Application of extracellular vesicles of ackermania muciniphila in preparation of trichina control product

Abstract

The invention relates to the technical field of biological medicines, in particular to application of extracellular vesicles of Ackermann muciniphila in preparation of trichina control products. Experiments prove that by supplementing the AKK bacterium extracellular vesicles, the intestinal flora of a mouse can be remarkably improved, the immune balance of a host is regulated, and the pathological change of the intestinal tract of the mouse can be improved. Experimental results show that the AKK bacterium extracellular vesicles have a certain protection effect on trichina infected mice and have the advantages of being efficient, safe, easy to produce on a large scale and the like, and a brand new technical solution is provided for prevention and treatment of trichinosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of extracellular vesicles of Akkermansia myxophilus in the preparation of trichinella control products. Background Technology

[0002] Trichinellosis is a zoonotic foodborne parasitic disease caused by the trichinella spiralis parasite. Currently, the prevention and treatment of this disease face many challenges: over-reliance on chemical drugs, which often have side effects, and slow development of trichinella vaccines due to the complexity of the trichinella spiralis' life cycle and its ability to evade immune clearance. Studies have shown that secretions or excretions produced during trichinella infection participate in the host's immune response, thus playing a role in evading host immune clearance. Currently, albendazole is the main drug used to treat trichinellosis, but its effectiveness is limited in the later stages of trichinella infection, and long-term use may lead to drug resistance and adverse reactions. Therefore, developing a safe and effective new strategy for the prevention or treatment of trichinellosis is urgently needed.

[0003] Recent studies have found that *AKK* bacteria and their extracellular vesicles can promote intestinal mucus secretion and maintain intestinal mucus homeostasis, thereby regulating intestinal mucosal barrier function. Current research on *AKK* bacteria and their extracellular vesicles mainly focuses on their role in regulating metabolic diseases and inflammatory bowel disease, such as obesity, diabetes, and colitis. However, a growing body of research is also finding that *AKK* bacteria and their extracellular vesicles participate in the regulation of infectious diseases.

[0004] The prior art (β-Glucan-triggered Akkermansia muciniphila expansion facilitates the expulsion of intestinal helminth via TLR2 in mice, Xuemin Jin et al., Carbohydrate Polymers 275 (2022) 118719) discloses that β-glucan (BG) is used globally and has significant health benefits; however, the effects and mechanisms of BG on host defense against nematode infection remain poorly understood. We observed that BG can trigger worm expulsion via the mucus layer in a non-immune-dependent manner, but in mice it is gut microbiota-dependent. BG restored the abundance of Bacteroidetes and Proobacteria induced by T. spiralis infection to control levels and significantly increased the relative abundance of Verrucomicrobia. Akkermansia (belonging to Verrucomicrobia) was significantly amplified in the BG T. spiralis group. Notably, daily oral administration of pasteurized A. muciniphila had a stronger anthelmintic effect than live bacteria and interacted with TLR2. The results of this study demonstrate an easy-to-implement strategy to facilitate the elimination of gastrointestinal nematodes. However, this prior art literature does not address external vesicles, and its conclusion is that pasteurized Akkermansia reduces Trichinella load via TLR2, while pasteurized Akkermansia is unable to reduce worm load in TLR2-deficient mice.

[0005] Our study found that different doses of Trichinella spiralis infection in mice resulted in varying changes in gut microbiota abundance. On day 7 after infection, the abundance of *Amycosis AKK* (AKK bacteria) significantly increased in the high-dose group, suggesting that *AKK* may play an important role in Trichinella spiralis infection. Therefore, based on the important role and potential value of *AKK* extracellular vesicles in regulating intestinal function and disease prevention, exploring their role in the prevention or treatment of Trichinella spiralis-related diseases has significant scientific implications and clinical application prospects. Summary of the Invention

[0006] This invention first provides the application of extracellular vesicles of Akkermansia myxophila in the preparation of trichinella control products.

[0007] In some embodiments, the prevention and control measures involve improving the gut microbiota of the host after Trichinella infection.

[0008] In some embodiments, the prevention and control measures involve modulating the host's immune response after Trichinella infection.

[0009] In some embodiments, the prevention and treatment refers to improving the intestinal pathological changes in the host after Trichinella infection.

[0010] In some embodiments, the product further comprises an overexpression vector of the MIF gene.

[0011] In some embodiments, the trichinella is Trichinella spiralis ISS534 (T. spiralis ISS534).

[0012] In some embodiments, the product comprises a pharmaceutically acceptable carrier, excipient, or diluent for AKK bacterial extracellular vesicles.

[0013] In some embodiments, the AKK bacterial extracellular vesicle content in the product is not less than 50 μg / mL.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: 1. It can effectively improve the intestinal flora imbalance caused by Trichinella infection: The extracellular vesicles of AKK bacteria in this invention can improve the diversity and imbalance of intestinal flora, and can alleviate the intestinal flora imbalance caused by Trichinella infection.

[0015] 2. Regulation of the host's immune response to Trichinella infection: The extracellular vesicles of the AKK bacteria of the present invention can induce Th1 and Th2-related cytokines, regulate the imbalance of the host's immune response caused by Trichinella infection, thereby enhancing the host's resistance to Trichinella infection.

[0016] 3. Improves intestinal pathological changes in hosts infected with Trichinella spiralis: The extracellular vesicles of the AKK bacteria of the present invention can increase the number of goblet cells and the expression of mucin, thereby improving intestinal lesions caused by Trichinella spiralis infection and enhancing the host's resistance to Trichinella spiralis infection.

[0017] 4. Immunomodulatory role of MIF in trichinosis infection: This invention evaluated the immunomodulatory effect of AKK bacterial extracellular vesicles on MIF-deficient mice, thus providing a new approach for the immunomodulatory prevention and control of trichinosis in the future.

[0018] 5. No toxic side effects in the control of Trichinella: The extracellular vesicles of the AKK bacteria of this invention, as probiotics in the intestine, have very low side effects and toxicity compared to chemical drugs, which may cause side effects and drug resistance, making them suitable for long-term use in the control of Trichinella.

[0019] 6. Diverse Applications: The extracellular vesicles of the AKK bacteria of this invention can be used for the prevention and treatment of Trichinella infection. They can be developed into various product forms such as pharmaceuticals and health foods.

[0020] This invention is the first to apply AKK bacterial extracellular vesicles to the prevention and treatment of trichinosis. By improving intestinal flora diversity, enhancing intestinal barrier function, and regulating immune response, it achieves an anti-trichinosis effect. Compared with traditional methods, this invention has advantages such as high efficiency and safety, providing a novel approach to the prevention and treatment of trichinosis. Attached Figure Description

[0021] Figure 1 This is a diagram showing the changes in the gut microbiota after Trichinella infection. Figure 1 A in the middle is a bar chart of relative species abundance; Figure 1 Map B shows the relative abundance distribution of species. Figure 1 C in the middle is a bar chart showing the distribution of LDA values; Figure 1 D in the diagram represents the species abundance clustering.

[0022] Figure 2 This is a diagram showing the results of external vesicle identification. Figure 2 Image A in the middle is the electron microscope result. Figure 2 B is the particle size result diagram.

[0023] Figure 3 This is a diagram showing the changes in the intestinal flora of mice after supplementing with AKK bacteria extracellular vesicles. Figure 3 In the middle section, A represents the relative abundance of Firmicutes in the mouse gut. Figure 3 B represents the relative abundance of Bacteroidetes in the mouse gut; Figure 3 In the middle, C represents the F / B ratio in the mouse intestine; Figure 3 D represents the relative abundance of AKK bacteria in the mouse gut.

[0024] Figure 4 This is a diagram of pathological changes in the mouse intestines analyzed by HE staining. Figure 4 Image A in the image is a representative HE image of intestinal tissue; Figure 4 In the middle, B represents the length of the intestinal villi; Figure 4 C represents the crypt depth.

[0025] Figure 5 This is a diagram of pathological changes in the mouse intestines analyzed by PAS staining. Figure 5 Image A in the image represents a PAS (particularly fibrous) image of intestinal tissue. Figure 5 B represents the number of goblet cells; Figure 5 C represents the expression level of mucin.

[0026] Figure 6 This is a graph showing the effect of AKK bacteria extracellular vesicles on cytokines in mice infected with Trichinella spiralis for 14 days. Figure 6 In the middle, A represents the level of IFN-γ in intestinal tissue; Figure 6 B represents the level of IL-4 in intestinal tissue; Figure 6 C represents the level of TNF-α in spleen tissue; Figure 6D represents the level of IFN-γ in spleen tissue; Figure 6 E represents the level of IL-10 in spleen tissue; Figure 6 The value of F represents the level of IL-4 in spleen tissue. Detailed Implementation

[0027] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0028] Example 1 Materials and Methods 1.1 Laboratory animals, strains and insect species The experimental animals were C57BL / 6 mice (6-8 weeks old), all purchased from the Experimental Animal Center of Hubei University of Medicine. The mice were housed in a specific pathogen-free SPF-grade laboratory at a room temperature of 21-25°C, with a 12-hour light / dark cycle. They had free access to food and water and were allowed to acclimatize for one week before the experiment.

[0029] The strain used in the experiment was *Akermansia muciniphila* 33894T (same as ATCC BAA-835), purchased from the Japan Microbiological Collection Center. The insect species used in the experiment was *Trichinella spiralis* (International Standard Species Number: ISS534), which was preserved through passage in our laboratory rats.

[0030] 1.2 Formulation Preparation AKK bacterial suspension: AKK bacteria were cultured in brain heart extract medium containing 1 g / L mucin and anaerobically incubated at 37°C for 2 days until the bacterial suspension became turbid. After centrifugation and washing, the precipitate was resuspended in anaerobic PBS and the concentration was adjusted to 1×10⁻⁶. 8 CFU / mL.

[0031] AKK bacterial extracellular vesicle extraction: AKK bacteria were cultured to an OD600 value of 1.0, centrifuged at 11,000 × g for 30 min at 4°C, and the supernatant was collected. This step was repeated twice. The collected supernatant was filtered sequentially through filter membranes with diameters of 0.45 μm and 0.22 μm. The filtered filtrate was injected into a fusion-sealed tube and centrifuged at 200,000 × g for 2 h 30 min at 4°C. The collected precipitate was resuspended in PBS buffer and stored in a -80°C freezer (for no more than 2 weeks) until use. The obtained AKK bacterial extracellular vesicles were observed and analyzed in detail for their morphology, diameter, and molecular size using techniques such as transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the isolated protein was quantitatively analyzed using a BCA protein concentration assay kit to determine the concentration of the isolated AKK bacterial extracellular vesicles. Finally, the concentration was prepared to 50 μg / mL using PBS solution.

[0032] 1.3 Experimental grouping and intervention plan The experimental mice were randomly divided into three groups: PBS + T. spiralis group, AKK inactivated bacteria (Pm) + T. spiralis group, and AKK extracellular vesicles (Aev) + T. spiralis group. One week before infection, each group was administered equal amounts of PBS, AKK inactivated bacteria, and AKK extracellular vesicles by gavage. Then, each group was infected with 250 Trichinella spiralis by gavage, and gavage was continued until day 14 after infection.

[0033] 1.4 Gut microbiota 16S rRNA sequencing Bacterial DNA was extracted and quantified from mouse feces. The V3-V4 hypervariable region of the 16S rRNA gene was then amplified, followed by library construction and sequencing to detect changes in the abundance of mouse gut microbiota. Data analysis was performed using Novogene's QIIME2 and R software on the cloud platform, comparing alpha diversity indices among groups, including the Chao1 and Shannon indices. LDA effect size (LefSe) analysis was then used to screen for statistically significant microbial biomarkers among multiple groups.

[0034] 1.5 Evaluation of Pathological Tissue Sections Intestinal tissue was collected from mice on day 35 of Trichinella infection. Samples were fixed with 4% paraformaldehyde, followed by graded dehydration with ethanol, clearing with xylene, and paraffin impregnation. Sections were prepared using a microtome and stained with hematoxylin and eosin (HE) and acetylene (PAS) (Wuhan Sewell). The stained sections were observed using an optical microscope (Olympus BX53F), and images were acquired using Olympus CellSens Standard 1.13 software. After HE staining, intestinal villus length and crypt depth were calculated from the acquired images; after PAS staining, goblet cell number and mucin expression levels were calculated from the acquired images.

[0035] 1.6 Assessment of relative cytokine expression levels Intestinal tissue was collected from mice on day 35 of Trichinella spiralis infection. The tissue samples were minced, and total RNA was extracted using the TRIzol method, followed by quantification. The extracted RNA was reverse transcribed into cDNA, and the expression levels of multiple cytokine genes (such as IL-4, IFN-γ, and TNF-α) were detected using real-time quantitative PCR. GAPDH was used as an internal control, and the relative expression levels of each cytokine mRNA in the tissue were calculated using the 2-ΔΔCt method. Differences between groups were assessed using t-tests or one-way ANOVA; p < 0.05 was considered statistically significant.

[0036] result 2.1 Effects of Trichinella spiralis infection on the abundance and diversity of gut microbiota in mice like Figure 1 As shown, changes in the gut microbiota of mice were detected on days 7 and 14 after Trichinella infection. Compared with the control group, the abundance of Verrucomicrobita (verrucomicrobial phylum) was significantly increased in the high-dose Trichinella infection group on day 7, while the abundance of Verrucomicrobita in the high, medium, and low-dose Trichinella infection groups showed no significant change on day 14. Figure 1 A). UPGMA cluster analysis showed that, at the phylum level, the abundance of each mouse verruciformis phylum was significantly increased in the high-dose group on day 7 after infection ( Figure 1 B). LEfSe analysis revealed that *A. muciniphila* was significantly enriched in the gut of the high-dose group on day 7 of infection, *Bacteroidales* was significantly enriched in the medium-dose group, and *Muribaculaceae* was significantly enriched in the low-dose group. Figure 1 (C) These results indicate that the abundance and diversity of gut microbiota in mice changed significantly after Trichinella infection, and that beneficial bacteria were significantly enriched in the gut of mice in the high-dose group.

[0037] like Figure 2As shown, extracellular vesicles derived from *AKK* bacteria in culture medium were collected and then purified by ultracentrifugation. These *AKK* bacteria extracellular vesicles were observed and identified by TEM and NTA, and the results showed that the *AKK* bacteria extracellular vesicles had a clear double membrane and exhibited a typical spherical structure. Figure 2 A), with an average diameter of 40-150 nm ( Figure 2 (B) These results demonstrate the basic structural features and protein composition of the extracellular vesicles of AKK bacteria, providing fundamental data for subsequent functional studies.

[0038] like Figure 3 As shown, one week before infection with Trichinella spiralis, mice in the experimental group were administered either inactivated AKK bacteria or AKK bacterial extracellular vesicles by gavage, while the control group was administered the same amount of PBS. On day 35 after infection, fecal samples were collected from the mice to analyze changes in the gut microbiota. The results showed that on day 35 after infection with Trichinella spiralis in wild-type (WT) mice, compared with the PBS control group, the abundance of *A. muciniphila* was significantly increased in the AKK inactivated bacteria and AKK bacterial extracellular vesicle groups. Figure 3 D), the abundance of Bacteroidetes in the AKK bacterial extracellular vesicle group decreased ( Figure 3 B); and MIF knockout of AKK extracellular vesicles Firmicutes ( Figure 3 The abundance of A) and the F / B ratio ( Figure 3 C), A. muciniphila abundance ( Figure 3 D) were significantly increased, while the abundance of Bacteroidetes was significantly decreased; these results indicate that gavage administration of AKK bacteria extracellular vesicles can significantly improve the intestinal flora of mice.

[0039] like Figure 4 As shown, HE staining results indicated that, compared with the PBS-infected control group, the pathological changes in the intestines of both the AKK inactivated bacteria and AKK extracellular vesicle groups were significantly improved. Figure 4 A), manifested in the length of the downy fibers ( Figure 4 B) and the increase in crypt depth ( Figure 4 C); and compared with wild-type mice (WT), the villus length of AKK inactivated bacteria and AKK bacterial extracellular vesicles in the MIF knockout group ( Figure 4 B) both decreased, while crypt depth ( Figure 4 C) Both increased, indicating that supplementation with AKK inactivated bacteria and AKK bacterial extracellular vesicles can improve intestinal lesions caused by Trichinella infection, and MIF knockout may reduce this effect.

[0040] like Figure 5 As shown, PAS staining results indicated that, compared with the PBS-infected control group, the pathological changes in the intestines of the AKK inactivated bacteria and AKK extracellular vesicle groups were improved to some extent. Figure 5 A), in which the number of goblet cells in the AKK bacterial extracellular vesicle group was significantly increased ( Figure 5 B), but the expression level of mucin did not change significantly ( Figure 5 C); and compared with wild-type mice (WT) in the extracellular vesicle group, the number of goblet cells in MIF knockout mice in the extracellular vesicle group (C); Figure 5 B) and mucin expression levels ( Figure 5 C) Both decreased, indicating that MIF knockout has a certain impact on the number of goblet cells and the expression of mucin; the above results show that supplementation with AKK inactivated bacteria and AKK bacterial extracellular vesicles can improve the intestinal mucosal barrier function.

[0041] like Figure 6 As shown, on day 35 after infection with Trichinella spiralis, intestinal and spleen tissues of mice were collected, and PCR analysis was performed to analyze changes in mouse cytokines. Compared with the PBS control group, the expression level of IFN-γ in the intestinal tissue of mice supplemented with wild-type AKK bacteria extracellular vesicles was decreased, but not significantly. Figure 6 A), IL-4 expression was significantly increased ( Figure 6 B); TNF-α levels in spleen tissue were decreased, but not significantly ( Figure 6 C), IL-10 Figure 6 E) and IL-4 ( Figure 6 F) expression was significantly decreased, and IFN-γ expression was significantly increased. Figure 6 D); and compared with wild-type mice in the AKK bacterial extracellular vesicle group, the expression levels of IFN-γ and IL-4 in the intestinal tissue of mice in the MIF knockout group were increased ( Figure 6 A), IFN-γ in spleen tissue ( Figure 6 D) and IL-4 ( Figure 6 F) expression levels decreased ( Figure 6 D); These results indicate that AKK bacteria can modulate the host's immune response, and that MIF plays an important immunomodulatory role in the process of Trichinella infection.

[0042] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of an extracellular vesicle of Akkermansia muciniphila in the preparation of a product for the prevention or treatment of trichinosis.

2. Use according to claim 1, characterized in that, The prevention or treatment is to improve the intestinal flora of a host after trichinosis infection.

3. Use according to claim 1, characterized in that, The prevention or treatment is to regulate the immune response of a host after trichinosis infection.

4. Use according to claim 1, characterized in that, The prevention or treatment is to improve the intestinal pathological changes of a host after trichinosis infection.

5. The use according to claim 1, characterized in that, The product further comprises an overexpression vector of the MIF gene.

6. Use according to any one of claims 1 to 5, characterized in that, The trichinosis is Trichinella spiralis ISS534 (T. spiralis ISS534).

7. Use according to any one of claims 1 to 5, characterized in that, The product comprises the extracellular vesicle of the AKK bacteria in a pharmaceutically acceptable carrier, adjuvant or diluent.

8. Use according to any one of claims 1 to 5, characterized in that, The content of the extracellular vesicle of the AKK bacteria in the product is not less than 50 μg / mL.

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