Use of AKKERMANSIA MUCINIPHILA in the preparation of a product for the prophylaxis, treatment and/or adjuvant treatment of cardiovascular diseasesTechnical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of AKKERMANSIA MUCINIPHILA in preparation of a product for preventing, treating and/or assisting in treating cardiovascular diseases.
Background
Cardiovascular disease (CVD) including coronary heart disease, hypertension, acute myocardial infarction, heart failure, and the like is a leading cause of death from non-infectious diseases worldwide. The incidence rate and the death rate of CVD are both the first in China, and with the increase of dangerous factors such as smoking, unhealthy diet and the like, the number of patients suffering from CVD is continuously increased and the incidence age tends to be younger. Acute Myocardial Infarction (AMI) is myocardial necrosis caused by acute and persistent ischemia and hypoxia of the coronary arteries. AMI is a cardiovascular disease that seriously jeopardizes human health, and despite the great advance in diagnosis and treatment technology, AMI and the heart failure that develops are still diseases with extremely high worldwide morbidity and mortality. For patients presenting with AMI, timely myocardial reperfusion using percutaneous coronary intervention (PPCI) is the most effective treatment. The myocardial reperfusion process itself can induce cardiomyocyte death, a phenomenon known as "myocardial reperfusion injury (IRI)", whose irreversible consequences include microvascular blockage and myocardial further infarction. Therefore, finding an effective therapeutic approach to prevent myocardial ischemia in AMI patients is an important research hotspot for cardiac protection.
In recent years, studies have shown that dysregulation of intestinal flora and changes in the levels of its metabolites are important factors in promoting the development of a number of systemic diseases, including CVD. Finding a strategy for CVD treatment from the intestinal flora point of view is a current research hotspot. Probiotics are living microorganisms which can bring various benefits to the organism, and have multiple functions of resisting inflammation, resisting oxidative stress, regulating intestinal flora, protecting intestinal barriers and the like. With the proposal of concepts such as the heart ⁃ intestinal axis, the brain ⁃ intestinal axis and the lung ⁃ intestinal axis, probiotics are widely applied to researches on cardiovascular systems, nervous systems, respiratory systems and the like.
Patent CN113330109a relates to the akkermansia muciniphila strain SNUG-61027 (accession No. KCTC13530 BP) and its use. In particular, there is provided a composition for appetite control or prevention, improvement, alleviation or treatment of metabolic disorders, which comprises as an active ingredient B2UM07 protein, or a culture broth or the like thereof, or isolated therefrom, for appetite control or prevention, improvement, alleviation or treatment of metabolic disorders, which are impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease or obesity, and a method of appetite control or prevention, improvement, alleviation or treatment of metabolic disorders using the composition.
No specific medicine is available for treating myocardial infarction, and comprehensive measures are mainly adopted for treatment at present. Therefore, there is a need to develop other effective therapeutic agents, which are not satisfied in the clinical needs of cardiovascular disease drugs, because of the lack of effective therapeutic means for cardiovascular disease.
The present invention is directed to autonomously developed microbial resources: AKKERMANSIA MUCINIPHILA (other patents have been submitted on the same day) with the preservation number of CGMCC No.20955, and the downstream application technology is developed.
After AKKERMANSIA MUCINIPHILA was obtained, the inventors performed various field application experiments including cardiovascular diseases, pain relieving, antitumor, cognitive diseases, metabolic diseases, inflammatory diseases, bone joint diseases, and the like, and obtained forward experimental results in some fields.
In view of the relevant regulations of patent law singleness, each indication is individually claimed where singleness is not present.
The invention is directed to the protection of application techniques for the treatment, co-treatment or prevention of cardiovascular diseases.
Since the related evidences of microorganism identification, performance test and the like are published in the microorganism patents submitted on the same day, the early work of the inventor is quickly known, and the related information of AKKERMANSIA MUCINIPHILA is briefly disclosed in the present invention.
Akkermansia muciniphila：
The conclusive information in this section is for facilitating the examination of the present invention and is not intended to limit the scope of the invention. The specific experimental process and experimental results are recorded in the microbial patents submitted on the same day as the invention.
1. AKKERMANSIA MUCINIPHILA is obtained by the separation and screening of the inventor.
2. AKKERMANSIA MUCINIPHILA preservation information: the preservation number is: CGMCC No. 20955.
3. AKKERMANSIA MUCINIPHILA 16srRNA: SEQ ID NO.1.
4. AKKERMANSIA MUCINIPHILA physiological and biochemical properties:
(1) Hydrophobicity: the rise with time is 30% or more at 60 min.
(2) Self-aggregation: the material rises with time, and is stable at 20 h and kept at about 52%.
(3) Gastrointestinal fluid tolerance:
the survival rate in gastric juice is in a decreasing trend along with the time extension, and the survival rate is about 85% at 240 min;
The survival rate in intestinal juice is overall reduced, and the survival rate is more than 80% at 240 min.
(4) Biofilm formation ability: weak nature.
(5) Acute toxicity experiment:
The result of a bacterial back mutation test shows that the strain has no mutagenicity;
Mice do not die through AKKERMANSIA MUCINIPHILA with high, medium and low doses of acute gastric lavage;
no significant difference was observed between the daily weight change of the mice in the test group and the control saline group;
During acute gastric lavage, there was no significant difference between daily changes in food intake of the mice tested and the mice in the control saline group;
during acute gastric lavage, there was no significant difference between daily blood glucose changes in the mice tested and the control saline group mice;
The blood indexes of the mice in the acute gastric lavage group and the control physiological saline group have no obvious difference;
In the liver, the triglyceride level in the liver of the tested mice is extremely lower than that of the mice in the control physiological saline group;
The triglyceride level in serum was not significantly different in the test mice from the control mice;
the cholesterol levels of the mice in the test group and the mice in the control group have no significant difference in serum and liver;
The bile acid levels of the mice in the tested group and the mice in the control group are not significantly different in serum and liver;
the glucose levels of the mice in the tested group and the mice in the control group are not significantly different in serum and liver;
the total protein levels of the mice in the tested group and the mice in the control group are not significantly different in serum and liver;
the glutamic pyruvic transaminase enzyme activities of the tested mice and the mice of the control group are not significantly different in serum and liver;
The glutamic-oxaloacetic transaminase enzyme activities of the mice in the tested group and the mice in the control group have no significant difference in serum and liver;
the creatinine content of the mice in the test group and the creatinine content of the mice in the control group are not significantly different in serum;
the urea nitrogen concentration of the mice in the test group and the mice in the control group are not significantly different in serum;
The weight of main organs such as heart, liver, spleen, kidney, thymus, brain, testis, lung, stomach, intestine and the like of the tested mice and the mice of the control group are basically not obviously different;
the liver and kidney of the mice in the tested group and the mice in the control group have no obvious pathological damage.
(6) Sub-chronic toxicity test:
Mice were perfused with high, medium, and low doses AKKERMANSIA MUCINIPHILA for 90 consecutive days without mortality;
no significant difference was observed between weekly weight changes in mice in the test group and the control saline group;
during the continuous gastric lavage period, no significant difference exists between the feed intake change of the tested mice and the mice in the control physiological saline group every week;
During continuous lavage, there was a significant difference between the blood glucose change of the weekly mid-dose lavage mice and the control saline group mice, and no significant difference between the other groups and the control group;
the blood indexes of the mice in the gastric lavage group and the control physiological saline group have no obvious difference;
the triglyceride levels of the mice in the test group and the mice in the control group have no significant difference in liver and serum;
The cholesterol levels of the mice in the test group and the mice in the control group have no significant difference in liver and serum;
the bile acid levels of the mice in the intragastric dosage and the mice in the control group have significant differences in the liver, while the other groups have no significant differences in serum and liver from the control group;
The glucose levels of the mice in the test group and the mice in the control group are not significantly different in liver and serum;
The total protein content of the mice in the tested group and the mice in the control group has no significant difference in liver and serum;
the glutamic pyruvic transaminase enzyme activities of the tested mice and the mice of the control group are not significantly different in serum and liver;
The glutamic-oxaloacetic transaminase enzyme activities of the mice in the tested group and the mice in the control group have no significant difference in serum and liver;
the creatinine content of the mice in the test group and the creatinine content of the mice in the control group are not significantly different in serum;
the urea nitrogen concentration of the mice in the test group and the mice in the control group are not significantly different in serum;
The weight of main organs such as heart, liver, spleen, kidney, thymus, brain, testis, lung, pancreas, stomach and intestine of the tested mice and the mice of the control group are basically not significantly different;
The liver and the kidney of the mice in the tested group and the mice in the control group have no obvious pathological damage;
The blood sugar regulating ability of the tested mice and the mice of the control group is not significantly different.
(7) Drug sensitivity analysis of AKKERMANSIA MUCINIPHILA:
The AKKERMANSIA MUCINIPHILA is sensitive to ampicillin, ceftriaxone, cefotaxime, meropenem, tetracycline, moxifloxacin and chloramphenicol.
Disclosure of Invention
In order to solve the above problems, the present invention provides AKKERMANSIA MUCINIPHILA, which is deposited in the China general microbiological culture Collection center, with the accession number: CGMCC No. 20955. The strain can effectively reduce myocardial infarction, remarkably inhibit expression of TNF-alpha, IL-1 beta and IL-18, and has good anti-inflammatory effect.
The present invention is directed to AKKERMANSIA MUCINIPHILA downstream application technology.
In particular to the application of AKKERMANSIA MUCINIPHILA in cardiovascular diseases.
Specifically, from the application field, the aforementioned applications include:
1. AKKERMANSIA MUCINIPHILA is applied to prevention, treatment, auxiliary prevention and auxiliary treatment of cardiovascular diseases in a treatment method and pharmaceutical application;
2. AKKERMANSIA MUCINIPHILA is applied to the prevention, treatment, auxiliary prevention and auxiliary treatment of cardiovascular diseases by the preparation and application of raw materials.
The application of AKKERMANSIA MUCINIPHILA in cardiovascular diseases is verified by taking a model mouse as an example, and the application range of the invention is not limited to a treatment method or pharmaceutical application based on the commonality of animal experiments in the field of medicines.
Specifically, from the category of cardiovascular diseases, the aforementioned applications include, but are not limited to: coronary heart disease, heart failure, hypertension, arrhythmia and/or cardiomyopathy.
The principle is based on the principle that various cardiovascular diseases occur because the diseases cause the generation or release of certain substances in the body, thereby affecting the balance of the cardiovascular system. Based on AKKERMANSIA MUCINIPHILA's experiments in a specific cardiovascular disease (coronary heart disease), it can be reasonably concluded or inferred that it can be equally applied or at least has application potential in other cardiovascular diseases.
More specifically, the coronary heart disease is angina pectoris and/or myocardial infarction.
Further specifically, the myocardial infarction is acute myocardial infarction, non-ST elevation myocardial infarction, right ventricular myocardial infarction, atrial myocardial infarction and/or pericarditis after myocardial infarction.
Preferably, the myocardial infarction is acute myocardial infarction.
More specifically, the heart failure may be left heart failure, right heart failure or total heart failure.
More specifically, the hypertension may be primary hypertension or secondary hypertension.
More specifically, the cardiac arrhythmia may be a sinus arrhythmia, an atrial arrhythmia, a ventricular arrhythmia, and/or a cardiac conduction block.
More specifically, the cardiomyopathy may be dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy and/or myocarditis.
In particular, the aforementioned uses include the use of AKKERMANSIA MUCINIPHILA fermentation broths, fermentation broths supernatants, fermentation broths precipitations, living bacteria, dead bacteria and/or any ingredients, components, compositions, metabolites derived directly or indirectly from AKKERMANSIA MUCINIPHILA, in terms of raw material forms; and with or without current, future auxiliary materials as permitted in a particular application area.
More specifically, the fermentation broth refers to a liquid obtained by inoculating a strain into a culture medium and culturing for a period of time.
More specifically, the fermentation broth supernatant refers to supernatant liquid of the fermentation broth after centrifugation; contains rich metabolites and a part of thallus fragments in the growth and propagation process of bacteria, and acid substances secreted by the bacteria and bacteriocins have antagonism and killing effects on harmful bacteria; amino acids after decomposing food by bacteria, and synthetic vitamins are in the culture solution, and also include enzymes secreted by bacteria useful for human body; and part of thallus components have immunity promoting effect on human body.
More specifically, the fermentation liquid sediment refers to liquid sediment obtained by centrifugation, and the liquid sediment comprises free protein, residual thalli, broken cells and residues of culture matrixes, namely the protein and the matrixes in the cells.
More specifically, the living bacteria are also called active bacteria group, can colonize and reproduce in intestinal tracts, and are beneficial to increasing the number of beneficial bacteria.
More specifically, the dead bacteria have lost viable microorganisms, which are unable to grow and reproduce, and the probiotics are rendered inactive by the production process, such as high temperature treatment or excessive drying.
In particular, from the pharmaceutical field, the medicament comprises:
(1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, viable and/or dead bacteria of AKKERMANSIA MUCINIPHILA;
(2) Pharmaceutically acceptable auxiliary materials.
More specifically, the pharmaceutically acceptable auxiliary materials are selected from one or more than two of buffering agents, excipients, diluents, preservatives, wetting agents, emulsifying agents, lubricants, fragrances, thickening agents, stabilizing agents, solubilizers, sweetener bacteriostats, suspending agents and antioxidants.
Preferably, the pharmaceutically acceptable auxiliary material is selected from at least one of alginate, water, syrup, methylcellulose, gelatin, calcium silicate, starch, acacia, mineral oil, cellulose, calcium phosphate, propyl hydroxybenzoate, fine crystalline cellulose, methyl hydroxybenzoate, magnesium lactose stearate, mannose, polyvinylpyrrolidone and talc.
More specifically, the dosage forms of the medicine are tablets, liquid, capsules, powder, suppositories and granules.
More specifically, the medicament comprises other medicaments for treating cardiovascular diseases.
More specifically, the auxiliary materials are selected from one or more than two of filling agents, capsule shell materials, solvents, stabilizers, flavoring agents, sweeteners and pigments.
Preferably, the filler is at least one selected from starch, corn flour and grape;
the capsule shell material is at least one of gelatin, hydroxypropyl methylcellulose and polyvinyl alcohol;
the solvent is at least one selected from water, alcohol, glycerol and ethanol;
The stabilizer is at least one selected from antioxidant and preservative;
the flavoring agent is at least one selected from natural perfume and artificial essence;
the sweetener is at least one selected from natural sweetener and artificial sweetener;
The pigment is at least one selected from natural pigment and artificial pigment.
Specifically, the viable count of AKKERMANSIA MUCINIPHILA is not lower than 1X 108 CFU/g or 1X 108 CFU/mL in terms of the active dose in the above fields.
Preferably, the number of viable bacteria of AKKERMANSIA MUCINIPHILA is 1X 108-1×1012 CFU/g or 1X 108-1×1012 CFU/mL.
Further preferably, the number of viable bacteria of AKKERMANSIA MUCINIPHILA is 1X 109-1×1012 CFU/g or 1X 109-1×1012 CFU/mL, such as 1×109CFU/g（CFU/mL）、2×109CFU/g（CFU/mL）、3×109CFU/g（CFU/mL）、4×109CFU/g（CFU/mL）、5×109CFU/g（CFU/mL）、6×109CFU/g（CFU/mL）、7×109CFU/g（CFU/mL）、8×109CFU/g（CFU/mL）、9×109CFU/g（CFU/mL）、10×109CFU/g, and other values within this range of values are selectable.
The invention also provides a method of treating cardiovascular disease by administering AKKERMANSIA MUCINIPHILA to a patient suffering from cardiovascular disease.
Specifically, the cardiovascular disease patient is a patient with coronary heart disease, heart failure, hypertension, arrhythmia and/or cardiomyopathy through clinical diagnosis.
In particular, the method of administration is selected from the group consisting of oral administration, intravenous administration, topical administration, intradermal administration and/or subcutaneous administration.
Specifically, a drug comprising the following (1) and (2) is administered to a patient suffering from cardiovascular disease:
(1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, viable and/or dead bacteria of AKKERMANSIA MUCINIPHILA;
(2) Pharmaceutically acceptable auxiliary materials.
More specifically, the pharmaceutically acceptable auxiliary materials are selected from one or more than two of wetting agents, emulsifying agents, preservatives, antioxidants, buffering agents, excipients, diluents, lubricants, bacteriostats, suspending agents, solubilizers, thickening agents, stabilizers, sweeteners and fragrances.
Preferably, the pharmaceutically acceptable auxiliary material is at least one selected from lactose, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.
More specifically, the medicament comprises other medicaments for treating cardiovascular diseases.
The invention also provides a treatment method of AKKERMANSIA MUCINIPHILA for cardiovascular diseases based on the derivative, derivative generated by the application.
In particular, based on AKKERMANSIA MUCINIPHILA application for treating or preventing cardiovascular diseases, the strain can be modified to generate, derive and derive AKKERMANSIA MUCINIPHILA with better effect for treating or preventing cardiovascular diseases, wherein the modification method is a traditional method and a genetic engineering method.
Further specifically, the conventional methods may be physical methods, chemical methods, and/or selection methods.
More specifically, the physical method is to induce mutation by using radiation or high pressure means, so as to realize strain improvement.
Preferably, the radiation method comprises ultraviolet radiation and/or X-ray radiation;
The high pressure method may be to expose the microorganism to a high pressure environment to produce an adaptive change.
More specifically, the chemical method is to induce mutation of strain by using chemical agent.
Preferably, the chemical agent may be nitrite, acetylcholine and/or nitrogen mustard.
More specifically, the selection method is to select microorganisms with excellent properties for propagation, so as to realize strain improvement.
Preferably, the selection method is used for improving the metabolic pathway and/or growth condition of the microorganism, so as to obtain AKKERMANSIA MUCINIPHILA with better effect of treating or preventing cardiovascular diseases.
Further specifically, the genetic engineering method may be gene cloning, gene knockout, gene editing and/or synthetic biology, thereby obtaining a strain having an excellent effect of treating or preventing cardiovascular diseases.
More specifically, the gene cloning refers to cutting the target gene from one cell and inserting the target gene into another cell, so as to realize the expression of the target gene in a new host, thereby enhancing the effect of treating or preventing cardiovascular diseases of the original strain.
More specifically, the gene knockout means that the target gene is deleted by a technical means, so that the regulation and control of the microbial property are realized, and the strain with better effect of treating or preventing cardiovascular diseases is obtained.
More specifically, the gene editing means that the target gene sequence is accurately modified by a technical means, so that the regulation and control of the microbial properties are realized, and the strain with better effect of treating or preventing cardiovascular diseases is obtained.
More specifically, the synthetic biology refers to constructing a novel gene sequence by using a chemical synthesis machine technology, introducing the novel gene sequence into microorganisms, and realizing the regulation and control of the properties of the microorganisms so as to obtain the strain with better effect of treating or preventing cardiovascular diseases.
The invention also provides a product containing AKKERMANSIA MUCINIPHILA which is generated, derived and derived based on the application.
In particular, a medicament for treating or preventing cardiovascular disease, said medicament comprising:
(1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, viable and/or dead bacteria of AKKERMANSIA MUCINIPHILA produced, derived;
(2) Pharmaceutically acceptable auxiliary materials.
More specifically, the pharmaceutically acceptable auxiliary materials are selected from one or more than two of buffering agents, excipients, diluents, preservatives, wetting agents, emulsifying agents, lubricants, fragrances, thickening agents, stabilizing agents, solubilizers, sweetener bacteriostats, suspending agents and antioxidants.
Preferably, the pharmaceutically acceptable auxiliary material is selected from at least one of alginate, water, syrup, methylcellulose, gelatin, calcium silicate, starch, acacia, mineral oil, cellulose, calcium phosphate, propyl hydroxybenzoate, fine crystalline cellulose, methyl hydroxybenzoate, magnesium lactose stearate, mannose, polyvinylpyrrolidone and talc.
More specifically, the dosage forms of the medicine are tablets, liquid, capsules, powder, suppositories and granules.
More specifically, the medicament comprises other medicaments for treating cardiovascular diseases.
More specifically, the auxiliary materials are selected from one or more than two of filling agents, capsule shell materials, solvents, stabilizers, flavoring agents, sweeteners and pigments.
Preferably, the filler is at least one selected from starch, corn flour and grape;
the capsule shell material is at least one of gelatin, hydroxypropyl methylcellulose and polyvinyl alcohol;
the solvent is at least one selected from water, alcohol, glycerol and ethanol;
The stabilizer is at least one selected from antioxidant and preservative;
the flavoring agent is at least one selected from natural perfume and artificial essence;
the sweetener is at least one selected from natural sweetener and artificial sweetener;
The pigment is at least one selected from natural pigment and artificial pigment.
Specifically, the number of viable bacteria of AKKERMANSIA MUCINIPHILA generated, derived and derived from the active dose in the above fields is not lower than 1×108 CFU/g or 1×108 CFU/mL.
Preferably, the number of viable bacteria of AKKERMANSIA MUCINIPHILA is 1X 108-1×1012 CFU/g or 1X 108-1×1012 CFU/mL.
Further preferably, the number of viable bacteria of AKKERMANSIA MUCINIPHILA is 1X 109-1×1012 CFU/g or 1X 109-1×1012 CFU/mL, such as 1×109CFU/g（CFU/mL）、2×109CFU/g（CFU/mL）、3×109CFU/g（CFU/mL）、4×109CFU/g（CFU/mL）、5×109CFU/g（CFU/mL）、6×109CFU/g（CFU/mL）、7×109CFU/g（CFU/mL）、8×109CFU/g（CFU/mL）、9×109CFU/g（CFU/mL）、10×109CFU/g, and other values within this range of values are selectable.
The invention has the technical effects that:
(1) The inner diameters of the end diastole and the end systole of the left ventricle of the mice in the acute myocardial infarction group are effectively reduced, the shortening Fraction (FS) and the Ejection Fraction (EF) are increased, and the function of the infarcted heart is effectively improved.
(2) Effectively reduces the serum cTnI level, CK-MB and LDH activity, and can obviously reduce myocardial damage.
(3) Remarkably inhibit the expression of TNF-alpha, IL-1 beta and IL-18, and has good anti-inflammatory effect.
Preservation description:
strain name: AKK PROBIO;
preservation number: CGMCC No. 20955;
classification naming: AKKERMANSIA MUCINIPHILA;
preservation date: 10 months and 26 days 2020;
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);
deposit unit address: the korean district North Star, beijing city, part No. 1, no. 3.
Drawings
FIG. 1 shows the levels of cardiac troponin I (cTnI) in the serum of 4 groups of experimental mice.
FIG. 2 shows the interleukin-1β (IL-1β) expression level in myocardial tissue of 4 groups of experimental mice.
FIG. 3 shows the interleukin-18 (IL-18) expression level in myocardial tissue of 4 groups of experimental mice.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Nouns and terminology:
Cardiac troponin i (Cardiac Troponin i, cTn i): is a regulating protein for myocardial muscle contraction, and is mainly used for examining myocardial injury or necrosis related diseases, such as myocardial infarction, viral myocarditis, acute heart failure, renal failure and myocardial anoxia.
Creatine kinase isozymes MB (CREATINE KINASE MB, CK-MB): the CK-MB index is mainly in cardiac muscle, and the increase of the CK-MB index can indicate myocardial damage, such as myocardial infarction, pericarditis, myocardial damage and other diseases.
Lactate dehydrogenase (Lactate Dehydrogenase, LDH): belongs to a glycolytic enzyme, is generally contained in myocardial zymogram examination and liver function examination, is a clinically common examination index, is generally used for judging the degree of myocardial injury and hepatic cell injury, and can assist in auxiliary diagnosis and identification of myocardial infarction, acute hepatitis and active liver diseases.
Tumor necrosis factor-alpha (Tumor necrosis factor-alpha TNF-alpha): is a pro-inflammatory cytokine produced mainly by macrophages and monocytes and is involved in normal inflammatory and immune responses. Alpha tumor necrosis factor production increases in a number of pathological conditions, including sepsis, malignancy, heart failure, and chronic inflammatory disease.
Interleukin-1 beta (Interleukin-1 beta, IL-1 beta): is produced from activated macrophages in the form of a pre-protein and is proteolytically processed into an active form by caspase-1 (CASP 1/ICE). IL-1β is an important mediator of the inflammatory response, involved in a variety of cellular activities including cell proliferation, differentiation and apoptosis.
Interleukin-18 (Interlukin-18, IL-18): is a protein which is produced in humans from the gene encoding IL 18. The protein encoded by the gene is a pro-inflammatory cytokine. Many cell types, both hematopoietic and non-hematopoietic, have the potential to produce IL-18.
AKKERMANSIA MUCINIPHILA in the present invention is AKK PROBIO.
Example 1 AKK PROBIO cultivation
(1) Culture medium
Solid plate culture: BHI (brain-heart extract (Brain Heart Infusion), OXOID, cat# CM 1032B)) +5% sheep blood
Weighing a certain volume of required BHI powder according to the requirement of a finished product, adding agar powder in a proportion of 20g/L, fixing the volume to the corresponding volume, and sterilizing at 121 ℃ for 20 min. After sterilization, cooling to 53 ℃ (hand held without scalding), adding 5% of aseptic defibrinated sheep blood, shaking uniformly, and pouring 15-20mL into a plate for standby.
(2) Culture conditions
Culture temperature: 37 ℃;
Culture conditions: completely anaerobic;
Culturing liquid fermentation liquid: BHI;
culture temperature: 37 ℃;
culture conditions: 8 layers of gauze are completely cultured anaerobically.
(3) Strain expansion culture
First-order: a2 mL tube of glycerol was inoculated into a tube containing 9mL of BHI at an inoculum size of 10%, and anaerobic cultured at 37℃for 24-48 hours.
And (2) second-stage: the primary fermentation tube was inoculated into a triangular flask containing 90mL of BHI at an inoculum size of 5%, and anaerobic cultured at 37℃for 24 hours.
Three stages: taking a secondary triangular flask fermentation broth, inoculating the secondary triangular flask fermentation broth into a triangular flask containing 300mL of BHI in an inoculum size of 5%, performing anaerobic culture at 37 ℃ for 13h, and performing freezing preservation at-80 ℃ on a 20% glycerol tube.
Example 2 animal experiments
2.1 Animals
Female C57BL/6 mice (Shanghai Nannon model biotechnology Co., ltd.) of 8 weeks old, total 40, body weight 18-22g, no specific pathogen. The feed is adaptively fed for one week before the experiment, and the feed and drinking water are freely fed, and the feeding environment meets the SPF (specific pathogen free) level requirement.
2.2 Reagents and instruments
Positive medicine, metoprolol succinate sustained release tablet, the main component is equivalent to 50mg of metoprolol tartrate, each tablet contains 47.5mg of metoprolol succinate, and the production enterprises: astraZeneca AB, product lot number: SYPB.
ELISA detection kit for mouse cardiac troponin I (cTnI), shanghai Xuan Biotechnology Co., ltd., cat: XY-SJH-XS1395.
Mouse creatine kinase isozyme MB (CK-MB) ELISA detection kit, shenzhen Ruiqing bioinformatics Co., ltd., product number: RQ-P-M0114.
Mouse Lactate Dehydrogenase (LDH) ELISA detection kit, shanghai initial Biotechnology Co., ltd., product number: CT68539.
Mouse TNF- α ELISA detection kit, shanghai, inc. Biotechnology, cat No.: XY-SJH-XS1204.
Interleukin 1 beta (IL-1 beta) ELISA detection kit, shang Xinyu Biotech Co., ltd., product number: XYA563Mu.
Mouse IL-18 ELISA assay kit, product number of Shanghai, jean biosciences limited: XY-SJH-XS1007.
2.3 Acute myocardial infarction modeling and pharmaceutical intervention
The molding method comprises the following steps: and constructing a myocardial infarction model by ligating the anterior descending branch of the left coronary artery.
8 Week old mice were randomly divided into 4 groups: the false operation group, the acute myocardial infarction group, the positive medicine group and AKK PROBIO groups of 10 patients in each group. After the mice are fasted and forbidden for 12 hours before operation, the mice are anesthetized, shaved and disinfected. The skin was incised, the muscle was peeled off with a blunt instrument, and the pericardium was incised to expose the heart. Ligature of left anterior descending branch of coronary artery, pale ligature end was observed, indicating successful ligature. The sham group passed the suture only through the lower edge of the left atrial appendage and did not ligate.
Drug intervention was performed 2 times every other day (once a day in the morning and evening) for 15 days. The metoprolol succinate sustained release tablet is given at the same time every other day after the positive medicine is successfully formed, and the stomach filling amount is 4mg/kg; AKK PROBIO groups, the gastric lavage amount at the same time every day is 1×109 CFU/unit; the sham operation group and the acute myocardial infarction group are filled with normal saline with the same amount of stomach. After the drug intervention is finished, mouse serum and myocardial tissue are taken.
2.4 Index detection
2.4.1 Echocardiography detection of cardiac function in mice
Ultrasonic cardiography detection is carried out on each group of mice by adopting a GE Voluson E four-dimensional color Doppler ultrasound instrument in the United states, and heart function related indexes including left ventricular end diastole (LVIDd), left ventricular end Systole (LVIDs), fractional Shortening (FS) and fractional Ejection (EF) are calculated.
2.4.2 Detection of cTnI levels, CK-MB and LDH Activity
After whole blood of each group of mice was left at room temperature for 1 hour, 1000 Xg was centrifuged for 20 minutes to obtain serum samples. Serum cTnI levels, CK-MB and LDH activity were assayed by reference to ELISA kit instructions.
2.4.3 Myocardial tissue inflammation index detection
Mouse myocardial tissue was homogenized in an ice bath, centrifuged at 1000 Xg for 15min, and the supernatant was used for detection. Referring to ELISA kit instructions, the expression levels of TNF-alpha, IL-1 beta and IL-18 in the myocardial tissue of the mice are detected.
2.5 Detection results
2.5.1 Cardiac function test results
The cardiac function test results are shown in Table 1, and compared with the sham operation group, the left ventricular end diastole inner diameter and the left ventricular end systole inner diameter of the acute myocardial infarction group mice are obviously increased, and the shortening Fraction (FS) and the Ejection Fraction (EF) are obviously reduced. The above indices of both the positive and AKK PROBIO groups showed a different degree of recovery compared to the acute myocardial infarction group, with the positive group slightly better than AKK PROBIO group.
TABLE 1
2.5.2 CTnI level, CK-MB and LDH Activity assay
The results of the cTnI level, CK-MB and LDH activity assays are shown in Table 2, and the serum cTnI level, CK-MB and LDH activity of mice in the acute myocardial infarction group are significantly increased compared to those in the sham operation group. The above indicators of both positive and AKK PROBIO groups were down-regulated to different extents compared to the acute myocardial infarction group, wherein AKK PROBIO group down-regulated ctni levels to a greater extent than the positive group. It is shown that AKK PROBIO groups and positive drug groups can effectively reduce myocardial injury.
TABLE 2
2.5.3 Myocardial tissue inflammation test results
The myocardial tissue inflammation test results are shown in Table 3, and compared with the sham operation group, the expression levels of TNF-alpha, IL-1 beta and IL-18 of the mice in the acute myocardial infarction group are obviously increased. The expression levels of the above indicators were significantly reduced in the positive and AKK PROBIO groups compared to the acute myocardial infarction group, with the two groups being equivalent in TNF- α expression levels; the AKK PROBIO group down-regulation was most pronounced at IL-1β, IL-18 expression levels, even near sham-operated groups.
TABLE 3 Table 3
Comparative example
Referring to the culture method of example 1 and the experimental method of central muscle tissue inflammation index detection of example 2, experiments were performed by replacing AKK PROBIO of the present invention with ATCC standard strain of ackermanni (ATCC BAA 835), and specific results are shown in table 4.
TABLE 4 Table 4
The above results indicate that the ATCC standard strain of ackermannia was down-regulated relative to the acute myocardial infarction group, but not as effective as the positive drug group, and less than AKK PROBIO of the present invention, as indicated in table 4. Therefore, AKK PROBIO of the invention can obviously inhibit the expression of TNF-alpha, IL-1 beta, IL-18 and cTnI, has better anti-inflammatory effect and can obviously reduce myocardial injury.
Application example 1: AKK PROBIO preparation method of viable bacteria
Culture method of AKK PROBIO live bacteria
(1) Culture medium
Solid plate culture: BHI+5% sheep blood
Weighing a certain volume of required BHI powder according to the requirement of a finished product, adding agar powder in a proportion of 20g/L, fixing the volume to the corresponding volume, and sterilizing at 121 ℃ for 20 min. After sterilization, cooling to 53 ℃ (hand held without scalding), adding 5% of aseptic defibrinated sheep blood, shaking uniformly, and pouring 15-20mL into a plate for standby.
(2) Culture conditions
Culture temperature: 37 ℃;
Culture conditions: completely anaerobic;
Culturing liquid fermentation liquid: BHI;
culture temperature: 37 ℃;
culture conditions: 8 layers of gauze are completely cultured anaerobically.
(3) Strain expansion culture
First-order: a2 mL tube of glycerol was inoculated into a tube containing 9mL of BHI at an inoculum size of 10%, and anaerobic cultured at 37℃for 24-48 hours.
And (2) second-stage: the primary fermentation tube was inoculated into a triangular flask containing 90mL of BHI at an inoculum size of 5%, and anaerobic cultured at 37℃for 24 hours.
Three stages: taking a secondary triangular flask fermentation broth, inoculating the secondary triangular flask fermentation broth into a triangular flask containing 300mL of BHI in an inoculum size of 5%, performing anaerobic culture at 37 ℃ for 13h, and performing freezing preservation at-80 ℃ on a 20% glycerol tube.
(4) Fermentation tank culture
And (3) placing the activated strain in a large-scale fermentation tank, and culturing the strain under the conditions as above. After the completion of the culture, the cells were centrifuged to obtain wet cells.
Application example 2: AKK PROBIO preparation method of dead bacteria
Killing the wet thalli prepared in application example 1 by a chemical or physical method to obtain dead bacteria;
The chemical method can be gas sterilization and/or liquid sterilization;
specifically, the gas sterilization can be ozone, ethylene oxide, formaldehyde, propylene glycol, glycerol or peracetic acid steam;
The liquid is sterilized to be a chemical agent such as ethanol;
the physical method can be thermal sterilization, illumination sterilization and/or microwave sterilization;
in particular, the thermal sterilization method can be a combustion method, a dry baking method, a boiling method and/or a pressure steam sterilization method;
the light sterilization can be sunlight exposure, ultraviolet irradiation and/or ionizing radiation.
Application example 3: oral medicament containing AKK PROBIO
Mixing the wet thalli prepared in application example 1 with auxiliary materials according to a certain proportion, and preparing the mixture into viable bacteria tablets or capsules, granules and the like through processes of freeze drying or spray drying, tabletting and the like;
Or subjecting the wet cell prepared in example 1 to cell disruption, and extracting the beneficial component by a proper technique; extraction modes comprise centrifugation, distillation, leaching, extraction and the like, 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 liquid, granules, injection and the like according to clinical use requirements.
Application example 4: a yogurt comprising AKK PROBIO.
Raw materials: the wet cells prepared in example 1, milk, yoghurt, soy milk or milk powder (fresh), yoghurt starter (lactobacillus bulgaricus and streptococcus thermophilus);
Taking milk preparation as an example:
Fresh milk, a yoghurt starter and wet thalli are mixed according to a certain proportion, stirred uniformly, covered by a preservative film and fermented in a warm place (preferably 30-40 ℃) for 6-8 hours (or overnight), and the fermented milk is refrigerated in a refrigerator.