Application of Akkermansia muciniphila in preparing drugs for improving and treating multiple sclerosis

Abstract

The Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 and the Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794 of the present invention can effectively treat and improve multiple sclerosis by enhancing the activity of microglia, improving weight loss and neurological damage caused by multiple sclerosis, inhibiting the expression of TLR4, thereby reducing central inflammatory response, and the above-mentioned effects are proved by relevant experiments in vivo and in vitro. In addition, the Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 and the Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794 can not only be used alone for the treatment of multiple sclerosis, but can also be used in combination with DMTs drugs such as other microorganisms and inflammatory mediator regulators, significantly improving the comprehensive therapeutic effect, reducing the side effects of DMTs drugs, and improving the quality of life of patients. This indicates that Akkermansia muciniphila AM06 and AM02 have the prospect of treating multiple sclerosis and have important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of Akkermansia muciniphila in the preparation of drugs for improving and / or treating multiple sclerosis. Background Art

[0002] Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system, with multiple inflammatory demyelination, axonal degeneration and glial scar formation as the main pathological features. Common sites of injury are located around the ventricles, optic nerves, brainstem, cerebellum and spinal cord. Patients often have symptoms and signs such as paralysis of one or more limbs, deep and shallow sensory impairment, decreased vision, ophthalmoplegia, diplopia and ataxia. MS usually occurs in people aged 20 to 40 years and is the main cause of disability in young people. The pathogenesis of MS is relatively complex, involving many processes such as innate immunity and adaptive immunity. It is currently believed that the onset of MS may be related to multiple factors such as autoimmune response, viral infection, individual genetics and environment. Among them, the mainstream view is that it is caused by axonal damage of central nervous cells caused by inflammation caused by immune cells (Aloisi F. Immune function of microglia [J]. Glia, 2001, 36 (2): 165-179).

[0003] Multiple sclerosis (MS) is clinically divided into three categories, namely: primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS) and relapsing-remitting multiple sclerosis (RRMS). Among them, 80% to 85% of MS patients belong to RRMS, and more than 50% of RRMS patients will slowly and progressively worsen their symptoms after 10 to 15 years, that is, they will progress to SPMS. Primary progressive multiple sclerosis (PPMS) accounts for about 10% of MS patients, with a course of more than 1 year. The disease is slowly and progressively aggravated without remission and relapse.

[0004] The treatment of MS is generally divided into acute phase treatment and remission phase treatment. The treatment of the acute phase is mainly to alleviate symptoms, shorten the course of the disease and prevent complications. Especially for patients with nerve and functional damage, drug treatment should be given in time. For acute phase treatment, regardless of its classification, the current consensus mainly recommends high-dose glucocorticoid shock as the first-line treatment to control inflammatory response and reduce neuritis damage, which has been basically popularized in clinical practice.

[0005] Treatment during the remission period is mainly aimed at regulating immunity, repairing damaged nerves, and controlling disease progression. Among them, disease modifying therapies (DMTs) are the recommended treatment methods during the remission period and are also a hot topic in the current treatment of MS. According to the mechanism of action of the drugs, they can be divided into five categories, namely: inflammatory mediator regulators represented by interferon β and prednisone acetate; immune cell migration inhibitors represented by fingolimod (gilenya), siponimod, and natalizumab; cell depletion / induction therapy strategies represented by cladribine, ocrelizumab, and rituximab; immune tolerance therapy represented by glatiramer acetate and glatiramer acetate; intracellular response regulators represented by teriflunomide and dimethyl fumarate.

[0006] The central and intestinal immune systems, intestinal flora, various levels of nervous systems and related endocrine systems form a network of interactions, forming the brain-gut-bacteria axis, in which the various levels of nervous systems regulate the intestines, so that the intestinal flora and the brain form a two-way regulation through the brain-gut axis. Studies have found that intestinal flora can affect brain function and promote the occurrence and development of neurological diseases, including multiple sclerosis, depression, neurodevelopmental disorders, Alzheimer's disease, Parkinson's disease, etc.

[0007] The intestinal mucosal layer plays an important role in immune response. As the main site for invasion and colonization of foreign pathogens and antigens, it forms the first line of defense of the body's immunity by combining with the lymphoid tissue present in it, and plays the role of mucosal barrier. The digestive tract is the largest immune tissue in the body. Under pathological conditions, when the intestinal mucosal barrier is damaged, pathogenic microorganisms are more likely to pass through the intestinal mucosa and enter the body, causing disease. Intestinal symbiotic bacteria play an important role in intestinal homeostasis and have a certain protective effect on intestinal integrity. Therefore, the use of probiotics to treat neurological diseases such as multiple sclerosis has become a hot topic in recent years.

[0008] In recent years, studies have found that MS is closely related to the abundance of Akkermansia. High abundance of Akkermansia muciniphila has been found in patients with MS and experimental autoimmune encephalomyelitis (EAE) mouse models (Berer K, Gerdes LA, Cekanaviciute E, et al. 2017. Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice. Proc Natl Acad Sci USA. 114(40): 10719–10724; Kivis Akk P, Pierre IV, Hrishikesh L, et al. 2018. A probiotic modulates the microbiome and immunity in multiple sclerosis. Ann Neurol. 83(6): 1147-1161; Al-Ghezi ZZ, Busbee PB, Alghetaa H, et al. 2019. Combination of Cannabinoids, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), mitigate experimental autoimmune encephalomyelitis (EAE) by altering the gut microbiome. Brain Behav Immun. 82: 25-35). However, the latest study has obtained different results, and found that certain subtypes of Akkermansia muciniphila have the effect of inhibiting the expression of RORγT and IL-17A in CD4+ or CD8+ T cells in mice and improving the symptoms of EAE in mice (Cox LM, Maghzi AH, Liu S, et al. 2021. The gut microbiome in progressive multiple sclerosis. Ann Neurol. 89 (6): 1195-1211.). Therefore, the increase in the abundance of Akkermansia in MS patients may be a compensatory effect. Therefore, it is necessary to further study and develop the application of Akkermansia muciniphila in the prevention and / or treatment of multiple sclerosis. Summary of the invention

[0009] In view of the above problems, the purpose of the present invention is to provide two types of Akkermansia muciniphila for use in improving and / or treating multiple sclerosis. The inventors unexpectedly found that Akkermansia muciniphila, especially Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 and Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794, can effectively improve and treat multiple sclerosis by enhancing microglial activity, improving weight loss and neurological damage caused by multiple sclerosis, inhibiting the expression of TLR4, thereby reducing central inflammatory response.

[0010] In order to achieve the above object, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides the use of Akkermansia muciniphila in preparing a product for treating and / or improving multiple sclerosis;

[0012] The Akkermansia muciniphila is at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No.22793 and Akkermansia muciniphila AM02 with a deposit number of CGMCC No.22794.

[0013] In some embodiments, the Akkermansia muciniphila is one, two or more of live bacteria of Akkermansia muciniphila AM02 or Akkermansia muciniphila AM06, inactivated bacteria with complete morphological structure or inactivated bacteria with incomplete morphological structure, and live bacteria lysate.

[0014] In some embodiments, the Akkermansia muciniphila is one, two or more of the following: live bacteria of Akkermansia muciniphila AM02 or AM06, genetically recombinant, transformed or modified, attenuated, chemically treated, physically treated or inactivated Akkermansia muciniphila AM02 or AM06, lysate of Akkermansia muciniphila AM02 or AM06, and liquid culture supernatant of Akkermansia muciniphila AM02 or AM06.

[0015] In some embodiments, the multiple sclerosis includes primary progressive multiple sclerosis, secondary progressive multiple sclerosis, and relapsing-remitting multiple sclerosis.

[0016] In some embodiments, the multiple sclerosis is relapsing multiple sclerosis.

[0017] In some embodiments, the relapsing multiple sclerosis is at least one of acute monophasic, relapsing-remitting, chronic progressive, and protracted-relapsing multiple sclerosis.

[0018] In some embodiments, the product is a pharmaceutical product.

[0019] In some embodiments, the dosage form of the drug includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations. The drug is for human or animal use.

[0020] In some embodiments, the drug further includes pharmaceutically acceptable carriers and excipients.

[0021] In some embodiments, the excipients include at least one of water, saline solution, alcohol (e.g., polyethylene glycol, propylene glycol), silicone, wax, petrolatum, vegetable oil, sugar (e.g., lactose), gelatin, amylose, maltodextrin, microcrystalline cellulose, magnesium stearate, talc, silicon dioxide, surfactants (e.g., monoglycerides, diglycerides, glycerol fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone), silicic acid, viscous paraffin, or aromatic oils.

[0022] In a second aspect, the present invention also provides a composition containing Akkermansia muciniphila, wherein the Akkermansia muciniphila is at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793, Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794, and Akkermansia muciniphila standard strain BAA-835.

[0023] In some embodiments, the composition further comprises a DMTs drug.

[0024] In some embodiments, the DMTs drug is selected from one, two or more of prednisone acetate, interferon β, fingolimod, siponimod, natalizumab, cladribine, ocrelizumab, rituximab, glatiramer acetate, glatiramer acetate, teriflunomide and dimethyl fumarate.

[0025] In some embodiments, the Akkermansia muciniphila is one, two or more of a live bacterium, a killed bacterium with intact morphology and structure, a killed bacterium with incomplete morphology and structure, and a live bacterium lysate.

[0026] In some embodiments, the Akkermansia muciniphila is one, two or more of live Akkermansia muciniphila, genetically recombinant, transformed or modified, attenuated, chemically treated, physically treated or inactivated Akkermansia muciniphila, Akkermansia muciniphila lysate, and Akkermansia muciniphila liquid culture supernatant.

[0027] In some embodiments, the composition further comprises other probiotics.

[0028] In some embodiments, the composition is a pharmaceutical composition.

[0029] Wherein, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0030] In some embodiments, the pharmaceutical composition may be in the form of pills, tablets, granules, capsules, powders, suspensions, oral solutions or enema. The pharmaceutical composition may be administered orally, by enema or parenterally, and the administration cycle may be intermittent administration, periodic administration, continuous administration or long-term administration.

[0031] In a third aspect, the present invention also provides use of the above composition in the preparation of medicines for treating and / or improving multiple sclerosis.

[0032] Beneficial effects of the present invention:

[0033] The inventor unexpectedly found that Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 and Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794 can effectively treat and improve multiple sclerosis by enhancing microglial activity, improving weight loss and neurological damage caused by multiple sclerosis, inhibiting the expression of TLR4, thereby reducing central inflammatory response, and the above-mentioned effects are proved by relevant experiments in vivo and in vitro. In addition, the Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 and Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794 can not only be used alone for the treatment of multiple sclerosis, but can also be used in combination with DMTs drugs such as other microorganisms and inflammatory mediator regulators, significantly improving the comprehensive therapeutic effect, reducing the side effects of DMTs drugs, and improving the quality of life of patients. This indicates that Akkermansia muciniphila AM06 and AM02 have the prospect of treating multiple sclerosis and have important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a colony characteristic diagram of Akkermansia muciniphila AM02 of Example 1;

[0035] Figure 2 This is a colony characteristic diagram of Akkermansia muciniphila AM06 of Example 1;

[0036] Figure 3 This is a microscopic observation picture of Akkermansia muciniphila AM02 after Gram staining in Example 1;

[0037] Figure 4 This is a microscopic observation picture of Akkermansia muciniphila AM06 in Example 1 after Gram staining.

[0038] Strain deposit information

[0039] AM02:

[0040] Classification name: Akkermansia muciniphila, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Road, preservation date: June 28, 2021, preservation number: CGMCC No.22794.

[0041] AM06:

[0042] Classification name: Akkermansia muciniphila, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Road, preservation date: June 28, 2021, preservation number: CGMCC No.22793.

[0043] In this application, "more than one" generally refers to three or more. DETAILED DESCRIPTION

[0044] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0045] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0046] Example 1 Fermentation culture of Akkermansia muciniphila (Akk bacteria)

[0047] The Akkermansia muciniphila strain was streaked onto the GlcNAc-heme BHA plate and cultured anaerobically for 3 days. The colony morphology, staining properties, size, club shape and distribution were observed.

[0048] Colony characteristics: After 3 days of culture on the above medium, Akkermansia muciniphila showed round, convex, neatly edged, opaque, white, and unevenly sized colonies, see Figure 1 and Figure 2 .

[0049] Microscopic appearance: Akkermansia muciniphila is a Gram-negative bacterium that is oval and arranged singly or in chains. Figure 3 and Figure 4 .

[0050] A single colony was selected and inoculated into BHI broth supplemented with GlcNAc for fermentation culture for 48 hours (temperature was 37°C), and the obtained bacterial solution was centrifuged at a speed of 16000×g for 30 minutes, the supernatant was removed, and the precipitate was collected to obtain Akkermansia muciniphila bacterial slurry. Akkermansia muciniphila AM02, AM06, and ATCC BAA-835 were cultured respectively.

[0051] Example 2 Preparation of live bacterial solution, inactivated bacterial solution and lysate of Akkermansia muciniphila

[0052] The corresponding Akk bacteria AM02, Akk bacteria AM06 and Akk bacteria ATCC BAA-835 were prepared according to the fermentation culture method of Example 1.

[0053] (1) The bacterial strain was streaked onto a GlcNAc-heme BHA plate and cultured anaerobically at 37°C for 3 days.

[0054] (2) Enrichment: A single colony was selected from step (1) and inoculated into BHI broth supplemented with GlcNAc for enrichment culture. The culture time was 48 hours at a temperature of 37° C. The obtained bacterial solution was stored for later use.

[0055] (3) Akk bacteria live bacterial solution: After counting the live bacteria in the bacterial solution prepared in step (2), dilute it to 10 with physiological saline. 7 CFU / mL and 10 9 CFU / mL, save for future use.

[0056] (4) Akk bacteria inactivated solution: The Akk bacteria inactivated solution is prepared by high temperature or ultraviolet irradiation. Specific steps: Take 10 7 CFU / mL, 10 9 5 mL of Akk bacteria live bacterial solution with a CFU / mL was placed in a beaker, and the beaker containing the bacterial solution was placed in a constant temperature water bath at 100°C for 20 to 30 minutes or placed in an ultraviolet environment for 30 to 60 minutes to prepare Akk bacteria inactivated bacterial solution 10 7 cells / mL, 10 9 cells / mL and save for future use.

[0057] (5) Akk bacterial lysate: Akk bacterial lysate was prepared by ultrasonic disruption. 7 CFU / mL, 10 9 5 mL of Akk bacterial suspension with a CFU / mL was lysed by ultrasonic instrument for 30 minutes, with the on time of 10 seconds and the off time of 10 seconds. The efficiency of bacterial lysis in this system reached 99%. After lysis, it was centrifuged at 6000 rpm and 4°C for 10 minutes, filtered with a 0.22 μm filter head, and the filtrate was collected to prepare 107 CFU / mL, 10 9 CFU / mL of Akk bacterial lysate, save for later use.

[0058] Example 3 Effect of Akkermansia muciniphila on the proliferation activity of microglia

[0059] Ethidium bromide can be inserted into DNA to affect its replication, leading to glial cell death and myelin shedding. Therefore, in this example, after inducing mouse microglial cell line N9 cells with ethidium bromide for 24 hours, the cells were treated with Akk bacteria lysate prepared in Example 2 for 24 hours, and finally the apoptosis of N9 cells was detected using CCK-8 reagent.

[0060] 1. Recovery and passaging of N9 cells

[0061] Take out the cryopreserved tube containing N9 cells from the liquid nitrogen tank, quickly place it in a 37°C water bath and gently shake it to melt. Transfer the thawed cell mixture into a centrifuge tube (15 mL) containing 4 mL of complete culture medium (90% DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin), gently pipette back and forth, and centrifuge at 1000 rpm for 5 minutes at room temperature. Discard the supernatant, add 1 mL of complete culture medium to resuspend the cell pellet, and transfer the resuspended liquid to a culture bottle containing 5 mL of complete culture medium and incubate at 37°C, 5% CO 2 Culture in an incubator. Observe the growth status of cells after recovery and change the medium the next day.

[0062] Cell subculture: If the cell density reaches 80%-90%, subculture can be performed.

[0063] a) Discard the culture supernatant and rinse the cells twice with PBS without calcium and magnesium ions.

[0064] b) Add 1 mL of digestion solution (0.25% trypsin-0.53 mM EDTA) to the culture bottle, place it in a 37°C incubator for 2 minutes, then take it out and quickly add an appropriate amount (4-8 mL) of complete culture medium to terminate the digestion.

[0065] c) Gently blow to blow off the attached cells and suspend them. Transfer all the cell fluid to a 15mL centrifuge tube and centrifuge at 1000rpm for 5min. Then carefully pour off the supernatant and add complete culture medium to resuspend the cell pellet. Inoculate into a new culture flask at a subculture ratio of 1:2 to 1:4 and continue culturing.

[0066] 2. Effect of Akkermansia muciniphila lysate on N9 cell viability

[0067] (1) 5×10 5cells / mL of N9 cell solution were inoculated into 96-well plates, 200 μL per well, i.e. 1×10 5 cells / well, 6 replicates per group (see Table 1 for specific experimental groups). After gentle shaking, incubate at 37°C, 5% CO 2 The cells were cultured in the incubator until they were completely attached. About 24 hours later, the blank group was replaced with 200 μL of fresh complete medium, and the other groups were replaced with 200 μL of complete medium containing 0.1% ethidium bromide solution for model induction. After 24 hours, the blank group and the model group were replaced with 200 μL of fresh complete medium, and the other groups were replaced with a solution with a volume ratio of 1:3 between Akkermansia muciniphila lysate and complete medium.

[0068] (2) CCK-8 assay to detect cell proliferation activity

[0069] After 24 hours of intervention with Akkermansia muciniphila lysate, the absorbance value of each duplicate well in each group was detected using a CCK-8 cell proliferation detection kit (Dojindo, catalog number: CK04). The specific steps are as follows:

[0070] a) Prepare 10% CCK-8 solution at a volume ratio of 1:10 for CCK-8 and complete medium, add 100 μL to each well, and incubate at 37°C with 5% CO 2 Incubate in incubator for 2 h;

[0071] b) Use an ELISA reader to measure the absorbance of each well at 450 nm;

[0072] c) Data analysis: The cell viability of each group was examined by cell viability. The higher the viability, the stronger the cell viability. The specific calculation formula for cell viability is as follows:

[0073]

[0074] As: Indicates the experimental well, which contains cells and substances to be tested.

[0075] Ab: Indicates blank well, which does not contain cells or test substances.

[0076] Ac: indicates the control well, which contains cells but not the substance to be tested.

[0077] Table 1 Experimental groups

[0078]

[0079]

[0080] 3. Experimental results

[0081] Table 2N9 cell viability (%) (mean ± standard deviation, n = 6)

[0082]

[0083] Note: a indicates a significant difference compared with the model group (P<0.05); b indicates a significant difference compared with the ATCC BAA-835 low-dose lysate group (P<0.05); c indicates a significant difference compared with the ATCC BAA-835 high-dose lysate group (P<0.05).

[0084] The effect of Akk on N9 cell viability is shown in Table 2. The cell survival rate in the model group was significantly lower than that in the control group (P<0.05), indicating that ethidium bromide can cause apoptosis of mouse microglia, and the model was successfully established. The lysates of Akkermansia muciniphila AM02 and AM06 both have the effect of enhancing microglial viability (P<0.05). In terms of affecting N9 cell viability, the low-dose lysate groups of AM02 and AM06 were compared with the low-dose lysate group of ATCC BAA-835, respectively, and the high-dose lysate groups of AM02 and AM06 were compared with the high-dose lysate group of ATCC BAA-835, respectively, and there were also significant differences (P<0.05). The results showed that the effects of AM02 and AM06 on the viability of N9 cells were significantly better than those of the standard strain ATCC BAA-835.

[0085] Example 4. Effects of Akkermansia muciniphila on EAE model rats

[0086] The Experimental Autoimmune Encephalomyelitis (EAE) model is widely used to study human multiple sclerosis.

[0087] (1) Test method

[0088] The experimental animals were female SPF Lewis rats aged 6 to 8 weeks; female guinea pigs weighing 300 to 350 g were only used for the preparation of immune antigens.

[0089] 1) Preparation of immune preparation: After anesthetizing guinea pigs by intraperitoneal injection of 10% (m / v) chloral hydrate (3.5 mL / kg body weight), the spinal cord was removed under sterile conditions and ground on ice at a ratio of 1 g spinal cord: 1 mL normal saline to prepare solution A; 60 mg of tuberculin was dissolved in 10 mL complete Freund's adjuvant (CFA) and mixed to prepare solution B; solution B was mixed with normal saline at a volume ratio of 1:1 and used as the injection solution for the control group. Solution B was mixed with solution A at a volume ratio of 1:1 and manually emulsified for more than 1 hour to prepare the complete spinal cord antigen.

[0090] 2) Animal grouping: All Lewis rats were randomly divided into 6 groups according to their body weight, with 10 rats in each group. The specific experimental grouping and dosing regimen are shown in Table 3. The rats in the control group were operated synchronously with the rats in the experimental group, and the same amount of saline was injected at the same time and in the same position.

[0091] 3) Modeling method: 0.1 mL of spinal cord whole antigen was subcutaneously injected into the foot pads of each limb and both sides of the dorsal spine of each rat in the experimental group, and the same amount of control group injection was injected at the same location to establish the EAE model. The day of modeling immunization was recorded as day 0 (D0).

[0092] 4) Drug intervention: Starting from the first day after immunization (D1), Akk live bacteria solution (prepared in Example 2) and 2 mL of 1.5 mg / mL glatiramer acetate solution (glatiramer acetate, 40 mg / vial, prepared as an injection using normal saline) were intraperitoneally administered daily according to the group. The control group and the model group were gavaged with an equal volume of normal saline for 14 consecutive days.

[0093] 5) Detection method and frequency

[0094] After Lewis rats were immunized with spinal cord whole antigen and given drugs, the body weight of all rats was weighed and the neurological function was scored and recorded every other day. After the experiment, the rats were dissected and pathological observation was performed.

[0095] The neurological function scoring criteria are as follows (Saiga, R., Hoshino, M., Takeuchi, et al. Synchrotronradiation microtomography of brain hemisphere and spinal cord of a mouse model of multiple sclerosis revealed a correlation between capillary dilation and clinical score [J]. Journal of Comparative Neurology, 2019, 527 (13): 2091-2100)

[0096] 0 points: no symptoms.

[0097] 1 point: loss of tail tone and lameness.

[0098] 2 points: The hind limbs are weak and can be turned over and recovered.

[0099] 3 points: The hind limbs are completely paralyzed and can be moved by touch.

[0100] 4 points: complete paralysis of the hind limbs and partial paralysis of the forelimbs.

[0101] 5 points, dying or dead.

[0102] Among them, those whose symptoms are between the two are calculated as ±0.5 points.

[0103] The specific experimental groups are shown in Table 3:

[0104] Table 3 Experimental groups and dosing regimen

[0105]

[0106] (2) Test results

[0107] Table 4 Comparison of average body weight of Lewis rats (mean ± standard deviation, n = 10)

[0108]

[0109] Note: a indicates that there is a significant difference compared with the model group (P<0.05); b indicates that there is a significant difference compared with the ATCC BAA-835 live bacteria liquid group (P<0.05).

[0110] Table 5 Comparison of neurological impairment scores in Lewis rats (mean ± SD, n = 10)

[0111]

[0112] Note: a indicates that there is a significant difference compared with the model group (P<0.05); b indicates that there is a significant difference compared with the ATCC BAA-835 live bacteria liquid group (P<0.05).

[0113] The results in Table 4 show that on the 7th day of immunization, the body weight of the model group was significantly lower than that of the control group (P < 0.05); on the 14th day (i.e., the end of the experiment), the body weight of the rats in the AM02 live bacteria group and the AM06 live bacteria group was significantly higher than that in the ATCC BAA-835 live bacteria group (P < 0.05). The results show that both the Akkermansia municiphila AM02 strain and the AM06 strain have the efficacy of restoring experimental autoimmune encephalomyelitis, and the effect is significantly better than that of the ATCC BAA-835 live bacteria group.

[0114] The results in Table 5 show that on the 7th day of immunization, the neurological function injury score of the model group was significantly higher than that of the control group (P < 0.05), while compared with the model group, the positive group (glatiramer acetate), AM02 live bacteria group, and AM06 live bacteria group were significantly lower than the model group (P < 0.05). On the 14th day (i.e., the end of the experiment), the neurological function injury scores of rats in the positive group (glatiramer acetate), AM02 live bacteria group, AM06 live bacteria group, and ATCC BAA-835 live bacteria group were significantly lower than those in the model group (P < 0.05), and the comparison between the AM02 live bacteria group and the AM06 live bacteria group and the ATCC BAA-835 live bacteria group was statistically significant (P < 0.05), indicating that the efficacy of AM02 and AM06 was significantly stronger than that of ATCC BAA-835.

[0115] It can be clearly seen from the results in Tables 4 and 5 that Akkermansia muciniphila has the effect of significantly improving weight loss and neurological damage caused by multiple sclerosis, and the AM02 and AM06 strains provided by the present invention have more significant therapeutic effects than the ATCC BAA-835 strain.

[0116] Example 5. Effects of Akkermansia muciniphila on EAE model mice

[0117] MOG (Myelin Oligodendrocyte Glycoprotein) is a glycoprotein distributed on the surface of oligodendrocyte membrane in nerve myelin. In this example, MOG35-55 peptide segment was used to sensitize C57BL / 6 mice, and pertussis toxin was injected intraperitoneally to establish acute and chronic progressive (CP) EAE multiple sclerosis animal models.

[0118] (1) Test method

[0119] 90 C57BL / 6 female mice aged 8-10 weeks and weighing 18-22 g were randomly divided into 9 groups (see Table 6 for specific experimental groups) according to their body weight, with 10 mice in each group. The mice in the blank group were not treated, and the mice in the other groups were prepared into EAE model mice.

[0120] Preparation of antigen: The antigen MOG 35-55 was diluted with physiological saline to a concentration of 10 mg / mL and mixed with complete Freund's adjuvant (CFA) in equal volumes to prepare a water-in-oil emulsion.

[0121] Antigen injection: Each mouse was given 0.1 mL of antigen by subcutaneous injection at 4 points on both sides of the spine.

[0122] Pertussis toxin injection: Immediately after immunization (i.e., immediately after antigen injection) and 48 hours after immunization, 0.1 mL of pertussis toxin (5 mg / mL) was injected intraperitoneally into each mouse to prepare EAE mice. Mice in the blank group (control group) were injected with an equal amount of saline at the same time and location.

[0123] Drug intervention: Starting from the first day (D1) after immunization, prednisolone acetate (positive drug) or the inactivated Akk bacteria solution and lysate prepared in Example 2 were administered by gavage daily, with the gavage dose being 0.2 mL. The control group and the model group were gavaged with an equal volume of normal saline for 30 consecutive days.

[0124] Testing method and frequency:

[0125] All mice were weighed and neurological function scores were recorded every 2 days since the immune model was established. The neurological function scoring standard was the same as that in Example 3. After the experiment, the mice were dissected and HE staining was performed for pathological observation.

[0126] The specific experimental groups are shown in Table 6:

[0127] Table 6 Experimental groups and dosing regimen

[0128]

[0129]

[0130] (2) Test results

[0131] Table 7 Comparison of neurological impairment scores (mean ± standard deviation, n = 10)

[0132]

[0133] Note: a indicates that compared with the model group, there is a significant difference (P<0.05); b indicates that compared with the ATCC BAA-835 inactivated bacteria solution group, there is a significant difference (P<0.05); c indicates that compared with the ATCC BAA-835 lysate group, there is a significant difference (P<0.05).

[0134] Neurological function injury scores showed that MOG-induced mice began to show neurological dysfunction 5 days after immunization (D5), and the symptoms gradually worsened over time, reaching a peak (about 3 points) between the 7th and 16th days (D7-15), and then maintained at about 2 points, without significant relief. Therefore, it is believed that the period from the 5th day of onset (D5) to the 15th day (D15) when the symptoms are most severe can be regarded as the acute phase of this model, and the period from the 16th day to the 30th day (D16-30) can be regarded as the chronic phase of this model.

[0135] As shown in Table 7, in the acute phase (D14) and chronic phase (D30), the neurological function injury scores of mice in the positive group (prednisone acetate), AM02 inactivated bacteria liquid group, AM02 lysed bacteria liquid group, AM06 inactivated bacteria liquid group, and AM06 lysed bacteria liquid group were significantly lower than those in the model group (P < 0.05). The neurological function injury scores of mice in the AM06 inactivated bacteria group, AM06 lysed bacteria group, and AM02 inactivated bacteria group and AM02 lysed bacteria group were lower than those in the corresponding ATCC BAA-835 groups, and there were significant differences between the AM06 and AM02 groups and the corresponding ATCC BAA-835 groups (P < 0.05). The results showed that the therapeutic effects of AM02 and AM06 were significantly stronger than those of ATCC BAA-835.

[0136] The results in Table 7 indicate that Akkermansia municiphila has a significant effect on improving neurological impairment caused by multiple sclerosis, and the AM02 and AM06 strains provided by the present invention have stronger therapeutic effects than the ATCC BAA-835 strain.

[0137] Example 6. Effects of Akkermansia muciniphila on dicyclohexanone oxalyldihydrazone model mice

[0138] Cuprizone is a neurotoxic copper ion chelator that can specifically induce mitochondrial complex IV dysfunction in oligodendrocytes of the central nervous system, ultimately causing apoptosis of oligodendrocytes, leading to axonal damage and demyelination in the central nervous system. Cuprizone-induced demyelination in mice is currently one of the recognized animal models of toxic demyelination and is often used to simulate the pathological process of human multiple sclerosis. The TLR4 receptor protein is widely distributed and expressed in a variety of cells in the central nervous system, such as microglia, astrocytes, and oligodendrocytes, and is mainly expressed in microglia. Microglia are the main cell type for TLR4 inflammatory response in the central nervous system and are more easily activated and induced.

[0139] (1) Test method

[0140] 110 C57BL / 6 female mice aged 8 to 10 weeks and weighing 18 to 21 g were randomly divided into 11 groups according to their weight, with 10 mice in each group. The specific experimental grouping is shown in Table 8. Except for the blank group (control group), which was fed normally and gavaged with 0.2 mL of normal saline every day, the mice in the other groups were fed with feed containing 0.2% of dicyclohexanone oxalyl dihydrazone by mass fraction and gavaged with 0.2 mL of drug every day for intervention. The body weight was recorded and the neurological function score was performed every week. The scoring criteria were the same as those in Example 3. After 6 weeks, the mice were dissected for pathological observation, and the expression of TLR4 protein in brain tissue was detected by Western blotting.

[0141] The specific experimental groups are shown in Table 8:

[0142] Table 8 Experimental groups and dosing regimen

[0143]

[0144]

[0145] (2) Test results

[0146] Table 9 Comparison of relative expression of TLR4 protein in mouse brain tissue

[0147] Group Relative expression of TLR4 protein (mean ± standard deviation, n = 10) Control group <![CDATA[0.1845±0.0693 a ]]> Model Group 1.3330±0.0875 Positive group <![CDATA[0.3665±0.1809 a ]]> AM02 live bacteria liquid group <![CDATA[0.5164±0.1528 ab ]]> AM02 Inactivated Bacteria Solution Group <![CDATA[0.5952±0.0889 ac ]]> AM02+prednisone acetate group <![CDATA[0.2280±0.0588 adf <!-- 12 -->]]> AM06 live bacteria liquid group <![CDATA[0.4348±0.1621 ab ]]> AM06 Inactivated Bacteria Solution Group <![CDATA[0.4697±0.0714 ac ]]> AM06+prednisone acetate group <![CDATA[0.2191±0.0643 aef ]]> ATCC BAA-835 live bacteria liquid group <![CDATA[0.9024±0.1382 a ]]> ATCC BAA-835 inactivated bacteria solution group <![CDATA[0.9712±0.1976 a ]]>

[0148] Note: a indicates that compared with the model group, there is a significant difference (P<0.05); b indicates that compared with the ATCC BAA-835 live bacteria liquid group, there is a significant difference (P<0.05); c indicates that compared with the ATCC BAA-835 inactivated bacteria liquid group, there is a significant difference (P<0.05); d indicates that compared with the AM02 live bacteria liquid group, there is a significant difference (P<0.05); e indicates that compared with the AM06 live bacteria group, there is a significant difference (P<0.05); f indicates that compared with the positive drug group, there is a significant difference (P<0.05).

[0149] The results in Table 9 show that the relative content of TLR4 protein in the model group was significantly higher than that in the control group (P < 0.05), and the positive group (prednisone acetate), AM02 live bacteria liquid group, AM02 inactivated bacteria liquid group, AM06 live bacteria liquid group, AM06 inactivated bacteria liquid group, ATCC BAA-835 live bacteria liquid group, and ATCC BAA-835 inactivated bacteria liquid group were significantly lower than those in the model group (P < 0.05). The relative content of TLR4 protein in the AM06 live bacteria group and the AM06 inactivated bacteria group and the AM02 live bacteria group and the AM02 inactivated bacteria group was lower than that in the corresponding groups of ATCC BAA-835, and there was a significant difference (P < 0.05). The results show that the therapeutic effects of AM02 and AM06 are stronger than those of ATCC BAA-835. Compared with the AM02 live bacteria group and the positive drug prednisone acetate group, the relative content of TLR4 protein in the combined group AM02+prednisone acetate group was significantly lower (P<0.05, P<0.05), and compared with the AM06 live bacteria group and the positive drug prednisone acetate group, the relative content of TLR4 protein in the combined group AM06+prednisone acetate group was significantly lower (P<0.05). The results showed that the combined use of AM02, AM06 and prednisone acetate had a synergistic effect, and the combined use effect was better than that of either alone.

[0150] From the results in Table 9, it can be clearly seen that Akkermansia muciniphila has the effect of inhibiting the expression of TLR4, thereby reducing central inflammatory response and improving multiple sclerosis. In addition, compared with the ATCC BAA-835 strain, the AM02 and AM06 strains provided by the present invention have stronger therapeutic effects. When used in combination with prednisone acetate, the results of improving multiple sclerosis are more obvious.

[0151] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of Akkermansia muciniphila in the preparation of products for treating and / or improving multiple sclerosis; The Akkermansia muciniphila is at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No.22793 and Akkermansia muciniphila AM02 with a deposit number of CGMCC No.22794.

2. The use according to claim 1, characterized in that: The Akkermansia muciniphila is one, two or more of live bacteria of Akkermansia muciniphila AM02 or Akkermansia muciniphila AM06, inactivated bacteria with complete morphological structure or inactivated bacteria with incomplete morphological structure, and live bacteria lysate.

3. The use according to claim 1 or 2, characterized in that: The multiple sclerosis includes primary progressive multiple sclerosis, secondary progressive multiple sclerosis and relapsing-remitting multiple sclerosis.

4. The use according to claim 1 or 2, characterized in that: The multiple sclerosis is relapsing multiple sclerosis.

5. The use according to claim 4, characterized in that: The relapsing multiple sclerosis is at least one of acute monophasic, relapsing-remitting, chronic progressive and protracted relapsing multiple sclerosis.

6. The use according to claim 1 or 2, characterized in that: The product described is a pharmaceutical product.

7. The use according to claim 6, characterized in that: The dosage form of the medicine includes pills, tablets, granules, capsules, oral liquid or tube feeding preparations.

8. The use according to claim 6, characterized in that: The medicine also includes pharmaceutically acceptable carriers and excipients.

9. The use according to claim 8, characterized in that: The auxiliary materials include at least one of water, saline solution, alcohol, silicone, wax, vaseline, vegetable oil, sugar, gelatin, magnesium stearate, talc, silicon dioxide, surfactant, silicic acid or aromatic oil.

10. The use according to claim 8, characterized in that: The auxiliary material includes at least one of amylose, maltodextrin, microcrystalline cellulose and viscous paraffin.

11. A pharmaceutical composition containing Akkermansia muciniphila and DMTs, characterized in that: The Akkermansia muciniphila is at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793, Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794, or Akkermansia muciniphila standard strain BAA-835; The pharmaceutical composition is used for treating and / or improving multiple sclerosis.

12. The pharmaceutical composition according to claim 11, characterized in that The DMTs drug is selected from one, two or more of prednisone acetate, interferon β, fingolimod, siponimod, natalizumab, cladribine, ocrelizumab, rituximab, glatiramer acetate, teriflunomide and dimethyl fumarate.

13. The pharmaceutical composition according to claim 11, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to any one of claims 11 to 13, characterized in that The dosage form of the pharmaceutical composition is pills, tablets, granules, capsules, powders, suspensions, oral liquids or enemas.

15. Use of the pharmaceutical composition according to any one of claims 11 to 14 in the preparation of a medicament for treating and / or improving multiple sclerosis.

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