Application of wormwood polysaccharide in preparation of medicine for improving intestinal barrier and preventing and treating inflammatory bowel disease

Abstract

The invention belongs to the technical field of medicines for treating inflammatory bowel diseases, and particularly relates to application of wormwood polysaccharide in preparation of medicines for improving intestinal barriers and preventing and treating inflammatory bowel diseases. Experimental research shows that the wormwood polysaccharide plays a role in the inflammatory bowel disease by improving the intestinal barrier, so that the application range of the wormwood polysaccharide is widened, and a new way is provided for treating the inflammatory bowel disease. In addition, the wormwood polysaccharide is found to increase the number of colon goblet cells, up-regulate the expression of mucoprotein (MUC-2) and tight junction protein (Occudin and ZO-1) and down-regulate the expression of proinflammatory cytokines (TNF-alpha, IL-1beta and INF-gamma mRNA), not only can play a role in inflammatory bowel diseases, but also is expected to provide a new way for the treatment of other intestinal health related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of new medical uses of wormwood polysaccharides, and specifically relates to the use of wormwood polysaccharides in the preparation of drugs for improving intestinal barriers and preventing and treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) mainly includes ulcerative colitis (UC) and Crohn's disease (CD). It is an intestinal immune dysfunction disease caused by chronic and excessive inflammation, and its main characteristics are repeated attacks of intestinal inflammation and intestinal mucosal damage. Although IBD has a low mortality rate, it has a high disability rate and is difficult to cure. In severe cases, it may even cause cancer. At present, the classical drugs commonly used in the clinical treatment of IBD include amino acid salicylic acid derivatives (ASA), glucocorticoids (GC), and immunosuppressants (ISD). However, these drugs can only relieve the symptoms of the disease, but cannot cure it, and are prone to relapse after discontinuation of the drug, and long-term use will produce more adverse reactions. Therefore, it is of great clinical significance to develop drugs that can prevent, deeply relieve, or even cure IBD.

[0003] Studies have shown that intestinal mucosal barrier function plays a key role in the occurrence and development of IBD. Clinical experiments have found that in the early stage of IBD, intestinal permeability increases, which can cause intestinal allergens to enter the intestinal tissue, destroy the tight junction structure between intestinal epithelial cells, trigger immune cell infiltration, damage intestinal epithelial cells, aggravate the inflammatory process on the mucosal surface, and cause repeated inflammation. Therefore, intestinal mucosal barrier dysfunction has become an important observation indicator for recurrence and complications in IBD patients. In addition, intestinal flora plays an important regulatory role in intestinal repair and regeneration. Probiotics can regulate the thickness of the intestinal mucus layer, reduce epithelial cell apoptosis, protect the integrity of the intestinal barrier, and enhance intestinal barrier function. Pathogenic bacteria in the intestine will directly invade intestinal epithelial cells and destroy the integrity of the intestinal epithelium. The enterotoxins secreted by them will increase the permeability of the intestinal epithelium, and the immunosuppressive proteins secreted will cause mucosal immune disorders, thereby inducing the occurrence of intestinal inflammation. Therefore, correcting immune dysfunction, improving inflammatory response, regulating intestinal flora, and protecting intestinal barrier function are new directions and targets for the prevention and treatment of IBD.

[0004] Artemisia argyi, also known as fragrant wormwood, mugwort, medicinal grass, ice platform, etc., is a perennial semi-shrubby herb of the genus Artemisia in the Asteraceae family. As a traditional Chinese herbal medicine in my country, the whole plant of wormwood can be used as medicine, with the effects of anti-inflammatory, hemostasis, cold dispelling, dampness removal, cough relief, and fetal protection. Artemisia argyi polysaccharides (AAP) are a kind of heteropolysaccharide contained in wormwood. As a natural biomacromolecule, its molecular weight is mainly concentrated above 6kDa, and it is mainly composed of monosaccharides such as arabinose, glucose, xylose, and mannose in different molar ratios. Luo Xuan et al. found that wormwood polysaccharides can promote the increase of the weight of mouse immune organs, enhance the phagocytic function of macrophages, promote the ability of B cells to produce antibodies, and enhance the proliferation effect of T lymphocytes. Li et al. reported that wormwood polysaccharides can significantly upregulate the relative mRNA expression of IL-6 and TNF-α in peritoneal macrophages and promote T cell immune response. Koo et al. found that mugwort polysaccharides can upregulate the relative mRNA expression of IL-2, IL-6 and IL-12, and promote the activation of T lymphocytes and their subsets CD4+ and CD8+ cells. Mugwort polysaccharides have a strong antibacterial effect. Sun Yixuan et al. used the filter paper method to determine the antibacterial activity and minimum inhibitory concentration (MIC) of mugwort polysaccharides. The results showed that mugwort polysaccharides have a strong inhibitory effect on Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis, and a weak inhibitory effect on Gram-negative bacteria Escherichia coli.

[0005] The patent document of CN113855699A discloses an application of wormwood polysaccharide in regulating the structure of intestinal flora. The wormwood polysaccharide extracted by it is composed of monosaccharides such as arabinose, glucose, galactose, mannose, and xylose, which significantly improves the structure of intestinal flora by significantly increasing the number of probiotics in the mouse intestine and reducing the number of pathogenic bacteria. In addition, the patent of CN114028414A discloses the effect of wormwood polysaccharide isolated from wormwood leaves in colitis mice. The results show that wormwood polysaccharide can increase the content of probiotic lactobacillus and short-chain fatty acid producing bacteria, and reduce the content of pathogenic bacteria Gluconococcus and Escherichia coli-Shigella. Both of the above patents found that wormwood polysaccharide has a regulatory effect on intestinal microorganisms, but the effect of wormwood polysaccharide on intestinal barrier function is still unknown.

[0006] The patent document of CN117257829A discloses the discovery and use of a polysaccharide of wormwood to improve the inflammatory response of polycystic ovary syndrome (PCOS); provides a polysaccharide of wormwood, which can regulate the expression of TLR4 / NF-κB inflammatory pathway in the ovaries of PCOS rats, effectively reduce the expression of TLR4, MyD88, NF-κB mRNA and protein, reduce the expression of pro-inflammatory factors IL-6 and TNF-α in rat serum, and increase the expression of anti-inflammatory factors IL-4 and IL-10. However, the patent document only reports the function of wormwood polysaccharide in improving inflammation of polycystic ovary syndrome (PCOS), but the effect of wormwood polysaccharide on intestinal barrier function is still unknown.

[0007] In summary, exploring the function and mechanism of wormwood polysaccharides in improving the intestinal barrier and preventing and treating inflammatory bowel disease will help to fully tap the natural polysaccharide resources of wormwood polysaccharides and expand the application of wormwood polysaccharides in protecting the intestinal barrier, which is of great significance. Summary of the invention

[0008] In order to solve the above problems existing in the prior art, the present invention provides the use of wormwood polysaccharide in the preparation of a drug for improving the intestinal barrier to prevent and treat inflammatory bowel disease. The present invention first discovered that wormwood polysaccharide plays a role in inflammatory bowel disease by improving the intestinal barrier, which is safe and effective, which broadens the medical application scope of wormwood polysaccharide and also provides a new way to treat inflammatory bowel disease.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides the use of wormwood polysaccharides in the preparation of a drug for improving the intestinal barrier and preventing and treating inflammatory bowel disease.

[0011] The research team of the present invention found that wormwood polysaccharides can improve the intestinal barrier and prevent inflammatory bowel disease (IBD). The experimental evidence and specific manifestations are as follows:

[0012] (1) The results of the experiment showed that: Pathological examination found that Artemisia argyi polysaccharides can reduce the destruction of the morphological integrity of colonic tissue in IBD, the disorder of colonic mucosal epithelial cells, the infiltration of a large number of inflammatory cells in the submucosal layer, the partial or even complete disappearance of the glandular structure, and the disappearance of the serosal layer.

[0013] (2) The experimental results show that wormwood polysaccharides can inhibit the expression level of proinflammatory factors (such as TNF-α, IL-1β, INF-γmRNA) and regulate intestinal immunity. Under normal physiological conditions, the secretion of proinflammatory and anti-inflammatory cytokines in the body's colon tissue is in a relatively stable state. IBD causes an imbalance in the expression of proinflammatory and anti-inflammatory cytokines, and the abnormal increase in the secretion of proinflammatory cytokines can further activate the body's immune system, thereby causing persistent inflammation and immune response, causing inflammatory tissue damage. Therefore, by inhibiting proinflammatory factors, wormwood polysaccharides can play a role in preventing and treating IBD.

[0014] (3) The experimental results show that wormwood polysaccharides can increase the number of goblet cells and the expression level of mucin-2 in colon tissue and regulate the thickness of the intestinal mucus layer. The mucus secreted by goblet cells in the intestinal epithelial barrier has the function of isolating the internal and external environment of the intestine and lubricating the intestinal mucosa, which can effectively block the invasion of intestinal pathogens and substances that potentially damage intestinal mucosal epithelial cells. Therefore, by increasing the number of goblet cells and the expression level of MUC-2, wormwood polysaccharides can play a role in preventing and treating IBD.

[0015] (4) The experimental results show that wormwood polysaccharides can increase the expression level of colon tight junction proteins (such as Occludin and Z0-1). Tight junction structures are located at the top of intestinal epithelial cells. Occuldin and ZO-1 are two core proteins that maintain tight junction structures and are key components of the intestinal epithelial barrier. They play a vital role in maintaining the integrity and selective permeability of the intestinal barrier. Therefore, by increasing the expression level of colon tight junction proteins, wormwood polysaccharides can alleviate intestinal mucosal barrier damage in IBD.

[0016] Furthermore, the inflammatory bowel disease is acute inflammatory bowel disease.

[0017] Furthermore, the drug is used to protect the integrity of the intestinal mucosal barrier or repair intestinal mucosal barrier damage, or / and to alleviate weight loss caused by inflammatory bowel disease, or / and to alleviate blood in the stool caused by inflammatory bowel disease.

[0018] Furthermore, the drug is used to inhibit pro-inflammatory factors, increase the number of goblet cells and the expression level of mucin in colon tissue, and increase colon tight junction proteins.

[0019] Furthermore, the pro-inflammatory factor is one or more of TNF-α, IL-1β or INF-γ mRNA.

[0020] Furthermore, the mucin is MUC-2.

[0021] Furthermore, the tight junction protein is one or more of Occuldin or ZO-1.

[0022] In a second aspect, the present invention provides the use of wormwood polysaccharides in the preparation of drugs for regulating goblet cells, mucins, proinflammatory factors and / or tight junction proteins in colon tissue.

[0023] The present invention found that wormwood polysaccharides can increase the number of goblet cells and the expression level of mucin in colon tissue, and can increase the expression of tight junction proteins. Studies have shown that the mucus secreted by goblet cells in the intestinal mucosal barrier can isolate the internal and external environments of the intestine, and the reduced expression of tight junction proteins can lead to increased intestinal permeability. When the intestinal epithelial barrier function is damaged and the permeability increases, foreign pathogenic metabolites, pathogenic bacteria or toxins enter the blood circulation and pose a serious threat to the health of the body. Therefore, in addition to preventing and treating inflammatory bowel disease, wormwood polysaccharides are expected to be used for a variety of diseases by improving the intestinal mucosal barrier.

[0024] Furthermore, the pro-inflammatory factor is one or more of TNF-α, IL-1β or INF-γ mRNA.

[0025] Furthermore, the mucin is MUC-2, and the tight junction protein is one or more of Occuldin or ZO-1.

[0026] The present invention has the following advantages and beneficial effects: The present invention has found through experimental research that wormwood polysaccharides play a role in inflammatory bowel disease by improving the intestinal barrier, which broadens the medical application scope of wormwood polysaccharides and provides a new way to treat inflammatory bowel disease. In addition, the present invention found that wormwood polysaccharides increase the number of colonic goblet cells, upregulate the expression of mucin (MUC-2) and tight junction proteins (Occuldin and ZO-1), and downregulate the expression of proinflammatory cytokines (TNF-α, IL-1β, INF-γmRNA), which can not only play a role in inflammatory bowel disease, but also provide a new way to treat other intestinal health-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The effect of AAP on the DAI score of colitis in mice, (A) Representative image of bloody stool in mice; (B) Bloody stool score in mice; (C) DAI score. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0028] Figure 2 The effect of AAP on the organ indexes of DSS-induced mice: (A) liver index; (B) spleen index; (C) kidney index; (D) heart index. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0029] Figure 3The effect of AAP on the colon length of DSS-induced mice, (A) Representative images of colon; (B) Colon length; (C) Cecum length. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0030] Figure 4 Effects of AAP on colonic histopathological damage induced by DSS in mice (A) Colon HE staining; (B) Colonic histopathological score; (C) Colonic crypt depth. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0031] Figure 5 Effects of AAP on DSS-induced colon cytokines in mice (A) TNF-α relative mRNA expression; (B) IL-1β relative mRNA expression; (C) IFN-γ relative mRNA expression; (D) IL-10 relative mRNA expression. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0032] Figure 6 Effects of AAP on colonic goblet cells and mucin expression in DSS-induced mice (A) AB-PAS staining of colonic tissue (B) Representative immunofluorescence images of each group; (C) Colonic goblet cells; (D) Relative mRNA expression of MUC-2; (E) Fluorescence analysis of MUC-2. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0033] Figure 7 Effects of AAP on the expression of tight junction proteins in the colon of mice induced by DSS (A) Representative immunofluorescence images of mouse colon ZO-1 and Occludin; (B) Fluorescence analysis of ZO-1; (C) Fluorescence analysis of Occludin. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0034] Figure 8 Analysis of colonic microbial composition (A) The top 10 species and analysis in relative abundance at the phylum level; (B) The top 10 species and analysis in relative abundance at the genus level. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0035] Fig. 9 is the colon dilution curve.

[0036] Fig.10Alpha diversity analysis (A) Goods_coverage index; (B) Chao1 index; (C) Pielou_e index; (D) Shannon index; (E) Simpson index; (F) Dominance index. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0037] Fig.11 Beta diversity analysis (A) principal coordinate analysis; (B) Simper analysis, the top 10 species and abundances of the difference contribution between the two groups. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0038] Fig.12 This is the LEfSe species difference analysis (LDA greater than 3) (A) Differential microbial community analysis between the CON group and the DSS group; (B) Differential microbial community analysis between the DSS group and the DSS+200AAP group; (C) Differential microbial community analysis between the CON group and the 200AAP group. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described in detail in conjunction with the following specific examples. It should be understood that the scope of protection claimed by the present invention is not limited to the scope of the examples, and that changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0040] Example 1: Efficacy test of wormwood polysaccharide on inflammatory bowel disease

[0041] 1.1 Experimental Materials

[0042] 1.1.1 Experimental animals and drugs

[0043] Experimental animals: 8-week-old healthy male Balb / c mice weighing 18-22 g and free of specific pathogens (SPF) were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing).

[0044] Artemisia argyi polysaccharide: the polysaccharide in Example 1, with a polysaccharide content of 90%, purchased from Shaanxi Jinrun Biotechnology Co., Ltd.

[0045] 1.1.2 Main reagents and instruments

[0046] Main reagents: physiological saline, double distilled water, xylene, anhydrous ethanol, neutral gum, 1% hydrochloric acid alcohol differentiation solution, hematoxylin-eosin staining solution, Alixin blue test kit, isopropanol, chloroform, DEPC treated water, RNAiso Plus (TaKaRa, Japan), reverse transcription kit (TaKaRa, Japan), Premix Ex Taq TM II (Tli RNaseHPlus), Luminaris Color HiGreen High POX qPCR Master Mix (Thermo Fisher, USA), sodium citrate antigen retrieval buffer (pH = 6.0), phosphate buffered saline PBS (pH = 7.4), 5wt% bovine serum albumin (BSA) blocking solution, DAPI staining solution, anti-fluorescence quenching reagent, Occludin antibody (primary antibody; Proteintech), ZO-1 antibody (primary antibody; Proteintech), Muc-2 antibody (primary antibody; Proteintech), and secondary antibody.

[0047] Main instruments: dehydrator (Leica, Germany), embedding machine, slicer (Leica, Henan Shujing Trading Co., Ltd.), oven, low-temperature centrifuge, vortex mixer (IKAVORTEX), instant centrifuge, real-time fluorescence quantitative PCR instrument QRTPCR (Thermo Fisher ABI 7300, USA), laser confocal microscope LSM (ZEISS LSM780, Germany), fluorescence microscope (ECLIPSE NI, Shanghai Nikon Precision Machinery Co., Ltd.), and ultrasonic cleaner.

[0048] 1.1.3 Drug configuration

[0049] (1) Preparation of Artemisia argyi polysaccharide suspension

[0050] Weigh 0.2 g of wormwood polysaccharide into a 15 mL centrifuge tube, add sterile water to make up to 10 mL, the concentration of wormwood polysaccharide solution is 20 mg / mL, mice are gavaged at a volume of 0.1 mL / 10 g, the dosage of wormwood polysaccharide for mice is 200 mg / kg, prepared before use.

[0051] (2)DSS solution preparation

[0052] Weigh 15 g of DSS powder, pour it into a 1000 ml beaker, add double distilled water to 500 mL, stir until completely dissolved, and prepare a DSS solution with a final concentration of 3%. Re-prepare the solution every two days and feed the animals with the new solution.

[0053] 1.2 Experimental design

[0054] After one week of pre-feeding of Balb / c mice, 40 8-week-old male mice were randomly divided into 4 groups: CON group, DSS group, DSS+200AAP group, and 200AAP group. The DSS+200AAP group and the 200AAP group were gavaged with 200 mg / kg of AAP solution dissolved in sterile water every day, and the CON group and DSS group were gavaged with the same volume of sterile water for 21 days. DSS modeling was started on the 22nd day for 7 days. The CON group and the 200AAP group were fed with sterile water, and the DSS group and the DSS+200AAP group were fed with sterile water containing 3wt% DSS. At the same time, sterile water and polysaccharide solution were gavaged according to the method before modeling. Mice can eat and drink freely, and all feeding items related to mice must be strictly sterilized before use. During the experiment, the activity status of the mice was observed every day, and their weight changes were recorded in detail.

[0055] 1.3 Sample collection

[0056] At the end of the experiment, the weight of the mice was recorded and blood was collected by eyeball removal. Serum was collected and stored at -20°C. After blood collection, the mice were killed by cervical dislocation. Disinfect with 75% ethanol by volume, cut the abdominal skin along the midline of the mouse abdomen to expose the abdominal cavity, separate the small intestine and large intestine in turn, weigh and measure the length, observe the pathological state of the intestinal tissue, and take pictures. The duodenum, jejunum, ileum, and colon were separated, and after rinsing with saline, two sections of intestinal tissue were cut from the middle part of each intestinal tissue, each section of intestinal tissue was about 3 cm, one section of the intestinal tissue was immersed in 4wt% paraformaldehyde fixative for intestinal morphological analysis; the other section of intestinal tissue was wrapped in tin foil and stored in liquid nitrogen, kept at a low temperature of -80°C for subsequent experiments to extract RNA from tissue samples and analyze the expression of related gene mRNA.

[0057] 1.4 Test methods

[0058] (1) Disease Activity Index (DAI) score

[0059] During the experimental phase, the weight changes, fecal characteristics and degree of blood in the feces of the mice were observed daily for DAI scoring. The scoring criteria are detailed in Table 1.

[0060] Table 1 DAI scoring criteria

[0061]

[0062] (2) Observation of intestinal tissue morphology

[0063] a. Paraffin embedding and sectioning

[0064] Take out the fixed intestinal segments, take 1cm of each, place them in an embedding box, and rinse them slowly with water overnight. Dehydrate them in different concentrations of ethanol (70% for 1h, 80% and 85% for 30min each, 90% and 95% for 10min each, 100% I and 100% II for 5min each). The dehydrated tissue needs to be transparent with xylene solution twice (xylene solution I for 4min, xylene solution II for 4min). After the tissue is transparent, dip it in wax twice (wax I for 1h, wax II for 1h). Use an embedding machine to embed and trim the tissue after waxing, and then put it in a freezer to cool naturally. Put the embedded wax block into a paraffin slicer to prepare 5μm thick slices, spread the slices in warm water at 40℃, gently use a slide to pick up the slices, and then dry them at 55℃.

[0065] b. HE staining

[0066] The dried tissue sections were immersed in xylene solution (xylene solution I for 10 minutes, xylene solution II for 10 minutes) for dewaxing. Sequentially immersed in 100% ethanol I, 100% ethanol II, 95% ethanol I for 1 minute each, 85% ethanol I and 75% ethanol I for 2 minutes each, and ddH2O for 3 minutes to rehydrate the sections. The sections were first stained with hematoxylin for 7 minutes, rinsed with running water for 5 minutes, and then differentiated with 1wt% hydrochloric acid ethanol for 25 seconds, and rinsed with running water for 10 minutes. Immersed in 75% and 85% ethanol II for 1 minute each, and stained with eosin for 4-6 seconds. After the sections were immersed in 85% ethanol III for 30 seconds, they were immersed in 95% ethanol II, 100% ethanol III, and 100% ethanol IV for 1 minute each, and then immersed in xylene solution (xylene solution III for 5 minutes, xylene solution IV for 10 minutes), and finally sealed with neutral resin. The images were taken under a fluorescence microscope, and then the colon crypt depth (Crypt Depth, CD) was measured using Image-Pro analysis software.

[0067] (3) Colon histological pathological scoring

[0068] The colon tissue HE staining results were used to perform colon histopathological scoring (Histopathological score of colon), and the scoring criteria are shown in Table 2.

[0069] Table 2 Histopathological scoring criteria

[0070]

[0071] (4) Alzheimer's blue-glycogen staining

[0072] The colon tissue wax block was cut into 4 μm thick slices. After dewaxing and rehydration, the slices were stained with Alpha Blue dye for 15 minutes and washed with distilled water for 30 seconds. Periodic acid staining solution was added to cover the sample, incubated at room temperature for 10 minutes, and then washed with distilled water for 2 minutes. When the slide was not completely dry, the Schiff reagent was added to cover the sample, incubated at room temperature for 5 minutes, and the stained slide was removed after the incubation, and the slide was slowly rinsed with running water for 5 minutes. The slide was removed, dried, and sealed with a neutral resin.

[0073] (5) Real-time fluorescence quantitative PCR (RT-PCR) analysis

[0074] a. Total RNA Extraction and cDNA Synthesis

[0075] The colon sample was ground into powder in a mortar with liquid nitrogen, and about 0.1 g was taken into a 1.5 mL RNase-free tube, and 1 mL of pre-cooled RNAiso Plus lysis solution (Takala, Japan) was added and shaken to mix. 200 mL of chloroform was added, mixed, allowed to stand for 5 min, and centrifuged at 12000 g / min and 4°C for 10 min. 400 μL of aqueous phase and 400 μL of isopropanol were aspirated into a new 1.5 mL RNase-free tube, shaken and allowed to stand for 5 min in an ice box, and centrifuged at 12000 g / min and 4°C for 10 min. The white precipitate obtained after discarding the supernatant was washed twice with 75% ethanol by volume, and after drying slightly, 20-40 μL of DEPC water was added to fully dissolve the RNA precipitate. The integrity of the total RNA was detected by 1 wt% agarose gel electrophoresis, and the RNA was judged to be degraded based on the brightness ratio of the 28S:18S band of ribosomal RNA shown in the electrophoresis diagram. If the quality is intact, the mRNA reverse transcription kit (PrimeScript TM Reverse transcription was performed according to the instructions of RT Master Mix, TaKaRa, Japan. cDNA was diluted with RNase-free ddH2O (volume ratio 1:4) for later use.

[0076] b. Primer design and RT-PCR reaction

[0077] Primer 5.0 software was used to design the experimental primers, which were synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are shown in Table 3. β-actin was selected as the internal reference gene, and 2 –ΔΔCt Method for analyzing gene expression.

[0078] Table 3 RT-PCR primer sequences

[0079]

[0080] According to TaKaRa Premix Ex Taq TMII (Tli RNaseH Plus) was used to configure the RT-PCR reaction system, with a total volume of 10 μL (see Table 4). Subsequently, the expression level of the target gene was detected by QuanStudio 6 real-time fluorescence quantitative PCR instrument, with 3 replicates set for each sample.

[0081] Table 4 RT-PCR reaction system and procedure

[0082]

[0083] (6) Immunofluorescence

[0084] The dried slices were placed in an oven (59% to 62°C) for 1 hour, and the slices were placed in different xylene and anhydrous ethanol in turn, namely xylene I 15 minutes-xylene II 15 minutes-xylene III 15 minutes-anhydrous ethanol I 5 minutes-anhydrous ethanol II 5 minutes-85% alcohol by volume 5 minutes-75% alcohol by volume 5 minutes-distilled water 5 minutes. The slices were immersed in sodium citrate antigen retrieval buffer with pH = 6.0, heated on medium and low heat for 10 minutes / time, stopped for 2 minutes, for a total of 3 times. After the heating was completed, the temperature was naturally cooled to room temperature (1.5 hours), and the slices were washed 3 times in PBS (pH = 7.4), each time for 8 minutes. 5wt% BSA was added dropwise to block at room temperature for 1.5 hours. The blocking solution was shaken off, and an appropriate amount of primary antibody diluted with 5wt% BSA was added, incubated at 4°C for 15 hours, and reheated in a 37°C incubator for 2 hours. The slices were washed 3 times in PBS (pH = 7.4), each time for 8 minutes. After the slices are slightly dry, add the secondary antibody of the same species as the primary antibody and incubate in a 37°C incubator away from light for 1 hour. Wash the slides in PBS (pH = 7.4) for 3 times, 8 minutes each time. After the slices are slightly dry, add DAPI staining solution and incubate in the dark for 8 minutes. Wash the slices in PBS (pH = 7.4) for 3 times, 8 minutes each time, and finally seal the slides with anti-fluorescence quenching sealing medium.

[0085] (7) 16S rDNA sequencing of colon contents

[0086] The colon content samples were sent to Tianjin Novogene Bioinformatics Technology Co., Ltd. for 16S rDNA sequencing. The specific analysis process included the following operations.

[0087] Genomic DNA extraction and sample detection: Total bacterial DNA was extracted using a DNA kit, and the DNA concentration and integrity were detected using an Agilent 5400.

[0088] PCR amplification: This test uses the V3-V4 hypervariable region primers of the 16S rDNA gene for PCR amplification. The primer sequences are as follows:

[0089] 515F:5'-CCTAYGGGRBGCASCAG-3'

[0090] 806R:5'-GGACTACNNNGGGTATCTAAT-3'

[0091] Purification of PCR products: PCR products were detected by electrophoresis using 2 wt% agarose gel; qualified PCR products were purified by magnetic beads.

[0092] Library construction and upstream sequencing: A small fragment library was constructed according to the characteristics of the amplified region. The constructed library was quantified by Qubit and Q-PCR. After the library was qualified, PE250 sequencing was performed using NovaSeq6000.

[0093] Sequencing data processing: The raw data of each sample was obtained by splitting according to the barcode, and the barcode and primer were removed. Then, the R1 and R2 sequence data were spliced ​​by FLASH software. The spliced ​​tags were quality controlled to obtain Clean Tags, and then chimeras were filtered to obtain effective data (Effective Tags) that can be used for subsequent analysis. Then, DADA2 was used to reduce noise and duplicate, and the sequences were clustered into ASVs with 100% similarity, and representative ASVs were annotated for species. Alpha diversity index (ACE, chao1, goods_coverage, observed_species, shannon, simpson) was calculated based on the annotation results of ASVs to analyze the diversity of microbial communities in the samples. According to species annotation and ASV abundance data, similar ASV information was merged to form a species abundance table. Beta diversity analysis was further performed using the ASV phylogenetic relationship to explore the differences in microbial community structure in different groups. PCoA analysis was performed to display the differences in community structure composition in two dimensions. LEfSe analysis was used to find biomarkers with statistical differences between different groups.

[0094] 1.5 Statistical analysis

[0095] The experimental data were statistically analyzed using SPSS22.0 software. One-way ANOVA was used for data from different groups, and multiple comparisons were performed in combination with Tukey's method. All data are presented in the form of mean and standard error (SEM). * indicates a significant difference at P<0.05, ** indicates an extremely significant difference at P<0.01, and *** indicates an extremely significant difference at P<0.001. GraphPadPrism 8.0.2 and Adobe Illustrator 2023 software were used for drawing.

[0096] 1.6 Experimental Results

[0097] 1.6.1 Effect of AAP on DAI score of colitis in mice

[0098] Depend on Figure 1 As shown in (A), during the modeling process, mice that drank 3 wt% DSS solution began to have blood in their stools on the 4th day, indicating that the intestinal inflammation modeling of mice was successful. Figure 1 As shown in (B), on the 7th day of DSS-induced mice, the bloody stool score in the DSS group was significantly increased compared with the CON group (P < 0.01); compared with the DSS group, the bloody stool score in the DSS+200AAP group was significantly reduced (P < 0.05), and the bloody stool score in the 200AAP group was significantly reduced (P < 0.01). Figure 1 As shown in (C), on the 7th day of DSS-induced mice, compared with the CON group, the DAI score in the DSS group was extremely significantly increased (P < 0.01), and the DAI score in the DSS+200AAP group was significantly increased (P < 0.05); compared with the DSS group, the DAI score in the DSS+200AAP group was significantly decreased (P < 0.05), and the DAI score in the 200AAP group was extremely significantly decreased (P < 0.01).

[0099] 1.6.2 Effects of AAP on organ indices of DSS-induced mice

[0100] Depend on Figure 2 As shown in (A), compared with the other three groups, the 200AAP group significantly increased the liver index of mice (P < 0.01). There was no significant difference in the heart index, kidney index, and spleen index among different groups.

[0101] 1.6.3 Effect of AAP on colon length in DSS-induced mice

[0102] Depend on Figure 3 In (B), compared with the CON group, the colon length in the DSS group was extremely significantly shortened (P < 0.01), and the colon length in the DSS+200AAP group was significantly shortened (P < 0.05); compared with the 200AAP group, the colon length in the DSS group was extremely significantly shortened (P < 0.001), and the colon length in the DSS+200AAP group was significantly shortened (P < 0.05). Figure 3 As shown in (C), the cecum length of mice in the 200AAP group was significantly increased compared with the DSS group and the DSS+200AAP group (P < 0.05).

[0103] 1.6.4 Effect of AAP on DSS-induced colonic pathological damage in mice

[0104] Depend on Figure 4As shown in (A), the colon tissue of the CON group and the 200AAP group was intact, the colon mucosal epithelial cells were neat and orderly, and the mucosal layer, submucosal layer, serosa layer and muscular layer were intact. The integrity of the colon tissue morphology in the DSS group was severely damaged, the colon mucosal epithelial cells were disordered, a large number of inflammatory cells infiltrated the submucosal layer, the glandular structure was partially or completely disappeared, and the serosa layer disappeared. Compared with the DSS group, the integrity of the colon tissue structure of the mice in the DSS+200AAP group was enhanced to varying degrees, and the inflammatory response was alleviated, mainly manifested in the basic integrity of the mucosal layer and submucosal layer of the colon tissue, and the infiltration of inflammatory cells was significantly reduced.

[0105] Depend on Figure 4 As shown in (B), compared with CON, the colon histological pathological score of mice in the DSS group was significantly increased (P < 0.001); compared with the DSS group, the colon histological pathological score of mice in the DSS+200AAP group was significantly reduced (P < 0.05), and the colon histological pathological score of mice in the 200AAP group was significantly reduced (P < 0.001). Figure 4 As shown in (C), compared with the CON group, the crypt depth of the colon of mice in the DSS group was significantly increased (P < 0.01); compared with the DSS group, the crypt depth of the colon of mice in the 200AAP group was significantly decreased (P < 0.001).

[0106] Example 2: Effect of AAP on DSS-induced colon cytokines in mice

[0107] Depend on Figure 5 As shown in (A), compared with the CON group, the relative mRNA expression of cytokine TNF-α in the DSS group was significantly upregulated (P < 0.001), and the relative mRNA expression of cytokine TNF-α in the DSS+200AAP group was significantly upregulated (P < 0.01); compared with the DSS group, the relative mRNA expression of cytokine TNF-α in 200AAP was significantly downregulated (P < 0.01). Figure 5 As shown in (B), compared with the CON group and the 200AAP group, the relative mRNA expression of cytokine IL-1β in the DSS group was significantly upregulated (P < 0.001); compared with the DSS+200AAP group, the relative mRNA expression of cytokine IL-1β in the 200AAP group was significantly downregulated (P < 0.01). Figure 5 As shown in (C), compared with the CON group and the 200AAP group, the relative mRNA expression of cytokine INF-γ in the DSS group was significantly upregulated (P < 0.001). Figure 5 As shown in (D), the relative mRNA expression of cytokine IL-10 in the DSS group was significantly upregulated compared with that in the 200AAP group (P < 0.05).

[0108] Example 3: Effects of AAP on DSS-induced colonic goblet cells and mucin expression in mice

[0109] Depend on Figure 6 As shown in (A), the number of goblet cells in the colon tissue of mice in the CON group and 200AAP group was relatively large, the number of goblet cells in the colon tissue of mice in the DSS group decreased sharply, and the number of goblet cells in the colon tissue of mice in the DSS+200AAP group decreased to a certain extent. The expression of the colon mucin MUC-2 protein in each group of mice was analyzed by immunofluorescence. Representative pictures of each group are shown in Figure 6 In (B). Figure 6 In (C), compared with the CON group, the number of goblet cells in the colon tissue of the DSS group was significantly reduced (P < 0.001), and the number of goblet cells in the colon tissue of the DSS+200AAP group was significantly reduced (P < 0.01); compared with the DSS group, the number of goblet cells in the colon tissue of the DSS+200AAP group was significantly increased (P < 0.05), and the number of goblet cells in the colon tissue of the 200AAP group was significantly increased (P < 0.001). Figure 6 As shown in (D), compared with the CON group, the relative mRNA expression of MUC-2 in the DSS group was significantly downregulated (P < 0.05), and the relative mRNA expression of MUC-2 in the 200AAP group was extremely significantly upregulated (P < 0.01). In addition, compared with the DSS group and the DSS+200AAP group, the relative mRNA expression of MUC-2 in the 200AAP group was extremely significantly upregulated (P < 0.001). Figure 6 As shown in (E), compared with the CON group, the fluorescence intensity of MUC-2 protein in the DSS group decreased significantly (P < 0.001), and the fluorescence intensity of MUC-2 protein in the DSS+200AAP group decreased significantly (P < 0.05). In addition, compared with the DSS group, the fluorescence intensity of MUC-2 protein in the DSS+200AAP group increased significantly (P < 0.05), and the fluorescence intensity of MUC-2 protein in the 200AAP group increased significantly (P < 0.001); compared with the DSS+200AAP group, the fluorescence intensity of MUC-2 protein in the 200AAP group increased significantly (P < 0.01).

[0110] Example 4: Effect of AAP on the expression of tight junction proteins in the colon of mice induced by DSS

[0111] The expression levels of tight junction protein Z0-1 and Occludin protein in the colon of each group of mice were analyzed by immunofluorescence. Representative pictures of each group are shown in Figure 7 In (A). Figure 7In (B), compared with the CON group, the fluorescence intensity of Z0-1 protein in the DSS group decreased significantly (P < 0.001), and the fluorescence intensity of Z0-1 protein in the DSS+200AAP group decreased significantly (P < 0.01). Compared with the 200AAP group, the fluorescence intensity of Z0-1 protein in the DSS group decreased significantly (P < 0.001), and the fluorescence intensity of Z0-1 protein in the DSS+200AAP group decreased significantly (P < 0.05). Figure 7 As shown in (C), compared with the CON group, the fluorescence intensity of Occludin protein in the DSS group was extremely significantly decreased (P < 0.01); compared with the DSS group, the fluorescence intensity of Occludin protein in the DSS+200AAP group was significantly increased (P < 0.05), and the fluorescence intensity of Occludin protein in the 200AAP group was extremely significantly increased (P < 0.01).

[0112] Example 5: Effect of AAP on the Colonic Microbial Composition of DSS-Induced Mice

[0113] (1) Analysis of colonic microbial composition

[0114] Depend on Figure 8 As shown in (A), at the phylum level, the microbial flora of the four groups was mainly composed of Firmicutes and Bacteroidota, and the sum of their relative abundances exceeded 90%. Compared with the DSS+200AAP group, the relative abundance of Proteobacteria in the CON group was extremely significantly reduced (P < 0.01), and the relative abundance of Proteobacteria in the DSS group was significantly reduced (P < 0.05). In addition, compared with the CON group, the relative abundance of Patescibacteria in the DSS group was extremely significantly reduced (P < 0.01); compared with the DSS group, the relative abundance of Patescibacteria in the DSS+200AAP group was significantly increased (P < 0.05).

[0115] Depend on Figure 8As shown in (B), at the genus level, the microbial flora of the four groups was mainly composed of Lactobacillus, Muribaculaceae, Bacteroides, Alistipes, Alloprevotella, Odoribacter, Clostridia_UCG-014, and Lachnospiraceae_NK4A136_group. Compared with the CON group, the relative abundance of Bacteroides and Odoribacter in the DSS group was significantly increased (P < 0.05), and the relative abundance of Bacteroides in the DSS+200AAP group was extremely significantly increased (P < 0.01); compared with the DSS group, the relative abundance of Bacteroides in the 200AAP group was significantly decreased (P < 0.05). Compared with the CON group, the relative abundance of Alistipes in the DSS group and the DSS+200AAP group was significantly decreased (P<0.05); compared with the DSS group and the DSS+200AAP group, the relative abundance of Alistipes in the 200AAP group was extremely significantly increased (P<0.001).

[0116] (2) Alpha diversity analysis

[0117] Depend on Fig. 9 It can be seen that the dilution curves of the CON group, DSS group, DSS+200AAP group, and 200AAP group tend to be stable. Fig.10 As shown in (A), the four sequencing depth indexes are close to 100%, which basically covers all species in the sample and is sufficient to reflect the species diversity in the sample. Fig.10 As shown in (E / F), compared with the CON group and 200AAP group, the Simpson index in the DSS group was significantly increased (P < 0.05), while the Dominance index was significantly decreased (P < 0.05).

[0118] (3) Beta diversity analysis

[0119] The analysis results based on the weighted unifrac distance principal coordinate analysis (PCoA, Principal Co-oridinatesanalysis) are as follows Fig.11As shown in (A), the first principal coordinate accounts for 44.95% and the second principal coordinate accounts for 17.95%. In addition, the distance of bacterial distribution in the CON group and the 200AAP group is not clearly distinguished, while the distance of bacterial distribution in the DSS group and the DSS+200AAP group is clearly distinguished. Simper analysis can quantify the contribution of each species to the difference between the two groups. The results show the top 10 species and their abundance that contribute to the difference between the CON group and the DSS group. Fig.11 As shown in (B), the three bacterial genera with the largest difference contributions between the CON group and the DSS group are Alistipes, Bacteroides, and Muribaculaceae; compared with the CON group, the relative abundance of Alistipes and Muribaculaceae in the DSS group is lower, while the relative abundance of Bacteroides is higher.

[0120] (4) LEfSe species difference analysis

[0121] Through linear discriminant analysis (LEFSE, LDAEffect Size), biomarkers with significant differences in the colonic microbial composition of four groups of mice with different treatments were screened out (the threshold of LDA was 3). Fig.12As can be seen from (A), compared with the CON group, in the DSS group, the Bacteroides genus, Bacteroidaceae family, Erysipelotrichales order, Erysipelatoclostridiaceae family, Erysipelatoclostridium genus, Candidatus_Soleaferrea genus, Lachnospiraceae_UCG_006 genus, Gemellaceae family, Gemella genus, Clostridium_leptum species, Staphylococcales order, Parabacteroides_distasonis species, Campilobacterota phylum, Campylobacteria class, Campylobacterales order, Helicobacteraceae family, Helicobacter genus, Campylobacter genus, Campylobacter family, Peptococcales order, Peptococcaceae family, Acutalibacter_muris species, Flavobacteriales order, and Azospirillum_sp family are biomarkers with significant differences. In the CON group, Marvinbryantia genus, Eubacterium_xylanophilum_group genus, Saccharimonadales order, Saccharimonadia class, Patescibacteria phylum, Saccharimonadaceae family, Candidatus_Saccharimonas species, Frankiales order, Saccharimonadales family, Saccharimonadales species, Muribaculum genus, Eubacterium_ventriosum_group genus, Cytophagales order, Sutterellaceae family, Geobacter genus, and PeM15 genus were biomarkers with significant differences.

[0122] Depend on Fig.12As can be seen from (B), compared with the DSS group, in the DSS+200AAP group, the Proteobacteria phylum, Burkholderiales order, Saccharimonadales order, Saccharimonadia class, Patescibacteria phylum, Comamonadaceae family, Saccharimonadaceae family, Methylophilaceae family, Candidatus_Saccharimonas genus, Actinobacteria class, Dechloromonas genus, Lacibacter genus, Roseomonas genus, Ferribacterium genus, and Rhizobiales order were biomarkers with significant differences. In the DSS group, Clostridia class, Lachnospiraceae family, Lachnospirales order, Oscillospirales order, Oscillospiraceae family, Lachnospiraceae_UCG_006 genus, Ruminococcaceae family, Deferribacterota phylum, Mucispirillum genus, Deferribacteraceae family, Deferribacterales order, Deferribacteres class, Mucispirillum_schaedleri species, Colidextribacter genus, Lachnospiraceae_FCS020_group genus, and Eubacterium_siraeum_group genus were the biomarkers with significant differences.

[0123] Depend on Fig.12 In (C), compared with the CON group, the Marinifilaceae family, Odoribacter genus, MBNT15 genus, Burkholderiales order, MBNT15 class, MBNT15 family, MBNT15 phylum, MBNT15 order, and Desulfovibrio genus in the 200AAP group were significantly different biomarkers. In the CON group, the Prevotellaceae_UCG_001S genus and Muribaculum genus were significantly different biomarkers.

[0124] The above description is only a preferred embodiment of the present invention, and does not impose any limitation on the present invention. Any simple modification, change and equivalent transformation made to the above embodiment according to the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Use of wormwood polysaccharide in the preparation of a drug for improving the intestinal barrier and preventing inflammatory bowel disease, the drug is used to increase the number of colon goblet cells, or / and to reduce the level of pro-inflammatory factors in colon tissue, or / and to increase mucin in colon tissue, or / and to increase the level of tight junction protein in colon tissue, wherein the pro-inflammatory factor is TNF-α, IL-1β or INF-γ One or more of the above, the mucin is MUC-2, and the tight junction protein is one or more of Occuldin or ZO-1.

2. The use according to claim 1, characterized in that The inflammatory bowel disease is acute inflammatory bowel disease.

3. The use according to claim 1 or 2, characterized in that: The polysaccharide content of the wormwood polysaccharide is 90%.

4. The use of Artemisia argyi polysaccharides in the preparation of drugs for regulating the number of goblet cells, mucin, pro-inflammatory factors and / or tight junction protein levels in colon tissue.

5. The use according to claim 4, characterized in that The pro-inflammatory factors are TNF-α, IL-1β or INF-γ mRNA, the mucin is MUC-2, and the tight junction protein is Occuldin or ZO-1.

Images