Bacteroides ovale NCU-34 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacteroides ovatus NCU-34 and application thereof. The Bacteroides ovatus NCU-34 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 6, 2024, the address is Institute of Microbiology, Chinese Academy of Sciences, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.32937. The invention further discloses a preparation method of the Bacteroides ovatus NCU-34. The invention provides a strain of bacteroides ovatus NCU-34, the bacteroides ovatus NCU-34 can be used for preparing a medicine for preventing and / or treating atherosclerosis, and according to the experimental data result of the embodiment of the invention, the bacteroides ovatus NCU-34 and the metabolite IAA of the bacteroides ovatus NCU-34 provided by the invention can improve the progress of AS.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to a strain of Bacteroides ovatus NCU-34 and its application. Background Art

[0002] Atherosclerosis (AS) is a disease of large and medium-sized arteries characterized by endothelial dysfunction, vascular inflammation, and accumulation of lipids, cholesterol, calcium ions, and cell debris in the intima of the vessel wall. This is a chronic inflammatory state with infiltration of macrophages, T cells, and other immune cells, which together with cholesterol form atherosclerotic plaques. The deposition of oxidized low-density lipoprotein (ox-LDL) further leads to increased endothelial permeability, endothelial cell dysfunction, and the secretion of a large number of inflammatory factors and adhesion factors, such as interleukins (IL). These factors promote the migration of smooth muscle cells to the intima, the adhesion of monocytes, and the entry of lipids into macrophages, which further take up lipids into foam cells to form atherosclerotic plaques.

[0003] A complex and diverse microbial community is formed in the human intestine to maintain symbiotic relationships and homeostasis. The increasing understanding of the intestinal microbiota and its role in host metabolism and immunity has promoted unprecedented interest in developing diagnostic and therapeutic targets for many diseases related to the intestinal microbiota. Evidence to date has shown that changes in the composition and diversity of the intestinal microbiota are associated with a high risk of developing cardiovascular diseases (CVDs), such as atherosclerosis, hypertension, heart failure, stroke, atrial fibrillation, and myocardial fibrosis. Therefore, it is very necessary to find an effective single or multi-species bacterial species for the treatment of atherosclerosis. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a strain of Bacteroides ovatus NCU-34 and its application, specifically adopting the following technical solutions: In a first aspect of the present invention, a strain of Bacteroides ovatus NCU-34 is provided, wherein the Bacteroides ovatus NCU-34 ( Bacteroides ovatus ) was deposited on December 6, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32937.

[0005] The gene sequence of 16S rDNA of the above-mentioned Bacteroides ovatus NCU-34 is shown in SEQ ID No: 1. The present invention identifies the new bacterium as Bacteroides ovatus NCU-34 by PCR amplification, sequencing, comparison and tree building of 16S rRNA.

[0006] SEQ ID No: 1:

[0007] The second aspect of the present invention provides a culture of Bacteroides ovatus NCU-34, which is prepared from the above-mentioned Bacteroides ovatus NCU-34.

[0008] Preferably, the above-mentioned Bacteroides ovatus NCU-34 culture contains at least one of live Bacteroides ovatus NCU-34 bacteria, bacterial components of Bacteroides ovatus NCU-34 and their derivatives, and metabolites of Bacteroides ovatus NCU-34.

[0009] More preferably, the above-mentioned Bacteroides ovatus NCU-34 metabolite includes indole-3-acetic acid.

[0010] The third aspect of the present invention provides use of the above-mentioned Bacteroides ovatus NCU-34 or a culture of Bacteroides ovatus NCU-34 in the preparation of a medicament for preventing and / or treating atherosclerosis.

[0011] The fourth aspect of the present invention provides a drug for preventing and / or treating atherosclerosis, wherein the drug comprises the above-mentioned Bacteroides ovatus NCU-34.

[0012] Preferably, the above-mentioned medicine also includes pharmaceutical excipients.

[0013] More preferably, the dosage form of the above-mentioned drug is powder, granule, capsule or tablet.

[0014] The fifth aspect of the present invention further provides a biological preparation, which comprises the above-mentioned Bacteroides ovatus NCU-34 or the above-mentioned Bacteroides ovatus NCU-34 culture.

[0015] The beneficial effects of the present invention are: The present invention provides a strain of Bacteroides ovatus NCU-34, which can be used to prepare a drug for preventing and / or treating atherosclerosis. According to the experimental data results of the embodiments of the present invention, the Bacteroides ovatus NCU-34 provided by the present invention and its metabolite IAA can improve the progression of AS. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1The figure shows the results of the in vitro probiotic property evaluation of Bacteroides ovatus NCU-34; a is the survival curve of B.ovatus; b is the drug resistance experiment of the bacterial disc diffusion method; c is the inhibition diameter quantification diagram; d is the bile salt resistance experiment and colony morphology; e is the schematic diagram of the antibacterial experiment; f is the quantification diagram of the antibacterial experiment, and the black dotted line is the Oxford cup diameter; g is the schematic diagram of the B.ovatus adhesion experiment; h is the average number of Bacteroides ovatus NCU-34 adhered to each cell (abbreviated as BO in the figure); Figure 2 The results of the experiment on improving atherosclerosis by transplantation of Bacteroides ovatus NCU-34 into mice are shown; a, schematic diagram of the relevant experimental design; b, changes in mouse weight; c, serum LPS content; d, serum inflammatory factor content: IL-6, IL-1β, TNF-α; e, serum cholesterol content: ldl-c, TC, TG; f, gross image of aorta stained with Oil Red "O"; g, representative image of liver stained with Oil Red "O"; h, heat map of fecal differences in bile acids in groups C, M, and BO, red represents positive correlation, and green represents negative correlation; i, liver gene q-PCR detection; j, representative images of colon and liver WB; k, quantification of relative protein expression levels of FXR1 and CYP27A1 in colon and liver; l, IHC quantitative results of intestinal NPC1L1; m, serum IAA content; n, fecal IAA content.

[0018] Figure 3 The results of the experiment of improving atherosclerosis by transplanting Bacteroides ovatus NCU-34 into mice are shown; a is the gross weight of the mouse aorta (left); the percentage of the aortic oil red staining area (middle); the percentage of the aortic HE plaque area (right); b, representative HE images of the aorta; c, the percentage of the liver oil red "O" staining area; d, gross weight of the liver (left); liver NAS score (right); e, representative HE images of the liver; f, quantification of colon length; quantification of colon crypt depth; g, representative HE images of the colon; h, IHC quantification results of colon Occludin and ZO-1; i, representative IHC images of colon ZO-1 and Occludin; j, quantification of spleen gross weight and length; k, gross weight of the epididymis; l, bacterial abundance in fecal samples detected by q-PCR; BL: Lactobacillus; BI: Bifidobacterium; BA: Bacteroides; BO: B.ovatus. m, representative IHC images of colon NPC1L1; n, differential tryptophan heat map of M and BO feces. Red represents positive correlation and green represents negative correlation.

[0019] Figure 4Figure 3 shows the results of an in vivo experiment on the improvement of atherosclerosis by indole-3-acetic acid, a tryptophan metabolite of Bacteroides ovatus NCU-34; a, schematic diagram of the experimental design; b, changes in mouse body weight; c, serum LPS content; d, serum inflammatory factor content: IL-6, IL-1β, TNF-α; e, serum cholesterol content, ldl-c, TC, TG; f, gross image of aorta oil red "O" staining; g, percentage of aorta oil red staining area (upper left); percentage of plaque area (upper right); percentage of plaque necrotic core area (lower left); h, schematic diagram of valve Masson staining, ICAM and VCAM IHC staining, and immunofluorescence M1 / M2 ratio; i, valve Masson staining; ICAM; VCAM; immunofluorescence M1 / M2 ratio quantification; j, liver oil red "O" staining area percentage; k, colon occludin; Claudin-1 relative protein expression level quantification; l, representative WB images of colon occludin and Claudin-1.

[0020] Figure 5 Shown are the results of the in vivo improvement of atherosclerosis by indole-3-acetic acid, a tryptophan metabolite of Bacteroides ovatus NCU-34; a, gross weight of aorta; b, representative images of valve HE staining; c, gross weight of liver; liver NAS score; d, representative images of liver HE; e, representative images of colon HE and AB-PAS staining; f, quantification of colon length; colon crypt depth; quantification g, gross weight of epididymis; h, gross weight and length of spleen; i, IAA content in mouse feces; j, q-PCR detection of aortic genes, IL-1β, TNF-α, IL-6, ABCA1, MCP-1, AHR, TLR4, iNOS, NLRP3, JAK2, AKT, mTOR, HIF-1α.

[0021] Figure 6The results of the in vitro regulation of macrophage M1 / M2-1 by indole-3-acetic acid are shown; a, experimental design of macrophage M1 polarization induction; b, representative images of macrophage M1 immunofluorescence CD86+AHR staining; c, quantification of relative fluorescence intensity of macrophage M1: CD86, AHR; d, representative WB images of TLR4 and MyD88 of macrophage M1; e, relative protein expression levels of macrophage M1: TLR4; MyD88; f, immunofluorescence CD206+A of macrophage M2 Representative images of HR staining; g, quantification of relative fluorescence intensity of macrophage M2: CD206, AHR; h, representative WB images of aortic TLR4, MyD88, PP65, P65, IL-6, AHR, Arg-1 and IL-10; i, relative protein expression levels of aortic M1: TLR4, MyD88, PP65, P65, IL-6; j, relative protein expression levels of aortic M2: AHR, Arg-1, IL-10; k, human serum IAA content.

[0022] Figure 7 Shown are the experimental results of indole-3-acetic acid regulating macrophage M1 / M2-2 in vitro; a, representative image of IAA intervention on macrophage M1 polarization; b, experimental design for inducing macrophage M2 polarization; c, experimental design for inducing macrophage high-fat model; d, representative image of Oil Red "O" staining of macrophage high-fat model; e, representative image of immunofluorescence CD206+AHR staining of macrophage M2; f, quantification of relative fluorescence intensity of macrophage M2.

[0023] Figure 8 Shown is the microscopic morphology of Bacteroides ovatus NCU-34.

[0024] Fig. 9 Shown is the analysis of tryptophan metabolites in feces of Bacteroides ovatus NCU-34 after oral administration.

[0025] Fig.10 Shown is the 16S sequencing of humanized feces of mouse intestinal flora. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0027] All animal experiment-related procedures were approved by the Experimental Animal Welfare and Ethics Committee of Nanchang University ( (approval number: NCULAE - 20221031130) ).

[0028] Example 1 This example provides a method for separating and purifying Bacteroides ovatus NCU-34 (B.ovatus), comprising the following steps: 1. Fecal Sample Collection and Bacterial Isolation (1) Sample collection and processing ①Pick a small portion of fresh stool sample and place it in a centrifuge tube (15mL specification) containing 5 mL of PBS solution. Immediately send it to the laboratory bench and resuspend the sample with a shaker and shake it thoroughly. Centrifuge the sample at 500 rpm for 3 min and obtain the supernatant for subsequent dilution and coating.

[0029] The stool samples were obtained from people with risk factors for atherosclerosis (hypertension, diabetes, or dyslipidemia) but without diagnosed coronary artery disease.

[0030] ②Package the remaining stool sample, take 500 µL of the sample into an EP tube (1.5 mL specification) containing 500 µL of glycerol, add cysteine ​​to the preservation tube, and freeze it in a -80℃ refrigerator.

[0031] (2) Preparation of culture medium BHI (plus 0.5% yeast powder, 0.5 mg / mL L-cysteine, 10% heme); (3) Dilute the coating The supernatant samples were serially diluted 10~10 8 For new samples, the dilution gradient can be 10, 10 3 , 10 4 , 10 5 , apply 50 µL; after freezing, the sample can be 10, 10 2 , 10 3 , 10 4 Apply 30 µL.

[0032] (4) Vaccination Take a plate with 100-300 colonies, and inoculate 5-10 single colonies into the corresponding 5 ml liquid culture medium for aerobic and anaerobic activation culture for 24-28 hours in a sterile clean bench according to the shape, size, color, neat edges, ridges and faster growing colonies on the plate.

[0033] 2. Purification of Fecal Bacteria The cultured sample is inoculated onto a specific selective medium (BHI) to inhibit the growth of unwanted microorganisms while promoting the growth of target bacteria.

[0034] The inoculated culture medium is incubated at an appropriate temperature (e.g., 36 ± 1°C) for a period of time (e.g., 24 ± 2 hours) to allow the bacteria to grow and form visible colonies.

[0035] Observe the colonies: After the culture is completed, observe the colony morphology on the culture medium and select colonies suspected of target bacteria for further isolation.

[0036] The morphology of Bacteroides ovatus NCU-34 under the microscope is ( Figure 8 ), is an anaerobic, Gram-negative bacterium that is oval in shape. Its size is usually 1-2 microns in length and 0.5-1.0 microns in width. In culture medium, this bacterium forms oval colonies that are gray in color and approximately 1.0-1.5 mm in diameter.

[0037] Isolation and purification: Use a sterile tool (such as an inoculation loop) to pick up a single colony and inoculate it onto a new culture medium for further culture and purification. Repeat this process until a pure culture is obtained.

[0038] 3. Identification of Bacteroides ovatus NCU-34 (B.ovatus) Polymerase chain reaction (PCR) was used to amplify the conserved 16S rRNA sequence of bacteria for rapid identification of bacteria isolated from feces.

[0039] The target colony was selected, inoculated into a 96-well plate containing BHI liquid medium, and cultured at 37°C under anaerobic conditions for 24 hours. Then, the bacterial solution was used as a template, and the universal primers 27F: 5′-TGCAAACTRAAGATGGC-3′ (SEQ ID No: 2), 1492R: 5′-CAAACTAATGGAACGCATC-3′ (SEQ ID No: 3) were used for PCR. The amplified product was observed and detected in a gel imaging system after 1.0% agarose gel electrophoresis. After the size was consistent with the expected size, the gel was cut and recovered, and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain the 16S rDNA gene sequence of the strain. The sequence was compared with the NCBI (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) database to determine the species of the screened strain, and finally determined that the strain provided by the present invention was Bacteroides ovatus NCU-34.

[0040] Example 2 The in vitro probiotic properties of Bacteroides ovatus NCU-34 B.ovatus (BO) isolated in Example 1 were evaluated, and the specific process was as follows: 1. Growth curve Inoculate B.ovatus into sterile BHI special culture medium and culture statically for 24 hours to prepare the mother solution. Use a sterile pipette to accurately draw 0.5 ml of liquid and add it to a 15m culture tube containing 10 ml of sterile BHI+ culture medium, for a total of 9 tubes. Culture at 37°C with an oscillation frequency set to 250 r / min. Take out one tube at 0, 6, 12, 18, 24, 30, 36, 42, and 48 hours, and draw 2 ml of culture medium. Use the uninoculated culture medium as the blank control, and compare the above culture medium at a wavelength of 600 nm (determine the OD value). The results are as follows Figure 1 As shown in a in .

[0041] 2. Bile salt tolerance test of B.ovatus (1) Cultivate B. ovatus in a 37°C constant temperature incubator for 36-72 h using the corresponding liquid culture medium and keep the strain for later use; (2) Take out 100 µL and dilute it with PBS buffer for 10 1 , 10 3 , 10 5 times, centrifuge at 4,000 rpm for 3 min and discard the supernatant; culture in a 37°C constant temperature incubator for 4 h with a culture medium containing 0.0%~0.3% ox bile salts, mix well, take 10 μL to plate, and culture in a 37°C constant temperature incubator or anaerobic incubator for 36~48 h, count the viable bacteria, and keep experimental records.

[0042] 3. B.ovatus drug resistance experiment (1) Select B.ovatus and prepare culture medium: Prepare appropriate culture medium according to experimental needs, such as BHI solid culture medium. Prepare antibiotics: Select a series of antibiotics and prepare appropriate drug sensitivity paper for testing the drug resistance of probiotics.

[0043] (2) Inoculate the probiotics onto an appropriate culture medium and culture them at appropriate temperature and conditions until they reach the logarithmic growth phase. Pick out pure culture colonies from the cultured probiotics, suspend them in sterile saline, and adjust the turbidity to an appropriate level (e.g., 1 McFarland unit).

[0044] (3) Apply culture medium: Use a sterile cotton swab to dip the bacterial solution and evenly apply it on the surface of the prepared culture medium. Place drug-sensitive paper: Gently place drug-sensitive paper containing different antibiotics on the culture medium, ensuring that the paper is in close contact with the culture medium. Incubation: Place the culture dish at an appropriate temperature and conditions for incubation, such as incubating in an anaerobic chamber at 37°C for 48 hours.

[0045] (4) Observe the inhibition zone: After incubation, observe whether an inhibition zone is formed around the drug-sensitive paper. The size of the inhibition zone reflects the sensitivity or resistance of the probiotics to the antibiotic. Measurement and recording: Use appropriate tools to measure the diameter of the inhibition zone and record the size of the inhibition zone corresponding to each antibiotic.

[0046] 4. Antibacterial test of B.ovatus (1) Inoculate B. ovatus into the corresponding liquid culture medium, culture in a CO2 incubator at 37°C for 36-72 h, and centrifuge at 6,000 g for 5 min. (2) Spread Candida albicans, Staphylococcus aureus, Shigella coli O157, Salmonella typhimurium, and ATCC monoculture on LB solid culture medium. Gently place the Oxford cup on the plate, pipette 250 μL of the probiotic culture supernatant into the Oxford cup (do two replicates for each group), culture at 37 ℃, observe the size of the inhibition zone every 2 hours, measure the diameter of the inhibition zone after 8 hours, and keep experimental records.

[0047] 5. Cell Adhesion Assay of B.ovatus (1) Inoculate B. ovatus into the corresponding liquid culture medium and culture it in a carbon dioxide incubator at 37°C until its OD value reaches 0.6, then terminate the culture and keep the strain for later use; (2) Wash the Hela cell culture plate with a coverage rate of 30% on the coverslip once with sterile PBS buffer, take 1 mL of the above bacterial culture solution, mix it with 1 mL of a small amount of Hela cell culture solution, add it to the six-well plate, and culture it in a cell culture incubator at 37°C; (3) After 1 to 1.5 hours of incubation, remove the six-well cell culture plate, aspirate the culture medium, wash the plate five times with PBS buffer, fix the plate with methanol, perform Gram staining, and observe the plate under an oil immersion microscope.

[0048] Evaluation of the probiotic properties of B. ovatus in vitro Figure 1 shown by Figure 1It can be seen that B. ovatus is sensitive to chloramphenicol (C) 2.5cm, tetracycline (TET) 4.6cm, gentamicin (GEN) 2.1cm, penicillin (PEN) 1.7cm, ampicillin (AMP) 1.8cm, lincomycin (MY) 2.5cm, ciprofloxacin (CIP) 1.7cm, succinimidyl (SXT) 3.2cm, erythromycin (E) 4cm, and resistant to ceftriaxone (CTR). The inhibition zones of Bacteroides ovatus NCU-34 against Staphylococcus aureus Cowan 1 (1.4cm), Listeria monocytogenes ATCC 19111 (1.5cm), Salmonella typhi ATCC 6539 (1.3cm), Candida albicans SC531 (1.2cm), Shigella flexneri ATCC 12022 (0.8cm), and Escherichia coli O157 (0.8cm). In general, drug resistance experiments and antibacterial experiments suggest that B. ovatus is resistant to some antibiotics and has an antibacterial effect on common intestinal pathogens. At the same time, B. ovatus can withstand 0.2% bile salts and adhere to intestinal cells, providing a basis for exerting its functions in the body.

[0049] Example 3 This embodiment provides a method for preparing a culture of Bacteroides ovatus NCU-34 (metabolite indole-3-acetic acid), comprising the following steps: (1) Use peeled potatoes and glucose (mass ratio of 10:1) or glucose and peptone (mass ratio of 4:1) as the culture medium, then add distilled water to 1 L and sterilize at 121°C for 20 minutes.

[0050] (2) Inoculation and fermentation: After the culture medium is cooled, inoculate B. ovatus and continue to culture and ferment at 30°C, 120-130 r / min, and in the absence of light for 8-16 days.

[0051] (3) Extraction and purification: After the fermentation is completed, the fermentation mycelium and the bacterial liquid are separated; the bacterial liquid is extracted using an organic solvent such as ethyl acetate, the extracts are combined and concentrated, and then further purification steps (such as crystallization, recrystallization, etc.) are performed to obtain indole-3-acetic acid with higher purity.

[0052] In order to prove that a large amount of indole-3-acetic acid can be produced, this example also carried out tryptophan-targeted metabolomics detection on the feces of B. ovatus after oral gavage. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to accurately quantify 31 common tryptophan metabolites. To facilitate data analysis, a customized MetWo database (MWDB) was developed using the established standards of Wuhan MetWo Biotechnology Co., Ltd. A triple quadrupole mass spectrometer was used to acquire mass spectrometry quantitative data in multiple reaction monitoring (MRM) mode. The mass spectrometry data were processed using Analyst 1.6.3 software, and the chromatographic peaks detected for different sample analytes were integrated and corrected with reference to the retention time and peak shape information of the standard. The integrated peak area ratio of the detected sample was input into the linear equation derived from the standard curve to calculate the concentration of the target compound in the sample. The acquired metabolome data underwent comprehensive statistical analysis, including principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and cluster analysis. Differential metabolite screening was performed to identify significant changes in the metabolite spectrum, and its structure is as follows Fig. 9 As shown (analysis of tryptophan metabolites of Bacteroides ovatus NCU-34): different colors are filled with different values ​​obtained after standardization of different contents (red represents high content, green represents low content). It can be seen that indole metabolites are more enriched in the BO group, such as IAA, IA, and IGA, and some kynurenine metabolites XA, KYNA, and QA are more enriched in the M group.

[0053] Example 4 This example is to explore the effect of humanization of mouse intestinal flora, and the specific process is as follows: 1. Experimental Materials (1) Experimental animals and feeding conditions Four-week-old ApoE- / - mice were housed in an SPF-grade experimental animal room and maintained under a 12-hour light-on and 12-hour dark-off cycle, with 4-5 mice per cage and free access to food and water. The mice were given a 7-day adaptive feeding period before the start of the experiment. The temperature, humidity, noise and other conditions of the animal room were controlled within the specified range and cleaned regularly every day.

[0054] (2) Experimental animal grouping MC group (ApoE- / -bacterial mice gavaged with FMT from patients with coronary heart disease), fed (HFD); MN group (control group FMT gavaged ApoE- / - bacteria-reduced mice), fed (HFD); HC group (feces from patients with coronary heart disease); HM group (feces of the control group).

[0055] The appropriate number of donors was determined based on previous fecal microbiota transplantation (FMT) experiments, and donor screening was performed according to the following criteria: patients with coronary heart disease referred to patients diagnosed with myocardial infarction because their clinical symptoms were more severe and typical; the control group referred to people with risk factors for atherosclerosis (hypertension, diabetes, or dyslipidemia) but without a diagnosis of coronary heart disease.

[0056] Mixed antibiotic application strategy: An antibiotic cocktail consisting of vancomycin (100 mg / kg), neomycin sulfate (200 mg / kg), metronidazole (200 mg / kg), and ampicillin (200 mg / kg) was administered by oral gavage once daily for 1 week before FMT. Mice were monitored for signs of dehydration. PBS and bacterial suspension were administered by oral gavage once daily for 6 weeks.

[0057] 2. Detection indicators 2.1 Mouse feces extraction and 16S rRNA sequencing Fecal samples were stored at −80°C, and genomic DNA was extracted using a genomic DNA extraction kit (Shanghai Personalbio Biotechnology Co., Ltd.) according to the manufacturer's protocol. The V1-V9 region of the 16S rRNA gene was amplified using universal primers under optimized thermal cycling conditions. The amplified DNA fragments were paired-end sequenced on an Illumina HiSeq 2500 platform. The raw sequencing data were quality controlled using Vsearch (v2.13.4_linux_x86_64) and cutadapt (v2.3) to remove adapters, primers, and low-quality sequences. Amplicon sequence variants (ASVs) or operational taxonomic units (OTUs) representative sequences were obtained, and the length distribution of high-quality sequences was evaluated. Species annotation was performed using the classify-sklearn algorithm in the QIIME2 package. Each ASV or OTU sequence was classified using a pre-trained Naive Bayes classifier, and the sequences were compared with reference databases such as Greengenes, NT, and UNITE. Taxonomic annotation was performed at multiple taxonomic levels (kingdom, phylum, class, order, family, genus, and species). Subsequent analyses included assessments of species composition, alpha diversity, and beta diversity.

[0058] The results are as follows Fig.10 As shown, β diversity ( Fig.10 a) and relative abundance of genus-level bacteria in feces of two groups of mice ( Fig.10b) There were differences. The results of HC (feces of patients with coronary heart disease) and MC were similar, and the results of HM (feces of the control group) and MN were similar, proving that the intestinal flora of human origin can colonize well in the mouse intestine.

[0059] Example 5 This example is to explore the effect of Bacteroides ovatus NCU-34 transplantation on improving atherosclerosis, and the specific process is as follows: 1. Experimental Materials (1) Experimental animals and feeding conditions Four-week-old C57BL / 6 mice and ApoE- / - mice were housed in an SPF-grade experimental animal room and maintained under a 12-hour light-on and 12-hour dark-off cycle, with 4-5 mice per cage and free access to food and water. The mice were given a 7-day adaptive feeding period before the start of the experiment. The temperature, humidity, noise and other conditions of the animal room were controlled within the specified range and cleaned regularly every day.

[0060] (2) Experimental animal grouping Group C (WT, C57BL / 6 mice), fed with a standard diet consisting of (10 kcal %) and an equal amount of distilled water; Group M (ApoE- / - mice), fed (HFD); MC group (ApoE- / -bacterial mice gavaged with FMT from patients with coronary heart disease), fed (HFD); BO group (Bacteroides ovatus NCU-34 in ApoE- / - mice, intragastrically administered with bacterial solution at a concentration of 1×10 9 CFU / mL), fed (HFD); Mixed antibiotic application strategy: An antibiotic cocktail consisting of vancomycin (100 mg / kg), neomycin sulfate (200 mg / kg), metronidazole (200 mg / kg), and ampicillin (200 mg / kg) was administered by oral gavage once daily for 1 week before FMT. Mice were monitored for signs of dehydration. PBS and bacterial suspension were administered by oral gavage once daily for 6 weeks.

[0061] 2. Detection indicators (1) Animal general condition and weight measurement a. Observe the coat color, mental state, activity, cumulative food intake, water intake, stool and survival status of mice in each group; b. During the experiment, the weight of mice was measured every week. In the early morning, the mice were slowly placed on the electronic balance, and the value was read after stabilization. Three stable values ​​were measured continuously, and the average value was taken as the final weight value. When the mice were killed, gross images were taken: aorta, liver, spleen, colon, and the weight of aorta, liver, epididymal fat, spleen, and colon length were measured.

[0062] (2) Aorta ① Paraffin section HE and oil red staining to observe lipid deposition: a. HE staining of the aortic root area of ​​atherosclerosis model mice (morphological damage and necrotic core area); b. Oil red O staining of longitudinally opened aortic arch, thoracic and abdominal areas.

[0063] ②Masson staining to observe the number of tissue elastic fibers: Collagen fibers in the aortic root area were stained to observe the morphology of the vascular wall.

[0064] ③Immunohistochemistry and immunofluorescence staining of paraffin sections: a. Immunofluorescence detection of aortic CD86 (M1 macrophage recruitment and activation) and CD206 (M2 macrophage recruitment and activation); b. Immunohistochemistry cell adhesion molecules: vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM). 3. Observation of lipid deposition by HE and oil red staining of liver 4. Colonic tissue permeability Multiple immunostaining of colon tissue for occludin (occludens), ZO-1 (tight junction protein), and the number of goblet cells stained with Alcianblue / periodic acid–Schiff (AB-PAS, mucin).

[0065] 5.ELISA detection of serum inflammatory factors and related indicators (1) Determine the concentrations of cytokines interleukin IL-6, IL-1β, TNF-α, and lipopolysaccharide (LPS, an important indicator of intestinal barrier damage) in the stored whole blood supernatant; (2) Plasma lipid profile: TC, TG, and ldl-c.

[0066] (3) Serum indole-3-acetic acid detection.

[0067] 6. Fecal qPCR Analysis The relative abundance of colonizing bacteria in feces was determined by qPCR on day 3 after 4 weeks of treatment. Bacterial DNA in mouse feces was extracted using the QIAamp DNA Feces Micro Kit (QIAGEN) and qPCR analysis was performed on a 7500 Fast Real-Time PCR System with different primers to verify the colonization effect of Bacteroides ovatus NCU-34 and indole-3-acetic acid detection.

[0068] The results are as follows Figure 2-Figure 3 As shown (where Figure 2 a in the figure is the relevant experimental design: C57BL / 6J and ND or ApoE- / - mice were fed HFD for 8 weeks, pretreated with diet for 8 weeks, and then given broad-spectrum Abx by gavage for 1 week. The experimental intervention was fecal suspension or Bacteroides ovatus NCU-34 (1×10 9 CFU / mL ) was intragastrically administered with 200 μL, once every 2 days for 8 weeks)). The results showed that intragastrically administered Bacteroides ovatus NCU-34 (BO group) could reduce body weight compared with the M group (P=0.0384) ( Figure 2 b). The BO group improved serum LPS and inflammatory factors (Figure 3c-d) and blood lipid levels ( Figure 2 e), compared with the M and MC groups. Similarly, the evaluation of aortic plaque is the core indicator, Oil Red "O" staining ( Figure 2 f) and area percentage ( Figure 3 a) It was shown that the MC group increased the plaque by 7%, while the BO group reduced the formation of aortic plaque by 16%. Liver Oil Red "O" staining image ( Figure 3 g) and NAS score ( Figure 3 d) showed that the BO group effectively reduced liver fat deposition and liver damage. Colon length and crypt depth ( Figure 3 fg) suggest that B. ovatus colonization can restore the depth of intestinal crypts, increase colon length, and restore the content of intestinal barrier proteins (ZO-1, Occludin) ( Figure 3 hi). Finally, the spleen and epididymis were evaluated for spleen mass weight and length ( Figure 3 j) and epididymal body weight ( Figure 3 k) proved that the BO group could improve the inflammatory state of mice to a certain extent. Although in vitro adhesion experiments were performed, it was necessary to use specific B. ovatus primers for quantitative PCR (qPCR) on feces. The analysis found that B. ovatus could colonize in the intestines of mice ( Figure 3 l) Example 6 This example explores the effect of culturing indole-3-acetic acid (IAA) in Bacteroides ovatus NCU-34 obtained in Example 3 on improving atherosclerosis. The specific process is as follows: 1. Experimental Materials (1) Experimental animals and feeding conditions Four-week-old C57BL / 6 mice and ApoE- / - mice were housed in an SPF-grade experimental animal room and maintained under a 12-hour light-on and 12-hour dark-off cycle, with 4-5 mice per cage and free access to food and water. The mice were given a 7-day adaptive feeding period before the start of the experiment. The temperature, humidity, noise and other conditions of the animal room were controlled within the specified range and cleaned regularly every day.

[0069] (2) Experimental animal grouping Group C (WT, C57BL / 6 mice), fed with a standard diet consisting of (10 kcal %) and an equal amount of distilled water; Group M (ApoE- / - mice), fed (HFD); MC group (ApoE- / - mice gavaged with FMT from patients with coronary heart disease), fed (HFD); IAA group (indole-3-acetic acid was gavaged into ApoE- / - mice, 50 mg / kg) and fed (HFD).

[0070] (3) Experimental animal treatment At the end of the 8-week experiment, mice were fasted and deprived of water for 6 hours before being killed, and were anesthetized with sodium pentobarbital (50 mg / kg). The eyeballs were removed to obtain whole blood, and the blood was allowed to stand at room temperature for 2 hours, centrifuged at 3000 rpm at 4°C for 15 minutes, and the supernatant was stored in a -80°C refrigerator for later use. The entire aorta, colon, and liver were separated, and the heart, colon, and liver containing the aortic root and aortic arch were frozen in liquid nitrogen and stored at -80°C for further analysis. The weight of the epididymal fat pad was measured.

[0071] 2. Detection indicators (1) Animal general condition and weight measurement ① Observe the coat color, mental state, activity, cumulative food intake, water intake, stool and survival status of mice in each group; ② During the experiment, the weight of mice was measured every week. In the early morning, the mice were slowly placed on the electronic balance, and the value was read after stabilization. Three stable values ​​were measured continuously, and the average value was taken as the final weight value. When the mice were killed, gross images were taken: aorta, liver, spleen, colon, and the weight of aorta, liver, epididymal fat, spleen, and colon length were measured.

[0072] (2) Aorta Paraffin section HE and oil red staining to observe lipid deposition: ① HE staining of the aortic root area of ​​atherosclerosis model mice (morphological damage and necrotic core area); ② Oil red O staining of the longitudinally opened aortic arch, chest and abdominal areas.

[0073] Masson staining to observe the number of tissue elastic fibers: Collagen fibers in the aortic root area were stained to observe the morphology of the vascular wall.

[0074] Immune infiltration in paraffin sections by immunohistochemistry and immunofluorescence staining: ① Immunofluorescence detection of aortic CD86 (M1 macrophage recruitment and activation) and CD206 (M2 macrophage recruitment and activation); ② Immunohistochemical cell adhesion molecules: vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM); RNA extraction and real-time fluorescence quantitative PCR, protein extraction and WB detection (3) Observation of lipid deposition by HE and oil red staining of liver (4) Colon tissue permeability: RNA extraction and real-time fluorescence quantitative PCR, protein extraction and WB detection Colon tissue occludin, ZO-1 (5) ELISA detection of serum inflammatory factors and related indicators ① Determine the concentration of cytokines interleukin IL-6, IL-1β, TNF-α, and lipopolysaccharide (LPS, an important indicator of intestinal barrier damage) in the stored whole blood supernatant; ② Plasma lipid profile: TC, TG, ldl-c; ③ Serum indole-3-acetic acid detection.

[0075] The experimental results are as follows Figure 4 As shown (where Figure 4 a in the figure is C57BL / 6J and ND or ApoE- / - mice fed with HFD for 8 weeks, pretreated with diet for 8 weeks, and then given broad-spectrum Abx by gavage for 1 week. The experimental intervention was fecal suspension or IAA gavage 200 μL, once every 2 days, for 8 weeks). IAA gavage (IAA group) can reduce body weight compared with the M group (P=0.0301) (Figure 4b). In addition, serum LPS content and inflammatory factors ( Figure 4 cd), as well as serum lipid content (Figure 4e), all proved that IAA has the effect of reducing the level of inflammation in mice and regulating lipid metabolism. Next, the aorta body weight ( Figure 5 a), Oil Red "O" staining area percentage (Figure fg) and other evaluation results have changed significantly, and the IAA group can reduce the formation of aortic plaques by 20%. At the same time, HE staining of the aortic valve also showed changes in the shape of the plaques ( Figure 5 b) Quantification of plaque area and percentage of plaque necrotic core area ( Figure 4 g), it can be concluded that IAA has a certain effect of stabilizing and reducing plaque formation. This effect was supported by the 37% increase in aortic collagen content and 43% and 38% decreases in inflammatory chemokines VCAM and ICAM in the IAA group compared with the M group, which was also supported by macrophage M1 / M2 immunofluorescence staining ( Figure 4 hi). In conclusion, we demonstrated that IAA can improve aortic atherosclerosis, mainly by improving inflammation.

[0076] In addition, the lipid profile of related organs was analyzed. Liver solid weight and NAS score ( Figure 5 c), Oil red “O” staining area percentage of liver ( Figure 4 j) decreased by 70%, further supporting the conclusion that IAA can reverse lipid deposition in mice. Obviously, the colon length and crypt depth ( Figure 5 f) AB-PAS staining proves ( Figure 5 e), IAA can restore the number of goblet cells and deepen the depth of intestinal crypts. At the same time, the WB results of Occludin and Claudin-1 showed that ( Figure 4 kl), IAA also has the ability to restore intestinal barrier proteins. Since it can improve systemic symptoms, we speculate that IAA can enter the blood, so we tested the IAA content in mouse serum ( Figure 5 i). It was found that the imbalance of IAA was improved, which also explained why oral gavage could directly affect the aorta.

[0077] Example 7 This example explores the role of indole-3-acetic acid in regulating macrophages to improve atherosclerosis 1. Macrophage Culture RAW264.7 mouse macrophages were cultured at 2 × 10 5 The cells were seeded in 6-well plates at a density of 100 cells / mL and cultured in a 37°C, 5% CO2 incubator using DMEM medium containing 10% fetal bovine serum, 100 mg / mL streptomycin, and 100 U / mL penicillin.

[0078] 2. M1 polarization induction and grouping Lipopolysaccharide (LPS) and interferon-γ (INFγ) were used to induce pro-inflammatory M1 polarization. The experimental groups were as follows: control group (C): PBS; LPS (100 ng / ml) + Infγ (10 ng / ml) group; LPS + INFγ + IAA group (0.25-1mM); IAA group (1 mM); IAA was added 4 h before LPS treatment and then continued to be incubated with LPS for 18h.

[0079] 3. M2 polarization induction and grouping Interleukin-4 (IL-4) was used to induce M2 polarization. Experimental groups: Control group (C): PBS; IL-4 group (20 ng / mL); IL-4 + IAA group (1 mM); IAA group (1 mM); After 4 h of IAA pretreatment, IL-4 was added and incubated for 18 h.

[0080] 4. Inducing Cell Hyperlipidemia Model and Grouping Palmitoleic acid was used to establish a cell high lipid model. The experiment was divided into a control group (C) and a PBS group; the model group was treated with palmitoleic acid (10 mM), LPS (100 ng / mL), and INFγ (10 ng / mL); the IAA group received IAA (1mM) combined with palmitoleic acid, LPS, and INFγ; and the other group was treated with IAA alone. After 4 h of IAA pretreatment, palmitoleic acid was added for 18 h, and then LPS and INFγ were added for 6 h.

[0081] 5. AHR inhibitor experiments and grouping In the AHR inhibitor experiment, CH223191 was used. Group C (PBS); other groups included IL-4 alone, IL-4 combined with IAA (1 mM), and IL-4 combined with IAA and CH223191 (10 µM). After 4 h of pretreatment with IAA and CH223191, IL-4 was added and cultured for more than 18 h.

[0082] Detection indicators: Immunofluorescence: M1 marker CD86, M2 marker CD206, AHR Macrophage Oil Red "O" staining Mouse aorta WB: TLR-4, Myd88, P65, PP65, IL-6, AHR, Arg-1, IL-10.

[0083] Detection of IAA content in human serum.

[0084] The results are as follows Figure 6-7 As shown in the literature, animal experiments have shown that the M1 / M2 balance of macrophages is unbalanced in high-fat model mice, and IAA can restore the balance. Therefore, IAA can improve AS by acting on macrophages. First, we used classic drugs to induce M1 polarization of RAW264.7 cells ( Figure 6 a). Immunofluorescence CD68+AHR double staining results showed ( Figure 6bc), IAA can inhibit LPS+INFγ-induced M1 polarization, and this effect is not dependent on ahr activation. Subsequently, WB experiments were performed on the LPS-induced classical pathway ( Figure 6 d) TLR4 and myd88 inflammatory proteins decreased with increasing IAA concentrations, suggesting that the reduction of M1 polarization may be through the TLR4 / myd88 pathway. Then, M2 polarization is induced ( Figure 7 b). Immunofluorescence CD206+AHR staining results show that ( Figure 6 fg), IAA can promote IL-4-induced M2 polarization, and it depends on the activation of ahr. Next, to further explore the reasons why IAA reduces atherosclerosis, we conducted experiments on the high-fat model of RAW264.7 cells induced by palmitic acid combined with LPS+INFγ ( Figure 7 c). The results suggest that IAA can reduce the uptake of lipids by cells to a certain extent ( Figure 7 d).

[0085] In order to clarify the mechanism of IAA's effect on AS, we used IAA to intervene in the aorta of mice in vivo. First, qPCR was performed on the typical inflammatory signaling pathways of the aorta. The results showed that the TLR4 / MyD88 / P65 pathway and inflammatory-related factors were significantly changed, but there were no significant differences in lipid metabolism, AKT / mTOR / HIF-1α, and JAK pathways ( Figure 5 j). Therefore, WB detection was performed to confirm the increase of TLR4 / MyD88 / P65 pathway and inflammatory factor IL-6 ( Figure 6 hi), and both were reversed by IAA. Combined with the changes in M2 in cells, we found that IAA could enhance the expression of anti-inflammatory factors Arg-1 and IL-10, which may be partly through the AHR receptor ( Figure 6 hi). Finally, our team tested the IAA content in human serum ( Figure 6 j), the results showed that the content of ASCVD group was lower than that of NP group. The above results proved that IAA is an important microbial metabolite that can improve the progression of AS.

[0086] The embodiments of the present application are described above in conjunction with the accompanying drawings. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of ​​the present application, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the inspiration of the present application, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present application and the claims, all of which are within the protection of the present application.

Claims

1. A strain of Bacteroides ovatus NCU-34, characterized in that The Bacteroides ovatus ( Bacteroides ovatus )NCU-34 was deposited on December 6, 2024 at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32937.

2. The Bacteroides ovatus NCU-34 according to claim 1, characterized in that The gene sequence of 16S rDNA of the Bacteroides ovatus NCU-34 is shown in SEQ ID No:

1.

3. A culture of Bacteroides ovatus NCU-34, characterized in that Prepared by the Bacteroides ovatus NCU-34 described in any one of claims 1-2.

4. The Bacteroides ovatus NCU-34 culture according to claim 3, characterized in that The Bacteroides ovatus NCU-34 culture contains at least one of live Bacteroides ovatus NCU-34 bacteria, bacterial components of Bacteroides ovatus NCU-34 and derivatives thereof, and metabolites of Bacteroides ovatus NCU-34.

5. The Bacteroides ovatus NCU-34 culture according to claim 4, characterized in that The Bacteroides ovatus NCU-34 metabolites include indole-3-acetic acid.

6. Use of the Bacteroides ovatus NCU-34 according to any one of claims 1 to 2 or the Bacteroides ovatus NCU-34 culture according to any one of claims 3 to 5 in the preparation of a medicament for preventing and / or treating atherosclerosis.

7. A drug for preventing and / or treating atherosclerosis, characterized in that: The drug includes the Bacteroides ovatus NCU-34 described in any one of claims 1-2.

8. The drug according to claim 6, characterized in that The medicine also includes pharmaceutical excipients.

9. The drug according to any one of claims 7-8, characterized in that The dosage form of the medicine is powder, granule, capsule or tablet.

10. A biological agent, characterized in that: The biological preparation includes the Bacteroides ovatus NCU-34 described in any one of claims 1-2 or the Bacteroides ovatus NCU-34 culture described in any one of claims 3-5.

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