Bacteroides uniformis capsular polysaccharide extract and its application in preventing and treating ulcerative colitis

Abstract

The present invention discloses a capsular polysaccharide extract of Bacteroides uniformis and its application in the prevention and treatment of ulcerative colitis. The Bacteroides uniformis extract is a capsular polysaccharide of Bacteroides uniformis. In the present invention, the inventors first discovered a capsular polysaccharide of Bacteroides uniformis and found that it can effectively prevent or treat DSS-induced colitis, and verified the effectiveness of this effect through a colitis mouse model, thus providing a new idea and means for the treatment of ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to extracts of capsular polysaccharides of Bacteroides uniformis and their application in the prevention and treatment of ulcerative colitis. Background Art

[0002] Inflammatory bowel disease (IBD) refers to a series of diseases with common features of chronic gastrointestinal inflammation, usually manifested as non-specific clinical features such as abdominal pain, recurrent or bloody diarrhea, weight loss, and fatigue. In the past few decades, IBD has become one of the major challenges facing global public health. Worldwide, approximately 6.8 million people suffer from IBD, especially in newly industrialized countries, where the incidence of IBD shows an upward trend. Although the etiology of IBD has not been fully elucidated, there is evidence that it is affected by complex interactions among genetic, gut microbiota, external environment, immunity and other factors.

[0003] A healthy gut microbiota ecosystem is considered essential for the normal health of the host. On the contrary, dysregulation of gut microbiota, including a decrease in the overall species diversity of the flora or certain probiotics, is associated with gut dysfunction and gut diseases. Gut microbiota dysbiosis is a typical feature of gut-related diseases (including IBD). Therefore, regulating the stable state and function of gut microbiota to achieve the preventive and / or therapeutic benefits of gut diseases has important clinical significance.

[0004] Postbiotics refer to preparations of inanimate microorganisms and / or their components that are beneficial to the health of the host. Capsular polysaccharide is the main component in the outer capsule of the cell wall and is also one of the main representatives of the new generation of postbiotics. Studies have found that compared with live single strains, capsular polysaccharides of certain postbiotic strains can better restore the diversity of gut microbiota, which avoids the safety hazards brought by introducing single strains and has better safety and effectiveness. At the same time, compared with live strains, postbiotics of strains with special activities can be better preserved and applied and promoted. Therefore, the development of new strain postbiotics has important market value. Summary of the Invention

[0005] The present invention aims to solve at least one of the above technical problems existing in the prior art. To this end, the present invention provides an extract of Bacteroides uniformis and its application in the prevention and treatment of ulcerative colitis. In the present invention, an extract of Bacteroides uniformis, especially capsular polysaccharide of Bacteroides uniformis, is first discovered for its preventive and therapeutic effects on ulcerative colitis, and the effectiveness of this effect is verified through a colitis mouse model, thus providing a new idea and means for the treatment of ulcerative colitis.

[0006] In the first aspect of the present invention, there is provided the use of Bacteroides uniformis extract in the preparation of a medicament for preventing and / or treating ulcerative colitis.

[0007] In some embodiments of the present invention, the Bacteroides uniformis extract is Bacteroides uniformis capsular polysaccharide.

[0008] In some embodiments of the present invention, the Bacteroides uniformis capsular polysaccharide is composed of arabinose, galactose, glucose, galacturonic acid and glucuronic acid, and the molar ratio of arabinose, galactose, glucose, galacturonic acid and glucuronic acid is 0.51:0.25:300.19:1.95:1.72.

[0009] In the present invention, through database comparison, the inventors did not find polysaccharides identical to the Bacteroides uniformis capsular polysaccharide in the present invention in the prior art. Moreover, there are differences in the types and proportions of monosaccharide compositions in the polysaccharides obtained from the existing Bacteroides genus (including Bacteroides fragilis) and the Bacteroides uniformis capsular polysaccharide in the present invention. Therefore, it may be a novel-structured extracellular heteropolysaccharide of bacteria.

[0010] In some embodiments of the present invention, the relative molecular mass of the Bacteroides uniformis capsular polysaccharide is 1.819×10 4 Da.

[0011] In some embodiments of the present invention, the total sugar content of the Bacteroides uniformis capsular polysaccharide is 99.83%.

[0012] In some embodiments of the present invention, the Bacteroides uniformis capsular polysaccharide has a strong absorption of hydroxyl groups at 3367 cm -1 There is a strong absorption of hydroxyl groups.

[0013] In some embodiments of the present invention, the Bacteroides uniformis capsular polysaccharide is derived from Bacteroides uniformis ATCC8492.

[0014] In some embodiments of the present invention, the method for extracting the Bacteroides uniformis capsular polysaccharide includes the following steps:

[0015] (1) Repeatedly freeze-thaw Bacteroides uniformis and then perform ultrasonic disruption. Centrifuge to collect the supernatant, concentrate under reduced pressure and then perform alcohol precipitation;

[0016] (2) Take the alcohol-precipitated precipitate for deproteinization treatment. After ultrafiltration and washing, perform chromatography using ion-exchange cellulose, elute with NaCl, and collect the eluate in the peak segment of 0 mol / L to obtain the product.

[0017] In some embodiments of the present invention, the deproteinization treatment includes using papain and Sevag reagent for deproteinization.

[0018] In some embodiments of the present invention, the protein removal treatment is as follows: first, enzymatic hydrolysis is carried out using papain, and then the Sevag reagent is used to further remove proteins.

[0019] In some embodiments of the present invention, a 3KD ultrafiltration tube is used for ultrafiltration.

[0020] In some embodiments of the present invention, the ion exchange cellulose is DEAE cellulose-52 ion exchange cellulose.

[0021] In some embodiments of the present invention, in the chromatography, the eluent used is an NaCl solution.

[0022] In some embodiments of the present invention, the elution concentrations of the NaCl solution are 0, 0.05, 0.1, 0.3, and 0.5 mol / L in sequence.

[0023] In some embodiments of the present invention, the extraction method further includes further purification using a Sephadex gel chromatography column.

[0024] In some embodiments of the present invention, the eluent for Sephadex gel chromatography is a 0.1 mol / L NaCl solution.

[0025] In some embodiments of the present invention, the drug further includes pharmaceutically acceptable excipients.

[0026] In some embodiments of the present invention, the excipients include but are not limited to: solvents, emulsifiers, softeners, antioxidants, preservatives, chelating agents, pH regulators, thickeners, penetration enhancers, and light blockers.

[0027] In some embodiments of the present invention, the content of the Bacteroides uniformis extract in the drug is 1-100 wt%.

[0028] In some embodiments of the present invention, the applicable objects of the drug are animals, and the animals are selected from humans, cats, cows, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice.

[0029] In some embodiments of the present invention, the applicable object of the drug is humans.

[0030] The second aspect of the present invention provides the use of the Bacteroides uniformis extract in the preparation of an immunomodulator.

[0031] In some embodiments of the present invention, the immunomodulator can increase the proportion of Treg cells, decrease the proportion of Th17 cells, inhibit macrophage generation, and regulate the intestinal flora abundance.

[0032] In the present invention, after analyzing the intestinal lamina propria cells of mice administered with the capsular polysaccharide of Bacteroides uniformis by flow cytometry, it was found that the capsular polysaccharide of Bacteroides uniformis could inhibit the proportion of pro-inflammatory Th17 immune cells and stimulate the proportion of Treg cells that inhibit inflammation. This indicates that the capsular polysaccharide of Bacteroides uniformis has a good function of inhibiting inflammation under inflammatory conditions and an impact on the differentiation of the important induced immune cell subset Treg / Th17. In addition, it can also inhibit the proportion of macrophages. This suggests that the capsular polysaccharide of Bacteroides uniformis can inhibit the formation of macrophages in innate immunity induced by inflammation, thereby inhibiting inflammation. At the same time, it was also found that the capsular polysaccharide of Bacteroides uniformis could alleviate the decrease in microbial diversity induced by DSS. Therefore, it can be stated that the capsular polysaccharide of Bacteroides uniformis may be used as an intestinal microbiota regulator to relieve intestinal inflammation by increasing microbial diversity.

[0033] The beneficial effects of the present invention are as follows:

[0034] The present invention first discovered a capsular polysaccharide of Bacteroides uniformis and found that it can effectively prevent or treat DSS-induced colitis, and verified the effectiveness of this effect through a colitis mouse model, thereby providing a new idea and means for the treatment of ulcerative colitis. Description of the Drawings

[0035] Figure 1 It is the gradient elution curve when different components of the crude capsular polysaccharide of Bacteroides uniformis are subjected to DEAE cellulose-52 ion exchange cellulose chromatography and NaCl solution is used as the eluent.

[0036] Figure 2 It is the elution curve after further purification with Sephacryl S-300HR dextran gel after DEAE cellulose-52 ion exchange cellulose chromatography.

[0037] Figure 3 It is the infrared spectrum of BU-CPS-1B.

[0038] Figure 4 It is the ultraviolet spectrum of BU-CPS-1B.

[0039] Figure 5 It is the ion chromatogram of the derivatization product of the mixed monosaccharide standard.

[0040] Figure 6 It is the ion chromatogram of the derivatization product of BU-CPS-B.

[0041] Figure 7 It is the experimental flow chart of the in vivo treatment of colitis mouse model with the capsular polysaccharide BU-CPS-B of Bacteroides uniformis.

[0042] Figure 8 Effect of Bacteroides uniformis capsular polysaccharide BU-CPS-B on body weight and DAI score of colitis mice (N = 7 - 8).

[0043] Figure 9 Effect of Bacteroides uniformis capsular polysaccharide BU-CPS-B on colon length of colitis mice (N = 7 - 8, * compared with NC group, *** indicates P < 0.05, **** indicates P < 0.01).

[0044] Figure 10 Effect of Bacteroides uniformis capsular polysaccharide BU-CPS-B on spleen weight of colitis mice (N = 7 - 8, * compared with NC group, ** indicates P < 0.05, **** indicates P < 0.01).

[0045] Figure 11 Effect of Bacteroides uniformis capsular polysaccharide BU-CPS-B on colon pathology of colitis mice.

[0046] Figure 12 Effect of Bacteroides uniformis capsular polysaccharide BU-CPS-B on the proportion of Treg cells in the colonic lamina propria of colitis mice.

[0047] Figure 13 Effect of Bacteroides uniformis capsular polysaccharide BU-CPS-B on the proportion of Th17 cells in the colonic lamina propria of colitis mice.

[0048] Figure 14 Effect of Bacteroides uniformis capsular polysaccharide BU-CPS-B on the proportion of macrophages in the colonic lamina propria of colitis mice. Specific implementation manner

[0049] The content of the present invention will be further described in detail through specific examples below. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or by existing technical methods without special instructions. Unless otherwise specified, the test or testing methods are conventional methods in the art.

[0050] In the following examples, the Bacteroides uniformis selected is the commercial standard strain Bacteroides uniformis ATCC 8492.

[0051] Culture and identification of Bacteroides uniformis

[0052] In a laminar flow hood, wipe the cryopreservation tube containing Bacteroides uniformis ATCC 8492 with absorbent cotton soaked in 75% alcohol. After air-drying the alcohol, untie the wrapped sealing film, heat around the tube opening with an alcohol lamp, then open the cryopreservation tube, and add 500 μL of sterile water to the freeze-dried strain powder. After dissolution and mixing, pick up the bacterial liquid with a sterile inoculation loop and streak it on a Columbia blood agar plate, and culture it under anaerobic conditions at 37°C for 48 h. After 48 h, pick a single colony from the streaked plate and transfer it to a new blood agar plate for culture, and culture it under anaerobic conditions at 37°C for 48 h.

[0053] Extraction of bacterial genomic DNA: Use a bacterial genomic DNA extraction kit (Tiangen) to extract the genomic DNA of Bacteroides uniformis ATCC 8492, and the specific operation refers to the instruction manual. After obtaining the DNA, detect its DNA concentration and purity, and an OD260 / OD280 ratio in the range of 1.7 - 1.9 is considered qualified.

[0054] PCR amplification: Use the qualified DNA as a template for PCR amplification.

[0055] The PCR amplification system is shown in Table 1.

[0056] Table 1 PCR amplification system

[0057]

[0058]

[0059] Among them, the upstream primer is 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO: 1);

[0060] The downstream primer is 1492R: 5’-GGTTACCTTGTTACGACTT-3’ (SEQ ID NO: 2).

[0061] The PCR amplification program is as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1.5 min, for 30 cycles; extension at 72°C for 10 min. The amplified product is entrusted to Sangon Biotech (Shanghai) Co., Ltd. for 16S rRNA sequencing to confirm the correct bacterial species.

[0062] Extraction and purification of capsular polysaccharide of Bacteroides uniformis

[0063] Inoculate the confirmed Bacteroides uniformis ATCC 8492 into BHI liquid medium and culture it at 37°C for 24 h. After two generations of activation, inoculate it into BHI liquid medium for scale-up culture at an inoculation amount of 2% (v / v). The culture temperature is 37°C and the culture time is 48 h.

[0064] After culturing for 48 h, the fermentation broth was centrifuged at 4500 rpm for 30 min at 4 °C to obtain the bacterial cells (precipitate), which were washed twice with PBS. The bacterial cell precipitate was repeatedly frozen and thawed 10 times. Then, 10 volumes of ddH2O were added, and ultrasonic disruption was performed for 20 min with an interval of 30 s. After centrifugation at 4500 rpm for 30 min, the supernatant was collected. The precipitation part was continued to repeat the above steps of ultrasonic treatment and centrifugation. All the supernatants were pooled and concentrated under reduced pressure to 1 / 5 of the original volume. While stirring, pre-cooled 95% ethanol was added to make the final ethanol concentration in the solution 80%, and left overnight at 4 °C. The next day, after centrifugation at 4000 rpm for 30 min, the crude extract of Bacteroides uniformis capsular polysaccharide after ethanol precipitation was obtained.

[0065] The crude extract of Bacteroides uniformis capsular polysaccharide was subjected to protein removal treatment. Papain combined with the Sevag method was used to remove proteins from the crude extract of Bacteroides uniformis capsular polysaccharide. The specific operation was as follows: The obtained crude extract of Bacteroides uniformis capsular polysaccharide was dissolved in an appropriate amount of ultrapure water, and papain was added to make the final concentration of papain in the solution 1 mg / mL. It was incubated in a water bath at 60 °C with stirring for 1 h, and then boiled at 100 °C for 15 min to fully inactivate the papain therein. After centrifugation at 4000 rpm for 30 min, the supernatant was taken, and 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) was added. After vortexing for 10 min, it was centrifuged at 4000 rpm for 20 min. The water layer was taken and the operation was repeated until the white emulsion between the water layer and the chloroform layer was completely removed. The water layer was collected, concentrated under reduced pressure to remove the residual organic reagents, and then freeze-dried to obtain the protein-free solid form of Bacteroides uniformis capsular polysaccharide.

[0066] The solid form of Bacteroides uniformis capsular polysaccharide was reconstituted with ultrapure water, ultrafiltered and washed with a 3KD ultrafiltration tube, and centrifuged at 4000 rpm, and the operation was repeated three times. A small amount of liquid was left for resuspension and then freeze-dried to obtain the crude extract of Bacteroides uniformis crude capsular polysaccharide.

[0067] The crude capsular polysaccharide extract of *Bacteroides uniformis* was purified using a DEAE cellulose-52 ion exchange cellulose chromatography column. Weigh 20 g of DEAE Cellulose 52 ion exchange cellulose into a glass beaker, add sufficient ultrapure water to swell until the volume remains unchanged. First, treat it with 0.5 mol / L NaOH solution for 1 h, then repeatedly wash it with ultrapure water until neutral, and then treat it with 0.5 mol / L HCl solution for 1 h, and wash it with ultrapure water until neutral. The chromatography column has a specification of 2.6×30 cm. After cleaning, fix it vertically on an iron stand, add 1 / 3 column volume of ultrapure water, and open the outlet. Then slowly pour the packing into the chromatography column along a glass rod, let it settle naturally, and gently tap the column body with a soft rod to remove air bubbles. Then repeatedly add the packing (DEAEcellulose-52 ion exchange cellulose) until it reaches 5 cm from the top of the chromatography column and stop column packing. Connect a constant flow pump and balance it with ultrapure water at a flow rate of 1.0 mL / min. Weigh 50 mg of the crude capsular polysaccharide extract of *Bacteroides uniformis*, dissolve it thoroughly in 10 mL of ultrapure water, centrifuge it at 4500 rpm for 15 min, take the supernatant and filter it through a 0.45 μm filter membrane. Load the sample and start elution by turning on the constant flow pump. The eluents are 0, 0.05, 0.1, 0.3, 0.5 mol / L NaCl solutions in sequence, with a flow rate of 1.0 mL / min, and collect 8 mL in each tube. The collected sample solutions were detected for absorbance at 490 nm using the phenol-sulfuric acid method and an elution curve was plotted. According to the elution curve, collect the components under the peak segment of 0 mol / L NaCl solution. Then the collected eluates were concentrated and washed with a 3 kD ultrafiltration tube respectively, and then freeze-dried to obtain BUCPS-1, which was stored in a desiccator for standby.

[0068] Further purification was carried out using Sephacryl S-300HR dextran gel. Sephacryl S-300HR is a pre-treated packing material, stored in 20% ethanol, and washed with ultrapure water before use. The chromatography column has a specification of 1.6×90 cm. After being cleaned, it is vertically fixed on an iron stand, 1 / 3 column volume of ultrapure water is added, and the outlet is opened. Subsequently, the packing material is slowly poured into the chromatography column along a glass rod, allowed to settle naturally, and the column body is gently tapped with a soft rod to remove air bubbles. Then, the packing material is repeatedly added to the appropriate height. After the liquid level of the packing material remains calm, a constant flow pump is connected, and the dextran gel column is rinsed with 5 column volumes of 0.1 mol / L NaCl solution to remove the residual ethanol in the packing material and further compact the packing material of the gel column. Then, 50 mg of each component purified by DEAE 52 is weighed separately, fully dissolved in 10 mL of ultrapure water, centrifuged at 4500 rpm for 15 min, and the supernatant is taken and filtered through a 0.45 μm filter membrane. The filtrate is used as a sample for loading, and the constant flow pump is turned on to start elution. The eluent is 0.1 mol / L NaCl solution, the flow rate is 0.5 mL / min, and 4 mL is collected in each tube. The collected sample solution is detected every other tube by the phenol-sulfuric acid method, and an elution curve is plotted. According to the elution curve, the eluent under the elution peak is collected. The eluent is concentrated, dialyzed, and freeze-dried to obtain purified Bacteroides uniformis capsular polysaccharide (BU-CPS). Among them, in this example, the BU-CPS-1B component was selected as the Bacteroides uniformis capsular polysaccharide for the following tests. The peak segment distribution of the BU-CPS-1B component is 16-32, and the yield is 84%.

[0069] The gradient elution curves corresponding to each component of BU-CPS are as Figure 1 and 2 shown.

[0070] An appropriate amount of dried BU-CPS-1B was taken and pressed into flakes, and infrared spectroscopy was performed using ATR-FTIR, and infrared scanning was carried out in the wavenumber range of 4000-400 cm -1 .

[0071] The results are as Figure 3 shown. Infrared determination shows that BU-CPS-1B has a strong absorption of hydroxyl groups at 3367 cm -1 .

[0072] Ultraviolet spectroscopy was used to analyze whether the purified polysaccharide sample contains nucleic acids and proteins. The specific operation is as follows: BU-CPS-1B was prepared into a 1 mg / mL solution with ultrapure water, and ultraviolet full-wavelength scanning was carried out in the wavelength range of 190-400 nm, and whether there are absorption peaks at 260 nm and 280 nm was observed to judge whether the purified polysaccharide sample contains nucleic acids and proteins.

[0073] The results are as Figure 4As shown, ultraviolet detection found no absorption peaks of proteins and nucleic acids at 260 / 280, indicating that the purified polysaccharide sample contains no nucleic acids and proteins.

[0074] Using glucose as a standard, the total sugar content of BU-CPS-1B was determined by the phenol-sulfuric acid method. Among them, the total sugar content in the sample can be calculated according to the standard curve:

[0075]

[0076] Among them, C is the concentration calculated according to the standard curve, M is the actual weighed mass, and V is the total volume of the extraction solution.

[0077] The specific operation is as follows: Weigh 2 - 5 mg of the sample, dissolve it in 1 mL of sterile water, ultrasonically extract for 10 min, and centrifuge at 12000 rpm for 10 min. Pipette 0.4 mL of the supernatant, dilute it with ddH2O and mix it with phenol-sulfuric acid. After the reaction ends, measure the absorbance at 490 nm, and calculate the total sugar content based on the obtained glucose standard curve.

[0078] The glucose standard curve obtained by the phenol-sulfuric acid method is y = 2.81x + 0.0551, R 2 = 0.9986. After calculation, it was found that the total sugar content of BU-CPS-1B was 99.83%, and its sugar purity was high.

[0079] Gel Permeation Chromatography (GPC) was used to detect the relative molecular weight of the polysaccharide of BU-CPS-1B and to test its purity at the same time. The specific operation is as follows: First, inject dextran standards with different molecular weights successively, record their retention times (TR), and plot a standard curve with the TR of each dextran standard as the abscissa and the logarithm of the corresponding molecular weight (Mw) as the ordinate to obtain the regression equation of Lg(Mw) and TR, specifically: lg(Mw) = -1.0977xTR + 12.271, and the correlation coefficient R 2 = 0.994. The chromatographic conditions are as follows: The high-performance liquid chromatograph is Shimadzu LC-20AT, the chromatographic column is PolySep-GFC-P 4000 chromatographic column (Phenomenex, 300×7.8 mm), the detector is an Evaporative Light Scattering Detector (ELSD), the set temperature of the detector is 60°C, the gain value is 10, the column temperature is 35°C, ultrapure water is used as the mobile phase, the flow rate is 1.0 mL / min, and the injection volume is 20 μL for detection.

[0080] The relative molecular mass of BU-CPS-1B was determined by HPGPC method. According to the linear regression equation, substituting its retention time TR = 7.59 min into the calculation, the relative molecular mass of BU-CPS-1B was obtained as 1.819×10 4 Da.

[0081] The monosaccharide composition of BU-CPS-1B was identified. The monosaccharide composition of BU-CPS-1B was determined by pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) - high performance liquid chromatography. The specific steps were as follows: Weigh 5 mg of BU-CPS-1B into a stoppered reaction tube, add 2 mL of trifluoroacetic acid (TFA), seal it and hydrolyze it in an oil bath at 135 °C for 3 h. After complete hydrolysis, cool it down. Add 5 mL of methanol and concentrate it under reduced pressure until it is dried by rotation. Repeat the treatment 3 times to remove the residual TFA. Then add 800 μL of deionized water to dissolve. Take 100 μL of the completely hydrolyzed polysaccharide solution, add 100 μL of 0.5 mol / L PMP methanol solution and 100 μL of 0.3 mol / L NaOH solution respectively. After mixing evenly, place it in a water bath at 70 °C and react for 30 min. After cooling, add 105 μL of 0.3 mol / L HCl solution. Add 200 μL of ultrapure water for dilution. Then add 600 μL of chloroform solution, vortex and mix evenly, and centrifuge (10000 rpm, 15 min). Discard the lower chloroform layer and repeat 3 times to remove the residual PMP. Collect the aqueous layer, filter it through a 0.45 μm filter membrane and then use HPLC for detection. The chromatographic conditions were as follows: The chromatographic system used was a Thermo ICS5000 ion chromatographic system (ICS5000, Thermo Fisher Scientific, USA), and an electrochemical detector was used to analyze and detect the monosaccharide components. The chromatographic column was Dionex TM CarboPac TMPA20 (150*3.0 mm, 10 μm) liquid chromatography column. The injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 ml / min; column temperature was 30 °C; elution gradient: 0 min, phase A / phase B / phase C (95:5:0, V / V); 26 min, phase A / phase B / phase C (85:5:10, V / V); 42 min, phase A / phase B / phase C (85:5:10, V / V); 42.1 min, phase A / phase B / phase C (60:0:40, V / V); 52 min, phase A / phase B / phase C (60:40:0, V / V); 52.1 min, phase A / phase B / phase C (95:5:0, V / V); 60 min, phase A / phase B / phase C (95:5:0, V / V). By comparing the peak times and peak areas of each standard monosaccharide (after derivatization), qualitative and quantitative analysis of the BU-CPS-1B component was carried out to obtain the monosaccharide composition and molar ratio of BU-CPS-1B.

[0082] The results are as Figure 5 and Figure 6 shown.

[0083] The ion chromatogram of the capsular polysaccharide ( Figure 5 ) was compared with the peak map of the derivatization product of the mixed monosaccharide standard ( Figure 6 ). According to the retention time, it was known that the capsular polysaccharide BU-CPS-1B of Bacteroides uniformis was mainly composed of arabinose, galactose, glucose, galacturonic acid and glucuronic acid. The molar ratio of arabinose, galactose, glucose, galacturonic acid and glucuronic acid was 0.51:0.25:300.19:1.95:1.72. And through database comparison, it was found that there were differences in the types and proportions of the monosaccharide compositions of the polysaccharides obtained from the existing Bacteroides (including Bacteroides fragilis) and the monosaccharide composition of BU-CPS-1B in the present invention. It was speculated that it might be a novel extracellular heteropolysaccharide with a new structure.

[0084] Therapeutic effect of Bacteroides uniformis capsular polysaccharide on colitis mouse model

[0085] (1) Preparation of Bacteroides uniformis capsular polysaccharide BU-CPS-1B solution:

[0086] Take 100 mg of the purified Bacteroides uniformis capsular polysaccharide BU-CPS-1B obtained in the above example, add PBS to prepare a 1 mg / mL Bacteroides uniformis capsular polysaccharide BU-CPS-1B solution, and filter it through a 0.22 μm microporous membrane for later use.

[0087] (2) Preparation of colitis mouse model:

[0088] Thirty female six-week-old C57BL / 6 mice were taken. After one week of adaptive feeding, they were randomly divided into 4 groups (7 - 8 mice in each group): normal control group (Control), model group (NC), 5-aminosalicylic acid group (5-ASA), and Bacteroides uniformis capsular polysaccharide group (BU-CPS). Mice in the normal group and the model group were gavaged with 200 μL of PBS. Mice in the 5-ASA group were gavaged with 5-aminosalicylic acid solution at a concentration of 6 mg / mL every day, and the gavage dose was 50 mg of 5-aminosalicylic acid dry weight / kg (i.e., 8.33 mL of 5-aminosalicylic acid solution). Mice in the BU-CPS group were gavaged with the above-mentioned Bacteroides uniformis capsular polysaccharide BU-CPS-1B solution every day, and the gavage dose was 200 μg / mouse / day (200 μL, concentration of 1 mg / mL). After 9 days of gavage for all groups of mice, the drinking water of the other three groups except the normal control group was changed to drinking water containing 2.5% DSS to induce colitis (lasting for one week to complete the modeling). After the modeling was completed, the drinking water was changed back to normal water. Two days later, the mice were sacrificed and samples (colon and spleen) were taken from the mice.

[0089] The experimental flow chart is as Figure 7 shown.

[0090] Meanwhile, the body weight changes of the mice during the experiment were recorded.

[0091] Based on the percentage of body weight loss and the disease activity index of the diseased animals, a comprehensive score was obtained to get the DAI value. Among them, if the body weight remained unchanged, it was 0 points; if it was 1 - 5, it was 1 point; if it was 5 - 10, it was 2 points; if it was 10 - 15, it was 3 points; if it was greater than 15, it was 4 points; if the stool viscosity was normal, it was 0 points; if the stool was loose, it was 2 points; if there was diarrhea, it was 4 points; in terms of stool bleeding, if it was normal, it was 0 points; if the occult blood was positive, it was 2 points; if there was overt bleeding, it was 4 points. The total score of the 3 results was divided by 3 to obtain the DAI value.

[0092] The obtained mouse specimens were subjected to HE staining.

[0093] Flow cytometry was used to detect the proportion of Treg cells, Th17 cells, and macrophages. The specific steps are as follows: After dissecting the mouse, the mesentery and Peyer's patches were removed with ophthalmic scissors, the colon was cut open, the contents were rinsed with pre-cooled PBS, cut into 0.5 cm pieces in 8 mL PBS, and manually shaken vigorously. Centrifuge and discard the supernatant. Cut the colon with scissors to facilitate enzymatic digestion, place it in 1640 culture medium containing 4 mL of enzyme (2 mg / mL collagenase II, 2 mg / mL collagenase IV, 1 mg / mL DNase I), digest at 37°C, 100r for 30 min (constant temperature shaking water bath), centrifuge at 650g for 5 min, remove the supernatant, resuspend in PBS, and pass through a 70 μm mesh screen. The resuspended cell suspension was separated by Percoll and centrifuged at 800 g for 20 min. The middle white turbid layer was aspirated and resuspended in 1640 medium containing 10% FBS and 1% double antibody. After counting, the cells were inoculated into a 96-well plate, with about 1×10 cells per well. 6 cells.

[0094] When detecting the proportion of Treg cells by flow cytometry, first resuspend the cells with 100μL PBS, add 50μL of the prepared CD4+ and CD25+ solution, and incubate at room temperature in the dark for 25min. After the cell extracellular staining incubation is completed, wash with 2mL PBS, centrifuge at 300g for 5min, remove the supernatant, add 500μL of membrane permeabilization solution to resuspend and mix, and incubate at 4℃ in the dark for 50min. Wash with 1mL of fixed membrane permeabilization buffer and centrifuge to remove the supernatant, repeat once, add 100μL PBS to resuspend the cells, add 50μl Anti-Mouse / RatFoxp3PE (FOXP3 diluted with PBS at 1:20), and incubate at 4℃ in the dark for 30min. Wash with 1mL of fixed membrane permeabilization buffer and centrifuge to remove the supernatant, resuspend and load on the machine.

[0095] When detecting the proportion of Th17 cells by flow cytometry, first add stimulation blocker to the cell sample in advance, then add 2mL complete medium and culture in the incubator for 6 hours, centrifuge at 800g for 10min, and remove the supernatant. Add 1mL PBS to the precipitate, vortex, and centrifuge. Remove the supernatant and add 100μL PBS to resuspend. Add Anti CD4 FITC (diluted at 1:200) and incubate at room temperature in the dark for 25min. After incubation, wash with 2mL PBS, centrifuge at 300g for 5min, and remove the supernatant. Add 500μL permeabilization fixative to resuspend and mix, and react at 4℃ in the dark for 50min. Wash with 1mL fixation permeabilization buffer and centrifuge at 300g for 5min. Remove the supernatant, add 100μL PBS to resuspend the cells, add Anti-Mouse / Rat IL-17A (FOXP3 diluted with PBS at 1:40), and incubate at 4℃ in the dark for 30min. Wash with 2 mL of fixation and permeabilization buffer, centrifuge to remove the supernatant, and resuspend on the machine.

[0096] When detecting the proportion of macrophages by flow cytometry, first incubate with FCR blocking reagent (FcRblock) at 4°C for 5 - 10 min, then perform surface staining with F4 / 80, CD11b, and CD86 at 4°C for 30 min. Wash the cells 2 - 3 times with PBS, add cell fixation solution, and fix at room temperature in the dark for 30 min. Centrifuge at 150 g for 5 min to discard the fixation solution, add 2 mL of 10×Intracellular Staining PermeabilizationWash Buffer diluted 10-fold with ddH2O to resuspend the cells, centrifuge at 150 g for 5 min, and discard the supernatant. Repeat steps 2 - 3 times. Resuspend the cells with 100 μL of 1×Intracellular Staining PermeabilizationWashBuffer, add CD206 antibody, and incubate at room temperature in the dark for 30 min. After incubation, wash the cells 2 - 3 times with 2 mL of 1×Intracellular Staining PermeabilizationWash Buffer, add 500 μL of cell staining buffer to resuspend the cells, and load onto the machine.

[0097] Meanwhile, take the intestinal contents of some mice in each group and perform 16S rRNA gene sequencing of the flora to analyze the effects of the solutions in each group on the intestinal flora. Use Stool DNA Kit to extract the DNA of the intestinal flora. The specific operation refers to the instruction manual. Identify the purity of the extracted DNA by 1% agarose gel electrophoresis. Detect the DNA concentration of the extracted sample on a Nanodrop 2000 nucleic acid analyzer. Dilute the DNA to 1 ng / μL with DEPC water according to the concentration.

[0098] The PCR amplification of 16S rRNA is the same as the above example, and the PCR amplification products are entrusted to Beijing Novogene Bioinformatics Technology Co., Ltd. for sequencing.

[0099] The results are as Figures 8 to 14 shown.

[0100] To explore the in vivo anti-inflammatory activity of Bacteroides uniformis OMV, the inventors used a classical DSS-induced colitis mouse model and observed indicators such as the body weight changes, inflammation scores, and pathological morphology of mice in each group during the administration period by prophylactically administering the drug 9 days in advance, so as to explore the effect of the capsular polysaccharide of Bacteroides uniformis on intestinal inflammation in colitis mice. The results showed that intragastric administration of 200 μg / animal / day of the capsular polysaccharide solution of Bacteroides uniformis could alleviate the weight loss and DAI intestinal inflammation score of colitis mice, and at the same time alleviate the colon shortening and spleen enlargement induced by DSS. The spleen is an important immune organ, and DSS induction can make the spleen larger, stimulate the immune response, and aggravate intestinal inflammation. The results confirmed that after administration of the capsular polysaccharide of Bacteroides uniformis, the spleen weight decreased, suggesting that Bacteroides uniformis OMV has a certain immunosuppressive function. At the same time, the HE staining results showed that compared with the model group, after administration of the capsular polysaccharide of Bacteroides uniformis, the degree of inflammatory infiltration in the mouse colon decreased, the number of goblet cells increased, and the intestinal barrier was restored, which also reflected the direct regulatory effect of the capsular polysaccharide of Bacteroides uniformis on intestinal pathological morphology.

[0101] In addition, to further explore the in vivo immune regulatory function of the capsular polysaccharide of Bacteroides uniformis, flow cytometry was used to analyze the composition of Treg / Th17 immune cells in the intestinal lamina propria of DSS-induced colitis mice. The results showed that the capsular polysaccharide of Bacteroides uniformis could inhibit the proportion of pro-inflammatory Th17 immune cells and stimulate the proportion of Treg cells that inhibit inflammation. This suggests that the capsular polysaccharide of Bacteroides uniformis has a good anti-inflammatory function under inflammatory conditions and an impact on the differentiation of the important induced immune cell subsets Treg / Th17.

[0102] At the same time, flow cytometry was also used to analyze the composition of macrophages in the intestinal lamina propria of DSS-induced colitis mice, and it was found that the capsular polysaccharide of Bacteroides uniformis could inhibit the proportion of macrophages. This suggests that the capsular polysaccharide of Bacteroides uniformis can inhibit the formation of macrophages in innate immunity induced by inflammation, thereby inhibiting inflammation.

[0103] A decrease in intestinal flora diversity is a typical feature of intestinal inflammation. By analyzing the α-diversity of the intestinal contents of DSS-induced colitis mice through 6S rRNA gene sequencing, the results showed that the capsular polysaccharide of Bacteroides uniformis could alleviate the decrease in flora diversity induced by DSS. Therefore, it can be shown that the capsular polysaccharide of Bacteroides uniformis may be used as an intestinal flora regulator to alleviate intestinal inflammation by increasing flora diversity.

[0104] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of the capsular polysaccharide of Bacteroides uniformis in the preparation of a drug for preventing and / or treating ulcerative colitis; The capsular polysaccharide of Bacteroides uniformis is composed of arabinose, galactose, glucose, galacturonic acid and glucuronic acid, and the molar ratio of arabinose, galactose, glucose, galacturonic acid and glucuronic acid is 0.51:0.25:300.19:1.95:1.72; The Bacteroides uniformis is Bacteroides uniformis ATCC 8492.

2. The application according to claim 1, characterized in that, The extraction method of the capsular polysaccharide of Bacteroides uniformis includes the following steps: (1) Repeatedly freeze-thaw Bacteroides uniformis and then perform ultrasonic fragmentation, centrifuge to collect the supernatant, concentrate under reduced pressure and then perform alcohol precipitation; (2) Take the alcohol-precipitated precipitate for protein removal treatment, ultrafilter and wash, then perform chromatography using ion exchange cellulose, elute with NaCl, and collect the eluate in the peak segment of 0 mol / L to obtain the product.

3. The application according to claim 2, wherein The extraction method further includes further purification.

4. The application according to claim 3, wherein The purification is performed by chromatography using Sephadex.

5. The application according to claim 1, wherein The drug also includes pharmaceutically acceptable excipients.

6. The application according to claim 1, wherein The content of the Bacteroides uniformis extract in the drug is 1-100 wt%.

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