Application of hairyvein agrimony polysaccharide in preparation of medicine for preventing or treating ulcerative colitis

Abstract

The invention relates to the technical field of medicines, in particular to application of hairyvein agrimony polysaccharide to preparation of a medicine for preventing or treating ulcerative colitis. Research finds that the hairyvein agrimony polysaccharide can effectively inhibit intestinal inflammation of mice with ulcerative colitis and promote mucous membrane healing. The further mechanism study finds that the hairyvein agrimony polysaccharide can improve the structure and composition of intestinal flora, especially the growth of enriched probiotics such as faecalibacillus, lactobacillus and bifidobacterium, and the growth of the enriched probiotics such as the faecalibacillus, the lactobacillus and the bifidobacterium can be improved, and the hairyvein agrimony polysaccharide can also be used for improving the intestinal flora and the growth of the enriched probiotics such as the faecalibacillus, the lactobacillus and the bifidobacterium. Meanwhile, the immune balance of Th17 / Treg cells of mice with ulcerative colitis can be adjusted, and the immune homeostasis is remodeled, so that the effect of treating ulcerative colitis is achieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of agrimony polysaccharide in preparing a medicine for preventing or treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disease of unknown etiology that primarily affects the mucosa and submucosa of the rectum and colon. Clinical manifestations of UC include persistent or recurrent diarrhea, bloody and mucous stools, abdominal pain, and tenesmus. Severe cases may also present with varying degrees of systemic symptoms, including weight loss, fever, tachycardia, fatigue, and even nausea and vomiting.

[0003] The incidence of UC is increasing year by year worldwide. Currently, a variety of treatments are available for UC, including aminosalicylic acid drugs, glucocorticoids, immunosuppressants, and biologics. These treatments can achieve certain efficacy in the short term, but they still face numerous challenges. Mesalazine is a first-line drug for the treatment of mild to moderate UC, but its clinical efficacy is limited. Approximately 30% of patients experience induction remission failure with mesalazine, and over time, more than half of patients lose their response. Patients who do not respond to mesalazine and those with more severe clinical symptoms should consider step-up treatment with steroids, immunosuppressants, or biologics. However, these treatments are subject to unstable efficacy and patient intolerance, and their efficacy and safety remain unclear. Therefore, there is an urgent need to find new, effective, and safe treatments.

[0004] Traditional Chinese medicine has unique advantages in the treatment of UC. It can significantly improve symptoms by regulating the body's immune function and improving the intestinal microecological environment. It also has relatively few adverse reactions and a low recurrence rate. As a traditional Chinese medicine, Agrimoniae has the effects of astringency, hemostasis, antimalarial, antidysentery, and detoxification. The "Compendium of Materia Medica" records that its above-ground parts can be used to treat white diarrhea. Modern pharmacological studies have found that Agrimoniae contains a variety of active ingredients, including flavonoids, triterpenoids, and phenolic compounds, which can be anti-inflammatory, analgesic, immune-regulating, and promote mucosal repair. Early studies have found that Agrimoniae polysaccharides in Agrimoniae have significant antioxidant effects and can be used to treat tumors. However, its role in UC is still unknown. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the use of Agrimoniae polysaccharide in the preparation of drugs for preventing or treating ulcerative colitis.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The present invention provides the use of Agrimoniae polysaccharide in preparing a medicine for preventing or treating ulcerative colitis.

[0008] Agrimony polysaccharide is extracted from the traditional Chinese medicine Agrimony and is a water-soluble acidic heteropolysaccharide. The Agrimony polysaccharide of the present application can be synthesized by existing conventional methods or can be purchased directly.

[0009] The synthesis method is as follows:

[0010] Weigh 500 g of crushed Agrimony Root, add 7.5 L of water, extract at 80°C for 12 h, centrifuge and collect the supernatant, concentrate to an appropriate volume, add 3 times the amount of anhydrous ethanol to precipitate, let stand at 4°C overnight, centrifuge and collect the precipitate, freeze-dry to obtain crude Agrimony Root polysaccharide.

[0011] Crude Agrimoniae polysaccharide was passed through a cellulose DE-52 anion exchange column (2.6 cm × 40 cm) using a 0-1 mol / L NaCl gradient elution at a flow rate of 1.0 mL / min. The eluate was collected at 3 mL / tube. The sugar-containing peak was detected by phenol-sulfuric acid. Dialysis and freeze-drying were performed to obtain the refined polysaccharide fraction. The fraction was further purified using an AKTA Basie 100 system using a Supemex-200 (XK16 / 60) column. The sample was dissolved in water and loaded onto the column. The eluate was eluted with water at a flow rate of 1.0 mL / min. The eluate was collected at 3 mL / tube. The sugar-containing fraction was then freeze-dried to obtain Agrimoniae polysaccharide.

[0012] The relative molecular mass of the isolated and purified Agrimonia polysaccharide was determined by gel filtration to be 1.90×10 5 .

[0013] The total sugar content, protein content, and uronic acid content of Agrimoniae polysaccharide were 81%, 3.4%, and 45%, respectively. Gas chromatography results showed that Agrimoniae polysaccharide contained galactose, glucose, rhamnose, arabinose, mannose, fucose, and xylose in a molar ratio of 5.65:2.67:2.61:1.86:1.12:1.09:1.

[0014] Furthermore, the drug is used to improve intestinal flora.

[0015] Furthermore, improving the intestinal flora refers to improving the structure and composition of the intestinal flora and increasing the abundance of probiotics.

[0016] Furthermore, the probiotics include one or more of the genus Faecalibaculum, the genus Lactobacillus, and the genus Bifidobacterium.

[0017] Furthermore, the drug is used to regulate the immune balance of helper T lymphocytes 17 / regulatory T lymphocytes.

[0018] Furthermore, regulating the helper T lymphocyte 17 / regulatory T lymphocyte immune balance refers to inhibiting the number of helper T lymphocyte 17 and promoting the differentiation of regulatory T lymphocytes.

[0019] The present invention also provides the use of Agrimoniae polysaccharide in preparing a Th17 cell differentiation inhibitor drug.

[0020] Furthermore, the drug also includes pharmaceutically acceptable excipients selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, polymer skeleton materials and film-forming materials.

[0021] Furthermore, the drug is prepared into any pharmaceutically acceptable dosage form using Agrimonia polysaccharide as the active pharmaceutical ingredient.

[0022] Furthermore, the dosage form is tablet, capsule, granule, oral solution, injection, sustained-release preparation, controlled-release preparation or nano preparation.

[0023] The technical solution of the present invention has the following advantages:

[0024] The present invention provides a method for preparing a drug for preventing or treating ulcerative colitis using agrimony polysaccharide. Studies have shown that agrimony polysaccharide significantly improves the coat color, food intake, water intake, bowel movement frequency, activity level, mental state, and daily disease activity index scores of UC mice. Furthermore, agrimony polysaccharide can also prolong the colon length and reduce the spleen-to-body weight ratio of UC mice. HE pathological staining shows that agrimony polysaccharide can also inhibit the infiltration of inflammatory cells and promote the recovery of glandular structure. This indicates that agrimony polysaccharide can effectively inhibit intestinal inflammation in UC mice and promote mucosal healing, and is superior to the positive drug mesalamine.

[0025] Further mechanism studies found that Herba Agrimoniae polysaccharides can improve the structure and composition of intestinal flora, especially enrich the growth of probiotics Faecalibaculum, Lactobacillus, and Bifidobacterium; at the same time, it can also inhibit the number of T helper 17 cells (Th17) in the spleen and mesenteric lymph nodes of UC mice, promote the differentiation of regulatory T cells (Treg), thereby regulating the Th17 / Treg cell immune balance.

[0026] Furthermore, Agrimoniae polysaccharide, derived from the traditional Chinese medicinal herb Agrimoniae, is a natural plant extract. Compared to chemically synthesized drugs, its metabolic process in the body is milder, significantly reducing the risk of toxic side effects and adverse reactions. Furthermore, Agrimoniae polysaccharide also possesses multiple effects, including antioxidant and immune regulation. Its multi-target mechanism of action makes it widely adaptable in the treatment of UC, enabling intervention in various pathological processes, offering promising prospects for its clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 is the daily body weight percentage of mice in each group in Experimental Example 1 of the present invention (Body weight);

[0029] Figure 2 is the daily disease activity index score (Disease Activity Index score) of each group of mice in Experimental Example 1 of the present invention;

[0030] Figure 3 This is a diagram of the entire colon of each group of mice in Experimental Example 1 of the present invention;

[0031] Figure 4 Statistical graph of colon length of mice in each group in Experimental Example 1 of the present invention;

[0032] Figure 5 is a picture of the spleen of each group of mice in Experimental Example 1 of the present invention;

[0033] Figure 6is the spleen / body weight ratio of each group of mice in Experimental Example 1 of the present invention;

[0034] Figure 7 This is the HE staining image of each group of mice in Experimental Example 1 of the present invention;

[0035] Figure 8 This is a comparison chart of the histopathology scores of mice in each group in Experimental Example 1 of the present invention;

[0036] Figure 9 This is the principal coordinate analysis diagram of the 16S bacterial community analysis in Experimental Example 1 of the present invention;

[0037] Figure 10 This is a histogram of the bacterial composition at the genus level of the intestinal flora of various mice in Experimental Example 1 of the present invention;

[0038] Figure 11 This is a linear discriminant analysis diagram of various mouse intestinal flora in Experimental Example 1 of the present invention;

[0039] Figure 12 Flow cytometry of Th17 cells and Treg cells in the spleen of each group of mice in Experimental Example 1 of the present invention;

[0040] Figure 13 This is an analysis of the Th17 cell content level in the spleen of each group of mice in Experimental Example 1 of the present invention;

[0041] Figure 14 This is an analysis of the Treg cell content level in the spleen of each group of mice in Experimental Example 1 of the present invention;

[0042] Figure 15 Flow cytometry of Th17 cells and Treg cells in the mesenteric lymph nodes of mice in each group in Experimental Example 1 of the present invention;

[0043] Figure 16 This is an analysis of the Th17 cell content level in the mesenteric lymph nodes (MLN) of each group of mice in Experimental Example 1 of the present invention;

[0044] Figure 17 This is an analysis of the Treg cell content levels in the mesenteric lymph nodes (MLN) of each group of mice in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0045] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0046] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0047] Experimental Example 1

[0048] 1. Experimental Animals

[0049] Nineteen 6-week-old, SPF-grade, healthy female C57BL / 6 mice weighing 18-20 g were provided by Spefoc Biotechnology (Beijing) Co., Ltd. Mice were housed in a quiet, suitable environment for 7 days. The room temperature was controlled at (22 ± 2)°C, the relative humidity was maintained at 40% to 65%, and a 12-h light cycle was maintained (8:00-20:00 during the day; dark from 20:00-8:00 at night). During the housing period, the mice were free to move around, had ample clean drinking water, and were fed a standard mouse diet.

[0050] 2. Experimental drugs

[0051] Agrimoniae Herba Polysaccharides (AHP) was purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., with UV ≥ 95%, stored at 4°C, and prepared into a solution with distilled water for later use. 100 μL / 10 g of mouse body weight was administered orally daily.

[0052] Mesalazine granules were purchased from Shanghai Aifa Pharmaceutical Co., Ltd. and prepared into a solution with distilled water. 100 μL / 10 g of mouse body weight was administered orally daily.

[0053] 3. Main reagents and instruments

[0054] Dextran sulfate sodium salt (DSS), average molecular weight 36,000-50,000 (purchased from MP Biomedicals); fecal occult blood test strips (Zhuhai Beisuo Biotechnology Co., Ltd.); RPMI1640 culture medium (purchased from Hyclone), FBS (purchased from Gibco); Percoll, red blood cell lysis buffer, 1× PBS, double-antibody antibodies, and bovine serum albumin (all purchased from Solebol); hematoxylin stain, eosin stain, hematoxylin differentiation solution, and hematoxylin bluing solution (purchased from Wuhan Sevier Biotechnology Co., Ltd.); flow cytometry antibodies CD3, CD4, F4 / 80, IL-17, and FOXP3 (purchased from Biolegend), and Trizol reagent (purchased from Invitrogen).

[0055] DP74 microscope (purchased from Olympus); flow cytometer (purchased from BD Biosciences), 7300 real-time fluorescence quantitative PCR instrument (purchased from Applied Biosystems).

[0056] 4. Animal grouping, modeling and drug administration

[0057] Grouping: C57BL / 6 mice (SPF grade), female, weighing 18-20 g, were randomly divided into blank group (Control) of 5 mice, model group (DSS) of 5 mice, Agrimoniae polysaccharide group (DSS+AHP) of 5 mice, and mesalazine group (DSS+Mesalazine) of 4 mice.

[0058] Modeling: The ulcerative colitis (UC) mouse model was established by freely drinking 2.5% DSS solution for 7 days and deionized water for 7 days, for three cycles (except for the blank group). The mice in the blank group had free access to food and water throughout the whole process.

[0059] Administration: Starting from the 6th day after modeling, the Agrimoniae polysaccharide group was gavaged with Agrimoniae polysaccharide solution at a dose of 100 mg / kg mouse body weight (meaning that the mass of Agrimoniae polysaccharide administered per kg mouse body weight was 100 mg); the mesalazine group was gavaged with mesalazine solution at a dose of 0.6 g / kg mouse body weight (meaning that the mass of mesalazine administered per kg mouse body weight was 100 mg); the blank group and the model group were gavaged with sterile drinking water at a volume of 100 μL / 10 g mouse body weight, once a day, for 31 days.

[0060] 5. Observation indicators and methods

[0061] (1) General observation

[0062] During the period, the mice's weight, diet, activity, mental state, hair condition, fecal occult blood, defecation frequency and characteristics were observed and recorded daily.

[0063] (2) Efficacy evaluation criteria

[0064] The disease activity index (DAI) was calculated according to the UC mouse model standard. DAI = (weight loss index + stool characteristics + fecal occult blood) / 3, and weight loss index = 100% - (weight on the day / initial weight before modeling * 100%). Specific evaluation criteria are shown in Table 1.

[0065] Table 1 Scoring criteria for the disease activity index

[0066]

[0067] Note: No decrease in weight loss index means that the weight on that day has not decreased compared to the initial weight, that is, the weight on that day is equal to or greater than the initial weight.

[0068] (3)Acquisition of materials

[0069] 31 days after administration, the colon tissue, spleen tissue and mesenteric lymph node tissue of each group of mice were collected respectively. The colon tissue was collected at 1 cm above the anus.

[0070] When sampling, the entire colon length of the mouse was measured, the spleen was removed and weighed, and the spleen-to-body weight ratio was calculated.

[0071] Colon tissues were collected for pathological morphology observation, and flow cytometry was used to detect CD4 + T cell subsets, the specific methods are as follows:

[0072] 1) Pathomorphological observation

[0073] Colon tissues were collected for pathological embedding and HE staining to evaluate the degree of tissue damage in mice with ulcerative colitis. The evaluation indicators included the number of crypts, damage to the intestinal epithelium, and damage to the mucosa and submucosa.

[0074] Specifically, when sampling, cut the colon tissue of the mouse 1 cm away from the anus, remove the feces, wash with PBS, and place it in 10% formalin for 24 hours. Then, remove it and place it in gradient alcohol for dehydration. After wax immersion, embedding, slicing with a thickness of 5μm, mounting, dewaxing, staining with hematoxylin-eosin (HE), and sealing, the morphological structure of the mouse colon tissue is observed under an optical microscope. During the production process, pay attention to quickly fix the animal tissue after sacrifice to ensure the original morphological structure. The scissors used to cut the tissue block should be sharp, and do not clamp the tissue too tightly to avoid deformation of the tissue and cells due to squeezing. Samples should be taken accurately according to the anatomical site and keep the material clean.

[0075] 2) Detection of CD4 in spleen of UC mice by flow cytometry + T cell subset content

[0076] After the spleen was collected, it was ground with a 70 μm cell sieve, lysed, centrifuged by density gradient and stained by flow cytometry. The CD4 + The main T lymphocyte subsets were determined by lysing the spleen of the mouse at 4°C. The tissue was gently minced using a sterile syringe with a rubber stopper, filtered through a 70μm cell sieve, and centrifuged (2000 rpm, 4°C, 5 min). The supernatant was discarded and resuspended in 2mL of cDMEM to obtain a single-cell suspension. Stimulant (Biolegend, catalog number: 423303) was added and the cells were placed in a 37°C 5% CO2 constant temperature incubator for 8 hours to stimulate the secretion of related factors. After the stimulation, the cells were collected and centrifuged (2000 rpm, 4°C, 5 min) and the supernatant was discarded. After blocking with 3% BSA solution for 20 minutes, the cells were centrifuged (2000 rpm, 4°C, 5 min) and the supernatant was discarded. CD3 and CD4 flow cytometry antibodies were added for surface antibody staining, and the cells were incubated in the dark at 4°C for 40 minutes to stain the cell membrane surface. The cells were fixed with fixative (invitrogen 00-512-43, 00-5223-56, configured at 1:3). After the membrane was broken for 40 minutes, IL-17 and FOXP3 flow cytometry antibodies were added for staining. The cells were incubated in the dark at 4°C for 40 minutes for staining. After washing and centrifugation (2000 rpm, 4°C, 5 min), the cells were resuspended and subjected to flow cytometry detection. The flow cytometry data were analyzed using Flowjo.

[0077] 3) Flow cytometry detection of CD4 in mesenteric lymph nodes of UC mice + T cell subset content

[0078] The mesenteric lymph nodes were collected and then ground, subjected to density gradient centrifugation and flow cytometry staining. The CD4 +The content of major T lymphocyte subsets was determined by collecting mouse mesenteric lymph nodes at 4°C throughout the entire process. The tissue was gently ground using a sterile syringe with a rubber plug, filtered through a 70μm cell sieve, and a mesenteric lymph node single-cell suspension was obtained. The suspension was centrifuged (2000 rpm, 4°C, 5 min), the supernatant was discarded, and the suspension was resuspended and blocked for 20 min. The cells were then resuspended after centrifugation at 4°C, 1600 rpm, and 5 min. Stimulant (Biolegend, Catalog No.: 423303) was added and the cells were placed in a 37°C 5% CO2 constant temperature incubator for 8 hours to stimulate the secretion of related factors. After the stimulation, the cells were collected and centrifuged (Biolegend, Catalog No.: 423303) and the supernatant was discarded. After blocking for 20 minutes, the cells were centrifuged (Biolegend, Catalog No.: 423303) and the supernatant was discarded. CD3 and CD4 flow cytometry antibodies were added for surface antibody staining, and the cells were incubated in the dark at 4°C for 40 minutes to stain the cell membrane surface. The cells were fixed with fixative (Invitrogen 00-512-43, 00-5223-56, 1:3 configuration). After the membrane was permeated for 40 minutes, IL-17 and FOXP3 flow cytometry antibodies were added for staining, and the cells were incubated in the dark at 4°C for 40 minutes for staining. The cells were washed and centrifuged (2000 rpm, 4°C, 5 minutes) and resuspended for flow cytometry detection. The flow cytometry data were analyzed using FlowJo.

[0079] (4) 16S rRNA sequencing

[0080] After 30 days of dosing for all mice (i.e., one day before sampling), the lower abdomen of the mice was gently pressed to promote defecation. Sterile tweezers were used to collect fresh feces into several sterile EP tubes with corresponding numbers, and then frozen in a -80°C refrigerator. The DNA from mouse feces was extracted using a DNA extraction kit, and the 16S rRNA gene V3-V4 region was amplified using primers 338-F and 806-R. The library was constructed according to the library construction kit. The PCR products were sequenced using the Illumina high-throughput sequencing platform. The raw data obtained by Illumina sequencing were sequenced and the sequence quality was controlled to obtain valid data. All data analysis was performed on the Meiji Bio Cloud Platform.

[0081] 6. Data Statistics

[0082] Data analysis and graphing were performed using SPSS 22.0 and GraphPad Prism 9.5 software. Measurement data were expressed as (x ± s). After homogeneity of variance was tested, one-way analysis of variance (ANOVA) was used to compare groups. When the variances were homogeneous, the least significant difference (LSD) method was used for comparisons between the two groups. When the variances were unequal, the Dunnett T3 test was used for comparisons. The overall efficacy rate was count data, and the chi-square test was used. P < 0.05 was considered statistically significant.

[0083] 7. Experimental Results

[0084] (1) General information of each group

[0085] All groups of mice tolerated the modeling and drug intervention well. Behavioral observations revealed that mice in the blank group showed no abnormalities, were energetic, active and well-fed, and had shiny, smooth fur. Compared with the blank group, mice in all groups with DSS-induced UC exhibited significant intestinal symptoms, including varying degrees of blood in the stool, diarrhea, and weight loss. They also exhibited lethargy, slowed movements, poor appetite, dull fur, and hunched backs. Administration of agrimony polysaccharide solution and mesalamine aqueous solution significantly alleviated the mice's mental state, behavioral performance, dull fur, and diarrhea and blood in the stool compared to the model group.

[0086] (2) Effect of Agrimoniae Polysaccharide on Body Weight in Mice with Ulcerative Colitis

[0087] like Figure 1 As shown in the results, compared with the blank group, the body weights of the UC mice in the model group, the Herba Agrimoniae Polysaccharides group, and the Mesalazine group decreased significantly; compared with the model group, the body weights of the mice in the Herba Agrimoniae Polysaccharides group and the Mesalazine group gradually increased significantly after treatment (Compared with the model group, ** P<0.01; compared with the blank group, ## P<0.01).

[0088] (3) Effect of Agrimoniae Polysaccharide on DAI Score in Mice with Ulcerative Colitis

[0089] like Figure 2 As shown in the results, compared with the blank group, the DAI scores of UC mice in the model group, the Herba Agrimoniae Polysaccharides group, and the Mesalazine group were significantly increased; after treatment, compared with the model group, the DAI scores of mice in the Herba Agrimoniae Polysaccharides group and the Mesalazine group gradually decreased (compared with the model group, * P<0.05).

[0090] (4) Effect of Agrimoniae Polysaccharide on Colon Length in Mice with Ulcerative Colitis

[0091] like Figure 3 、 4 As shown, compared with the blank group, the colon length of the model group was significantly shortened ( ## P<0.01), colon stiffness and intestinal fibrosis were obvious; compared with the model group, after treatment with Agrimoniae polysaccharide and mesalazine, the colon length of the two groups was significantly restored ( ** P<0.01), among which the colon length of UC mice in the Agrimoniae polysaccharide group tended to be longer than that in the mesalazine group.

[0092] (5) Effects of Agrimoniae Polysaccharide on the Spleen and Body Weight Ratio of Mice with Ulcerative Colitis

[0093] like Figure 5 、 6 As shown in the figure, compared with the blank group, the spleen size and length of the model group increased significantly, and the spleen-to-body weight ratio increased significantly ( ## P<0.01); Compared with the model group, after treatment with Agrimoniae polysaccharide, the spleen size and length of UC mice showed a trend of recovery, and the spleen weight was significantly reduced compared with the model group ( * P<0.05), among which the spleen weight ratio of UC mice in the Agrimoniae polysaccharide group tended to be lower than that in the mesalazine group.

[0094] (6) Improvement of colon pathology in mice with ulcerative colitis by Agrimoniae polysaccharide

[0095] like Figure 7 、 8 As shown in the figure, under the light microscope, compared with the blank group, the colon mucosa of the model group showed obvious edema, intestinal epithelial shedding, villi disappearance, glandular deformation, local erosion and bleeding, and inflammatory cell infiltration. Compared with the model group, after treatment with Agrimoniae polysaccharide and mesalazine, the glandular structure of the mice in the Agrimoniae polysaccharide group was significantly restored, the degree of edema was reduced, the inflammatory cell infiltration was improved, and no obvious erosion and epidermal cell shedding were observed ( * The glandular structure in the mesalazine group recovered to a certain extent, the edema was reduced, the inflammatory cell infiltration was improved, and the erosion was alleviated compared with the model group (P=0.06). Among them, the degree of mucosal and glandular recovery and edema reduction in the Agrimoniae polysaccharide group was slightly better than that in the mesalazine group (HE staining, ×200).

[0096] (7) Effects of Agrimoniae Polysaccharide on Intestinal Microflora in Mice with Ulcerative Colitis

[0097] like Figure 9-11As shown in the results, principal coordinate analysis (PCoA) at the operational taxonomic unit (OTU) level revealed significant differences in the colonic intestinal flora composition between the model and normal groups. After intervention with Agrimoniae polysaccharide, the flora composition of UC mice shifted and showed a trend toward that of the normal group. Regarding bacterial composition, genus-level results showed that Agrimoniae polysaccharide significantly increased the abundance of the probiotic genera Faecalibaculum, Lactobacillus, and Bifidobacterium in UC mice. Linear discriminant analysis (LEfSe) results indicated that the probiotic Faecalibaculu rodentium may be a specific marker flora in UC mice after Agrimoniae intervention. These results suggest that Agrimoniae polysaccharide can alter the intestinal flora structure of UC mice and increase the abundance of probiotic bacteria, with Faecalibaculu rodentium being a key genus in its role.

[0098] (8) Effect of Agrimoniae Polysaccharide on Splenic CD4 in Mice with Ulcerative Colitis + The impact of T cells

[0099] like Figure 12-14 As shown in the figure, flow cytometry showed that compared with the model group, the Th17 cells (CD4 + IL-17 + ) content decreased significantly ( * P<0.05; compared with the model group, Treg cells (CD4 + FOXP3 + ) content showed a clear upward trend ( ** P<0.01).

[0100] (9) Effect of Agrimoniae Polysaccharide on CD4 in Mesenteric Lymph Nodes of Mice with Ulcerative Colitis + The impact of T cells

[0101] like Figure 15-17 As shown in the figure, the mesenteric lymph nodes of mice were collected, single cell suspensions were prepared for staining, and flow cytometry was used to detect that compared with the model group, the Th17 cells (CD4 + IL-17 + ) content decreased significantly ( * P<0.05); Compared with the model group, Treg cells (CD4 + 、FOXP3 + ) content increased significantly ( ** P<0.01).

[0102] The present invention used sodium dextran sulfate to construct a chronic UC mouse model. After intervention, it was found that Agrimoniae polysaccharide significantly improved the coat color, food intake, water intake, bowel movement frequency, activity level, mental state, and DAI scores of UC mice. Furthermore, Agrimoniae polysaccharide can also prolong the colon length and reduce the spleen-to-body weight ratio of UC mice. HE pathological staining showed that Agrimoniae polysaccharide can also inhibit the infiltration of inflammatory cells and promote the recovery of glandular structure. These results all indicate that Agrimoniae polysaccharide can effectively inhibit intestinal inflammation in UC mice and promote mucosal healing, and is superior to the positive drug mesalamine.

[0103] The present invention analyzed the intestinal flora structure of UC mice and found that Agrimoniae polysaccharide can improve the structure and composition of intestinal flora, especially enrich the growth of probiotics Faecalibaculum, Lactobacillus, and Bifidobacterium. In addition, considering the close relationship between T cell differentiation and ulcerative colitis, the present invention collected the spleen and mesenteric lymph nodes of UC mice respectively and detected the CD4 + The levels of Th17 and Treg, the main subsets of T cells. The results showed that Agrimoniae polysaccharide can inhibit the number of Th17 cells in the spleen and mesenteric lymph nodes of mice with ulcerative colitis, promote the differentiation of Treg, and thus regulate the Th17 / Treg cell immune balance.

[0104] In summary, Agrimoniae polysaccharide can effectively inhibit the intestinal inflammatory response of ulcerative colitis and promote mucosal healing. Its mechanism of action is related to regulating the structure of intestinal flora and regulating the immune balance of Th17 / Treg cells, which provides new ideas and strategies for targeted immunotherapy of ulcerative colitis.

[0105] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Application of Agrimoniae polysaccharide in the preparation of medicines for preventing or treating ulcerative colitis.

2. The use according to claim 1, characterized in that The drug is used to improve intestinal flora.

3. The use according to claim 2, characterized in that Improving the intestinal flora refers to improving the structure and composition of the intestinal flora and increasing the abundance of probiotics.

4. The use according to claim 3, characterized in that The probiotics include one or more of the genus Faecalibaculum, the genus Lactobacillus, and the genus Bifidobacterium.

5. The use according to claim 1, characterized in that The drug is used to regulate the immune balance of helper T lymphocytes 17 / regulatory T lymphocytes.

6. The use according to claim 5, characterized in that The regulation of helper T lymphocyte 17 / regulatory T lymphocyte immune balance refers to inhibiting the number of helper T lymphocyte 17 and promoting the differentiation of regulatory T lymphocytes.

7. Application of Agrimoniae polysaccharide in the preparation of drugs as Th17 cell differentiation inhibitors.

8. The use according to any one of claims 1 to 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

9. The use according to any one of claims 1 to 7, characterized in that: The medicine uses Agrimonia polysaccharide as a medicine active ingredient and is prepared into any pharmaceutically acceptable dosage form.

10. The use according to claim 9, characterized in that The dosage form is tablet, capsule, granule, oral solution, injection, sustained-release preparation, controlled-release preparation or nano preparation.

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