Use of akkermansia muciniphila and compositions thereof for the preparation of a medicament for improving and / or treating parkinson's disease with depression

Abstract

The application belongs to the technical field of biological medicine, and relates to application of mucinophilic Akkermansia in preparation of a medicine for improving and / or treating Parkinson's disease with depression, the application applies the mucinophilic Akkermansia to the clinical comorbidity scene of Parkinson's disease with depression, and the curative effect of the bacteria in improving the behavior of PD with depression is verified in multiple PD animal models and depression-related mouse models. The application further discloses that the mucinophilic Akkermansia improves the depression-like behavior under the background of aging, and the mucinophilic Akkermansia has different regulating effects on the peripheral and central 5-HT systems for different genetic backgrounds, which provides a theoretical basis for precise intervention targeting different PD subtypes, and it is clear that the mucinophilic Akkermansia plays a role in improving the depression-like behavior by remodeling the intestinal flora, which provides a new microbial target for treating PD depression targeting the metabolites of the flora.

Description

Technical Field

[0001] This invention relates to the use of Akkermansia myxophilus and its compositions in the preparation of medicaments for improving and / or treating Parkinson's disease with depression, and belongs to the field of biomedical technology. Background Technology

[0002] Parkinson's disease (PD) is the second most common progressive neurodegenerative disease, with the number of patients worldwide projected to reach 25.2 million by 2050. The pathological features of PD include the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain and the abnormal aggregation of α-synuclein (α-syn) to form Lewy bodies. In addition to motor symptoms (resting tremor, rigidity, and bradykinesia), non-motor symptoms such as depression, anxiety, and gastrointestinal dysfunction severely impact patients' quality of life. The incidence of depression is as high as 40%-50%, and this comorbid depression in Parkinson's disease often appears in the prodromal stage of the disease.

[0003] Currently, clinical treatment for Parkinson's disease (PD) with depression mainly includes drug therapy such as selective serotonin reuptake inhibitors (SSRIs, such as sertraline and venlafaxine), serotonin-norepinephrine reuptake inhibitors (SNRIs, such as amitriptyline and imipramine), and dopamine receptor agonists (such as pramipexole), as well as psychotherapy such as cognitive behavioral therapy (CBT). However, drug therapy has problems such as high drug tolerance (approximately 25%) and anticholinergic side effects (cognitive impairment, orthostatic hypotension, and worsening constipation). Therefore, developing novel treatment strategies with fewer side effects and better patient compliance is of significant clinical importance.

[0004] The Braak hypothesis proposes that the pathology of Parkinson's disease (PD) may originate in the gut, with misfolded α-synuclein cells spreading retrogradely to the central nervous system via the vagus nerve. Clinical studies have found that gastrointestinal dysfunctions such as constipation can appear up to twenty years earlier than the motor symptoms of PD, further supporting the role of the gut in PD. Research shows that PD patients exhibit characteristic gut microbiota dysbiosis: a decrease in beneficial bacteria that produce short-chain fatty acids (SCFAs) (such as Lachnospiraceae and Faecalibacterium) and an increase in pro-inflammatory bacteria (such as Enterobacteriaceae). The gut microbiota metabolites SCFAs (mainly acetic acid, propionic acid, and butyric acid) can affect central nervous system inflammation and the neurotransmitter system through mechanisms such as regulating intestinal barrier function, inhibiting histone deacetylases (HDACs), and activating G protein-coupled receptors (GPR41 / GPR43). The gut-brain axis, through the synergistic action of three major mechanisms—microbial metabolites, the HPA axis, and vagal nerve signaling—connects the gastrointestinal tract, gut microbiota, and central nervous system, serving as a "communication bridge" for bidirectional communication between the gut and the brain. Numerous studies have shown that the gut microbiota can influence host mood, cognition, and behavior by producing short-chain fatty acids (SCFAs), regulating the tryptophan-serotonin pathway, affecting vagal nerve activity, or inhibiting nervous system inflammation. Therefore, targeting the gut microbiota has become a novel therapeutic strategy for neuropsychiatric disorders such as depression.

[0005] Akkermansia myxophilus ( Akkermansia muciniphila AKK (Acinetobacter alkaloids) is a Gram-negative anaerobic bacterium that colonizes the intestinal mucus layer and uses mucin as a metabolic substrate. Various active components derived from AKK, including the outer membrane protein Amuc_1100, bacterial membrane lipids, secretory protein P9, and extracellular vesicles (AmEVs), play a crucial role in maintaining intestinal barrier integrity and regulating inflammatory responses. In the field of neurological diseases, AKK has shown some protective effects. In an Alzheimer's disease (AD) model, AKK reduces neuroinflammation and β-amyloid deposition by regulating the tryptophan metabolism-aromatic hydrocarbon receptor (AhR) pathway; in an amyotrophic lateral sclerosis (ALS) model, AKK can slow disease progression. However, in a malignant intestinal environment, excessive proliferation of AKK leads to excessive depletion of mucin, exacerbating intestinal barrier damage and allowing inflammatory factors such as LPS to enter the bloodstream and ultimately reach the brain, thereby exacerbating neuroinflammation.

[0006] Current research on the role of AKK in PD with depression has the following limitations: 1. Unclear Mechanism of Action: The role and mechanism of AKK in age-related psychosomatic disease with depression remain unclear, and systematic studies on differences across different genetic backgrounds are lacking. It is also unclear whether AKK can affect central neurotransmitters (such as 5-HT) by regulating intestinal metabolites (such as SCFAs).

[0007] 2. Peripheral-central relationship unclear: The correlation between peripheral plasma and changes in 5-HT and SCFAs in the central nervous system also lacks in-depth research.

[0008] 3. Model limitations: Existing studies have mostly focused on young or male animals, lacking attention to aging female individuals, while PD is highly prevalent in the elderly population and exhibits gender differences. Summary of the Invention

[0009] The present invention provides the use of Akkermansia myxophilus and its composition in the preparation of medicaments for improving and / or treating Parkinson's disease with depression, thereby solving the technical problems existing in the prior art as described above.

[0010] The technical solution provided by this invention is as follows: One of the objectives of this invention is to provide the use of Akkermansia myxophilus in the preparation of medicaments for improving and / or treating Parkinson's disease with depression.

[0011] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the Parkinson's disease with depression refers to age-related or Parkinsonian depressive-like behaviors associated with α-synucleosis.

[0012] Furthermore, the *Ackermania* strain is ATCC BAA-835. Furthermore, as described above, the application can regulate peripheral-central 5-HT levels or enhance the absorption of intestinal SCFAs through Akkermansia myxophilus.

[0013] Furthermore, the dosage form of the drug is lyophilized powder, liquid bacterial suspension, tablet or capsule.

[0014] A second objective of this invention is to provide a mixed bacterial agent, including *Ackermania glutinis* as described above in the application.

[0015] Furthermore, the mixed bacterial agent also includes intestinal butyric acid-producing bacteria.

[0016] Furthermore, the intestinal butyric acid-producing bacteria is at least one strain from the Trichophyceae family.

[0017] Furthermore, the intestinal butyric acid-producing bacteria are one or more of Lachnospiraceae COE1, Lachnospiraceae sp000403215, Lachnospiraceae UBA3402 sp910588505, and Lachnospiraceae UBA3282 sp009774655.

[0018] A third objective of this invention is to provide the use of the mixed bacterial agent described above in the preparation of a medicament for improving and / or treating Parkinson's disease with depression.

[0019] A fourth objective of this invention is to provide a composition comprising Akkermansia myxophila as described above and a butyric acid precursor.

[0020] Furthermore, the butyric acid precursor is dietary fiber or resistant starch.

[0021] A fifth objective of this invention is to provide the use of the composition described above in the preparation of a medicament for improving and / or treating Parkinson's disease with depression.

[0022] The technical solution provided by this invention has the following advantages compared with the prior art: This invention applies *Ackermania myxophilus* to the clinical comorbidity of Parkinson's disease (PD) with depression, and systematically validates its efficacy in improving depressive behaviors in PD in multiple PD animal models and depression-related mouse models. This invention further reveals that *Ackermania myxophilus* improves depressive-like behaviors in an aging context; and that *Ackermania myxophilus* exerts different regulatory effects on the peripheral and central 5-HT systems depending on different genetic backgrounds, providing a theoretical basis for precise intervention targeting different PD subtypes; it clarifies that *Ackermania myxophilus* exerts its effect in improving depressive-like behaviors by remodeling the gut microbiota, providing a new microbial target for treating PD depression by targeting gut microbiota metabolites. Attached Figure Description

[0023] Figure 1 The experimental flowcharts and results of Examples 1-4 of the present invention are shown; the experimental flowchart of AKK improving depressive-like behavior in C57 mice and A53T α-syn transgenic mice is shown. Figure 1 (A) Immobility time in the forced swimming test of mice ( Figure 1 (B) Sucrose preference rate in mouse sucrose preference experiment ( Figure 1(C) Data are expressed as Mean ± SEM, and Student's t-test was used for statistical analysis. Compared with the PBS group, *P<0.05, C57-PBS: n=5 animals / group, C57-AKK: n=6 animals / group, Tg-PBS: n=4 animals / group, Tg-AKK: n=6 animals / group.

[0024] Figure 2 This is a graph showing the experimental results of Example 5 of the present invention; the concentration of 5-HT in plasma ( Figure 2 Concentration of 5-HT in the hippocampus (A) Figure 2 (Image B) Immunofluorescence staining of 5-HT in the mouse hippocampus (Image B) Figure 2 (C) Statistical graph of 5-HT expression in the hippocampus ( Figure 2 D), plasma propionic acid concentration ( Figure 2 E), plasma butyrate concentration ( Figure 2 (F): Data are expressed as Mean ± SEM, and statistical analysis was performed using Student's t-test. Compared with the PBS group, *P<0.05, **P<0.01, C57-PBS: n=4 animals / group, C57-AKK: n=5 animals / group, Tg-PBS: n=3 animals / group, Tg-AKK: n=3 animals / group.

[0025] Figure 3 The experimental results are shown in Example 6 of the present invention; α-diversity (observed) analysis ( Figure 3 (A) α-diversity analysis ( Figure 3 B), α-diversity-Shannon analysis ( Figure 3 (C). Statistical analysis was performed using the Wilcoxon rank-sum test (*P<0.05). phylum-level analysis of gut bacterial diversity ( Figure 3 (D) Species-level analysis of gut bacterial diversity ( Figure 3 E and LEfSe analyses showed enriched bacterial taxa in the C57-PBS and C57-AKK groups. Figure 3 Analysis of the F and LEfSe groups showed enriched bacterial taxa in mice in the Tg-PBS and Tg-AKK groups. Figure 3 (Middle G). C57-PBS: n=4 animals / group, C57-AKK: n=6 animals / group, Tg-PBS: n=3 animals / group, Tg-AKK: n=3 animals / group. Statistical analysis was performed using the Wilcoxon rank-sum test (*P<0.05).

[0026] Figure 4Figure 7 shows the experimental results of Example 7 of the present invention; Functional annotation and classification of the C57-AKK group and the C57-PBS group based on the KEGG orthologous system (KO). Figure 4 Functional annotation and classification of the C57-AKK group and the Tg-AKK group based on the KEGG orthologous system (KO). Figure 4 Differential abundance analysis of KEGG metabolic modules between the C57-B, C57-PBS and C57-AKK groups ( Figure 4 Differential abundance analysis of KEGG metabolic modules between the C, Tg-PBS and Tg-AKK groups ( Figure 4 Differential abundance analysis of KEGG metabolic modules between the D), Tg-AKK group and C57-AKK group ( Figure 4 (E). Detailed Implementation

[0027] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0028] Animal housing: The A53T α-synuclein transgenic mouse strain M83 (Tg(SNCA)83Vle) was purchased from Shanghai Southern Model Biotechnology Co., Ltd., and C57BL / 6J mice were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. All animals were housed in temperature- and humidity-controlled facilities (23-25℃, 50-60% humidity) with a 12-hour light / dark cycle and free access to food and water. All animal experiments were conducted in accordance with the guidelines approved by the Shandong Provincial Laboratory Animal Care and Use Committee for New Drug Development in Yantai. Mice were allowed 30 minutes to acclimatize in the testing room before all behavioral tests.

[0029] Example 1: Culture of Akkermansia myxophilus AKK strain (ATCC BAA-835) was cultured in brain heart infusion broth (purchased from Millipore Sigma) under strictly anaerobic conditions at 37°C, with a gas composition of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide. After 12 hours of anaerobic culture, the culture was centrifuged at 4000 rpm / min for 15 min at 4°C. After washing and resuspending with deoxyphosphate (PBS) buffer, the culture was centrifuged again under the same conditions to adjust the bacterial concentration to 1×10⁻⁶. 9 CFU / mL was administered to mice via gavage.

[0030] Example 2: Intestinal flora depletion and oral administration experiment Mice were divided into the C57 group (C57BL / 6J mice) and the Tg group (A53T α-synuclein transgenic mice) based on their genotype (C57BL / 6J mice and A53T α-synuclein transgenic mice). They were then randomly assigned to an AKK-treated experimental group and a PBS control group, resulting in four groups: C57-PBS, C57-AKK, ​​Tg-PBS, and Tg-AKK. The four groups of mice were administered a quadruple antibiotic regimen (ampicillin 0.25 mg / mL, neomycin 0.25 mg / mL, metronidazole 0.25 mg / mL, and vancomycin 0.125 mg / mL) via drinking water for 7 consecutive days. Subsequently, each mouse in the C57-AKK and Tg-AKK groups was administered 200 μL of the bacterial culture prepared in Example 1 (containing 2 × 10⁻⁶ cells / mL) daily via gavage. 8 Mice in both the C57-PBS and Tg-PBS groups were administered 200 μL of deoxygenated PBS buffer daily via gavage. Antibiotics were introduced via drinking water feeding, and the experiment lasted 63 days.

[0031] Example 3 Forced Swimming Experiment The forced swimming test is a commonly used test to assess depressive-like behavior in mice. Mice from Example 2 were subjected to the forced swimming test 28 days after the initial experiment. Figure 1 (A)

[0032] The forced swimming experiment was conducted in a transparent glass experimental tank filled with water (temperature 25±2℃). The tank dimensions were: height 25 cm, diameter 12 cm, and water level 15 cm. Mice were gently placed in the tank, and a camera recorded their activities for 6 minutes. EthoVision® XT behavioral software was used to analyze and statistically determine the time the mice remained immobile in the last 4 minutes. The length of immobility reflected the degree of despair in the mice.

[0033] The results showed that, compared with the PBS control group, mice treated with AKK for 28 days exhibited a significant reduction in immobility time (C57-AKK). vs C57-PBS, P=0.0252; A53T-AKK vs A53T-PBS (P=0.0339) indicates that behavioral despair was significantly improved in both genotype mice after AKK administration. Figure 1 (B)

[0034] Example 4 Sugar Water Preference Experiment Measuring the mice's preference for sucrose solution, the anhedonia caused by a decrease in sucrose water preference is one of the important indicators of depressive-like behavior. Mice from Example 2 were subjected to a sucrose water preference test 40 days after the experiment. Figure 1(A)

[0035] The sucrose preference experiment was conducted as follows: During the first 24 hours of the experiment, mice were given two bottles of solution containing 1% sucrose. In the following 24 hours, for the first 12 hours, mice were given one bottle of water and one bottle of 1% sucrose solution; for the next 12 hours, the positions of the two bottles were swapped to prevent positional preference. During the third 24-hour period, mice were subjected to a 12-hour fasting and water restriction. The consumption of each solution was recorded for the next 12 hours to calculate the sucrose preference rate. The sucrose preference rate (%) = (sucrose solution intake / total fluid intake) × 100%, where total fluid intake = sucrose solution intake + pure water intake.

[0036] The results showed that, compared with the PBS control group, both C57 and A53T mice in the AKK-treated experimental groups showed a greater preference for sucrose water (C57-AKK group vs. C57-PBS group, P=0.0485; A53T-AKK group vs. A53T-PBS group, P=0.0498), indicating that after AKK administration, the reward stimuli were restored in both C57 and A53T mice. Figure 1 (C)

[0037] Example 5: Akkermansia myxophilus improves depressive-like behavior by upregulating 5-HT levels in the hippocampus of Tg mice. All mice following the experiments in Examples 2-4 were anesthetized and perfused with phosphate-buffered saline (PBSS) via cardiac perfusion. Brain tissue was fixed in 4% paraformaldehyde at 4°C for 24 hours. After dehydration in 30% sucrose, the tissue was embedded in OCT embedding medium and cut into 15 μm thick coronal sections using a cryostat. Based on mouse brain stereotaxic atlases, brain sections between -2.06 mm and -2.30 mm in the anterior fontanelle were selected for immunofluorescence staining of the hippocampus. Sections were washed with PBS buffer, permeabilized with 0.3% Triton X-100 for 15 min, and then washed again with PBS buffer. Non-specific binding was blocked with 10% goat serum (diluted in PBS buffer) for 2 hours, followed by incubation overnight at 4°C with a primary antibody (1:200, YA6451, MCE) diluted with 10% goat serum. Sections were washed three times with PBS (5 min each time) and then incubated for 2 hours with secondary antibodies diluted in 10% goat serum (1:500, donkey anti-rabbit IgG 488; donkey anti-mouse IgG 555). After washing with PBS buffer, slides were mounted with anti-fluorescence quenching mounting medium containing DAPI. At least two brain slices from each mouse were taken for quantitative analysis. Images were acquired using a laser scanning confocal microscope and analyzed using ImageJ software.

[0038] The levels of 5-HT in plasma and hippocampus of anesthetized mice were detected using liquid chromatography-mass spectrometry. Results are as follows: Figure 2The results showed that AKK administration significantly increased plasma 5-HT levels in C57 mice compared to the PBS control group (P=0.0043). In contrast, there was no significant difference in plasma 5-HT levels between the AKK and PBS groups in the Tg group. Figure 2 The results showed that AKK administration had no significant effect on hippocampal 5-HT levels in C57 mice compared to the PBS control group. Conversely, in the Tg group, AKK treatment significantly increased hippocampal 5-HT levels (P=0.0301). Simultaneously, the distribution and intensity of 5-HT in the hippocampus could be detected by immunofluorescence staining, as shown in the results below. Figure 2 Tables C and D show that AKK treatment significantly increased 5-HT levels in the hippocampus of Tg group mice compared to the PBS control group (P=0.0034). Conversely, in C57 mice, there was no significant difference in hippocampal 5-HT levels between the AKK and PBS groups. This suggests that regional differences in 5-HT content may be intrinsically linked to a specific genetic background of α-syn overexpression.

[0039] Then, the levels of SCFAs in the plasma of mice with different genotypes were further examined to investigate the role of AKK in regulating 5-HT by modulating metabolites in peripheral plasma.

[0040] Methods for detecting SCFAs in plasma: Sample pretreatment: Weigh hippocampal tissue samples, add 100 μL of double-distilled water, and place in a pre-cooled 4℃ tissue homogenizer. Set the program to 60 Hz, 60 s, 5 times, with 15 s intervals, and homogenize the tissue until no obvious tissue precipitation is observed. Add 400 μL of pre-cooled acetonitrile and sonicate for 15 min. Centrifuge at 12000 rpm, 4℃, for 15 min. Take 200 μL of the supernatant, dry it in a nitrogen stream, and redissolve it in 50 μL of acetonitrile / water (1:9, v / v) solution for LC-MS / MS analysis of neurotransmitters. Take another 200 μL of the supernatant and mix it with 50 μL of acetonitrile / 50 mM NH4HCO3 aqueous solution (1:1, v / v) containing 20 mM EDCI, 20 mM NHS, and 20 mM o-nitrophenylhydrazine. React at 600 rpm and 40 °C. The resulting mixture was diluted 10-fold with 10% acetonitrile aqueous solution, and then centrifuged at 14000 × g for 15 min at room temperature. The supernatant was then transferred to an autosampler for LC-MS / MS analysis of short-chain fatty acids.

[0041] 20 μL of plasma was mixed with 80 μL of pre-cooled acetonitrile and sonicated at 0°C for 15 min, followed by centrifugation at 14000 × g for 20 min at 4°C. 40 μL of the supernatant was transferred to an autosampler vial for LC-MS / MS analysis of neurotransmitters. Another 40 μL of the supernatant was mixed with 10 μL of an acetonitrile / 50 mM NH4HCO3 aqueous solution (1:1, v / v) containing 20 mM EDCI, 20 mM NHS, and 20 mM o-nitrophenylhydrazine, and reacted at 600 rpm and 40°C. The resulting mixture was diluted 10-fold with 10% acetonitrile aqueous solution, followed by centrifugation at 14000 × g for 15 min at room temperature. The supernatant was transferred to an autosampler vial for LC-MS / MS analysis of short-chain fatty acids.

[0042] LC-MS / MS detection protocol: Samples were analyzed using a Nexera LC-40 UPLC system (Shimadzu) coupled with a Triple Quad™ 7500 system (Sciex). Chromatographic separation of neurotransmitters and SCFAs was performed using an HSS T3 column (1.8 μm particle size, 2.1 mm inner diameter × 100 mm length, Waters) and a BEH C18 column (1.7 μm particle size, 2.1 mm inner diameter × 100 mm length, Waters), respectively. The injection volume was 2 μL, and the column temperature was maintained at 40°C. Mobile phases A and B were water and acetonitrile containing 0.1% formic acid, respectively. The flow rate of neurotransmitters was 0.2 mL / min, with the following gradient elution program: 0–2 min, 2% B; 2–7 min, 2–85% B; 7–9 min, 85–90% B; 9–9.1 min, 90–2% B; 9.1–12 min, 2% B. The flow rate of SCFAs was 0.3 mL / min, with the following gradient elution program: 0–2 min, 10%–15% B; 2–11 min, 15–55% B; 11–11.5 min, 55–95% B; 11.5–13 min, 95% B; 13–13.1 min, 95–10% B; 13.1–15 min, 10% B. To avoid contamination of the mass spectrometry system with excessive derivatization reagent, the valve was switched to waste liquid 3 min prior to the event. Data acquisition was performed using multiple reaction monitoring (MRM) mode in positive ion ionization mode (ESI+). The ion source parameters are set as follows: 1) Neurotransmitters: spray voltage 5.5 kV, source temperature 480°C, nebulizer gas 45 psi, auxiliary heating gas 45 psi, curtain gas 40 psi; 2) Short-chain fatty acids: spray voltage 5.5 kV, source temperature 500°C, nebulizer gas 50 psi, auxiliary heating gas 60 psi, curtain gas 40 psi.

[0043] like Figure 2 As shown in Figure E, compared with their respective control groups (C57-AKK) vs C57-PBS, A53T-AKK vs A53T-PBS), AKK increased the concentration of propionic acid in the plasma of mice in the C57 group (P>0.05) and significantly upregulated the concentration of propionic acid in the plasma of A53T α-syn transgenic mice in the Tg group (P=0.0221). Figure 2 As shown in Figure F, AKK increased the concentration of butyrate in the plasma of mice in the C57 group and A53T α-syn transgenic mice in the Tg group.

[0044] Example 6: The remodeling of gut microbiota by *Ackermania myxophilus* is gene-dependent. To further investigate whether AKK regulates central neurotransmitter function by remodeling the gut microbiota, metagenomic sequencing analysis was performed on mouse fecal samples. In Example 2, after 63 days of experimentation, significant changes were observed in the microbial diversity of the mouse gut (e.g., Figure 3 (As shown in AC), the changes observed were particularly pronounced in A53T α-syn transgenic mice in the Tg-PBS and Tg-AKK groups. AKK treatment remodeled the gut microbiome α-diversity of A53T α-syn transgenic mice. Specifically, compared to the Tg-PBS control group, the Tg-AKK group showed significantly increased observed species index and Chao1 index, as well as a significantly increased Shannon index. These changes indicate that AKK intervention increased species richness, estimated total number of taxa, community diversity, and evenness. In contrast, no significant changes were observed in the C57 group mice.

[0045] The composition of the gut microbiota was analyzed at the phylum level (e.g. Figure 3 (D). In C57 mice, AKK treatment significantly enriched Bacteroidota and Actinomycetota. In A53T mice, AKK induced different remodeling patterns, with Bacillota significantly enriched after AKK treatment. Notably, Desulfobacterota and Campylobacterota, involved in sulfur metabolism, were enriched in the Tg-AKK group. Given that Campylobacterota is primarily a sulfur-oxidizing bacterium, while Desulfobacterota reduces sulfate to hydrogen sulfide, this indicates an enhanced sulfur cycling flux in the gut of mice treated with the Tg-AKK group.

[0046] The composition of the gut microbiota was analyzed at the species level (e.g. Figure 3 (Middle E). In C57 mice, AKK treatment significantly enriched […]. Lactobacillus taiwanensis, Lepagella muris and Phocaeicola sartorii. L. taiwanensis (Bacillota phylum, consistent with the overall decline of this phylum but expansion in specific ecological niches) and Bifidobacterium animalis The enrichment of (Actinomycetot phylum) is consistent with the increase in their respective phyla or specific functional niches. L. taiwanensis Known to regulate tryptophan metabolism and produce neuroactive substances, it may be associated with the increased peripheral 5-HT levels observed in C57 mice. In A53T mice, the gut microbiota is predominantly Bacillota. AKK treatment significantly upregulated Lachnospiraceae. COE1 This is a promising producer of butyric acid. Additionally, species from the Bacillota phylum possess anti-inflammatory properties. Lactobacillus It remains highly abundant.

[0047] To identify specific bacterial taxa enriched by AKK in each genotype, we performed LEfSe analysis ( Figure 3 (F and G). In C57 mice ( Figure 3 In the middle F group, pathogenic bacteria were enriched in the control group. Duncaniella dubosii However, this bacterium was inhibited by AKK. The AKK-treated group was enriched with... Lactobacillus taiwanensis, Phocaeicola sartorii and Lachnospira sp Related bacterial species. These groups include known SCFA-producing bacteria and tryptophan-metabolizing bacteria. L. taiwanensis The specific enrichment of [a specific substance] was associated with elevated plasma 5-HT levels in C57 mice, suggesting that the mechanism is driven by peripheral tryptophan conversion. In A53T mice ( Figure 3 In the AKK treatment group (red column), biomarker characteristics showed significant differences. Multiple butyrate-producing Lachnospiraceae (including COE1 sp000403215, UBA3402 sp910588505, and UBA3282 sp009774655) and other organisms involved in polysaccharide degradation were observed. Duncaniella Both *D. muris* and *D. dubosii* were significantly enriched. Conversely, the Tg-PBS control group (blue column) was enriched. Muribaculum Notably, the significant enrichment of Lachnospiraceae in Tg-AKK mice provides a microbiological basis for the observed elevation of hippocampal 5-HT. Butyrate, a major metabolite of these bacteria, can cross the blood-brain barrier and has been shown to stimulate central serotonin synthesis and reduce neuroinflammation. Compared to C57 mice, the latter's mechanism appears to be more peripherally dependent. Lactobacillus Mediated tryptophan metabolism.

[0048] The above results indicate that the effects of AKK on the gut microbiota are genotype-dependent. In C57 mice, AKK promotes a microbiota predominantly composed of Bacteroides and enriched with Lactobacillus, potentially driving peripheral 5-HT synthesis. In A53T transgenic mice, AKK enhances a microbiota predominantly composed of Bacteroidota and specifically enriches with butyrate-producing Lachnospiraceae.

[0049] Example 7 Genotype-dependent remodeling of gut microbiota functional potential and metabolic pathways by *Ackermania myxophilus* To elucidate the metabolic mechanism by which AKK improves host neurological function through gut microbiota remodeling, a multidimensional comparative analysis of KEGG orthologs and metabolic modules was conducted based on metagenomic sequencing data among different genotypes (wild-type C57 and transgenic A53T) and treatment groups (PBS and AKK). The results showed that AKK-mediated functional remodeling is strictly genotype-dependent, triggering different metabolic reprogramming strategies under healthy and pathological backgrounds.

[0050] In wild-type C57 mice, AKK administration by gavage for 56 days induced widespread and significant alterations in transcriptional function. Figure 4 (A and C in the middle). At the genetic level ( Figure 4 Compared with the PBS control group, the C57-AKK group showed significant enrichment of several key metabolic genes, including K05523 (D-lactate dehydrogenase) and K01744 (aspartate aminolysin), indicating enhanced lactate utilization and replenishment of tricarboxylic acid cycle intermediates. Simultaneously, the upregulation of threonine-3-dehydrogenase (K15789) and dipeptidyl peptidase 4 (K01278, DPP4) suggests a redirection of nitrogen metabolism and potential activation of the GLP-1 signaling axis. At the pathway level ( Figure 4 These gene-level changes translate into a clear redirection of metabolic pathways: the tricarboxylic acid cycle module (M00011), representing typical aerobic oxidation, is significantly suppressed, while the aspartate degradation module (MF0012) and neurotransmitter synthesis modules, including the serotonin synthesis module (MGB001) and the GABA synthesis module (MGB022), are significantly suppressed. Figure 4 AKK was significantly enriched in the gut microbiota (E). These data indicate that, under healthy physiological conditions, AKK drives a shift in the gut microbiota from an "energy oxidation mode" to a "neurometabolic signal transduction mode," promoting the synthesis of neuroactive substances. This is consistent with previous observations of elevated peripheral 5-HT levels and increased tryptophan metabolism in C57 mice. L. taiwanensis The enrichment results are consistent.

[0051] In contrast, A53T α-syn transgenic PD mice exhibited a different response pattern, which can be summarized as "gene-level silencing and pathway-level specific activation." Notably, in intragroup comparisons of A53T mice (Tg-PBS vs. Tg-AKK), no significantly differentially enriched KEGG orthologs were detected, a stark contrast to the widespread gene expression changes observed in C57 mice. This suggests that, under pathological conditions, AKK does not function by significantly altering the abundance of individual functional genes; its regulatory mechanism may be more refined, involving functional redundancy or synergistic changes below the statistical threshold for single genes.

[0052] However, although no significant fluctuations have been observed at the single-gene level, highly specific functional reprogramming has been detected at the level of higher-order metabolic modules. Figure 4 (D). It is worth noting that, Figure 4 Figure B demonstrates the inherent functional profile differences between C57 and A53T mice under the same intervention (AKK treatment), highlighting the decisive influence of genotype on the baseline of microbial function. In the A53T model's specific functional response ( Figure 4 The Tg-AKK group (blue bar) characteristically exhibited significant enrichment of the purine degradation modules M00958 (adenine ribonucleotide degradation, AMP → uric acid) and M00959 (guanine ribonucleotide degradation, GMP → uric acid). These two modules are responsible for the final conversion of AMP and GMP into uric acid. This finding reveals a key biological phenomenon: in PD models, AKK may achieve systemic activation of specific metabolic pathways (purine degradation) without causing drastic statistical changes in overall KO gene abundance by finely regulating the expression ratios of existing microbial genes, activating low-abundance key strains, or promoting metabolic synergy. Given the oxidative stress induced by high levels of α-synuclein aggregation in A53T mice, enhanced purine metabolism may lead to elevated uric acid levels. Uric acid, as a potent endogenous antioxidant, may play a neuroprotective role here and synergistically interact with the aforementioned increase in hippocampal 5-HT levels, jointly constituting the metabolic basis of antidepressant activity.

[0053] Furthermore, unlike the C57-AKK group, which is enriched in neurotransmitter synthesis modules, the overall metabolic profile of the Tg-AKK group is dominated by basal metabolism and short-chain fatty acid production modules, especially propionic acid production module (MF0123), as well as extensive degradation of amino acids such as tyrosine, methionine, and glutamic acid. Figure 4 (E). This result is consistent with... Figure 3The highly correlated specific enrichment of butyrate / propionic acid-producing bacteria (such as Trichophytonceae) observed in the Tg-AKK group further confirms that short-chain fatty acids may be key effector molecules mediating changes in blood-brain barrier permeability and central 5-HT synthesis in this model. Cross-genotype comparisons also revealed that the DNA repair-related gene K10979 (Ku protein) showed a relative enrichment trend only in the Tg-AKK group. Figure 4 (B) This further suggests the adaptive response of microorganisms to genotoxic stress under pathological conditions.

[0054] In summary, functional enrichment analysis revealed distinct metabolic reprogramming between the two genotypes after AKK intervention. In wild-type C57 mice, AKK primarily enriched pathways involved in tryptophan metabolism and serotonin biosynthesis, which is consistent with observed... L. taiwanensis The abundance increase was consistent. In contrast, in A53T transgenic mice, AKK was primarily enriched in pathways associated with short-chain fatty acid production (particularly propionate), which was associated with the amplification of Trichophyceae species and subsequent increases in 5-HT levels in the hippocampus.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 drugs for improving and / or treating Parkinson's disease with depression.

2. The application according to claim 1, characterized in that, The Parkinson's disease with depression mentioned refers to age-related or Parkinsonian depressive-like behaviors associated with α-synucleosis.

3. The application according to claim 1, characterized in that, The *Ackermania* strain described is ATCC BAA-835.

4. The application according to claim 1, characterized in that, Akkermansia mycotoxin can regulate peripheral-central 5-HT levels or enhance the absorption of intestinal SCFAs.

5. The application according to claim 1, characterized in that, The dosage form of the drug is lyophilized powder, liquid bacterial suspension, tablet or capsule.

6. A mixed microbial agent, characterized in that, This includes *Ackermania glutinis* and *butyric acid-producing bacteria* in the applications described in any one of claims 1 to 5.

7. The mixed microbial agent according to claim 6, characterized in that, The intestinal butyric acid-producing bacteria are at least one strain from the Trichophyceae family.

8. The use of the mixed bacterial agent as described in claim 6 in the preparation of a medicament for improving and / or treating Parkinson's disease with depression.

9. A composition, characterized in that, Includes *Ackermania gravidarum* and butyrate precursors in the applications described in any one of claims 1 to 5.

10. Use of the composition of claim 9 in the preparation of a medicament for improving and / or treating Parkinson's disease with depression.

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