Application of ackermania muciniphila and recombinant trichina serine protease carried by ackermania muciniphila derivative in preparation of medicine for treating intestinal inflammation symptoms

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of ackermania muciniphila and recombinant trichina serine protease carried by a derivative of the ackermania muciniphila to preparation of a medicine for treating intestinal inflammation symptoms. According to the present invention, the ackermann muciniphila and the ackermann muciniphila derivative carrying recombinant trichina serine are firstly disclosed, such that the physical barrier of the intestinal mucosa is repaired through the synergistic effect, the expression of TNF-alpha, IL-6, IL-1beta and other proinflammatory factors is specifically regulated and inhibited, the intestinal flora structure is remodeled, and the intestinal inflammation state is significantly improved; experimental results show that the Ackerman muciniphila and the derivative thereof carrying the recombinant trichina serine protease can significantly relieve weight loss of mice with intestinal inflammation induced by DSS, reduce DAI score, and reverse the phenomena of shortened colon and abnormal swelling of spleen. According to the invention, the probiotics and the parasite-derived protein are combined, so that the method has the advantages of high efficiency, safety and easiness in large-scale production, and a new biological preparation strategy is provided for accurate treatment of IBD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Akkermansia myxophila and its derivatives loaded with recombinant Trichinella serine protease in the preparation of drugs for treating enteritis symptoms. Background Technology

[0002] Inflammatory bowel disease (IBD), primarily including ulcerative colitis and Crohn's disease, is a global disease characterized by chronic, relapsing intestinal inflammation. The pathogenesis of IBD is complex, involving multiple factors such as genetic susceptibility, environmental factors, gut microbiota dysbiosis, and abnormal activation of the host immune system. While existing drugs (such as aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and biologics) have some efficacy, they generally suffer from inconsistent response rates, secondary failure, increased risk of infection, and potential side effects.

[0003] Relevant technical searches revealed that gut microbiota dysbiosis is also considered an important environmental factor in the occurrence and development of IBD. The gut microbiota not only participates in the development and regulation of the immune system, but its structural abnormalities can also directly or indirectly exacerbate intestinal inflammatory responses. Akkermansia muciniphila (A. muciniphila), as a core symbiotic bacterium of the gut, has shown a protective effect in various IBD disease models, but its regulatory mechanism is singular, relying solely on a complex inter-species regulatory approach. Summary of the Invention

[0004] To address the issues of weak efficacy and limited regulatory mechanisms in related technologies and drugs, this invention first provides the application of Akkermansia myxophila and its derivatives loaded with recombinant Trichinella serine protease in the preparation of drugs for treating enteritis symptoms, and briefly introduces its effects.

[0005] The inventors discovered through research that the incidence of IBD exhibits significant geographical differences. It is more common in developed countries with better healthcare systems; while in developing countries and regions with higher rates of parasitic infection, its incidence is generally lower, suggesting a possible negative correlation between parasitic infection and IBD risk. This, combined with existing research supporting the hygiene hypothesis that parasitic worms and their metabolites are crucial for the development and regulation of the immune system, suggests that their absence may be related to immune system imbalances and an increased incidence of autoimmune diseases.

[0006] Therefore, based on the ability of *Akermansia myxophilus* to maintain intestinal mucosal barrier function, improve host metabolism, and regulate immune balance, the inventors combined it with a parasite-derived functional protein, namely recombinant trichinella serine protease. Trichinella spiralisIt organically combines with serine protease (rTsSP) to restore intestinal homeostasis and improve IBD symptoms through the synergistic effect of bidirectional immune regulation.

[0007] The specific technical solution is as follows: According to one aspect of the present invention, the use of Akkermansia myxophila and its derivatives loaded with recombinant Trichinella serine protease in the preparation of a drug for treating symptoms of enteritis is first disclosed.

[0008] Furthermore, the Akkermansia myxophilus derivative is selected from one of the following: supernatant of bacterial culture, outer membrane vesicles derived from Akkermansia myxophilus, and protein extracted from Akkermansia myxophilus.

[0009] In some embodiments, the *Ackermania* strain is a genetically engineered strain; the modification is intended to enhance the colonization ability, acid production capacity, or immunomodulatory function of the strain; the strain has the accession number ATCCBAA-835.

[0010] Furthermore, the recombinant Trichinella serine protease is obtained by recombinant expression and purification of a serine protease derived from Trichinella in a gene engineering expression system; the gene engineering expression system is selected from either a prokaryotic expression system or a eukaryotic expression system.

[0011] Furthermore, the Ackermansia myxophila and its derivatives, carrying recombinant Trichinella serine protease, exert their effects through at least one of the following mechanisms: repairing the intestinal mucosal physical barrier, inhibiting the expression of pro-inflammatory factors, and promoting the expression of anti-inflammatory cytokines.

[0012] Furthermore, the pro-inflammatory factors include TNF-α, IFN-γ, IL-6, IL-17, or IL-1β; the anti-inflammatory factors include IL-4, IL-10, IL-22, or TGF-β.

[0013] In some embodiments, the Ackermansia myxophila and its derivatives, loaded with recombinant Trichinella serine protease, exert their effects by inhibiting the expression of pro-inflammatory cytokines TNF-α, IL-6, or IL-1β.

[0014] In some embodiments, the Ackermansia myxophila and its derivatives, loaded with recombinant Trichinella serine protease, exert their effects by promoting the expression of the anti-inflammatory factor IL-10.

[0015] According to another aspect of the present invention, a pharmaceutical composition for treating symptoms of enteritis is provided, comprising an effective therapeutic amount of Akkermansia myxophilus and its derivatives, a therapeutically effective amount of recombinant Trichinella serine protease, and pharmaceutically acceptable excipients; the dosage form of the pharmaceutical composition is an oral formulation, an enema formulation, or a suppository.

[0016] Further, the *Ackermania pseudomallei* and its derivatives are mixed with the recombinant *Trichinella spiralis* serine protease in a single formulation at a fixed dosage ratio; the dosage range is *Ackermania pseudomallei* (or equivalent derivatives): recombinant *Trichinella spiralis* serine protease = (1 × 10⁻⁶) / ( ... 7 -1×10 11 CFU / mL): (1-1000 μg / mL).

[0017] Furthermore, the Ackermansia myxophila and its derivatives and the recombinant Trichinella serine protease are prepared into separate formulations and used sequentially or simultaneously.

[0018] Finally, the present invention also provides a method for preparing the pharmaceutical composition, comprising the following steps: S1. Under anaerobic conditions, Akkermansia myxophilus is cultured and centrifuged to collect the bacterial cells for freeze-drying protection to prepare live bacterial preparations, or inactivated bacterial cells are prepared by heat treatment, irradiation or other methods, or its derivatives are collected by ultracentrifugation or other methods to prepare Akkermansia myxophilus active ingredients. S2. The gene encoding Trichinella spiralis serine protease was cloned into an expression vector, transformed into an expression host for induced expression, and high-purity recombinant Trichinella spiralis serine protease was obtained by cell disruption, centrifugation, and chromatography purification. S3. The active ingredient of Akkermansia myxophila obtained in step S1 and the recombinant Trichinella serine protease obtained in step 2 are mixed with pharmaceutical excipients in a certain proportion to prepare the desired dosage form.

[0019] The application of Akkermansia myxophilus and its derivatives, as disclosed in this invention, in the preparation of drugs for treating enteritis symptoms by carrying recombinant Trichinella serine protease, organically combines probiotics with intestinal repair and immune regulation functions with parasite-derived proteins with specific immune regulation potential, thereby exerting a synergistic effect of multiple targets and pathways. This solves the problems of weak efficacy and single regulatory mechanism of related technologies and provides a new technical means with great application prospects for the clinical treatment of IBD.

[0020] Compared with related technologies, the present invention has at least the following beneficial effects: I. Synergistic Repair of Intestinal Barrier: Akkermansia myxophilus and its derivatives can promote mucin secretion and enhance the expression of tight junction proteins, thereby directly repairing the damaged physical barrier of the intestinal mucosa; recombinant Trichinella serine protease can regulate the inflammatory environment to create suitable conditions for barrier repair, thereby synergistically repairing the intestinal barrier with Akkermansia myxophilus.

[0021] II. Bidirectional Immune Regulation: Both *Ackermania myxophilus* and its derivatives, and recombinant *Trichinella spiralis* serine proteases, can reduce the secretion of pro-inflammatory factors or increase the production of anti-inflammatory factors to varying degrees. This invention leverages the synergistic effect of combining these two agents to upregulate the expression of the anti-inflammatory factor IL-10 while significantly downregulating the expression of pro-inflammatory factors IL-6, IL-1β, and TNF-α, thereby reversing the imbalanced intestinal immune response towards an anti-inflammatory tolerance state.

[0022] III. Improved efficacy and safety: By using engineered recombinant proteases to avoid the risk of infection by directly using live parasites, the structure of the intestinal microbiota is regulated. Through endogenous probiotics, multi-target intervention is achieved, which effectively prolongs the effect, thereby reducing the dosage of active ingredients, reducing potential toxic side effects, and improving human safety. Attached Figure Description

[0023] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0024] Figure 1 Characterization diagram of *Ackermania myxophilus* and its derivatives with recombinant *Trichinella spiralis* serine protease. In the diagram, A represents extracellular vesicles of *Ackermania myxophilus*. Akk B is an electron microscopy image of the recombinant Trichinella serine protease (scale bar = 200 nm); B is an SDS gel electrophoresis protein map of the recombinant Trichinella serine protease.

[0025] Figure 2 The intervention effect of Akkermansia myxophilus and its derivatives combined with recombinant Trichinella serine protease on DSS-induced inflammatory bowel disease in mice. In the figures, A is a graph showing the trend of mouse body weight change; B is a line graph of the disease activity index (DAI); C is a schematic diagram of changes in mouse colon length; D is a bar graph of changes in mouse colon length; E is a schematic diagram of changes in mouse spleen mass; and F is a bar graph of changes in mouse spleen mass.

[0026] Figure 3 Image showing the H&E staining results of mouse colon tissue sections. In the image, A is a schematic diagram of H&E staining of colon tissue sections; B is a bar chart showing the statistical scores of colon tissue pathology.

[0027] Figure 4 Image showing PAS staining results of mouse colon tissue sections. In the image, A is a schematic diagram of PAS staining of colon tissue sections; B is a bar chart of quantitative analysis of colon goblet cells.

[0028] Figure 5The results of quantitative analysis of cytokines are shown in the following figures. A represents the statistical bar chart of IL-6 activity; B represents the statistical bar chart of IL-10 activity; C represents the statistical bar chart of IL-1β activity; and D represents the statistical bar chart of TNF-α activity.

[0029] Figure 6 Bar chart of species abundance in mouse gut microbiota.

[0030] Figure 7 Schematic diagram of mouse gut microbiota diversity analysis. A represents the analysis of the Alpha diversity index Chao1; B represents the Alpha diversity index Shannon; C represents the Alpha diversity index Simpson; and D represents the PcoA analysis based on the Bray-Curtis distance.

[0031] Figure 8 A schematic diagram of the LefSe analysis results. Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0033] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification, claims and accompanying drawings of this invention are intended to cover non-exclusive inclusion, such as a process, method, system, product or device that includes a series of steps or units, which is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products or devices.

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0035] Example 1: Preparation of extracellular vesicles of *Ackermania pseudomallei* and recombinant *Trichinella spiralis* serine protease 0. Culture of Akkermansia muciniphila: Akkermansia muciniphila (A. muciniphila 33894T, same as ATCC BAA-835, purchased from the Japan Microbiological Collection Center) was inoculated into synthetic medium and cultured anaerobically at 37°C until the logarithmic growth phase (OD200). 600After measuring approximately 0.6-0.8 g, the cells were centrifuged at 5000 × g for 15 min. The cells were then collected, resuspended in anaerobic PBS, and the concentration adjusted to 1 × 10⁻⁶ g. 8 CFU / mL. A portion of the bacterial suspension was pasteurized by heating it in a 70℃ constant temperature water bath for 30 min to obtain pasteurized Akkermansia myxophilia.

[0036] It should be noted that the formulation of the synthetic culture medium is as follows: 38.5 g / L brain heart extract broth, 16 g / L soybean peptone, 11.3 g / L anhydrous glucose, 5.5 g / L N-acetylglucosamine, 4 g / L L-threonine, and 0.5 g / L L-cysteine.

[0037] 1. Ackermansia muciniphila external vesicles ( Akk Preparation of -Evs): The supernatant of *Akermansia myxophilus* culture was centrifuged at 6000 g for 15 min at 4°C in a refrigerated centrifuge. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtrate was centrifuged at 100000 g for 2 h in a refrigerated centrifuge at 4°C, and the resulting precipitate was collected as Evs. Akk -Evs. Transmission electron microscopy (TEM) was used to examine... Akk -Evs was used for observation, and the results were as follows Figure 1 As shown in Figure A, it exhibits a typical double-membrane vesicle structure. After correct identification, it was prepared into a 50 μg / mL solution using PBS.

[0038] 2. Preparation of recombinant Trichinella spiralis serine protease (rTsSP): Trichinella spiralis serine protease genes were screened from the NCBI database, cloned into the pET28b expression vector, and a His tag was introduced before transformation into *E. coli* BL21. The expression conditions were then optimized, with the optimal conditions being 37°C and induction with 0.5 mM IPTG for 4 h. Based on this, the culture was expanded, and the bacterial cells were collected, sonicated, and purified using nickel column affinity chromatography. Endotoxins were removed using the Triton-114 phase separation method, finally obtaining the rTsSP protein suitable for subsequent experiments. The rTsSP protein was verified by SDS-PAGE electrophoresis as follows. Figure 1 As shown in B, there is a distinct single band at 35 kDa.

[0039] Example 2 Akk -Evs with rTsSP protein for synergistic treatment of acute IBD in mice 3. Animal Adaptation and Grouping: Fifty-six SPF-grade male C57BL / 6 mice (6-8 weeks old) were purchased from the Experimental Animal Center of Hubei University of Medicine. All animal experiments followed the guidelines of the institution's Animal Care and Use Committee. The mice were acclimatized for one week in an SPF-grade animal facility (12 / 12 h light / dark cycle, free access to water and food, room temperature 21-25℃). Subsequently, the 56 male mice were randomly divided into 5 groups (8-12 mice per group): Control group; DSS enteritis model group (DSS); DSS enteritis model + recombinant Trichinella serine protease treatment group (DSS + rTsSP); DSS enteritis model + recombinant Trichinella serine protease + Akk -Evs processing group (DSS + rTsSP + Akk -Evs); DSS enteritis model + recombinant Trichinella serine protease + pasteurized inactivated A. muciniphila treatment group (DSS + rTsSP + A. muciniphila).

[0040] 4. Model Establishment and Dosing Regimen: 21, 14, and 7 days prior to DSS modeling, DSS + rTsSP and DSS + rTsSP + [other drugs / treatments] were administered [to the target group / treatment]. Akk Mice in three groups—Evs, DSS + rTsSP + A. muciniphila—were intraperitoneally immunized three times with recombinant Trichinella spiralis serine protease (20 μg per mouse, dissolved in 100 μL PBS). The remaining groups received an equal volume of PBS solution. Mice were given 3% dextran sulfate sodium (DSS) solution for modeling, with free access to drinking water for 7 consecutive days (except for the control group). The normal control group received only regular drinking water throughout the process. Akk The Evs group and the DSS + rTsSP + A.muciniphila group were administered daily via gavage at regular intervals. Akk -Evs (50 μg / animal) and pasteurized inactivated A. muciniphila (1× 10⁻⁶) 8 CFU / each).

[0041] 5. Clinical Symptom Assessment (DAI Score): During the observation period, the mouse's body weight, fecal shape, and fecal blood loss were recorded daily. The specific scoring criteria are as follows: weight loss (no decrease 0, 1%-5% 1, 5%-10% 2, 10%-20% 3, >20% 4); fecal consistency (dry 0, formed but not sticky 1, loose 2, loose stool 3, watery stool 4); fecal blood loss (no 0, occult blood 1, visible bloodstains 2, obvious bleeding 4).

[0042] DAI scoring results statistics are as follows: Figure 2As shown, compared with the DSS model group, the DSS+rTsSP group, DSS+rTsSP+ Akk Mice in the -Evs group and the DSS+rTsSP+A. muciniphila group recovered faster after weight loss and had significantly lower Disease Activity Index (DAI) scores. The DSS model group showed significant colonic and splenomegaly, compared to the DSS+rTsSP group. Akk The colon length of mice in the -Evs group and the DSS+rTsSP+A. muciniphila group was significantly increased and the compensatory splenic swelling was significantly reduced, indicating that the systemic inflammatory state was significantly improved.

[0043] Example 3 Histopathological Analysis 6. Sample Processing: After DSS modeling was completed, all mice were given regular drinking water and kept in the water for 3 days before being euthanized. Colon tissue from each group of mice was collected, fixed in 4% paraformaldehyde, and then dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin.

[0044] 7. Staining procedure: After sectioning, H&E staining (Wuhan Sewell, GP1136) and PAS staining (Wuhan Sewell, catalog number GP1039) were performed respectively. The stained sections were then observed under an optical microscope (Olympus BX53F) and images were acquired using Olympus cellSens Standard 1.13 software.

[0045] 8. Results Analysis: H&E-stained sections were scored histopathologically based on the degree of epithelial damage and inflammatory infiltration. The results are as follows: Figure 3 As shown, it can be seen Figure 3 In group A, the DSS group exhibited obvious characteristics of enteritis, with disordered crypt structure and extensive infiltration of inflammatory cells such as neutrophils; however, the inflammatory condition improved after the addition of rTsSP; DSS+rTsSP+ Akk Compared with the DSS+rTsSP+A. muciniphila group, the DSS group showed significant repair of intestinal mucosal damage, with a marked reduction in the depth and extent of neutrophil infiltration and reconstruction of crypt structure. Its histopathological score was as follows: Figure 3 The results showed that B was significantly lower than that of the rTsSP group alone, which effectively demonstrated that Akkermansia myxophilus and recombinant Trichinella serine protease have a significant synergistic effect in repairing physical damage to colonic tissue and have a positive intervention effect on DSS-induced inflammatory bowel disease in mice.

[0046] PAS staining analysis results are as follows: Figure 4 As shown in Figure A, the number of goblet cells in the DSS model group is greatly reduced, and the mucus layer is thinned or even absent. Figure 4B is a goblet cell count statistical graph, showing DSS+rTsSP+ Akk Compared with the DSS+rTsSP+A. muciniphila group, the goblet cell density in the DSS group was significantly increased, and the number of goblet cells was higher than that in the rTsSP group alone. This indicates that Akkermansia mucinophilus and recombinant Trichinella serine protease can effectively promote mucin secretion, reshape the intestinal mucus barrier, and prevent pathogen invasion, thus ensuring the safety of the intestinal environment.

[0047] Example 4: Analysis of relative expression levels of cytokines 9. RNA Extraction: Mouse colon tissue samples were cleaved and placed in 1.5 mL EP tubes. 1 mL of Trizol lysis buffer was added, and the mixture was homogenized. 200 μL of chloroform was added, and the mixture was vigorously vortexed and incubated at room temperature (25°C) for 10 min. The mixture was then centrifuged at 12000 g for 15 min at 4°C. The supernatant was transferred to a new EP tube, and an equal volume of isopropanol was added. The mixture was vortexed and incubated at room temperature (25°C) for 10 min. The mixture was then centrifuged at 12000 g for 15 min at 4°C, and the supernatant was discarded. 1 mL of 75% ethanol was added to wash and precipitate the RNA. The mixture was then centrifuged again at 12000 g for 10 min at 4°C, and the supernatant was discarded. This process was repeated once. After air-drying for 5 min, 20 μL of ddH2O was added to obtain the RNA.

[0048] 10. Reverse transcription: Prepare the mixture in a 200 μL EP tube as shown in Table 1: Table 1. 10 μL reverse transcription system After mixing by pipetting, centrifuge and set the PCR program to 42℃ for 2 min. Prepare the mixture in 200 μL E tubes as shown in Table 2.

[0049] Table 2 cDNA synthesis reaction solution 20 μL system After mixing by pipetting, centrifuge and set the PCR program to 37℃ for 15 min, then 85℃ for 5 s.

[0050] 11. Quantitative Detection of Cytokines: The expression levels of multiple cytokine (IL-6, IL-10, IL-1β, Tnf) genes were detected by real-time quantitative PCR using the SYBR Green qPCR kit (Beijing Zhuangmeng). The PCR reaction system was prepared according to the instructions, as shown in Table 3.

[0051] Table 3 20 μL q-PCR reaction system The PCR program was set to 95℃, 30 s → (95℃, 3 s → 60℃, 30 s) cycles for 35-40 cycles. GAPDH was used as an internal control, and the relative expression levels of each cytokine mRNA were calculated using the 2-ΔΔCt method. Intergroup comparisons were performed using t-tests or one-way ANOVA; p < 0.05 was considered statistically significant.

[0052] 12. Results Analysis: Gene expression levels, such as... Figure 5 As shown, DSS induction significantly upregulated the expression of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the mouse colon while downregulating the expression of IL-10. The rTsSP group, rTsSP+A. muciniphila group, or rTsSP+ Akk The results from the -Evs group showed that intervention with Akk and rTsSP significantly reversed the high expression of the aforementioned pro-inflammatory factors, restoring them to normal levels. It also promoted, but not significantly, the upregulation of IL-10 levels. This effectively demonstrates that Akkermansia myxophilus combined with recombinant Trichinella serine protease can exert a protective effect against DSS-induced colitis by inhibiting the expression of pro-inflammatory factors.

[0053] Example 5: 16S rRNA sequencing of gut microbiota 13. Sample Preparation: Mix mouse fecal samples with PBS solution at a ratio of 1:9, centrifuge at 3000×g for 3 min, and collect the supernatant to obtain bacterial culture. Extract bacterial DNA using a plasmid mini-prep kit (Beijing Tiangen). Add the above bacterial culture to an EP tube, centrifuge at 12000 rpm for 1 min, discard the supernatant and retain the bacterial precipitate. Add 150 µL of P1 reagent and gently pipette the mixture with the colonies at the bottom of the tube until it turns a light pink color. Add 150 µL of P2 reagent and gently shake the tube 3-5 times until a purple color reaction occurs. Add 350 µL of P5 reagent and quickly invert the tube 3-5 times until the liquid in the tube turns yellow and flocculent precipitate appears. Centrifuge at 12000 rpm for 5 min. Place the plasmid extraction column into a 2 mL recovery tube, pour the supernatant into the column, incubate at 37°C for 5 min, centrifuge at 12000 rpm for 1 min, and discard the lower layer. Add 300 µL of PWT washing buffer to the column, centrifuge at 12000 rpm for 1 min, and discard the lower layer. Add 700 µL of DNA washing buffer, centrifuge at 12000 rpm for 1 min, discard the lower layer, and incubate at room temperature (25°C) for 5 min. Take a 1.5 mL EP tube and place the dried adsorption column into it. Evenly add 30 μL of ddH2O to the adsorption column membrane, incubate at 37°C for 5 min, centrifuge at 12000 rpm for 5 min, and collect the lower layer.

[0054] 14. 16S rRNA Sequencing: PCR amplification was performed on the V3-V4 hypervariable region of the 16S rRNA gene, followed by library construction and NovaSeq 6000 sequencing. After quality control, data analysis was conducted using the Novogene Cloud platform (QIIME2 software, R software). Tukey's test was used to compare differences in alpha diversity indices (including Chao1 and Shannon indices) among groups; principal coordinate analysis (PcoA) was performed based on Bray-Curtis distance. Furthermore, LDA effect size (LefSe) analysis was used to screen for statistically significant microbial biomarkers among multiple groups.

[0055] 15. Results Analysis: The bar chart of species abundance in the mouse gut microbiota is shown below. Figure 6As shown, at the phylum level, the main gut microbiota in each group were Firmicutes (Bacillota), Bacteroidetes, Proteobacteria, Campylobacterota, and Thermodesulfobacteriota, accounting for a total of 95%. Compared with the Control group, the proportions of Bacteroidetes and Campylobacteria were decreased in the DSS group, while the proportions of Proteobacteria and Thermodesulfobacteriota were increased. Intervention with rTsSP reversed these changes, effectively inhibiting the decrease in Bacteroidetes and the increase in Proteobacteria and Thermodesulfobacteriota. Based on the DSS group, rTsSP combined with... Akk -Evs intervention further reduced the proportion of Bacteroidetes; while the effect of rTsSP combined with pasteurized A. muciniphila intervention was similar to that of TsSP alone, inhibiting the spread of conditionally pathogenic bacteria in the gut and maintaining the safety of the intestinal environment.

[0056] Analysis of gut microbiota diversity in mice, such as Figure 7 As shown, significant differences exist between the Control group and the DSS group, and between the Control group and each intervention group, in multiple gut microbiota α diversity indices. Compared with the DSS group, rTsSP combined with Akk - The Evs intervention further significantly reduced the Shannon index. PcoA analysis based on Bray-Curtis distance indicated that the Control group was significantly separated from the DSS group and each intervention group in terms of gut microbiota structure, while there was some overlap between the DSS group and each intervention group. This suggests that the combined intervention significantly alleviated the reduction in gut microbiota diversity caused by intestinal inflammation.

[0057] LefSe analysis such as Figure 8 As shown, 14 ASVs in the Control group exhibited significant differences, with significant enrichment in the families Muribaculaaceae, Prevotellaceae, and Akkermansiaceae. The six ASVs playing a major regulatory role in the DSS were primarily concentrated in the families Lachnospiraceae and Erysipelatoclostridiaceae. After rTsSP intervention, the families Bacteroidaceae and the genera Bacteroides were most prominent; rTsSP+ AkkIn the -Evs group, the Enterobacteriaceae family and the Escherichia-Shigella genus were most significantly enriched; in the rTsSP+A. muciniphila group, the Mycoplasmataceae family was significantly enriched. The enrichment of beneficial bacteria genera indicates that Akkermansia myxophilus carrying recombinant Trichinella serine protease can not only improve the mucosal barrier and cytokine expression levels through specific immunomodulation, but also improve the intestinal microecological environment and provide long-term protection.

[0058] The above description represents the preferred embodiments of the present invention. It should be noted that all reagents and materials used in the embodiments of the present invention, unless otherwise specified, are standardized products that can be obtained through conventional commercial channels. However, it should be understood that those skilled in the art may choose equivalent products from other suppliers, which does not depart from the scope of protection of the present invention. For those skilled in the art, several improvements and modifications can be made without departing from the principles described in the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Akkermansia myxophilus and its derivatives in the preparation of drugs for treating enteritis symptoms by loading recombinant Trichinella serine protease.

2. The application according to claim 1, characterized in that, The Akkermansia myxophilus derivative is selected from one of the following: supernatant of bacterial culture, outer membrane vesicles derived from Akkermansia myxophilus, and protein extracted from Akkermansia myxophilus.

3. The application according to claim 1, characterized in that, The recombinant Trichinella serine protease is obtained by recombinant expression and purification of a serine protease derived from Trichinella in a gene engineering expression system; the gene engineering expression system is selected from either a prokaryotic expression system or a eukaryotic expression system.

4. The application according to claim 1, characterized in that, The Ackermansia myxophila and its derivatives, carrying recombinant Trichinella serine protease, exert their effects through at least one of the following mechanisms: repairing the intestinal mucosal physical barrier, inhibiting the expression of pro-inflammatory factors, and promoting the expression of anti-inflammatory cytokines.

5. The application according to claim 4, characterized in that, The pro-inflammatory factors include TNF-α, IFN-γ, IL-6, IL-17, or IL-1β; the anti-inflammatory factors include IL-4, IL-10, IL-22, or TGF-β.

6. A pharmaceutical composition for treating symptoms of enteritis, characterized in that, The pharmaceutical composition comprises an effective therapeutic amount of Akkermansia myxophilus and its derivatives, a therapeutically effective amount of recombinant Trichinella serine protease, and pharmaceutically acceptable excipients; the dosage form of the pharmaceutical composition is an oral preparation, an enema preparation, or a suppository.

7. The pharmaceutical composition according to claim 6, characterized in that, Said Akkermansia muciniphila and its derivatives are mixed in a single preparation with said recombinant Trichinella serine proteinase in a fixed ratio of doses; said doses range from Akkermansia muciniphila (or equivalent derivative): recombinant Trichinella serine proteinase = (1 x 10 7 -1 x 10 11 CFU / mL): (1-1000 μg / mL).

8. The pharmaceutical composition according to claim 6, characterized in that, The Ackermansia myxophila and its derivatives, along with the recombinant Trichinella serine protease, are prepared as separate formulations and used sequentially or simultaneously.

9. A method for preparing a pharmaceutical composition according to any one of claims 6-8, characterized in that, Includes the following steps: S1. Under anaerobic conditions, Akkermansia myxophilus is cultured and centrifuged to collect the bacterial cells for freeze-drying protection to prepare live bacterial preparations, or inactivated bacterial cells are prepared by heat treatment, irradiation or other methods, or its derivatives are collected by ultracentrifugation or other methods to prepare Akkermansia myxophilus active ingredients. S2. The gene encoding Trichinella spiralis serine protease was cloned into an expression vector, transformed into an expression host for induced expression, and high-purity recombinant Trichinella spiralis serine protease was obtained by cell disruption, centrifugation, and chromatography purification. S3. The active ingredient of Akkermansia myxophila obtained in step S1 and the recombinant Trichinella serine protease obtained in step 2 are mixed with pharmaceutical excipients in a certain proportion to prepare the desired dosage form.

Images