria, broken cells, and residues of the culture matrix, mainly protein and intracellular matrix.

[0092] More specifically, the live bacteria, also called active flora, can colonize and multiply in the intestine, which is beneficial to increase the number of beneficial bacteria.

[0093] More specifically, the dead bacteria are microorganisms that have lost their vitality and are unable to grow and reproduce. The probiotics lose their vitality due to the production process, such as high temperature treatment or excessive drying.

[0094] The present invention also provides a method for relieving pain, which comprises administering to a patient suffering from pain Akkermansiamuciniphila.

[0095] Specifically, the pain patients are patients clinically diagnosed with nociceptive pain, inflammatory pain, neuropathic pain, cancer pain and / or psychiatric pain.

[0096] Specifically, the method of administration is selected from oral administration, intravenous injection, topical administration, intradermal administration and / or subcutaneous administration.

[0097] Specifically, a medicament comprising the following (1) and (2) is administered to a patient suffering from pain:

[0098] (1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila;

[0099] (2) Pharmaceutically acceptable excipients.

[0100] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0101] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0102] The dosage form of the medicine is tablet, capsule, granule or injection.

[0103] More specifically, the medicine comprises an analgesic medicine.

[0104] The present invention also provides Akkermansia muciniphila produced, derived, and derived based on the above application. Methods used to relieve pain.

[0105] Specifically, based on the analgesic use of Akkermansia muciniphila, the strain can also be improved to produce, derive, and derive Akkermansia muciniphila with better analgesic effect, and the improvement method is a traditional method and a genetic engineering method.

[0106] More specifically, the conventional method may be a physical method, a chemical method and / or a selection method.

[0107] More specifically, the physical method refers to the use of radiation or high pressure to induce mutations, thereby achieving strain improvement.

[0108] Preferably, the radiation method comprises ultraviolet radiation and / or X-ray radiation;

[0109] The high pressure method may be to expose the microorganisms to a high pressure environment to cause them to undergo adaptive changes.

[0110] More specifically, the chemical method refers to the use of chemical agents to induce mutation of bacterial strains.

[0111] Preferably, the chemical agent may be nitrite, acetylcholine and / or nitrogen mustard.

[0112] More specifically, the selection method refers to the process of selecting microorganisms with excellent traits for reproduction, thereby achieving strain improvement.

[0113] Preferably, the selection method is used to improve the metabolic pathway and / or growth conditions of the microorganism to obtain Akkermansia muciniphila with better analgesic effect.

[0114] More specifically, the genetic engineering method may be gene cloning, gene knockout, gene editing and / or synthetic biology, so as to obtain strains with better analgesic effects.

[0115] More specifically, the gene cloning refers to cutting out the target gene from one cell and inserting it into another cell to achieve the expression of the target gene in the new host to enhance the analgesic effect of the original strain.

[0116] More specifically, the gene knockout refers to deleting the target gene through technical means to achieve the regulation of microbial traits to obtain strains with better analgesic effects.

[0117] More specifically, the gene editing refers to the precise modification of the target gene sequence through technical means to achieve the regulation of microbial traits in order to obtain strains with better analgesic effects.

[0118] More specifically, the synthetic biology refers to the use of chemical synthesis machine technology to construct new gene sequences and introduce them into microorganisms to achieve regulation of microbial traits to obtain strains with better analgesic effects.

[0119] The present invention also provides a method for generating, deriving, or developing a method based on the above application. Products of Akkermansiamuciniphila .

[0120] Specifically, a method for preparing analgesic drug , the drug comprises:

[0121] (1) The fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila produced, derived or derived;

[0122] (2) Pharmaceutically acceptable excipients.

[0123] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0124] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0125] The dosage form of the medicine is tablet, capsule, granule or injection.

[0126] More specifically, the medicine comprises other analgesic drugs.

[0127] Specifically, in terms of active dose, the viable count of Akkermansia muciniphila produced, derived, or derived is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

[0128] Preferably, the viable count of Akkermansia muciniphila is 1×10 8 -1×10 12 CFU / g or 1×10 8 -1×10 12 CFU / mL.

[0129] More preferably, the viable count of Akkermansia muciniphila is 1×10 9 -1×10 12 CFU / g or 1×10 9 -1×10 12 CFU / mL, such as 1×10 9 CFU / g (CFU / mL), 2×10 9 CFU / g (CFU / mL), 3×10 9 CFU / g (CFU / mL), 4×10 9 CFU / g (CFU / mL), 5×10 9 CFU / g (CFU / mL), 6×10 9 CFU / g (CFU / mL), 7×10 9 CFU / g (CFU / mL), 8×10 9 CFU / g (CFU / mL), 9×10 9 CFU / g (CFU / mL), 10×10 9 CFU / g, etc. Other point values ​​within this numerical range can be selected.

[0130] Specifically, for pain relief Pharmaceutical Raw Materials The drug raw materials are the produced, derived, and derived Akkermansia muciniphila fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria.

[0131] The technical effects achieved by the present invention are:

[0132] (1) In the hot plate test, the AKK PROBIO group showed an upward trend from 30 to 130 minutes and reached a level comparable to that of the positive drug group at 130 minutes.

[0133] (2) In the acetic acid writhing test, the number of writhing times in the AKK PROBIO group was comparable to that in the positive drug group, or even slightly lower than that in the positive drug group, indicating that Akkermansia muciniphila has a good analgesic effect.

[0134] Storage Instructions:

[0135] Strain name: AKK PROBIO;

[0136] Deposit number: CGMCC No.20955;

[0137] Taxonomic nomenclature: Akkermansia muciniphila;

[0138] Deposit date: October 26, 2020;

[0139] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0140] Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1 It is the inhibition rate of mouse writhing times in the acetic acid writhing test. DETAILED DESCRIPTION

[0142] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0143] The Akkermansia muciniphila in the present invention is AKK PROBIO.

[0144] Example 1 Cultivation of AKK PROBIO

[0145] (1) Culture medium

[0146] Solid plate culture: BHI (Brain Heart Infusion, OXOID, Catalog No.: CM1032B)) + 5% sheep blood

[0147] Weigh a certain volume of BHI powder according to the finished product requirements, add agar powder at a ratio of 20g / L, dilute to the corresponding volume, and sterilize at 121℃ for 20min. After sterilization, add 5% sterile defibrinated sheep blood when it cools to 53℃ (not hot to hold), shake well and pour 15-20mL into a plate for later use.

[0148] (2) Culture conditions

[0149] Culture temperature: 37°C;

[0150] Culture conditions: completely anaerobic;

[0151] Liquid fermentation broth culture: BHI;

[0152] Culture temperature: 37°C;

[0153] Culture conditions: Completely anaerobic culture on 8 layers of gauze.

[0154] (3) Expansion culture of bacterial strains

[0155] Level 1: Take a 2 ml glycerol tube and inoculate it into a test tube containing 9 mL BHI at a 10% inoculum volume, and culture anaerobically at 37°C for 24-48 hours.

[0156] Secondary: Take the primary fermentation test tube and inoculate it into a Erlenmeyer flask containing 90 mL of BHI at a 5% inoculation rate, and culture anaerobically at 37°C for 24 h.

[0157] Level 3: Take the fermentation liquid from the second-stage Erlenmeyer flask and inoculate it into a Erlenmeyer flask containing 300 mL of BHI at a 5% inoculation rate. Incubate anaerobically at 37°C for 13 h and store in a 20% glycerol tube at -80°C.

[0158] Example 2 Hot Plate Experiment

[0159] 2.1 Animals

[0160] A total of 70 female C57BL / 6 mice (Shanghai Model Organisms Technology Co., Ltd.) weighing 18-22 g and free of specific pathogens were used. They were adaptively fed for one week before the experiment, with free access to food and water, and the breeding environment met the SPF level requirements.

[0161] 2.2 Reagents and instruments

[0162] Physiological saline (Beijing Biolab Technology Co., Ltd.); Fenbid capsules (Sino-US Tianjin SmithKline Pharmaceutical Co., Ltd., national medicine standard number H10900089); intelligent hot plate instrument (manufacturer: Beijing Haifuda Technology Co., Ltd., model RG26-YLS-6B).

[0163] 2.3 Hot plate experiment

[0164] The time required for the mouse to produce a pain response (i.e. licking the hind paw) when the mouse's foot contacts the hot plate and is stimulated by heat is the pain threshold. The analgesic effect of the drug is reflected by measuring the change in the pain threshold of the drug-treated mice. At room temperature of 22°C, the temperature of the hot plate was adjusted to (55±0.5)°C, and the mouse was placed on the hot plate. The time (s) required for the beginning of licking the hind paw was taken as the reaction time, and the average of the two reaction times was taken as the basal pain threshold before drug administration. Those mice whose licking time was <5s or >30s were discarded. 30 qualified mice were selected and randomly divided into a blank control group (normal saline), a positive drug group (Fenbid capsules, dosage 90mg / kg), an AKK PROBIO group (dosage 1×10 9 CFU / mouse), 10 mice per group. All drugs or saline were gavaged once a day at the corresponding dose for 15 days. The gavage volume was calculated according to the dose of the group and the actual weight of the mice. The pain threshold was measured at 30min, 80min, and 130min after the last gavage. The experimental results are shown in Table 1.

[0165] Table 1

[0166]

[0167] The results showed that the pain threshold of the blank control group did not fluctuate significantly from 30 to 130 minutes after the drug was administered. The pain threshold of the positive drug group showed a significant upward trend from 30 to 80 minutes; it slightly decreased at 130 minutes, but also maintained at a fairly high level. The AKKPROBIO group showed an upward trend from 30 to 130 minutes, and reached a level comparable to that of the positive drug group at 130 minutes. The pain thresholds of both groups at each time point after drug administration were significantly higher than those of the blank control group.

[0168] Example 3 Acetic acid writhing test

[0169] 3.1 Animals

[0170] Same as 2.1.

[0171] 3.2 Reagents

[0172] Glacial acetic acid (Shandong Maojun Chemical Technology Co., Ltd.).

[0173] 3.3 Acetic acid writhing test

[0174] The injection of acetic acid solution into mice caused a persistent pain that stimulated the peritoneum and intermittent motor reaction, which was manifested by the abdomen being concave, the anterior abdominal wall being close to the cage bottom, the buttocks being twisted, and the hind limbs being stretched out, showing a special posture. 30 mice were selected and randomly divided into a blank control group (normal saline), a positive drug group (Fenbid capsule, dosage 90mg / kg), an AKK PROBIO group (1×10 9CFU / mouse), 10 mice per group. All drugs or saline were gavaged once a day at the corresponding dose for a total of 21 days. The gavage volume was calculated according to the dose of the group and the actual weight of the mice. 30 minutes after the last gavage, 0.6% glacial acetic acid (0.1mL / 10g) was injected intraperitoneally, and the mice were immediately placed in a cage individually to observe the number of writhing reactions in the mice within 15 minutes. The inhibition rate of writhing times (%) = (mean number of writhing in the blank control group - mean number of writhing in the drug intervention group) / mean number of writhing in the blank control group × 100%. The experimental results are shown in Table 2.

[0175] Table 2

[0176]

[0177] The results showed that the number of writhings in the positive drug group and AKK PROBIO group was significantly lower than that in the blank control group, and the number of writhings in the AKKPROBIO group was equivalent to that in the positive drug group, or even slightly lower than that in the positive drug group; the inhibition rate of the number of writhings in the positive drug was 31.67%, and the inhibition rate of the number of writhings in the AKK PROBIO group was 34.66%, indicating that AKK PROBIO has a good analgesic effect.

[0178] Comparative Example

[0179] Referring to the culture method of Example 1 and the experimental method of Example 3, the analgesic effect of the ATCC standard strain of Akkermansia muciniphila (ATCC BAA 835) was tested, and the results are shown in Table 3:

[0180] Table 3

[0181] Group Number of twists (times) Twist inhibition rate (%) ATCC standard strains 32.80±3.39 18.20％

[0182] The results showed that the ATCC standard strain reduced the number of writhing times compared with the control group, but the inhibition rate of writhing times was only half of that of the AKK PROBIO group, which was significantly inferior to the AKK PROBIO of the present invention. Therefore, the analgesic effect of the AKK PROBIO of the present invention is comparable to that of the positive drug, and is even slightly stronger than that of the positive drug in the acetic acid writhing test.

[0183] Application Example 1: Preparation of AKK PROBIO live bacteria

[0184] AKK PROBIO live bacteria culture method

[0185] (1) Culture medium

[0186] Solid plate culture: BHI + 5% sheep blood

[0187] Weigh a certain volume of BHI powder according to the finished product requirements, add agar powder at a ratio of 20g / L, dilute to the corresponding volume, and sterilize at 121℃ for 20min. After sterilization, add 5% sterile defibrinated sheep blood when it cools to 53℃ (not hot to hold), shake well and pour 15-20ml into a plate for later use.

[0188] (2) Culture conditions

[0189] Culture temperature: 37°C;

[0190] Culture conditions: completely anaerobic;

[0191] Liquid fermentation broth culture: BHI;

[0192] Culture temperature: 37°C;

[0193] Culture conditions: Completely anaerobic culture on 8 layers of gauze.

[0194] (3) Expansion culture of bacterial strains

[0195] Level 1: Take a 2 ml glycerol tube and inoculate it into a test tube containing 9 mL BHI at a 10% inoculum volume, and culture anaerobically at 37°C for 24-48 hours.

[0196] Secondary: Take the primary fermentation test tube and inoculate it into a Erlenmeyer flask containing 90 mL of BHI at a 5% inoculation rate, and culture anaerobically at 37°C for 24 h.

[0197] Level 3: Take the fermentation liquid from the second-stage Erlenmeyer flask and inoculate it into a Erlenmeyer flask containing 300 mL of BHI at a 5% inoculation rate. Incubate anaerobically at 37°C for 13 h and store in a 20% glycerol tube at -80°C.

[0198] (4) Fermentation tank culture

[0199] The activated bacteria were placed in a large fermentation tank and cultured under the same conditions as above. After the culture was completed, the wet bacteria were obtained by centrifugation.

[0200] Application Example 2: Preparation of AKK PROBIO dead bacteria

[0201] Preparation method of AKK PROBIO dead bacteria:

[0202] The wet bacteria prepared in Example 1 are killed by chemical or physical methods to obtain dead bacteria;

[0203] The chemical method may be gas sterilization and / or liquid sterilization;

[0204] Specifically, the gas sterilization may be ozone, ethylene oxide, formaldehyde, propylene glycol, glycerin or peracetic acid vapor;

[0205] The liquid sterilization agent is a chemical reagent, such as ethanol;

[0206] The physical method may be heat sterilization, light sterilization and / or microwave sterilization;

[0207] Specifically, the thermal sterilization method may be a burning method, a dry baking method, a boiling method and / or a pressure steam sterilization method;

[0208] The light sterilization can be sunlight exposure, ultraviolet irradiation and / or ionizing radiation.

[0209] Application Example 3: An oral medicine containing AKK PROBIO

[0210] The wet bacteria prepared in Example 1 are mixed with auxiliary materials in a certain ratio, and the mixture is made into live bacteria tablets, capsules, granules, etc. by spray drying, tabletting, etc.;

[0211] Alternatively, the wet bacteria prepared in Example 1 are subjected to cell disruption, and then the beneficial components are extracted by appropriate techniques; the extraction methods include centrifugation, distillation, leaching, extraction, etc., and the specific selection depends on the components contained in the probiotics; the extracted components can be prepared into different dosage forms such as capsules, oral liquids, granules, injections, etc. according to clinical use needs.

Claims

1. Use of Akkermansia muciniphila in the preparation of a drug or pharmaceutical raw material for preventing, treating and / or assisting in the treatment of pain, characterized in that: The Akkermansia muciniphila is deposited with CGMCC No.20955.

2. The use according to claim 1, characterized in that: The medicine or pharmaceutical raw material contains live bacteria of Akkermansia muciniphila.

3. The use according to claim 1, characterized in that: The pain is nociceptive pain, inflammatory pain, neuropathic pain, cancer pain and / or psychogenic pain.

4. The use according to claim 3, characterized in that: The nociceptive pain is pain caused by damage to the skin, muscles, ligaments, fascia, and bones.

5. The use according to claim 4, characterized in that: The noxious pain is burns.

6. A pharmaceutical raw material, characterized in that: The invention comprises live bacteria of Akkermansia muciniphila; the preservation number of the Akkermansia muciniphila is: CGMCC No.20955.

7. A pain-relieving drug, characterized in that: The medicine comprises: (1) Live bacteria of Akkermansia muciniphila; (2) Pharmaceutically acceptable excipients; The Akkermansia muciniphila is deposited with CGMCC No.20955.

8. The drug according to claim 7, characterized in that The dosage form of the medicine is tablet, capsule, granule or injection.

9. The drug according to claim 7, characterized in that The viable count of Akkermansia muciniphila in the drug is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

10. The drug according to claim 7, characterized in that The medicines described include other analgesic drugs.

11. The drug according to any one of claims 7 to 10, characterized in that The pharmaceutically acceptable excipients are selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, solutes that make the preparation isotonic with the recipient's blood, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

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