Application of Akkermansia muciniphila and its products in the prevention and treatment of diabetes

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the application of Akkermansia muciniphila and its products in the prevention and treatment of diabetes. The Akkermansia muciniphila is AKK ONE, and its deposit number is CGMCC No. 20954. The AKK ONE provided by the present invention can effectively improve blood sugar tolerance, alleviate liver damage caused by diabetes, provide technical support for the prevention or treatment of diabetes and the improvement of lipid metabolism, and expand the bacterial seed resources.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to application of Akkermansia muciniphila and products thereof in the prevention and treatment of diabetes. Background Art

[0002] Diabetes is a group of metabolic diseases caused by multiple factors and characterized by chronic hyperglycemia. It is caused by defects in insulin secretion and / or utilization. Long-term high blood sugar levels can cause damage to multiple systems, leading to chronic progressive lesions, functional impairment and failure of tissues and organs such as the eyes, kidneys, nerves, heart and blood vessels. When the disease is severe or stressful, it can cause acute and severe metabolic disorders, such as diabetic ketoacidosis and hyperosmolar hyperglycemic syndrome.

[0003] Diabetes is a metabolic disease, which is mainly manifested by high blood sugar levels. High blood sugar can cause diabetic patients to have symptoms such as polydipsia, polyphagia, polyuria, and weight loss. Lipid metabolism is also an important aspect of human metabolism, which is mainly manifested by abnormal metabolism of cholesterol, triglycerides, and low-density lipoprotein. Cholesterol metabolism disorder is the main cause of diabetes. Hypercholesterolemia is a characteristic lesion of diabetes. The main reason is that patients consume too much fat, leading to fatty liver. Excessive fat accumulation in the liver will hinder the liver's clearance, thereby causing increased cholesterol levels. Triglyceride levels are also one of the causes of diabetes. Severe triglyceridemia can cause arteriosclerosis, thereby increasing the risk of cardiovascular and cerebrovascular diseases. Low-density lipoprotein can promote the synthesis of cholesterol in the body, and cholesterol can be oxidized into low-density lipoprotein in the body. Therefore, the level of low-density lipoprotein is closely related to the onset of diabetes, and it is also a risk factor for cardiovascular and cerebrovascular diseases in diabetic patients. In summary, diabetes can cause many metabolic abnormalities in the body, and lipid metabolism is one of them. Therefore, for diabetic patients, it is recommended to maintain a high dietary fiber diet and reduce fat intake, which will help control the blood sugar and blood lipid levels of diabetic patients.

[0004] At present, the drugs for treating diabetes are mainly Western medicine preparations, including insulin preparations, sulfonylurea preparations, biguanide preparations, insulin resistance improvers, α-glucosidase inhibitors, etc. These drugs act on a certain link of blood sugar regulation to play a role in blood sugar control, with rapid onset of action, but with large side effects, single efficacy, and easy to produce resistance.

[0005] Chronic inflammation caused by endotoxins entering the blood due to dysbiosis is one of the important factors in the development of metabolic diseases such as obesity and diabetes. Imbalanced bacterial flora can cause chronic systemic inflammation, leading to pancreatic β-cell damage and decreased insulin resistance. It also affects the absorption of sugar by other cells in the body for energy conversion, making it difficult for the human body to function normally, and finally causing a series of complications. Studies have shown that probiotics can effectively inhibit the development of prediabetes into type 2 diabetes. Probiotics help the human body lower blood sugar concentration, thereby inhibiting the symptoms of prediabetes.

[0006] Patent CN113330109A relates to Akkermansia muciniphila SNUG-61027 strain (preservation number KCTC13530BP) and its use. Specifically, a composition for appetite control or prevention, improvement, alleviation or treatment of metabolic diseases, a use for appetite control or prevention, improvement, alleviation and treatment of metabolic diseases, and a method for appetite control or prevention, improvement, alleviation and treatment of metabolic diseases using the composition are provided, wherein the composition comprises the strain, or its culture fluid, or B2UM07 protein separated therefrom as an active ingredient, and the metabolic disease is impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease or obesity.

[0007] Therefore, there is an urgent need to develop more safe and effective probiotics to meet the huge clinical needs.

[0008] 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.20954 (other patents have been submitted on the same day).

[0009] After obtaining Akkermansia muciniphila, the inventors conducted application experiments in multiple fields including improving diabetes, anti-infection, improving respiratory diseases, regulating lipid metabolism, improving enteritis, etc., and obtained positive experimental results in some fields.

[0010] Taking into account the relevant provisions of the Patent Law on unity, protection is requested separately for each indication that does not have unity.

[0011] The present invention is directed to the protection of application technology for improving diabetes.

[0012] 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.

[0013] 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.

[0014] 1. Akkermansia muciniphila was obtained by separation and screening by the present inventors.

[0015] 2. The deposit information of Akkermansia muciniphila: The deposit number is: CGMCC No.20954.

[0016] 3. Physiological and biochemical properties of Akkermansia muciniphila:

[0017] (1) Hydrophobicity: The hydrophobicity of the strain increased with time, reaching more than 30% at 60 min.

[0018] (2) Self-aggregation: The self-aggregation of the strain increased with time and tended to be stable at 20 hours, maintaining at about 50%.

[0019] (3) Gastrointestinal fluid tolerance:

[0020] The survival rate of the strain in gastric juice showed a downward trend with the extension of time, and the survival rate was about 80% at 240 minutes;

[0021] The survival rate of the strains in the intestinal fluid showed a downward trend as a whole, and the survival rate was above 80% at 240 minutes.

[0022] (4) Biofilm formation ability: weak.

[0023] (5) Bacterial reverse mutation test: The results of the bacterial reverse mutation test showed that the strain was not mutagenic.

[0024] (6) Acute toxicity:

[0025] The mice were acutely gavaged with high, medium and low doses of AKK ONE, and no mortality occurred. The daily weight changes of the mice in the test groups were compared with those in 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] There was no significant difference in triglyceride levels in serum and liver between the test mice and the control mice;

[0030] There was a very significant difference in liver cholesterol levels between mice given high doses orally and mice in the control group, but no significant differences were found in serum and liver in other groups.

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

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

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

[0034] There was no significant difference in the activity of alanine aminotransferase in serum and liver between the test mice and the control mice.

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

[0036] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0037] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0038] 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;

[0039] There was no significant pathological damage in the liver and kidneys of the test mice and the control group mice.

[0040] (7) Subchronic toxicity test:

[0041] Mice were gavaged with high, medium and low doses of AKK ONE for 90 consecutive days without death. There was no significant difference in the weekly weight change between the test group and the control saline group;

[0042] 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;

[0043] During the continuous gavage period, there was no significant difference in the blood glucose changes between the mice gavaged with a medium dose per week and the mice in the control saline group;

[0044] There were no significant differences in the blood parameters between the mice in the gavage group and the control saline group;

[0045] There was no significant difference in triglyceride levels in the liver and serum between the test mice and the control mice;

[0046] There was no significant difference in cholesterol levels in the liver and serum between the test mice and the control mice;

[0047] 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.

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

[0049] There was no significant difference in the total protein content in the liver and serum between the test mice and the control mice;

[0050] There was a significant difference in the activity of alanine aminotransferase in the serum of mice given the medium dose and the control group, but no significant difference was found in the serum and liver of the other groups.

[0051] There was a significant difference in the activity of alanine aminotransferase in the serum of mice given the medium dose and the control group, but no significant difference was found in the serum and liver of the other groups.

[0052] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0053] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0054] 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;

[0055] There was no significant pathological damage in the liver and kidneys of the test mice and the control mice;

[0056] There was no significant difference in blood sugar regulation ability between the test mice and the control group mice. Summary of the invention

[0057] In order to solve the above problems, the present invention provides a new strain of Akkermansia muciniphila, named AKK ONE, with a deposit number of CGMCC No. 20954. The strain can be effectively used for the prevention and treatment of diabetes and improve metabolic abnormalities.

[0058] In one aspect, the present invention provides the use of Akkermansia muciniphila in preparing a product for preventing and / or treating diabetes; the deposit number of the Akkermansia muciniphila is CGMCC No.20954.

[0059] Specifically, the product comprises fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, lyophilized powder and / or cell lysate of Akkermansia muciniphila.

[0060] More specifically, the fermentation liquid refers to the liquid obtained by inoculating the bacteria into the culture medium and culturing it for a period of time.

[0061] To be more specific, 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.

[0062] 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.

[0063] 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.

[0064] More specifically, the dead bacteria are microorganisms that have lost their vitality and are unable to grow and reproduce, and the probiotics lose their vitality due to the production process.

[0065] More specifically, the inactivation method includes, but is not limited to: any one or more of high temperature inactivation, radiation inactivation or chemical inactivation;

[0066] The high-temperature inactivation may be dry heat inactivation or wet heat inactivation; the radiation inactivation may be light radiation or ionizing radiation, the light radiation may be ultraviolet or infrared radiation, the ionizing radiation includes but is not limited to microwave radiation; the chemical inactivation includes but is not limited to gas sterilization or liquid sterilization, the gas sterilization is carried out using a gaseous sterilant, such as ozone or formaldehyde, and the liquid sterilization is carried out using a liquid sterilant.

[0067] More specifically, the lyophilized powder is obtained by freeze-drying the aforementioned culture solution. The lyophilized powder generally also includes a lyophilization protectant. The lyophilization protectant includes, but is not limited to, pH buffer, filler, sugar, nonionic surfactant, ligand, etc. The pH buffer includes, but is not limited to, any one or more of Tris, amino acids or their salts, citric acid or its salts, acetic acid or its salts. The filler includes, but is not limited to, any one or more of mannitol, glycine, and bovine serum albumin. The sugar may be a disaccharide, such as any one or more of sucrose or trehalose. The nonionic surfactant includes, but is not limited to, Tween, and the Tween may be Tween-20, Tween-60, Tween-80, etc. The lyophilization protectant may also include antioxidants, etc. The lyophilization protectant may also specifically include albumin, polyethylene glycol, etc.

[0068] More specifically, the cell lysis solution can be obtained by lysing the bacteria obtained by the aforementioned culture. The lysis can be physical lysis or chemical lysis. The physical lysis includes but is not limited to grinding, ultrasonic disruption, etc. The chemical lysis includes but is not limited to chemical reagent lysis and enzymatic hydrolysis, and the enzymatic hydrolysis can be hydrolase or oxidase. The lysis can also be performed by increasing the intracellular pressure to cause the cells to rupture by themselves.

[0069] Specifically, the product is a medicine or a pharmaceutical raw material.

[0070] More specifically, the product is a drug, and the drug also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include but are not limited to: any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0071] Preferably, the pharmaceutically acceptable excipient can be selected from at least one of 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.

[0072] In yet another aspect, the present invention provides use of Akkermansia muciniphila in preparing a product for improving abnormal lipid metabolism.

[0073] Specifically, the deposit number of the Akkermansia muciniphila is CGMCC No.20954.

[0074] More specifically, the product comprises fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, lyophilized powder and / or cell lysate of Akkermansia muciniphila.

[0075] Specifically, the product is a medicine or a pharmaceutical raw material.

[0076] More specifically, the product is a medicine, and the medicine also includes pharmaceutically acceptable excipients.

[0077] More specifically, the drug also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0078] Preferably, the pharmaceutically acceptable excipient can be selected from at least one of 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.

[0079] In yet another aspect, the present invention provides a method for preparing a medicament for preventing and / or treating diabetes.

[0080] Specifically, the method comprises culturing Akkermansia muciniphila; the deposit number of the Akkermansia muciniphila is CGMCC No.20954.

[0081] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0082] More specifically, the drug also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0083] Preferably, the pharmaceutically acceptable excipient can be selected from at least one of 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.

[0084] In yet another aspect, the present invention provides a method for preparing a drug for improving abnormal lipid metabolism.

[0085] Specifically, the method comprises culturing Akkermansia muciniphila; the deposit number of the Akkermansia muciniphila is CGMCC No.20954.

[0086] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0087] More specifically, the drug also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0088] Preferably, the pharmaceutically acceptable excipient can be selected from at least one of 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.

[0089] The technical effects achieved by the present invention are:

[0090] (1) AKK ONE has the ability to improve blood sugar and glucose tolerance in T2DM mice.

[0091] (2) AKK ONE can effectively reduce the serum insulin content of T2DM mice.

[0092] (3) AKK ONE alleviates HFD / STZ-induced liver damage in mice.

[0093] (4) AKK ONE can improve the phenomenon of increased lipids in diabetic mice.

[0094] Storage Instructions:

[0095] Strain name: AKK ONE;

[0096] Deposit number: CGMCC No.20954;

[0097] Taxonomic nomenclature: Akkermansia muciniphila;

[0098] Deposit date: October 26, 2020;

[0099] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0100] Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 This is the blood glucose change curve of mice OGTT.

[0102] Figure 2 The effect of AKK ONE on insulin in T2DM mice.

[0103] Figure 3 The effect of AKK ONE on serum AST and ALT in T2DM mice, where A is the AST content in serum; B is the ALT content in serum.

[0104] Figure 4 This is a histopathological section of mouse liver.

[0105] Figure 5 This is a histopathological section of mouse pancreas.

[0106] Figure 6 is the gene expression level in mouse liver tissue.

[0107] Nouns and terms:

[0108] Glutamic oxaloacetic transaminase (AST) is mainly distributed in the myocardium, followed by the liver, kidneys, and skeletal muscles. The content of AST in normal serum is very low, but if the corresponding cells are damaged, the cell membrane permeability increases, and the AST in the cells will be released into the blood, causing its increase. It is generally used as an auxiliary examination for organ damage.

[0109] Alanine aminotransferase (glutamic-pyruvic transaminase, ALT) is a transferase that reflects the patient's liver function. It refers to alanine aminotransferase. When the alanine aminotransferase is elevated, it indicates that the patient's liver cells are damaged and that there is a problem with the patient's liver function.

[0110] Serum insulin (INS) is a protein hormone. Insulin in the body is secreted by pancreatic β cells. Serum insulin is the only hormone in the body that lowers blood sugar and promotes the synthesis of glycogen, fat, and protein at the same time.

[0111] Triglyceride (TG), also known as neutral fat, is a lipid formed by the reaction of glycerol molecules and fatty acids. It is one of the components of blood lipids and has the function of providing energy for cell metabolism. Triglyceride determination is an important part of blood lipid examination. It is the sum of triglycerides contained in each lipoprotein in plasma.

[0112] Total cholesterol (TC) is the sum of cholesterol contained in various lipoproteins in the blood. Cholesterol is the main component of cell membranes and is also an important raw material for synthesizing physiologically active substances such as adrenal cortex hormones, sex hormones, bile acids and vitamin D. The total cholesterol concentration in human blood can be used as an indicator of lipid metabolism to assess the risk of atherosclerosis and ischemic cardiovascular and cerebrovascular diseases.

[0113] High-density lipoprotein cholesterol (HDL-C) is an anti-atherosclerotic lipoprotein that can transport cholesterol from extrahepatic tissues to the liver for metabolism and excrete it from the body in bile. Its plasma level is negatively correlated with the risk of metabolic diseases.

[0114] Low density lipoprotein cholesterol (LDL-C) is the main lipoprotein in fasting plasma, accounting for about 2 / 3 of plasma lipoproteins, and is the main carrier for transporting cholesterol to extrahepatic tissues. Its concentration is significantly positively correlated with the incidence of metabolic diseases.

[0115] PPAR-α is mostly expressed in tissues with active fatty acid metabolism, such as the liver, heart, skeletal muscle, and kidney. It is also the most expressed PPAR subtype in human endothelial cells. It participates in blood glucose and blood lipid metabolism, maintains energy metabolism balance, and regulates inflammatory responses.

[0116] CPT-1 is the first member of the CPT gene family and plays a key role in lipid metabolism. CPT-1 is mainly located in the liver and muscle cells, and the liver controls how fat in food is converted into energy.

[0117] C / EBP-α is a DNA-binding protein with a leucine zipper structure. As a eukaryotic transcriptional regulatory factor, it participates in the transcriptional regulation of cells rich in this protein, such as hepatocytes and adipocytes.

[0118] The SCD-1 gene encodes the enzyme stearoyl-CoA desaturase, which converts saturated fatty acids into monounsaturated fatty acids.

[0119] HSL is mainly present in adipose tissue, adrenal glands, macrophages, placenta, ovarian cells, and in lower amounts in myocardium and skeletal muscle. The expression of the HSL gene has a significant inhibitory effect on the deposition of TG in adipocytes.

[0120] LPL is present in large quantities in the capillary endothelium of the myocardium, kidney, brain, skeletal muscle, adipose tissue, etc., with the highest content in the adrenal glands and adipose tissue. LPL can hydrolyze TG carried and transported by VLDL. The absence and low activity of LPL can cause serious fat metabolism disorders in the body.

[0121] The SREBPs family regulates fat production. SREBP-1 mainly regulates the expression of FA biosynthesis genes and LDLR, while SREBP-2 preferentially controls the expression of cholesterol biosynthesis genes.

[0122] The expression regulation of HMGCR gene can affect the accumulation of cholesterol in the body. DETAILED DESCRIPTION

[0123] 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.

[0124] Example 1

[0125] The invention provides an Akkermansia muciniphila named AKK ONE, with a deposit number of CGMCC No.20954.

[0126] 1 Experimental methods

[0127] 1.1 Preparation of experimental strain resuspension

[0128] The frozen strain was inoculated into BHI liquid culture medium, cultured at 37°C for 15 hours, centrifuged at 4°C and 5000r / min for 5 minutes, and the supernatant was discarded to obtain bacterial sludge. The bacterial sludge was inoculated into BHI liquid culture medium at a 3% inoculum and activated at 37°C for 15 hours. A small amount of bacterial liquid was taken with an inoculation loop to make three-zone lines on BHI solid culture medium, cultured at 37°C for 48 hours, and a single colony was picked and cultured in BHI liquid culture medium for 15 hours. After three consecutive activations, it was used as a bacterial suspension. The bacterial suspension was diluted and coated on a plate for counting, and the concentration of the bacterial suspension was adjusted to 10 with sterilized PBS liquid culture medium. 9 CFU / mL.

[0129] BHI medium: BHI (Brain Heart Infusion, OXOID, catalog number: CM1032B)) + 5% sheep blood.

[0130] Culture conditions: Completely anaerobic culture on 8 layers of gauze.

[0131] 1.2 Experimental Animal Grouping and Dosing

[0132] Forty male C57BL / 6J 6-week-old mice (average weight 21.1±0.3 g) were purchased from the Animal Experiment Center of Chongqing Medical University. After one week of adaptive feeding, the mice were randomly divided into a normal group, a model group, a metformin group, and an AKK ONE group (N=8). The mice in the normal group were fed with a standard diet (Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., catalog number: 1010001), and the mice in the model group and the experimental group were fed with a 60% high-fat diet (Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., catalog number: XTHF60). During this period, all mice had free access to water, and the experiment lasted for 4 weeks. Starting from the first day of the fifth week, 50 mg / kg·bw of STZ (Sigma-Aldrich, catalog number: V900890-1G) was dissolved in sodium citrate buffer, and the mice in the model group and the experimental group were intraperitoneally injected for 5 consecutive days. At the same time, the mice in the normal group were used as controls and only injected with sodium citrate buffer. One week later, the fasting blood glucose (FBG) of the mice was measured (fasting for 8 hours). If it exceeded 11.1 mmol / L, the mice were considered to have type 2 diabetes (T2DM) mice. Subsequently, one group of mice in the experimental group was gavaged with fresh AKK ONE bacterial solution (1×10 10 CFU / kg·bw), one group was gavaged with metformin (100mg / kg·bw; Shandong Sibonde Pharmaceutical Co., Ltd., catalog number: H20060230) daily for intervention, and the rest of the mice were gavaged with 10mL / kg·bw normal saline once a day for 4 weeks. During the experiment, the body weight of the mice was measured every week. During the treatment period, the food and water intake of the mice was recorded every 2 days. In the 10th week, the mice were fasted overnight, and the FBG and oral glucose tolerance test (OGTT) of the mice were measured. At the end of the 10th week, the experiment was terminated, the mice were fasted for 12h, and the mice were killed. Blood samples were collected, and the serum was separated by centrifugation at 4℃ and 3000r / min for 10min, and then transferred to -80℃ for use. Tissue samples (including liver, spleen, and kidney) were quickly processed and weighed, and some tissues were fixed in 4% paraformaldehyde for 48h, and the remaining tissues were frozen in liquid nitrogen and then transferred to -80℃ for storage.

[0133] 1.3 Oral glucose tolerance test (OGTT)

[0134] At week 0, mice were subjected to OGTT, fasted for 12 h, and allowed to drink water freely. The mice were weighed before the experiment, and their blood glucose was measured immediately (recorded as the blood glucose value at 0 min). Subsequently, 2 g / kg·bw glucose solution was gavaged, and the blood glucose level was measured once at 0 min, 30 min, 60 min, and 120 min, and the blood glucose curve was drawn.

[0135] 1.4 Determination of serum indicators

[0136] The collected blood samples were placed in a refrigerator for 30 minutes to prevent blood coagulation, and then centrifuged in a 4°C low-temperature centrifuge. The centrifugation conditions were: 3000r / min, 10min. The upper serum was taken and the Nanjing Jiancheng blood lipid determination kit was used to determine the contents of AST (Cat. No.: C010-2-1), ALT (Cat. No.: C009-2-1), INS (Cat. No.: E-EL-M1382c), TC (Cat. No.: A111-1-1), TG (Cat. No.: A110-1-1), LDL-C (Cat. No.: A113-1-1) and HDL-C (Cat. No.: A113-1-1) in mouse serum.

[0137] 1.5 HE pathological analysis of liver and pancreatic tissues

[0138] The size of mouse liver and pancreas tissue was about 1cm. 2 The tissue was fixed in a tissue fixative for 24 hours, then cleared, waxed, sliced, and finally stained with HE to observe the morphology and structure using a biological microscope.

[0139] 1.6 Real-time fluorescence quantitative PCR

[0140] The liver tissues of the experimental group mice were taken, genes were extracted, and the expression levels of PPAR-α, CPT-1, C / EBP-α, SCD-1, HSL, LPL, SREBP-1, SREBP-2, and HMGCR were determined. Total RNA extraction: The liver tissues of the mice in each group were used to extract total cellular RNA according to the instructions of the TRLzol kit (Invitrogen-cons, catalog number: 15596026). The RNA quality was detected by agarose gel electrophoresis, and its concentration was determined by a microplate reader. Reverse transcription: The first chain of cDNA was synthesized according to the instructions of the reverse transcription kit. RT-PCR: The above-reversed cDNA was amplified by RT-PCR using the SYBR-Green quantitative PCR method. The amplification conditions were 95°C for 60s; 95°C for 15s, 40 cycles; 55°C for 30s; 72°C for 35s; 95°C for 30s; 55°C for 35s. The β-actin gene was used as the internal reference gene in the experiment, and 2 -ΔΔCt The relative quantitative method was used to calculate the gene expression in each group. The primer sequences are shown in Table 1.

[0141] Table 1 Primer sequences

[0142]

[0143] 1.7 Data Analysis

[0144] The experimental data were expressed as mean ± standard deviation (SD), and Graph Pad Prism 8.0 software was used for statistical analysis and plotting.

[0145] 2 Experimental results

[0146] Effect of AKK ONE on oral glucose tolerance in T2DM mice

[0147] The blood glucose change curve in the oral glucose tolerance test (OGTT) experiment of mice is as follows Figure 1 As shown. 30 minutes after oral administration of glucose, the blood glucose levels of mice in each group reached the highest value. Subsequently, during the period of 30-120 minutes, the blood glucose levels of mice in each group showed a downward trend to varying degrees. Among them, compared with the normal group, the blood glucose levels of mice in the model group increased rapidly 30 minutes after oral administration of glucose, and maintained a high blood glucose level within 30-120 minutes. 120 minutes after oral administration of glucose, the blood glucose levels of mice in the model group failed to return to the blood glucose level before oral administration of glucose, indicating that diabetic mice had impaired glucose tolerance. The blood glucose levels of mice in the metformin and AKK ONE groups were significantly lower than those in the model group within 30 minutes after oral administration of glucose, especially the blood glucose level of the AKK ONE group at 120 minutes was slightly lower than that of the metformin group. The results showed that AKK ONE has the ability to improve blood glucose and glucose tolerance in T2DM.

[0148] 2.2 Effect of AKK ONE on insulin in T2DM mice

[0149] like Figure 2 The results showed that the serum insulin levels of the model group mice were significantly higher than those of the normal group mice, indicating that the abnormal islet cell function of diabetic mice led to excessive secretion of insulin. After 4 weeks of intervention with the probiotic AKK ONE and metformin, the serum insulin content decreased.

[0150] Effect of AKK ONE on serum AST and ALT in T2DM mice

[0151] In case of liver damage, ALT and AST can be used as key markers. Figure 3 The results showed that compared with the normal group of mice, the ALT and AST levels in the serum of the model group mice were significantly higher, indicating that long-term T2DM would cause damage to the liver tissue of mice. After the intervention of AKK ONE and metformin, the levels of both were significantly reduced, but there was no significant difference between the groups, indicating that the strain can alleviate the liver damage of mice caused by HFD / STZ.

[0152] Effect of AKK ONE on blood lipids in T2DM mice

[0153] The effects of AKK ONE on blood lipid indexes of mice are shown in Table 2. The levels of TG, TC and LDL-C in the model group mice fed with a high-fat diet were significantly higher than those in the normal group mice, while the levels of HDL-C were significantly lower than those in the normal group mice, indicating that diabetes can cause lipid disorders in the serum. Oral administration of AKK ONE and metformin significantly inhibited the increase in blood lipids induced by a high-fat diet. After oral administration of AKK ONE and metformin, the levels of TG, LDL-C and TC in the serum of diabetic mice were significantly reduced, indicating that AKK ONE and metformin can improve the phenomenon of increased lipids in diabetic mice.

[0154] Table 2 Levels of TG, TC, HDL-C and LDL-C in mouse serum

[0155]

[0156]

[0157] 2.5 Pathological analysis of AKK ONE in mouse liver and pancreas

[0158] like Figure 4 As shown in the figure, the liver tissue of the normal group mice showed no fatty degeneration, no fat infiltration, abundant hepatocyte cytoplasm, clear cell membrane, neat arrangement, large and normal nucleus in the center, and clear tissue structure. The hepatocytes of the model group mice induced by high fat were swollen, with obvious fatty degeneration, and most of the hepatocytes contained lipid droplets. After intervention with AKK ONE and metformin, the fat droplets in the hepatocytes were improved to varying degrees, among which the number and density of fat droplets in the AKK ONE group were less, and the fatty degeneration in the liver was significantly improved. This shows that AKK ONE improves the accumulation of lipids in the liver and prevents the absorption of fat in the body.

[0159] like Figure 5 As shown. The pancreatic islets of mice in the normal group were complete in structure and full in morphology, and the number of pancreatic β cells was abundant and evenly distributed. The fat droplets in the pancreas of mice in the model group were saturated, the size of the pancreatic islets was significantly reduced, and the number of pancreatic β cells was also significantly reduced compared with the normal group. Compared with the model group, metformin and AKK ONE intervention for 4 weeks can significantly alleviate the atrophy of pancreatic islets in diabetic mice and reduce the phenomenon of massive apoptosis of pancreatic β cells. The above results show that metformin and AKK ONE can effectively improve pancreatic tissue damage caused by HFD and STZ and protect the pancreatic islet morphology of diabetic mice.

[0160] Effect of AKK ONE on the expression of lipid metabolism genes in the liver of T2DM mice

[0161] like Figure 6, the mRNA expression intensity of C / EBPα, SCD-1, LPL, SREBP1 and HMGCR in the liver tissue of the normal group mice was the weakest, and the expression of PPAR-α, CPT-1, HSL and SREBP2 was the strongest; while the expression of PPAR-α, CPT-1, HSL and SREBP2 in the diabetic model group mice was the weakest, and the expression of C / EBPα, SCD-1, LPL, SREBP1 and HMGCR was the strongest. AKK ONE and metformin can downregulate the expression of C / EBPα, SCD-1, LPL, SREBP1 and HMGCR in the liver tissue of diabetic mice and upregulate the expression of PPAR-α, CPT-1, HSL and SREBP2. In summary, the probiotic AKK ONE can regulate the expression of lipid metabolism-related genes in the liver tissue of diabetic mice.

[0162] 3 Conclusion

[0163] In this study, a T2DM mouse model was established by inducing HFD combined with STZ, and the hypoglycemic effect of the probiotic AKK ONE was evaluated by measuring the mouse OGTT, insulin and other indicators, and the effect of the probiotic AKK ONE on the organs of T2DM mice was evaluated by measuring the liver blood lipid levels, AST and ALT levels, and the expression of liver lipid metabolism genes. AKK ONE significantly improved the blood glucose level of T2DM mice. Compared with the D group, after 4 weeks of AKK ONE intervention, the oral glucose tolerance level of T2DM mice was alleviated, the insulin level was improved, and the blood lipid level was also improved, indicating that AKK ONE has the effect of significantly lowering blood sugar and regulating lipid metabolism.

[0164] Comparative Example

[0165] A comparative example was set up with reference to the experimental method of Example 1. The strain AKK ONE in Example 1 was replaced with AKKPROBIO (deposit number CGMCC No. 20955). The relative expression of the lipid metabolism gene PPAR-α in the liver of mice after intervention with AKK PROBIO was detected, and the detection results are shown in Table 3.

[0166] Table 3

[0167] Differences from Example 1 Relative expression of PPAR-α mRNA Comparative Example Replace AKK ONE dosing with an equal amount of AKK PROBIO 1.52±0.21

[0168] The above results indicate that AKK ONE provided by the present invention is significantly superior to AKK PROBIO in upregulating PPAR-α.

Claims

1. Akkermansia muciniphila Akkermansia muciniphila ) in the preparation of a drug or pharmaceutical raw material for preventing and / or treating type 2 diabetes, characterized in that: The Akkermansia muciniphila The deposit number is CGMCC No. 20954.

2. The use according to claim 1, characterized in that: The medicine or pharmaceutical raw material comprises Akkermansia muciniphila Fermentation broth, live bacteria and / or lyophilized powder.

3. The use according to claim 2, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

4. A method for preparing a drug for preventing and / or treating type 2 diabetes, characterized in that: The method comprises culturing Akkermansia muciniphila ; Akkermansia muciniphila The deposit number is CGMCC No.20954.

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