Use of akkermansia muciniphila and pharmaceutical compositions thereof for improving and treating neurodegenerative diseases

Abstract

The application provides application of mucinophilic Akkermansia in improving and / or treating neurodegenerative diseases, in particular, AM06 with a preservation number of CGMCC No. 22793 and AM02 with a preservation number of CGMCC No. 22794 can improve the learning and memory ability of rats with Alzheimer's disease, reduce the inflammatory response of brain tissue, improve the behavior of rats with Parkinson's disease, increase the levels of DA, DOPAC and HVA, enhance the coordination ability, and improve the motor disorder ability of SOD1 mutant transgenic mice.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, specifically relating to the application of Akkermansia myxophilus in improving and / or treating neurodegenerative diseases. Background Technology

[0002] Neurodegenerative diseases are a group of diseases caused by the gradual loss or even death of neuronal structure or function, resulting in dysfunction of the nervous system. These include various types such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), neuronal ceroid lipofuscinosis (NCL), and frontotemporal lobar dementia (FLTD). Except for a few familial neurodegenerative diseases with clearly identified causative genes, the pathogenesis of most diseases remains unclear, thus lacking effective treatments.

[0003] Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by progressive cognitive and behavioral impairments, including memory loss, language and motor function decline, and personality and behavioral abnormalities. These symptoms are primarily caused by the death of numerous neurons and the loss of synapses. Amyloid plaques resulting from the accumulation of Aβ in the brain and neurofibrillary tangles formed by the hyperphosphorylation of Tau protein are typical pathological features of AD and are considered possible causes of neuronal death. The pathogenesis of Alzheimer's disease is not fully understood, but it is generally believed to be related to free radical damage, inflammatory responses, and neurotoxic damage. Cerebral ischemia and hypoxia can also cause cognitive abnormalities. Currently, there is no specific drug to completely cure Alzheimer's disease. Treatment focuses on improving symptoms, preventing further progression of dementia, maintaining residual brain function, and reducing complications. The main treatment drugs fall into two categories: 1) Cholinesterase inhibitors: These increase the concentration and activity of acetylcholine in the central nervous system by inhibiting acetylcholinesterase. They are often used to treat mild to moderate AD patients and provide moderate improvement in cognitive function, mood, behavioral symptoms, and daily living functions. Commonly used drugs include donepezil, rivastigmine, and galantamine. 2) Excitatory amino acid receptor antagonists, such as memantine hydrochloride tablets.

[0004] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD), with tremor and paralysis as its main symptom. PD is caused by the death of dopaminergic neurons in the substantia nigra of the midbrain. However, apart from a small percentage of familial PD cases caused by mutations in several related genes, the pathogenesis of most sporadic PD cases remains unclear. The accumulation of α-synuclein and the presence of Lewy bodies are typical pathological features of PD, indicating abnormal protein homeostasis. Currently, the main treatments for Parkinson's disease include medication and surgery. Major medications for Parkinson's disease include levodopa (Madopamine), as well as some receptor agonists such as sifrol, tadalafil, MAO-B inhibitors, midopyleb, trihexyphenidyl, and amantadine. While amantadine is a relatively common choice, ester-based medications are the primary treatment for Parkinson's disease. Although medication can temporarily alleviate symptoms, once the initial "honeymoon period" is over, the irritant and side effects of Western medicine can have a significant impact on the body. Long-term use of medications such as levodopa and sifrol can lead to drug resistance, resulting in end-of-dose phenomena and on / off reactions. Long-term use of this medication can lead to increased stiffness, high blood pressure, low platelet count, and other adverse effects, thus exacerbating physical discomfort. Therefore, long-term use does more harm than good.

[0005] Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a mutation in the huntingtin gene. Symptoms include impaired motor coordination and cognition, dystonia, and choreiform movements, hence it is also commonly known as Huntington's disease. Current treatments are primarily symptomatic, lacking effective disease-modifying therapies. Ideal patient management involves a team of healthcare professionals from various specialties. The only FDA-approved treatment is bubenazine, a synaptic vesicle monoamine transporter inhibitor. Other investigational treatments include deep brain stimulation (DBS) and deuterated bubenazine molecules.

[0006] Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease or Lou Gehrig's disease, is characterized by progressive loss of motor neurons, muscle weakness, and atrophy, typically leading to death within 3-5 years of onset. The pathogenesis of most ALS cases remains unclear. Currently, there is no cure for ALS. However, several treatments can help control symptoms and prevent complications. Treatment medications include rilutek, edaravone, baclofen, diazepam, benzhexol hydrochloride, and amitriptyline hydrochloride.

[0007] Research has discovered a pathway connecting the gut and brain nerves, closely related to the gut microbiota, known as the gut-brain axis. The gut microbiota can regulate neuroinflammation and digestive symptoms through this axis, playing a crucial role in bidirectional communication between the gut and brain. It significantly impacts neurological function not only through neurotransmitter secretion but also through immunity and neural synapses. In-depth research into the interaction mechanisms between the gut microbiota and the neuroimmune system, as well as complex neurodegenerative diseases, can provide new insights for the prevention and treatment of these conditions.

[0008] Probiotics are a class of live microorganisms that play a positive role in human immunity, development, and nutrient absorption. They colonize the human digestive system in large numbers and improve the host's microecological balance. Probiotics have beneficial effects on the treatment of many diseases. For example, *Lactobacillus rhamnosus* and *Lactobacillus fermentum* can improve the altered microbiota and cognitive impairment induced by ampicillin administration in mice. A randomized, double-blind clinical trial showed that a combination of *Lactobacillus* subspecies (i.e., acidophilus, casein, bifidobacteria, and fermentative bacteria) can improve cognitive function to some extent in patients with Alzheimer's disease (AD). In various animal models, *Lactobacillus* and bifidobacteria can reduce the occurrence of anxiety and depression-related symptoms and have a positive impact on memory, learning, and cognition. Furthermore, some clinical trial results are consistent with those obtained from animal models, providing further support for the use of probiotics in the treatment of neuropsychiatric disorders. Therefore, probiotics may be a potential treatment for neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD).

[0009] Akkermansia myxophilus is a strictly anaerobic, Gram-negative bacterium that colonizes the mucus layer of the gastrointestinal tract and specifically degrades mucin. First isolated from human feces by Derrien in 2004 using mucin-containing media, it is widely colonized in the human gut and is one of the most abundant members of the human gut microbiota, accounting for approximately 3% to 5% of the total. It plays an important role in the treatment of various diseases and holds promise as a potential next-generation probiotic. Therefore, it is necessary to develop probiotic formulations using Akkermansia myxophilus for the treatment of neurodegenerative diseases. Summary of the Invention

[0010] The purpose of this invention is to provide the application of Akkermansia myxophilus in improving and / or treating neurodegenerative diseases, particularly strains AM06 (CGMCC No. 22793) and AM02 (CGMCC No. 22794), which can improve the learning and memory abilities of Alzheimer's rats, reduce brain tissue inflammation, improve the behavior of Parkinson's rats, increase DA, DOPAC, and HVA levels, enhance coordination, and improve the motor impairment of SOD1 mutant transgenic mice.

[0011] In a first aspect, the present invention provides the use of Akkermansia myxophilus in the preparation of medicaments for improving and / or treating neurodegenerative diseases, wherein the Akkermansia myxophilus strain is at least one of AM06 strain with accession number CGMCC No. 22793 and AM02 strain with accession number CGMCC No. 22794.

[0012] In some embodiments, the Akkermansia myxophilus is one, two or more of the following: live bacteria, inactivated bacteria with intact morphology and structure, inactivated bacteria with incomplete morphology and structure, bacterial lysate, and bacterial culture supernatant.

[0013] In some embodiments, the neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease.

[0014] In some embodiments, the dosage form of the drug includes pills, tablets, granules, capsules, oral liquids, or tube feeding preparations. The drug is administered to humans or animals.

[0015] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier.

[0016] In some embodiments, the drug is administered via injection or oral administration.

[0017] The present invention also provides a composition comprising Akkermansia myxophilus and other drugs that can improve and / or treat neurodegenerative diseases;

[0018] The other medications that can improve and / or treat neurodegenerative diseases include at least one of the following: medications for treating Alzheimer's disease, medications for treating Parkinson's disease, medications for treating amyotrophic lateral sclerosis (ALS), and medications for treating Huntington's disease.

[0019] The medications used to treat Alzheimer's disease include one, two, or more of donepezil, galantamine, and rivastigmine;

[0020] The medications used to treat Parkinson's disease include at least one of the following: anticholinergic drugs, amantadine, dopamine (e.g., dopasilidine), DR agonists, monoamine oxidase-B inhibitors, and COMT inhibitors.

[0021] The medications used to treat amyotrophic lateral sclerosis include at least one of edaravone, baclofen, diazepam, trihexyphenidyl, amitriptyline, and riluzole.

[0022] The drug used to treat Huntington's disease is selected from bubenazine.

[0023] In one embodiment, the Akkermansia myxophilus is at least one of AM06 with accession number CGMCC No. 22793, or AM02 with accession number CGMCC No. 22794, or the standard strain of Akkermansia myxophilus ATCC BAA-835.

[0024] In a preferred embodiment, the pharmaceutical composition is a combination of Akkermansia myxophilus AM06 with accession number CGMCC No. 22793 or Akkermansia myxophilus AM02 with accession number CGMCC No. 22794 and donepezil, the pharmaceutical composition being used to alleviate and / or treat Alzheimer's disease.

[0025] In a preferred embodiment, the pharmaceutical composition is a combination of Akkermansia myxophilus AM06 with accession number CGMCC No. 22793 or Akkermansia myxophilus AM02 with accession number CGMCC No. 22794 and dopaminergic azithromycin, the pharmaceutical composition being used to relieve and / or treat Parkinson's disease.

[0026] In a preferred embodiment, the pharmaceutical composition is a combination of Akkermansia myxophilus AM06 with accession number CGMCC No. 22793 or Akkermansia myxophilus AM02 with accession number CGMCC No. 22794 and edaravone, the pharmaceutical composition being used to relieve and / or treat amyotrophic lateral sclerosis (ALS).

[0027] Thirdly, the present invention also provides the use of the above-described composition in the preparation of medicaments for improving and / or treating neurodegenerative diseases.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The inventors of this application unexpectedly discovered that Akkermansia myxophilus, particularly Akkermansia myxophilus AM06 with accession number CGMCC No. 22793 and Akkermansia myxophilus AM02 with accession number CGMCC No. 22794, can improve the learning and memory abilities of Alzheimer's rats, reduce the levels of hs-CRP, TNF-α, and IL-4, reduce the inflammatory response in brain tissue, improve the behavior of Parkinson's rats, increase the levels of DA, DOPAC, and HVA, enhance coordination, improve the motor impairment ability of SOD1 mutant transgenic mice, and improve limb coordination and muscle strength, etc.

[0030] Furthermore, the Acerobacterium tumefaciens AM06 and AM02 of the present invention can be used in combination with other therapeutic drugs, and the combination can produce a synergistic effect, more significantly alleviating and / or treating neurodegenerative diseases such as AD, PD or ALS. Attached Figure Description

[0031] Figure 1 This is a colony characteristic diagram of Akkermansia AM02 obtained by the method in Example 1;

[0032] Figure 2 This is a colony characteristic image of Akkermansia AM06, which was cultured using the method in Example 1.

[0033] Figure 3 Microscopic observation of Akkermansia AM02, a bacterophilic bacteria cultured using the method in Example 1, after Gram staining;

[0034] Figure 4 This is a microscopic observation of Akkermansia AM06, a bacterophilic bacteria cultured using the method in Example 1, after Gram staining.

[0035] Strain Preservation Information

[0036] 1) AM02:

[0037] Classification and nomenclature: Akkermansia muciniphila, depositary institution: China General Microbiological Culture Collection Center, depositary address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, deposit date: June 28, 2021, accession number: CGMCC No. 22794.

[0038] 2) AM06:

[0039] Classification and nomenclature: Akkermansia muciniphila, depositary institution: China General Microbiological Culture Collection Center, depositary address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, deposit date: June 28, 2021, accession number: CGMCC No. 22793.

[0040] In this application, "more than three" means three or more types. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0042] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0043] Experimental methods not specifically described in the following examples are generally performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0044] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0045] Example 1: Culture of Akkermansia myxophilus

[0046] Akkermansia myxophilus was streaked onto GlcNAc heme BHA plates and anaerobic incubated for 3 days. Colony morphology, staining characteristics, size, coccoid shape, and distribution were observed.

[0047] Colony characteristics: After culturing for 3 days on the above-mentioned medium, *Ackermania pseudomallei* presents as round, raised, well-defined, opaque, white colonies of varying sizes. See [link to relevant documentation]. Figure 1 and Figure 2 .

[0048] Microscopic morphology: Gram-stained Akkermansia muciniphila are Gram-negative bacteria, appearing as oval, singly, or in chains. (See also...) Figure 3 and Figure 4 .

[0049] Single colonies were inoculated into BHI broth supplemented with GlcNAc and fermented for 48 hours (at 37℃). The resulting bacterial solution was centrifuged at 16000×g for 30 min, the supernatant was discarded, and the precipitate was collected to obtain Akkermansia mycelium sludge. AM02, AM06, and ATCC BAA-835 Akkermansia mycelium were then cultured separately.

[0050] Example 2: Test of Alzheimer's disease treatment with Akkermansia myxophilus

[0051] 1. Experimental Methods

[0052] (1) Model preparation

[0053] One hundred and forty healthy male SD rats, aged 7–9 months and weighing 220–238 g, were selected and acclimatized for one week. One hundred and twenty rats were randomly selected to establish an Alzheimer's disease model: 45 mg / kg sodium nitrite and 120 mg / kg D-galactose were injected subcutaneously into the neck and back of the rats once daily until the rats exhibited dementia. Successful modeling was defined as increased spontaneous activities such as scratching the ears and cheeks, and the appearance of dementia symptoms.

[0054] (2) Grouping and administration

[0055] The Alzheimer's disease model rats were randomly divided into 12 groups, with 10 rats in each group. The specific grouping is as follows:

[0056] a) Monotherapy group

[0057] Model group, positive control group (donepezil, 0.88 mg / kg·d);

[0058] AM02 live bacteria group (10 10 CFU / animal); AM06 live bacteria group (10 10 CFU / each);

[0059] BAA-835 live bacteria group (10) 10 CFU / animal), AM02 inactivated bacterial group (10 10 (cell / each)

[0060] AM06 inactivated bacteria group (10) 10 cell / each), BAA-835 inactivated bacterial group (10) 10 (cell / rat). Ten healthy rats were selected as a normal control group.

[0061] b) Combination drug treatment group

[0062] AM02 live bacteria (10) 10 The combination group of CFU / animal + donepezil (0.88 mg / kg·d) and AM06 live bacteria (10 10 The combination group of CFU / animal + donepezil (0.88 mg / kg·d) and AM02 inactivated bacteria (10 10 The combination of cell / animal and donepezil (0.88 mg / kg·d) and AM06 inactivated bacteria (10 10 The combination of donepezil (0.88 mg / kg / day) and donepezil (0.88 mg / kg / day) was used. In addition, a control group was set up with donepezil alone at a dose of 0.88 mg / kg / day.

[0063] c) Drug administration: The normal control group and the model group were administered 0.5 mL of normal saline by gavage daily; the drug administration groups were given the corresponding dose of drug. The corresponding drug was administered by gavage once a day for 4 consecutive weeks.

[0064] The experimental process and research were conducted in accordance with the principles and requirements set forth by the Animal Ethics Committee of the Chinese Association for Laboratory Animal Science.

[0065] (3) Morris water maze experiment

[0066] The experiment included an adaptation test to a water maze (day 1), a navigation test (days 2-5), and a spatial exploration test (day 6). One day prior to the experiment, rats were placed in the laboratory for acclimatization to reduce fear. For the navigation test, each rat was placed in the water four times daily, head facing the pool wall, from the midpoint of the edge of each of the four different quadrants. After finding a platform, the rat was allowed to remain on it for 10 seconds (the escape latency period). If the rat failed to find the platform within 90 seconds, it was guided to find it and remain there for 10 seconds. The rat's movement trajectory and the distance swam when finding the platform were recorded. The spatial exploration test was conducted on day 6. The platform was removed, and the rat was placed in the opposite quadrant of the navigation test platform. The distance swam in the target quadrant (the quadrant where the navigation test platform was placed) within 90 seconds, the total distance swam in the pool, and the number of times the target quadrant was crossed were recorded. The percentage of the total distance swum in the target quadrant within 90 seconds and the number of times the target quadrant was crossed were used as evaluation indicators for spatial learning and memory.

[0067] (4) Detection of serum IL-4, hs-CRP, and TNF-α levels by enzyme-linked immunosorbent assay (ELISA)

[0068] After the behavioral experiment was completed, blood was collected from the eyeballs and centrifuged at 3000 r / min for 2 min at 4℃ to separate the serum.

[0069] Dilute the antigen with 50 mm carbonate buffer and add it to the reaction wells of polystyrene. After capping, incubate at 4°C for 24 h. Wash three times the next day and dry. Add 0.1 mL of the test sample diluted with diluent (pH 7.4, 0.02 mol / L Tris-HCl buffer) to each well, along with positive and negative control samples. Incubate at 42°C for 60 min. Remove the liquid and wash three times, then dry. Add 0.1 mL of IL-4, hs-CRP, and TNF-α antibody to each well. Incubate again for 60 min, remove the liquid and wash three times, then dry. Add low-potential solution (0.1 mol / L Na2HPO4, 0.05 mol / L citric acid) to each well and mix well. Add 0.1 mL of o-phenylenediamine, shield from light for 20 min, and then add 0.05 mL of 2 mol / L H2SO4 to each well to terminate the reaction. The A450 value was measured using an ELISA reader to analyze the levels of IL-4, hs-CRP, and TNF-α.

[0070] (5) Data Statistics

[0071] SPSS 20.0 software was used for statistical analysis. The results are expressed as (x±s). The t-test was used for comparisons between groups, and P<0.05 was considered statistically significant.

[0072] 2. Experimental Results

[0073] 1) Morris Water Maze

[0074] The learning and memory abilities of AD rats were assessed using the Morris water maze test.

[0075] Table 1 Comparison of learning and memory abilities among rats in different groups (x±s, n=10)

[0076]

[0077] Compared with the model group, * This indicates a statistically significant difference (P<0.05). ** This indicates a highly significant difference (P<0.01);

[0078] Compared with the BAA-835 live bacteria group, a This indicates a significant difference (P<0.05). aa This indicates a highly significant difference (P<0.01); compared with the BAA-835 inactivated bacteria group, b This indicates a significant difference (P<0.05). bb This indicates a highly significant difference (P<0.01); compared with the positive group, c This indicates a significant difference (P<0.05). ccThis indicates a highly significant difference (P<0.01).

[0079] Compared with the live bacteria group of Akkermansia myxophilus AM06, d This indicates a significant difference (P<0.05). dd This indicates a highly significant difference (P<0.01);

[0080] Compared with the live bacteria group of Akkermansia myxophilus AM02, e This indicates a significant difference (P<0.05). ee This indicates a highly significant difference (P<0.01);

[0081] Compared with the inactivated group of Akkermansia amphiphila AM06, f This indicates a significant difference (P<0.05). ff This indicates a highly significant difference (P<0.01);

[0082] Compared with the inactivated group of Akkermansia amphiphila AM02, g This indicates a significant difference (P<0.05). gg This indicates a highly significant difference (P<0.01).

[0083] The time to reach the platform, the number of times the rats crossed the platform, and the percentage of the total distance swam in the target quadrant within 90 seconds reflected the rats' learning and memory abilities. Table 1 shows that the normal control group had statistically significant differences compared to the model group (P<0.05 or P<0.01), indicating successful Alzheimer's disease modeling in rats. There were significant differences in donepezil, and in the treatment groups with live or inactivated Akkermansia myxophilus AM06 and AM02, both individually and in combination (P<0.05 or P<0.01). Compared to the standard strain BAA-835, the effects of live and inactivated Akkermansia myxophilus AM06 and AM02 treatments alone were significantly improved, with statistically significant differences (P<0.05). Compared with the positive group (donepezil), the combination therapy of *Akkermansia myxophilus* AM06 and AM02 with donepezil showed a more significant improvement in treatment efficacy, with statistically significant differences (P<0.05 or P<0.01). Compared with the AM02 and AM06 live bacteria groups respectively, the AM02 live bacteria (10 10 CFU / animal) + donepezil (0.88 mg / kg·d), AM06 live bacteria (10 10The rats in the combination group treated with CFU / rat + donepezil (0.88 mg / kg·d) showed a significantly shorter time to reach the platform (P<0.05 or P<0.01), and significantly increased the number of platform crossings and the percentage of swimming distance in the target quadrant within 90 seconds (P<0.01 or P<0.05). Compared with the AM06 and AM02 inactivated bacteria groups, the AM06 inactivated bacteria (10 10 cell / animal) + donepezil (0.88 mg / kg·d), AM02 inactivated bacteria (10 10 The rats in the combination group (cell / rat) + donepezil (0.88 mg / kg·d) had significantly shorter time to reach the platform (P<0.05 or P<0.01), and significantly increased the number of times they crossed the platform and the percentage of swimming distance in the target quadrant within 90 seconds (P<0.01 or P<0.05).

[0084] 2) Detect IL-4, hs-CRP, and TNF-α levels.

[0085] The development of Alzheimer's disease symptoms is often accompanied by an inflammatory response in the body's brain tissue, and many pro-inflammatory cytokines (such as TNF-α) are key factors in neuroinflammation. hs-CRP, TNF-α, and IL-4 are commonly used clinical indicators for evaluating the body's inflammatory response. hs-CRP is a C-reactive protein mainly synthesized by liver tissue and is a marker of a broad-spectrum inflammatory response. TNF-α is a multifunctional inflammatory cytokine that can stimulate the production of factors such as IL-6 and IL-8, thereby inducing a persistent inflammatory response in the body. IL-4 can stimulate mast cells to release inflammatory mediators and is closely related to the body's inflammatory response. It mainly plays a role in inhibiting the inflammatory response in rat brain tissue, thus exerting an intervention effect.

[0086] Table 2 Comparison of serum levels of hs-CRP, TNF-α, and IL-4 in rats from different groups (x±s, n=10)

[0087]

[0088]

[0089] Compared with the model group, * This indicates a statistically significant difference (P<0.05). ** This indicates a highly significant difference (P<0.01);

[0090] Compared with the BAA-835 live bacteria group, a This indicates a significant difference (P<0.05). aa This indicates a highly significant difference (P<0.01); compared with the BAA-835 inactivated bacteria group, bThis indicates a significant difference (P<0.05). bb This indicates a highly significant difference (P<0.01); compared with the positive group, c This indicates a significant difference (P<0.05). cc This indicates a highly significant difference (P<0.01);

[0091] Compared with the live bacteria group of Akkermansia myxophilus AM06, d This indicates a significant difference (P<0.05). dd This indicates a highly significant difference (P<0.01);

[0092] Compared with the live bacteria group of Akkermansia myxophilus AM02, e This indicates a significant difference (P<0.05). ee This indicates a highly significant difference (P<0.01);

[0093] Compared with the inactivated group of Akkermansia amphiphila AM06, f This indicates a significant difference (P<0.05). ff This indicates a highly significant difference (P<0.01);

[0094] Compared with the inactivated group of Akkermansia amphiphila AM02, g This indicates a significant difference (P<0.05). gg This indicates a highly significant difference (P<0.01).

[0095] Table 2 shows the results of hs-CRP, TNF-α, and IL-4 levels in the normal mouse group and the treated mice. Compared with the model group, the levels of hs-CRP, TNF-α, and IL-4 in the normal control group were all significantly different (P<0.01), indicating successful model establishment. The donepezil group, the Akkermansia myxophilus monotherapy group, and the combined dopezil group all downregulated hs-CRP, TNF-α, and IL-4 levels, showing significant differences (P<0.05 or P<0.01). Compared with the standard strain BAA-835, live and inactivated AM06 and AM02 bacteria significantly reduced hs-CRP, TNF-α, and IL-4 levels, showing statistical differences (P<0.05). Compared with the donepezil monotherapy group, the combined dopezil group with Akkermansia myxophilus AM06 and AM02 showed statistical differences (P<0.05), significantly reducing the levels of inflammatory cytokines. Compared with the AM02 and AM06 live bacteria groups respectively, the AM02 live bacteria (10 10 CFU / animal) + donepezil (0.88 mg / kg·d), AM06 live bacteria (10 10The combination of CFu (0.88 mg / kg / day) and donepezil (0.88 mg / kg / day) downregulated hs-CRP, TNF-α, and IL-4 levels, with statistically significant differences (P<0.05); compared with the AM06 and AM02 inactivated bacteria groups, the AM06 inactivated bacteria (10 10 cell / animal) + donepezil (0.88 mg / kg·d), AM02 inactivated bacteria (10 10 The combination of *Akkermansia myxophilus* AM06 and AM02 (0.88 mg / kg / day) and donepezil downregulated hs-CRP and IL-4 levels, with statistically significant differences (P<0.05). Therefore, both live and inactivated *Akkermansia myxophilus* AM06 and AM02 can downregulate the levels of inflammatory factors hs-CRP, TNF-α, and IL-4, thereby inhibiting the inflammatory response in rat brain tissue and achieving an interventional effect. The combined use of AM06 and AM02 with donepezil has a synergistic effect, which can further improve the therapeutic effect on inflammation, i.e., alleviating and / or treating Alzheimer's disease.

[0096] In summary, intervention with Akkermansia myxophilus AM06 and AM02 in Alzheimer's disease model rats can improve their learning and memory abilities and reduce inflammatory responses in brain tissue. Akkermansia myxophilus AM06 and AM02 show good potential in improving and treating Alzheimer's disease, and their synergistic effects with other therapeutic drugs are significant.

[0097] Example 3: Test of Akkermansia myxophilus in the treatment of Parkinson's disease

[0098] 1. Experimental Methods

[0099] (1) Model preparation

[0100] One hundred and fifty clean-grade male SD rats, weighing (200±20) g, were acclimatized for one week. After examination to confirm the absence of rotational behavior, 140 rats were randomly selected, anesthetized with chloral hydrate, and fixed on a stereotaxic instrument. A 1.5 cm incision was made along the midline of the top of the head, precisely located according to the "Rats Brain Stereotaxic Atlas," and a hole was drilled 4.4 mm posterior to the anterior fontanelle and 1.3 mm lateral to the sagittal suture. 6-OHDA solution was drawn into a microsyringe and fixed to the stereotaxic instrument. The drug was slowly injected into the right striatum at a depth of 8.5 mm. After a 10-minute delay, the microsyringe was slowly withdrawn, the skin was sutured, and gentamicin was injected intraperitoneally at a dose of 2 U daily for three consecutive days to prevent infection. The rats were then returned to their normal cages for regular feeding. On the 30th day post-surgery, rats were intraperitoneally injected with apomorphine at a dose of 0.5 mg / kg to induce rotational behavior. The number of counterclockwise rotations within 30 minutes was recorded. A rotation rate of 7 r / min or higher was considered a successful model.

[0101] (2) Grouping and administration

[0102] Parkinson's disease model rats were randomly divided into 12 groups, with 10 rats in each group. The specific grouping is as follows:

[0103] a) Monotherapy group

[0104] Grouping: Model group, positive control group (dopacaridine, 100 mg / kg), AM02 live bacteria group (10 10 CFU / animal), AM06 live bacteria group (10 10 CFU / animal), BAA-835 live bacteria group (10 10 CFU / animal), AM02 inactivated bacterial group (10 10 cell / each), AM06 inactivated bacterial group (10 10 cell / each), BAA-835 inactivated bacterial group (10) 10 (cell / rat). Ten healthy rats were selected as the normal control group.

[0105] b) Combination drug treatment group

[0106] Groups include: AM02 live bacteria (10 10 The combination of CFU / animal and dopamine (100mg / kg) and AM06 live bacteria (10 10 The combination of CFU / animal and dopamine (100mg / kg) and AM02 inactivated bacteria (10 10 The combination of cell / animal) + dopaminergic hydrazine (100mg / kg) and AM06 inactivated bacteria (10 10 The combination of cell / animal and dopaminergic hydrazine (100mg / kg) was used.

[0107] c) Drug administration: The normal control group and the model group were administered 2 mL of normal saline by gavage daily; each drug administration group was given the corresponding dose of drug. The corresponding drug was administered by gavage once a day for 24 consecutive days.

[0108] The experimental process and research were conducted in accordance with the principles and requirements set forth by the Animal Ethics Committee of the Chinese Association for Laboratory Animal Science.

[0109] (3) Behavioral evaluation

[0110] Two hours after the end of drug administration, rats were intraperitoneally injected with apomorphine at a dose of 0.5 mg / kg to induce rotational behavior. The number of rotations in each group of rats from the 5th to the 30th minute was recorded, and the rotation frequency was calculated. The improvement of the drug on the behavior of the Parkinson's disease model rats was observed.

[0111] (4) Detection of serum biochemical indicators

[0112] After behavioral evaluation, rats in each group were anesthetized by intraperitoneal injection of 10% chloral hydrate. Blood was collected from the abdominal aorta at 4℃ and 3500 rpm. -1 Centrifuge for 10 min, aliquot the serum, and store at -80℃. High-performance liquid chromatography (HPLC) was used to detect the levels of the neurotransmitters dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the serum.

[0113] (5) Data Statistics

[0114] SPSS 20.0 software was used for statistical analysis. The results are expressed as (x±s). The t-test was used for comparisons between groups, and P<0.05 was considered statistically significant.

[0115] 2. Experimental Results

[0116] (1) Behavioral evaluation

[0117] The behavioral records of rats in each group were recorded, and the statistical results are shown in Table 3. Compared with the normal control group, the lateral circling frequency of PD rats in the model group was significantly increased, indicating successful modeling. After 24 days of administration, compared with the model group, the lateral circling frequency of rats in the dopaminergic sildenafil group, the AM06 and AM02 groups was significantly reduced (P < 0.05 or P < 0.01). Compared with the live or inactivated groups of the standard strain BAA-835, the AM06 and AM02 live and inactivated groups were all significantly different (P < 0.05), indicating that the effect of improving circling behavior was better. Compared with the dopaminergic sildenafil group, the AM06 and AM02 combined dopaminergic sildenafil groups were both highly significant (P < 0.01). Compared with the AM02 and AM06 live groups respectively, the AM02 live bacteria (10 10 CFU / animal) + donepezil (0.88 mg / kg·d), AM06 live bacteria (10 10 The combination of CFU / rat and donepezil (0.88 mg / kg·d) significantly reduced the circling frequency in rats, with statistically significant differences (P<0.01); compared with the AM06 and AM02 inactivated bacteria groups, the AM06 inactivated bacteria (10 10 cell / animal) + donepezil (0.88 mg / kg·d), AM02 inactivated bacteria (10 10 The combination of AM06 live bacteria (10 cells / rat) and donepezil (0.88 mg / kg·d) significantly reduced the circling frequency in rats, with a statistically significant difference (P<0.01). 10 CFU / animal) + donepezil (0.88 mg / kg·d), AM02 live bacteria (10 10 The combination of CFU / rat and donepezil (0.88 mg / kg·d) showed a synergistic effect, which could greatly regulate the circling behavior of rats.

[0118] Table 3. Circling behavior of rats in each group (x±s)

[0119]

[0120]

[0121] "-" indicates no spinning behavior;

[0122] Compared with the model group, * This indicates a statistically significant difference (P<0.05). ** This indicates a highly significant difference (P<0.01);

[0123] Compared with the BAA-835 live bacteria group, a This indicates a significant difference (P<0.05). aa This indicates a highly significant difference (P<0.01); compared with the BAA-835 inactivated bacteria group, b This indicates a significant difference (P<0.05). bb This indicates a highly significant difference (P<0.01); compared with the positive group, c This indicates a significant difference (P<0.05). cc This indicates a highly significant difference (P<0.01);

[0124] Compared with the live bacteria group of Akkermansia myxophilus AM06, d This indicates a significant difference (P<0.05). dd This indicates a highly significant difference (P<0.01);

[0125] Compared with the live bacteria group of Akkermansia myxophilus AM02, e This indicates a significant difference (P<0.05). ee This indicates a highly significant difference (P<0.01);

[0126] Compared with the inactivated group of Akkermansia amphiphila AM06, f This indicates a significant difference (P<0.05). ff This indicates a highly significant difference (P<0.01);

[0127] Compared with the inactivated group of Akkermansia amphiphila AM02, g This indicates a significant difference (P<0.05). gg This indicates a highly significant difference (P<0.01).

[0128] (2) Detection of DA, DOPAC and HVA content

[0129] Dopamine loss can lead to motor dysfunction symptoms, namely impaired coordination, a common symptom in Parkinson's disease.

[0130] Table 4. Serum levels of DA, DOPAC, and HVA in rats of each group

[0131]

[0132]

[0133] Compared with the model group, * This indicates a statistically significant difference (P<0.05). ** This indicates a highly significant difference (P<0.01). *** This indicates a highly significant difference (P<0.001);

[0134] Compared with the BAA-835 live bacteria group, a This indicates a significant difference (P<0.05). aa This indicates a highly significant difference (P<0.01); compared with the BAA-835 inactivated bacteria group, b This indicates a significant difference (P<0.05). bb This indicates a highly significant difference (P<0.01); compared with the positive group, c This indicates a significant difference (P<0.05). cc This indicates a highly significant difference (P<0.01);

[0135] Compared with the live bacteria group of Akkermansia myxophilus AM06, d This indicates a significant difference (P<0.05). dd This indicates a highly significant difference (P<0.01);

[0136] Compared with the live bacteria group of Akkermansia myxophilus AM02, e This indicates a significant difference (P<0.05). ee This indicates a highly significant difference (P<0.01);

[0137] Compared with the inactivated group of Akkermansia amphiphila AM06, f This indicates a significant difference (P<0.05). ff This indicates a highly significant difference (P<0.01);

[0138] Compared with the inactivated group of Akkermansia amphiphila AM02, g This indicates a significant difference (P<0.05). gg This indicates a highly significant difference (P<0.01).

[0139] The results of the detection of DA, DOPAC, and HVA levels in rats are shown in Table 4. Compared with the normal control group, the serum levels of DA, DOPAC, and HVA in the model group rats were significantly decreased, indicating that the model was successfully established.

[0140] Compared with the model group, the single-dose groups of DOPA, Akkermansia myxophilus AM06 and AM02, and the groups of Akkermansia myxophilus AM06 and AM02 combined with DOPA all increased the serum DA, DOPAC, and HVA levels in rats (P<0.05, P<0.01, or P<0.001), as shown in Table 4. Among them, the live and inactivated AM06 and AM02 groups showed statistically significant differences in increasing DA, DOPAC, and HVA levels in rats compared with the standard strain BAA-835 (P<0.05); the combined groups showed significant differences compared with the DOPA single-dose group (P<0.05). Compared with the live AM02 and AM06 groups, the live AM02 group (10... 10 CFU / animal) + donepezil (0.88 mg / kg·d), AM06 live bacteria (10 10 The combination of CFU / rat and donepezil (0.88 mg / kg·d) significantly increased the serum levels of DA, DOPAC, and HVA in rats, with statistically significant differences (P<0.05); compared with the AM06 and AM02 inactivated bacteria groups, the AM06 inactivated bacteria (10) group significantly increased the serum levels of DA, DOPAC, and HVA in rats. 10 cell / animal) + donepezil (0.88 mg / kg·d), AM02 inactivated bacteria (10 10 The combination of donepezil (0.88 mg / kg / day) and DA (cell / rat) significantly increased the serum levels of DA, DOPAC, and HVA in rats, with statistically significant differences (P<0.05 or P<0.01). This indicates that the combination therapy produced a synergistic effect, greatly increasing the serum levels of DA, DOPAC, and HVA in rats, thus better improving motor coordination and alleviating and / or treating Parkinson's disease symptoms, demonstrating good application potential.

[0141] Example 4: Test of Ackermania mutans for the treatment of amyotrophic lateral sclerosis (ALS)

[0142] 1. Experimental Methods

[0143] (1) Preparation of model mice

[0144] SOD1 G93AThe mice are transgenic mice carrying multiple copies of a human SOD1 mutant. This mSOD1 gene can change glycine to alanine at position 93 of the human SOD1 gene, leading to ALS. The transgenic mice were purchased from Jackson Laboratory, USA (B6SJL-Tg-SOD1*G93A-1Gur / J, Stock no. 002726). By mating, breeding, and reproducing the purchased transgenic male mice with wild-type female mice of the same background, the offspring can include wild-type normal mice and SOD1 mutant mice exhibiting ALS symptoms. G93A Transgenic mice were identified as positive mice using standard PCR technology.

[0145] (2) Grouping and administration

[0146] SOD1, which manifests as ALS symptoms G93A The transgenic mice were randomly divided into 12 groups, with 10 mice in each group. The specific grouping is as follows:

[0147] a) Monotherapy group

[0148] Grouping: Model group, positive control group (edaravone, 100 mg / kg), AM02 live bacteria group (10 10 CFU / animal), AM06 live bacteria group (10 10 CFU / animal), BAA-835 live bacteria group (10 10 CFU / animal), AM02 inactivated bacterial group (10 10 cell / each), AM06 inactivated bacterial group (10 10 cell / each), BAA-835 inactivated bacterial group (10) 10 (cell / mouse). Ten wild-type normal mice were selected as the control group.

[0149] b) Combination drug treatment group

[0150] Groups include: AM02 live bacteria (10 10 The combination group of CFU / animal + edaravone (15mg / kg) and AM06 live bacteria (10 10 The combination group of CFU / animal + edaravone (15mg / kg) and AM02 inactivated bacteria (10 10 The combination of cell / animal) + edaravone (15mg / kg) and AM06 inactivated bacteria (10 10 The combination of cell / animal and edaravone (15mg / kg) was used.

[0151] c) Drug administration: The normal control group and the model group were administered 2 mL of normal saline by gavage daily; each drug administration group was given the corresponding dose of drug. The corresponding drug was administered by gavage once a day for 24 consecutive days.

[0152] The experimental process and research were conducted in accordance with the principles and requirements set forth by the Animal Ethics Committee of the Chinese Association for Laboratory Animal Science.

[0153] (3) Behavioral testing methods

[0154] SOD1 G93A Transgenic mice develop symptoms at 12 to 14 weeks of age, exhibiting motor dysfunction, motor neuron degeneration, and excessive activation of glial cells, and become paralyzed and die at 19 to 23 weeks of age. Paw gripping strength, pole climbing, tumbling block, and open field tests were performed on 12-week-old mice.

[0155] (a) Limb gripping test

[0156] The severity of disease and the degree of muscle damage in mice were assessed by testing their grip strength in their limbs. Mice were gently placed on the central platform of a gripping board, and their tails were gently pulled to encourage them to grasp the board. As soon as the mouse gripped the gripping net with force, the board was pulled horizontally backward. The data was recorded when the instrument displayed the maximum grip strength value. After the start of drug administration, the grip strength of the mice was tested every two weeks, with each mouse measured three times. The maximum value among the three measurements was taken as the mouse's maximum grip strength value.

[0157] (b) Pole Climbing Experiment

[0158] A pole-climbing test was used to assess the motor coordination and muscle strength of mice. A homemade wooden pole, approximately 50 cm long and 1 cm in diameter, was wrapped with medical gauze to increase friction. The pole was placed vertically on a horizontal table. The mouse was held by its tail with its head down, its limbs gripping the top of the pole. After releasing the tail, the time was started, ensuring the mouse crawled downwards without external force. The time taken for the mouse to climb from the top of the pole to the bottom platform was recorded (using the hind limbs as the reference). Mice were trained for this behavior for 3 consecutive days before drug administration. Each mouse underwent three repeated trials, and mice that did not meet the standard were excluded. After drug administration began, the behavior was tested every two weeks. The maximum test time was no more than 15 seconds; any result exceeding 15 seconds was recorded as 15 seconds. The average of the three pole-climbing times was calculated as the final pole-climbing time.

[0159] (c) Rotating bar test

[0160] The motor abilities of mice were assessed using a Rotador instrument (4 cm in diameter, 20 rpm). For the first 7 days, mice practiced on the rotador for 5 minutes daily, followed by three consecutive runs, to acclimatize them to the exercise and establish a baseline for their motor abilities. Starting at 77 days of age, the time spent on the rotador was recorded (not exceeding 5 minutes), repeated three times each session, and the best performance was recorded. Afterward, rotador exercise was performed every two days.

[0161] (d) Open field test

[0162] The open field test primarily tracks the total path of mice's autonomous crawling within an open chamber over 5 minutes, assessing their motor autonomy and ability. The open field test chamber measures 200mm x 200mm x 200mm. Mice are placed in the chamber, and a high-definition camera tracks and records their movements over 5 minutes. An open field video analysis system is used to record the mice's movement trajectories and to statistically analyze the total crawling path.

[0163] (4) Data Statistics

[0164] SPSS 20.0 software was used for statistical analysis. The results are expressed as (x±s). The t-test was used for comparisons between groups, and P<0.05 was considered statistically significant.

[0165] 2. Experimental Results

[0166] When ALS model mice develop the disease, they exhibit motor dysfunction, motor neuron degeneration, and excessive activation of glial cells. Behavioral tests were performed on mice in each group, and the results are shown in Table 5.

[0167] Table 5. Behavioral test results of mice in each group

[0168]

[0169] Compared with the model group, * This indicates a statistically significant difference (P<0.05). ** This indicates a highly significant difference (P<0.01);

[0170] Compared with the BAA-835 live bacteria group, a This indicates a significant difference (P<0.05). aa This indicates a highly significant difference (P<0.01); compared with the BAA-835 inactivated bacteria group, b This indicates a significant difference (P<0.05). bb This indicates a highly significant difference (P<0.01); compared with the positive group, c This indicates a significant difference (P<0.05). cc This indicates a highly significant difference (P<0.01);

[0171] Compared with the live bacteria group of Akkermansia myxophilus AM06, d This indicates a significant difference (P<0.05). dd This indicates a highly significant difference (P<0.01);

[0172] Compared with the live bacteria group of Akkermansia myxophilus AM02, eThis indicates a significant difference (P<0.05). ee This indicates a highly significant difference (P<0.01);

[0173] Compared with the inactivated group of Akkermansia amphiphila AM06, f This indicates a significant difference (P<0.05). ff This indicates a highly significant difference (P<0.01);

[0174] Compared with the inactivated group of Akkermansia amphiphila AM02, g This indicates a significant difference (P<0.05). gg This indicates a highly significant difference (P<0.01).

[0175] As shown in Table 5, compared with the normal control group mice, the model group mice had significantly weaker claw gripping force, increased climbing time (indicating slower crawling speed), significantly reduced time spent on the rotating bar, and significantly reduced total distance crawled autonomously in the open box, indicating that the ALS mouse model was successfully established.

[0176] Using a grip strength meter to measure the grasping strength of mice can assess the degree of nerve damage and limb muscle damage. Compared with the model group, the grasping strength of mice in each single-drug group and the combined-drug group was increased to a certain extent (P<0.05 or P<0.01), indicating that administration of edaravone and Akkermansia myxophilus AM06 and AM02 can improve or treat ALS symptoms. Compared with the standard Akkermansia myxophilus BAA-835, AM06 and AM02 of the present invention have significantly better therapeutic effects (P<0.05); the combination of AM06 and AM02 with edaravone significantly enhanced the grasping strength of mice compared with AM06, AM02 and edaravone alone, showing a synergistic effect (P<0.05).

[0177] The pole climbing test can assess the crawling speed of mice and evaluate their limb coordination. Table 5 shows that, compared to the model group, the crawling speed of mice in both the single-drug and combined-drug groups improved to some extent after drug administration, with AM06 and AM02 showing significantly better effects than BAA-835 (P<0.05). The combined-drug group crawled significantly faster than the AM06, AM02, and edaravone single-drug groups (P<0.05).

[0178] Balance, coordination, and muscle strength in mice were assessed using a rotarod test. Table 5 shows that, compared to the model group, mice in both the single-drug and combined-drug groups exhibited significantly increased dwell time on the rotarod after drug administration (P<0.05 or P<0.01), with AM06 and AM02 showing significantly better efficacy than BAA-835 (P<0.05). The combined-drug groups had significantly longer dwell times than the single-drug groups (AM06, AM02, and edaravone alone) (P<0.05), demonstrating a certain synergistic effect.

[0179] The open field test recorded the total autonomous crawling distance of mice in an open box over 5 minutes, which can assess the mice's motor autonomy and motor ability. Compared with the model group, the total crawling distance of mice in each single-drug group and the combination-drug group was significantly increased (P<0.05 or P<0.01), and AM06 and AM02 were significantly better than BAA-835 treatment (P<0.05). The residence time in the combination-drug group was significantly longer than that in the AM06, AM02 and edaravone single-drug groups (P<0.05), showing a certain synergistic effect.

[0180] The results above show that the AM06 and AM02 strains of the present invention have a more significant effect on improving the coordination ability of mice, and when used in combination with edaravone, they have a synergistic effect, which can further improve the therapeutic effect.

[0181] In summary, the *Ackermania* AM06 and AM02 strains and their pharmaceutical compositions of the present invention can improve and treat neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Furthermore, they exhibit synergistic effects when used in combination with other drugs.

[0182] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of Akkermansia myxophilus in the preparation of medicaments for improving and / or treating Alzheimer's disease, wherein the Akkermansia myxophilus is at least one of strain AM06 with accession number CGMCC No. 22793 or strain AM02 with accession number CGMCC No. 22794.

2. The application according to claim 1, characterized in that, The *Ackermania* species mentioned are one or two of the following: live bacteria and inactivated bacteria with intact morphology and structure.

3. The application according to claim 1 or 2, characterized in that, The dosage forms of the drug include pills, tablets, granules, capsules, oral liquids, or tube feeding preparations.

4. A pharmaceutical composition comprising Akkermansia myxophilus and donepezil; The Akkermansia myxophilus is either AM06 with accession number CGMCC No. 22793 or AM02 with accession number CGMCC No. 22794. The pharmaceutical composition is used to treat Alzheimer's disease.

5. Use of the pharmaceutical composition of claim 4 in the preparation of a medicament for improving and / or treating Alzheimer's disease.

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