Clostridium praeckii vesicle targeting inflammatory bowel disease tissue and preparation and application of clostridium praeckii vesicle

Abstract

The invention discloses a clostridium praeparatum vesicle targeting inflammatory bowel disease tissues as well as preparation and application of the clostridium praeparatum vesicle. The vesicle is secreted by clostridium praeparatum ATCC27766, and the average particle size of the vesicle is 100-250 nm. Experimental data show that the vesicles have intestinal disease site targeting, can effectively remodel the intestinal immune microenvironment of ulcerative colitis patients, and inhibit the inflammation process through chemotactic factor secretion of macrophages. The clostridium praeparatum vesicle and the preparation can effectively overcome the defect that clostridium praeparatum and butyrate secreted by the clostridium praeparatum are prone to failure, the treatment means of ulcerative colitis are enriched, and the application prospect of anaerobic probiotics is widened.

Description

Technical Field

[0001] The present invention relates to bacterial vesicles, and in particular to Faecalibacterium prausnitzii vesicles targeting inflammatory bowel disease tissues and the preparation and application thereof. Background Art

[0002] Inflammatory Bowel Disease (IBD) is a complex disease characterized by chronic, recurrent intestinal inflammation, including ulcerative colitis (UC) and Crohn's disease (CD). Among them, ulcerative colitis is an important manifestation of inflammatory bowel disease, and its incidence has continued to rise worldwide in recent years, especially in my country. This disease seriously affects the quality of life of patients, poses a major challenge to the public health system, and also increases the burden on social medical resources. The pathogenesis of UC has not yet been fully clarified, and usually involves complex interactions between host genetic susceptibility, environmental factors, and intestinal microbiota. Among them, the intestinal flora plays a key bridging role in maintaining intestinal homeostasis and mediating the interaction between the host and the environment, and has become a research hotspot.

[0003] Current strategies for adjuvant treatment of UC mostly use exogenous probiotics, such as bifidobacteria and lactobacilli, to help regulate intestinal flora, and some patients have achieved good results in clinical practice. However, there is no clear conclusion on the dynamic changes and mechanism of action of endogenous probiotics that maintain intestinal microecological balance in a healthy state in a diseased state. Studies have shown that the intestinal flora structure of IBD patients is significantly imbalanced, manifested as species imbalance and decreased diversity, among which the reduction of Faecalibacterium prausnitzii is particularly obvious. Faecalibacterium prausnitzii belongs to the phylum Firmicutes and is an obligate anaerobic Gram-negative clostridium that accounts for about 5%-15% of healthy human feces. It produces butyrate through metabolism, participates in regulating immune balance, stimulates intestinal mucosal mucus secretion, and upregulates the expression of intestinal tight junction proteins, which plays an important role in maintaining intestinal homeostasis.

[0004] Although Faecalibacterium prausnitzii exhibits significant probiotic properties, it has many limitations in clinical applications. Because it is highly sensitive to oxygen, it is difficult to survive in an oxygen-exposed environment; at the same time, the butyrate it produces is susceptible to enzymatic hydrolysis in the small intestine and cannot effectively reach the lesion site. These characteristics limit the practical application of Faecalibacterium prausnitzii as a traditional oral probiotic preparation and cannot fully realize its potential therapeutic value. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a stable Faecalibacterium prausnitzii vesicle and preparation targeting macrophages in the lamina propria of inflammatory bowel disease tissues; the second purpose is to provide its use in inflammatory bowel disease drugs.

[0006] Technical solution: The Faecalibacterium prausnitzii vesicles targeting inflammatory bowel disease tissues described in the present invention are secreted by Faecalibacterium prausnitzii ATCC27766 and have an average particle size of 100-250 nm.

[0007] The method for preparing the Faecalibacterium prausnitzii vesicles of the present invention comprises:

[0008] (1) inoculating Faecalibacterium prausnitzii into sterile BHI medium and culturing at 36-38°C;

[0009] (2) When the culture of F. prausnitzii reaches OD 600 At the threshold, the culture was removed and the supernatant was collected after centrifugation;

[0010] (3) filtering the supernatant obtained in step 2 through a 0.22 μm filter, adding 0.2-0.4 times the volume of the supernatant with a vesicle extraction reagent, mixing well, incubating in a low-temperature shaker for 0.5-1.5 hours, and collecting the precipitate after centrifugation;

[0011] (4) The precipitate obtained in step 3 was resuspended in sterile PBS and stored at 2-8°C for a short period of time.

[0012] Preferably, the sterile BHI medium in step 1 is a modified LYHBHI medium, which contains brain heart perfusion fluid, yeast extract, cellobiose, maltose, cysteine ​​and heme; the OD in step 2 is 600 The threshold is 1.85-1.95.

[0013] The Faecalibacterium prausnitzii preparation of the present invention comprises the culture supernatant and / or vesicles of Faecalibacterium prausnitzii ATCC27766.

[0014] The invention relates to the use of the Faecalibacterium prausnitzii vesicles or Faecalibacterium prausnitzii preparations in medicines targeting intestinal lamina propria cells for the prevention, treatment, adjuvant treatment or prognostic care of inflammatory bowel disease.

[0015] Preferably, the application is application in a drug for improving colonic crypt atrophy and colonic epithelial necrosis defect.

[0016] Preferably, the application is application in drugs for alleviating intestinal barrier dysfunction.

[0017] Preferably, the application is application in drugs that inhibit colon immune cells and reduce pro-inflammatory chemokines in the intestinal microenvironment.

[0018] The pharmaceutical preparation of the present invention comprises the aforementioned Faecalibacterium prausnitzii vesicles or Faecalibacterium prausnitzii preparation, and other pharmaceutically acceptable carriers or excipients.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. It can effectively overcome the shortcomings of Faecalibacterium prausnitzii and the butyrate it secretes that are easily ineffective; 2. It has intestinal disease-targeting properties and can effectively reshape the intestinal immune microenvironment of patients with ulcerative colitis and curb the inflammatory process; 3. It enriches the treatment methods for ulcerative colitis and broadens the application prospects of anaerobic probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a transmission electron micrograph of the Faecalibacterium prausnitzii vesicle;

[0021] Figure 2 This is the distribution map of Faecalibacterium prausnitzii vesicles in mice;

[0022] Figure 3 This is the distribution map of Faecalibacterium prausnitzii vesicles in mouse colon tissue;

[0023] Figure 4 This is the distribution map of Faecalibacterium prausnitzii vesicles in the mouse colon lamina propria;

[0024] Figure 5 This is a flow cytometric analysis of mouse colon lamina propria cells;

[0025] Figure 6 Analytical graphs for mouse body weight, disease activity score, and colon length;

[0026] Figure 7 HE staining of mouse colon tissue;

[0027] Figure 8 This is the result of Faecalibacterium prausnitzii vesicles inhibiting the secretion of chemokines by macrophages. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below.

[0029] Example 1: Isolation of Faecalibacterium prausnitzii vesicles

[0030] The exosome extraction kit (Henan Beibei Technology Co., Ltd.) was used to extract the vesicles in the secretion supernatant of Faecalibacterium prausnitzii.

[0031] 1. Clostridium prausnitzii was cultured in LYHBHI medium at 37°C. The LYHBHI medium contained 37 g / L brain heart perfusion fluid, 5 g / L yeast extract, 1 g / L cellobiose, 1 g / L maltose, 1 g / L cysteine ​​and 5 mg / L heme.

[0032] 2.OD600 =1.9, centrifuge at 4000 rpm for 10 min, collect the supernatant, filter with a 0.22 μm filter, add 1 / 3 volume of vesicle extraction reagent, mix well, and incubate at 4 °C shaker for 1 h;

[0033] 3. Centrifuge at 12000 rpm for 25 min, collect the precipitate, add sterile PBS, place in a 37°C water bath to facilitate resuspension, and store at 4°C for a short term to obtain Faecalibacterium prausnitzii vesicles.

[0034] 4. The BCA protein quantification method measured the vesicle protein concentration to be 1 μg / μL. Dynamic light scattering (DLS) was used to characterize the particle size of the F. prausnitzii vesicles. The morphological characteristics of the F. prausnitzii vesicles were visualized by transmission electron microscopy. Figure 1 As shown, electron microscopy analysis showed vesicle morphology with a particle size of 110.1-238.5 nm.

[0035] Example 2: Distribution of Faecalibacterium prausnitzii vesicles in the body and colon tissues of normal and colitis mice

[0036] 1. Use dextran sulphate sodium (DSS) to induce colitis model in mice;

[0037] 2. Use near-infrared dye IR780 as a fluorescent probe to label vesicles, administer the drug by gavage, and perform in vivo imaging of mice or in vitro imaging of colon tissue 3h, 6h, 12h, and 24h after administration;

[0038] 3. Distribution of Faecalibacterium prausnitzii vesicles in mice Figure 2 As shown, 24 hours after administration, Faecalibacterium prausnitzii vesicles were still abundantly enriched and retained in the intestinal tract of colitis mice, while they were almost completely metabolized in normal mice;

[0039] 4. Distribution of Faecalibacterium prausnitzii vesicles in mouse colon tissue Figure 3 As shown, more Faecalibacterium prausnitzii vesicles accumulated in the intestinal tissues of colitis mice compared with normal mice.

[0040] Example 3: Uptake results of Faecalibacterium prausnitzii vesicles into different immune cells in the colonic lamina propria

[0041] 1. Use dextran sulphate sodium (DSS) to induce colitis model in mice;

[0042] 2. Use near-infrared dye IR780 as a fluorescent probe to label vesicles and administer the drug by gavage. After 12 hours, take the mouse colon and slice it longitudinally for in vitro fluorescence imaging. Figure 4As shown, F. prausnitzii vesicles can cross the damaged intestinal epithelium to reach the lamina propria and co-localize more with F4 / 80-labeled macrophages.

[0043] 3. Use near-infrared dye IR780 as a fluorescent probe to label vesicles and administer the drug by gavage. After 12 hours, take the mouse colon tissue, extract the intestinal lamina propria, and use flow cytometry to detect the distribution of Faecalibacterium prausnitzii vesicles in different immune cells. Figure 5 As shown, F. prausnitzii vesicles were mainly taken up by F4 / 80+CD11b+ macrophages.

[0044] Example 4: Faecalibacterium prausnitzii vesicles improve colitis in mice

[0045] 1. Starting from 3 days before DSS administration, mice were gavaged with PBS or 100μg, 200μg, or 300μg of Faecalibacterium prausnitzii vesicles every day. After 3 days, 2.5% DSS was added to the drinking water of mice for 5 days.

[0046] 2. After DSS administration, various indicators of mice were evaluated every day, such as Figure 6 The results showed that oral administration of Faecalibacterium prausnitzii vesicles could significantly alleviate the weight loss, disease activity score (DAI), and colon length of colitis mice in a dose-dependent manner;

[0047] 3. The colon tissues of mice in the Control group, DSS group, DSS+100μg Fp EVs (Faecalibacterium prausnitzii) group, DSS+200μg F.pEVs and DSS+300μg Fp EVs groups were taken out for HE staining to observe the integrity of colon crypts, the degree of colon epithelial damage and the degree of inflammatory cell infiltration. Figure 7 As shown, compared with the DSS group mice, the colon tissue crypts of the mice in the drug group were intact, without a large number of inflammatory cell infiltrations, and the colon tissue damage was significantly improved.

[0048] Example 6: Faecalibacterium prausnitzii vesicles inhibit the secretion of chemokines by macrophages in vitro

[0049] 1. Femoral bone marrow was obtained from 6-8 week old mice and stimulated with 20 ng / mL M-CSF for 7 days to obtain mouse bone marrow-derived macrophages (BMDM);

[0050] 2. After treating BMDM with different concentrations of Faecalibacterium prausnitzii vesicles for 24 h, 1 μg / mL LPS was added for 6 h, and the levels of chemokines secreted by BMDM were detected, such as Figure 8 As shown, Faecalibacterium prausnitzii vesicles can significantly inhibit the secretion of various monocyte chemokines by BMDM in a dose-dependent manner.

Claims

1. A Faecalibacterium prausnitzii vesicle targeting inflammatory bowel disease tissues, characterized in that: The vesicles are secreted by Clostridium prausnitzii ATCC27766 and have an average particle size of 100-250 nm.

2. A method for preparing the Faecalibacterium prausnitzii vesicles according to claim 1, characterized in that: include: (1) inoculating Faecalibacterium prausnitzii into sterile BHI medium and culturing at 36-38°C; (2) When the culture of F. prausnitzii reaches OD 600 At the threshold, the culture was removed and the supernatant was collected after centrifugation; (3) filtering the supernatant obtained in step 2 through a 0.22 μm filter, adding 0.2-0.4 times the volume of the supernatant with a vesicle extraction reagent, mixing well, incubating in a low-temperature shaker for 0.5-1.5 hours, and collecting the precipitate after centrifugation; (4) The precipitate obtained in step 3 was resuspended in sterile PBS and stored at 2-8°C for a short period of time.

3. The method for preparing the Faecalibacterium prausnitzii vesicles according to claim 2, characterized in that: The sterile BHI medium in step 1 is a modified LYHBHI medium, which contains brain heart perfusion fluid, yeast extract, cellobiose, maltose, cysteine ​​and heme.

4. The method for preparing the Faecalibacterium prausnitzii vesicles according to claim 3, characterized in that: In step 2, OD 600 The threshold is 1.85-1.

95.

5. A Faecalibacterium prausnitzii preparation, characterized in that: The preparation includes the culture supernatant and / or vesicles of Faecalibacterium prausnitzii ATCC27766.

6. Use of the Faecalibacterium prausnitzii vesicles of claim 1 or the Faecalibacterium prausnitzii preparation of claim 4 in a drug targeting intestinal lamina propria cells for the prevention, treatment, adjuvant treatment or prognostic care of inflammatory bowel disease.

7. The use according to claim 6, characterized in that: The application is application in medicines for improving colon crypt atrophy and colon epithelial necrosis defects.

8. The use according to claim 6, characterized in that: The application is application in drugs for alleviating intestinal barrier dysfunction.

9. The use according to claim 6, characterized in that: The application is application in drugs that inhibit colon immune cells and reduce pro-inflammatory chemokines in the intestinal microenvironment.

10. A pharmaceutical preparation, characterized in that: The pharmaceutical preparation comprises the Faecalibacterium prausnitzii vesicles of claim 1 or the Faecalibacterium prausnitzii preparation of claim 4, and other pharmaceutically acceptable carriers or excipients.

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