Preparation and application of dextran for relieving ulcerative colitis

Abstract

The invention belongs to the field of biological medicine, and particularly relates to preparation and application of dextran for relieving ulcerative colitis. The dextran GS128 is formed by directly catalyzing and synthesizing dextransucrase Gtf128 generated by heterologous expression in vitro by taking sucrose as a substrate, and the product is obtained by ethanol precipitation, centrifugation, dissolution and dialysis. The compound shows high anti-digestion performance in the process of simulating gastrointestinal tract digestion, and can relieve the symptoms of ulcerative colitis by inhibiting oxidative stress, adjusting the balance of proinflammatory / anti-inflammatory factors and improving the colon barrier.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the preparation and application of dextran for relieving ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease characterized by symptoms including abdominal pain, diarrhea, bloody mucus stools, and weight loss, often presenting as recurrent or cyclical flare-ups. Chronic inflammation of the colon induces oxidative stress and DNA damage, significantly increasing the risk of colon cancer. In recent years, the global prevalence of UC has continued to rise, evolving into a major public health challenge. Pathologically, UC patients primarily exhibit impaired intestinal barrier function, immune imbalance, and gut microbiota dysbiosis, with dietary factors being particularly crucial. Traditional medications and surgery have shown some efficacy in treating UC, but often come with serious side effects such as infection, malignancy, and hormone dependence. Therefore, adjunctive interventions have become an important strategy for preventing inflammatory responses, alleviating UC symptoms, and mitigating adverse effects.

[0003] Microbial extracellular polysaccharides exhibit multiple benefits in the intervention of ulcerative colitis, including reducing inflammation, enhancing intestinal barrier function, and regulating the immune response in the gastrointestinal tract. The bioactivity of microbial polysaccharides is influenced by their chemical structure, including glycosidic bond composition, molecular weight, type and number of monosaccharides, branching degree, side chain length, and functional groups. Dextran is a highly water-soluble α-glucan, mainly composed of D-glucose residues linked by (α1→6) glycosidic bonds (>50%). Furthermore, different numbers of (α1→3), (α1→2), and (α1→4) glycosidic bonds can form branches. Dextran sucrase is a type of glucan sucrase that transfers glucose groups produced from sucrose cleavage to acceptor molecules, gradually forming α-glucan chains, a process that does not consume ATP or cofactors. Dextran exhibits high structural heterogeneity due to differences in molecular weight, glycosidic bond type, linkage mode, and branching degree. This structural complexity and diversity lead to a variety of physicochemical properties and biological functions in dextran. Dextran has been reported to have prebiotic functions, promoting the growth of beneficial bacteria and the production of short-chain fatty acids in the gut, thereby improving gut health. Therefore, exploring the role of dextran in alleviating ulcerative colitis is of great significance. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a method for preparing and applying dextran to alleviate ulcerative colitis. The technical solution is as follows:

[0005] One type of dextran is synthesized in vitro by the dextran sucrase Gtf128 produced by heterologous expression. The amino acid sequence corresponding to this enzyme is the Dsr128 amino acid sequence reported in the invention patent "A strain of Leuconostoc mesenteroides and its application" (ZL202211703027.8).

[0006] The engineered bacteria used for heterologous expression include Escherichia coli, Bacillus subtilis, and Bacillus licheniformis.

[0007] The dextran sucrase was incubated overnight at 30°C in 50 mmol / L acetate buffer (containing 500 mmol / L sucrose solution, 1.0 mmol / L CaCl2, pH 5.5) to obtain the product.

[0008] The dextran is obtained by adding three times the volume of pre-cooled anhydrous ethanol to the product obtained in claim 2, followed by centrifugation, dissolution, and dialysis.

[0009] The dextran GS128 is predominantly composed of α-1,6 glycosidic bonds, and has a molecular weight of 1.06 × 10⁻⁶. 8 Da.

[0010] The degree of hydrolysis of the dextran GS128 during simulated gastrointestinal digestion is less than 0.5%.

[0011] The efficacy of dextran GS128 in alleviating ulcerative colitis is influenced by its polysaccharide structure, with the specific effects as follows:

[0012] (1) The α-glucan intervention reduced colon tissue damage in mice with ulcerative colitis and improved the infiltration of inflammatory cells.

[0013] (2) This dextran can reduce the production of the inflammatory marker MDA in the colon tissue of mice with ulcerative colitis, and increase the activity of antioxidant enzymes SOD and CAT and the level of antioxidant GSH.

[0014] (3) The dextran can reduce the levels of pro-inflammatory factors IL-1β, IL-6 and TNF-α in the colon tissue of mice with ulcerative colitis and increase the level of anti-inflammatory factor IL-10.

[0015] Beneficial effects:

[0016] 1. The dextran described in this invention uses inexpensive and readily available sucrose as the sole substrate and is obtained directly by dextran sucrase catalysis. The synthetic route is simple and has high conversion efficiency.

[0017] 2. The dextran extraction method described in this invention is simple, and the crude polysaccharide obtained has high purity;

[0018] 3. The dextran described in this invention has a strong ability to resist gastrointestinal digestion;

[0019] 4. The dextran described in this invention can alleviate the symptoms of ulcerative colitis to a certain extent, and can exert a potential synergistic effect when used in combination with existing therapeutic drugs. Attached Figure Description

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0021] Figure 1 GS128 synthesized in vitro

[0022] Figure 2 Changes in the degree of hydrolysis of GS128 under simulated in vitro digestion

[0023] Figure 3 Effects of different dextran levels on body weight in mice with dextran sulfate sodium (DSS)-induced ulcerative colitis

[0024] Figure 4 Effects of different dextran on colon length in DSS-induced ulcerative colitis mice

[0025] Figure 5 Effects of different dextran levels on disease activity index in DSS-induced ulcerative colitis mice

[0026] Figure 6 Pathological sections of colon tissue stained with hematoxylin and eosin by different dextran

[0027] Figure 7 Effects of different dextran levels on the levels of SOD(A), CAT(B), GSH(C), and MDA(D) in the colon of mice with DSS-induced ulcerative colitis.

[0028] Figure 8 Effects of different dextran on colonic cytokine secretion in mice with DSS-induced ulcerative colitis. Levels of inflammatory factors IL-1β (A), IL-6 (B), TNF-α (C), and IL-10 (D). Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0030] Example 1: Synthesis of Dextran

[0031] The amino acid sequence of dextran sucrase Gtf128 is the same as the Dsr128 amino acid sequence reported in the invention patent "A strain of Leuconostoc mesenteroides and its application" (ZL202211703027.8).

[0032] The target gene fragment was ligated into the linearized vector pETDuet-1 using homologous recombination cloning to obtain the ligation product. The ligation product was added to *E. coli* DH5α competent cells and incubated on ice for 30 min. Then, the cells were heat-shocked at 42°C for 90 s, and 0.9 mL of Amp-free LB liquid medium was added. The mixture was gently mixed and incubated at 37°C for 1 h on a shaker. Subsequently, 200 μL of the culture was plated onto Amp-resistant LB solid medium. After overnight incubation at 37°C, single colonies were randomly picked, and positive transformants were screened by colony PCR. After liquid culture, the recombinant plasmid was extracted using a plasmid miniprep kit. The correctly identified expression plasmid was transformed into competent *E. coli* BL21(DE3). After inducing protein expression with isopropyl-β-D-thiogalactoside, the cultured bacterial cells were resuspended in buffer, sonicated, centrifuged at 12000 rpm for 20 min, and the target protein was purified using a magnetic bead protein purification kit.

[0033] 50 U of recombinant dextran sucrase was added to 100 mL of 50 mmol / L acetate buffer (containing 500 mmol / L sucrose solution, 1.0 mmol / L CaCl2, pH 5.5), and incubated overnight at 30°C to obtain the dextran GS128 product. Figure 1 As shown, the synthesized GS128 is soluble in water. Based on the amino acid sequences of dextran sucrase Gtf1365 and Gtf836 (the amino acid sequences of Dsr1365 and Dsr836 reported in the invention patent "A strain of Leuconostoc mesenteroides and its application" (ZL202211703027.8)), dextran GS1365 and GS836 were synthesized in vitro following the same steps as above.

[0034] Example 2: Glycosidic bond composition of dextran

[0035] Accurately weigh 5 mg of polysaccharide into a 15 mL centrifuge tube, add 3 mL of anhydrous DMSO, and protect under nitrogen. Place the centrifuge tube in a 50°C water bath and sonicate for 3 hours, removing and shaking several times during this period to ensure the polysaccharide is completely dissolved in the DMSO. Then, add 150 mg of finely ground, dry NaOH powder, seal under nitrogen, and continue sonicating for 20 minutes, again removing and shaking by inversion several times. Next, place in an ice bath until the solution is completely solidified, quickly add 1 mL of iodomethane, seal again under nitrogen, and sonicate at 18-20°C in the dark for 30 minutes; repeat this low-temperature step 4 times. Add 2 mL of ultrapure water to terminate methylation, and neutralize to neutral with 1 mol / L acetic acid. Extract three times with chloroform, combine the chloroform phases, wash three times with ultrapure water, and dry under nitrogen to obtain the methylated polysaccharide.

[0036] Add 3 mL of 2 mol / L trifluoroacetic acid to the methylated sample and hydrolyze at 110 °C for 6 h; after cooling, dry under nitrogen. Add 0.5 mL of methanol and evacuate under nitrogen again to remove moisture. Then add 2 mL of freshly prepared sodium borodeuteride solution and reduce at room temperature for 2 h; after the reaction is complete, neutralize with acetic acid until no bubbles are generated in the solution. Add 0.5 mL of methanol and evacuate under nitrogen, repeating twice. Add 2 mL of freshly prepared pyridine / acetic anhydride (1:1, v / v) to the product and acetylate at 120 °C for 1 h; after cooling to room temperature, dry under nitrogen. Quickly add 1 mL of methanol and dry, repeating three times to remove residual reagents. Finally, dissolve the acetylated product in 4 mL of dichloromethane, concentrate under nitrogen, filter through a 0.22 μm organic filter membrane, and analyze by gas chromatography-mass spectrometry (GC-MS). Mass spectrometry collects fragment ion peaks of each PMAA and assigns the spectral data to the Complex Sugar Database of the Center for Complex Carbohydrates (CCRC) at the University of Georgia.

[0037] As shown in Table 1, GS128, GS1365, and GS836 have the same glycosidic bond linkage types, including Glcp-(1→, →3)-Glcp-(1→, →6)-Glcp-(1→ and →3,6)-Glcp-(1→), but their molar ratios differ. In particular, the glycosidic bond composition of GS128 is mainly α-1,6 glycosidic bonds, with a molar ratio of 87.74%.

[0038] Table 1. Glycosidic bond composition of dextran

[0039]

[0040] Example 3: Molecular weight of dextran GS128

[0041] 10 mg of dextran was weighed and dissolved in 2 mL of ultrapure water. After centrifugation at 12000 rpm for 10 min, the solution was filtered through a 0.22 μm aqueous filter membrane. A BRT105-104-102 tandem gel column (8 × 300 mm) was used for detection, coupled with a high-performance liquid chromatography (HPLC) system equipped with a differential refractive index detector. 0.05 mol / L NaCl solution was used as the mobile phase at a flow rate of 0.8 mL / min; the column temperature was 40 °C; and the injection volume was 20 μL. The molecular weight of dextran GS128 was determined to be 1.06 × 10⁻⁶ by HPLC. 8 Da.

[0042] Example 4: In vitro digestion of dextran GS128

[0043] Simulated oral digestion stage: A 15.0 mg / mL dextran solution was thoroughly mixed with simulated saliva at a 1:1 (v / v) ratio and incubated in a 37°C constant-temperature magnetic stirrer (150 rpm). At 1 min, 3 min, and 5 min, the reaction mixture was immediately aspirated and heated in a boiling water bath for 10 min to inactivate the enzyme, thus terminating the reaction. A blank control group was established, containing only simulated saliva without dextran.

[0044] Simulated gastric digestion stage: The polysaccharide mixture after oral digestion was mixed with simulated gastric juice at a 1:1 (v / v) ratio and incubated at 37℃ and 150 rpm. Samples were taken at 2h, 4h, and 6h, and the enzymes were immediately inactivated by boiling in a water bath for 10min after sampling. The pH was adjusted to 7.0 using 0.1mol / L NaHCO3. A blank control group was set up, which only added simulated gastric juice and no dextran.

[0045] Simulated small intestinal digestion stage: The sample after gastric digestion was mixed with simulated small intestinal digestion fluid at a ratio of 10:3 (v / v) and incubated at 37℃ and 150 rpm. Samples were taken at 2h, 4h, and 6h, and enzyme activity was immediately terminated by boiling in a water bath for 10min. A blank control group was set up, which only added simulated intestinal fluid and no dextran.

[0046] The reducing sugar content in samples at different time points was determined using the 3,5-dinitrosalicylic acid (DNS) method. The ratio of reducing sugar content to dextran content represented the degree of polysaccharide hydrolysis. Figure 2 As shown, dextran did not undergo hydrolysis within 5 minutes of oral digestion, indicating its high oral stability. During simulated gastric and intestinal digestion, the degree of hydrolysis of dextran did not change significantly. At 6 hours of simulated intestinal digestion, the degree of hydrolysis of GS128 was only 0.31%, demonstrating strong resistance to digestion.

[0047] Example 5: Effect of dextran on the improvement of ulcerative colitis induced by sodium dextran sulfate in mice.

[0048] (1) Animal experimental design and sample collection

[0049] Eight-week-old male C57BL / 6J mice were housed in an SPF-grade animal facility with a 12-hour light and dark cycle. They were allowed free access to sterile drinking water and standard feed. The mice were randomly divided into five groups as follows: (1) Control group: Distilled water was administered by gavage for 14 consecutive days starting from day 1. The drinking water was regular drinking water without DSS. (2) Model group: Regular drinking water was administered from day 1 to day 7. However, from day 8 to day 14, the mice were given drinking water containing 3% DSS and were administered distilled water by gavage for 14 consecutive days. (3) GS128 group (300 mg / kg / day): Regular drinking water was administered from day 1 to day 7. However, from day 8 to day 14, the mice were given drinking water containing 3% DSS and were administered a solution containing GS128 by gavage for 14 consecutive days. (4) GS1365 group (300mg / kg / day): From day 1 to day 7, the patient drank regular drinking water, but from day 8 to day 14, the patient drank drinking water containing 3% DSS, and was also given a solution containing GS1365 by gavage for 14 consecutive days. (3) GS836 group (300mg / kg / day): From day 1 to day 7, the patient drank regular drinking water, but from day 8 to day 14, the patient drank drinking water containing 3% DSS, and was also given a solution containing GS836 by gavage for 14 consecutive days.

[0050] At the end of the experiment, all mice had free access to water and were fasted overnight. On day 15, the mice were anesthetized with sodium pentobarbital and euthanized by cervical dislocation. The entire colon was removed using anatomical tools, and the cecum and colonic end were dissected separately using surgical scissors. Weight loss is the main clinical symptom of ulcerative colitis; on day 7 after DSS administration, the model group mice experienced a 12.85% decrease in body weight, significantly lower than the control group mice. Figure 3 After 14 days of dextran intervention via gavage, the weight loss in mice was alleviated to some extent, and the weight of mice in the GS128 group was higher than that in the GS1365 and GS836 groups. Furthermore, with dextran intervention, the colon length of mice with ulcerative colitis was significantly greater than that of mice in the model group, especially in the GS128 group. Figure 4 ).

[0051] (2) Assessment of disease activity index

[0052] Daily recordings of mouse weight, fecal morphology, and occult blood levels were performed. Mice were placed in beakers, and their weight changes were recorded daily using an analytical balance. Fecal occult blood levels were analyzed using a fecal occult blood test kit. The Disease Activity Index (DAI) of the mice was scored based on a combination of weight changes, fecal morphology, fecal staining time and results from the occult blood test kit, and the degree of weight loss. The DAI score was calculated based on fecal condition, occult blood levels, and the degree of weight loss. Figure 5 The DAI score can directly reflect the severity of clinical symptoms of DSS-induced ulcerative colitis in mice; a higher score indicates more severe symptoms. Mice in the model group exhibited symptoms such as loose stools, bloody stools, and blood around the anus. Mice treated with dextran showed significantly lower DAI scores than the model group (p<0.05), particularly the GS128 and GS1365 groups. This indicates that GS128 can effectively improve colitis symptoms such as weight loss, diarrhea, and fecal bleeding in mice with ulcerative colitis.

[0053] (3) Histopathological analysis of colon tissue

[0054] After rinsing the colon surface with phosphate buffer to remove residual paraformaldehyde, the tissue was sequentially dehydrated with a series of ethanol solutions for 1 hour each. It was then cleared using xylene-anhydrous ethanol (1:1, v / v) and pure xylene. After clearing, the tissue was immersed in molten paraffin for 30 minutes. 3 μm thick sections were cut using a paraffin microtome, flattened in a 42°C water bath, and then baked at 60°C. After treatment with xylene for 20 minutes, the sections were treated with a series of ethanol solutions for 5 minutes each, followed by rinsing with distilled water. The sections were then stained with hematoxylin for 5 minutes, rinsed with running water until they turned blue, differentiated with 1% hydrochloric acid ethanol solution for a few seconds, rinsed with running water, treated with 1% ammonia solution for a blue return, rinsed with running water, and then stained with eosin for 5 minutes. The stained sections were dehydrated stepwise with a series of ethanol solutions, cleared with xylene, and then mounted with neutral resin. Morphological observation was performed using an optical microscope, and images were acquired and analyzed using a scanner. Figure 6 As shown, the colonic structure of the control group mice was intact, with regular and uniform colonic epithelial cells, and no obvious inflammatory cell infiltration or intestinal epithelial cell necrosis was observed. The DSS-induced model group mice showed significant intestinal mucosal damage and submucosal edema in their colonic tissue, with abundant inflammatory cell infiltration in the muscularis mucosae and submucosa. Dextran intervention alleviated colonic tissue damage and improved inflammatory cell infiltration in mice with ulcerative colitis, with the GS128 intervention group showing the best effect.

[0055] (4) Measurement of oxidative stress indicators and inflammation-related cytokines

[0056] 100 mg of colon tissue was weighed and minced using pre-chilled sterile ophthalmic scissors. The minced tissue was then placed in a glass homogenizer containing 1 mL of physiological saline and thoroughly homogenized. The homogenate was transferred to a centrifuge tube and centrifuged at 12000 g for 15 min at 4°C. The supernatant was kept on ice for later use. The protein concentration in the colon tissue homogenate supernatant was determined using a Bradford protein assay kit. The levels of SOD, CAT, GSH, and MDA in mouse serum and colon tissue were determined using a total superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and malondialdehyde (MDA) assay kit. The levels of IL-1β, IL-6, TNF-α, and IL-10 in mouse colon were determined using an enzyme-linked immunosorbent assay (ELISA) kit.

[0057] like Figure 7 As shown, compared with the model group, the levels of SOD, CAT, and GSH, as well as the level of the anti-inflammatory factor IL-10, were increased in the colonic tissue of mice with ulcerative colitis in the dextran group, while the levels of MDA and pro-inflammatory factors IL-1β, IL-6, and TNF-α were decreased. Specifically, the IL-6 level in the colonic tissue of mice in the GS128 group was significantly lower than that in other dextran-treated groups. In summary, GS128 intervention can reduce the production of the inflammatory marker MDA in the colonic tissue of mice with ulcerative colitis, increase the activity of antioxidant enzymes SOD and CAT, and increase the level of the antioxidant GSH, and exert anti-inflammatory activity by regulating the balance of pro-inflammatory / anti-inflammatory factors.

Claims

1. A dextran GS128 is synthesized in vitro by dextran sucrase Gtf128 produced by heterologous expression.

2. The heterologous expression according to claim 1, characterized in that, The sources of engineered bacteria include Escherichia coli, Bacillus subtilis, and Bacillus licheniformis. The in vitro catalytic synthesis is characterized by incubating dextran sucrase overnight at 30°C in 50 mmol / L acetate buffer (containing 500 mmol / L sucrose solution, 1.0 mmol / L CaCl2, pH 5.5) to obtain the product. The dextran GS128 is characterized by adding three volumes of pre-cooled anhydrous ethanol to the product obtained in claim 2, followed by centrifugation, dissolution, and dialysis; and exhibiting strong resistance to digestion during simulated gastrointestinal digestion, with a degree of hydrolysis below 0.5%.

3. The use of dextran GS128 as described in claim 1 in the relief of ulcerative colitis.

4. The application according to claim 3, characterized in that, The alleviating effect of dextran GS128 on ulcerative colitis is influenced by its polysaccharide structure. This polysaccharide can alleviate colonic inflammation by preventing excessive oxidative stress in the colon of ulcerative colitis mice, regulating the balance of pro-inflammatory / anti-inflammatory factors, and improving the colonic barrier.

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