Application of Tripterygium wilfordii polysaccharide in the preparation of drugs for the prevention and treatment of inflammatory bowel disease

Abstract

The present invention relates to the field of medical technology, and discloses the application of Tripterygium wilfordii polysaccharide in the preparation of drugs for the prevention and treatment of inflammatory bowel disease. The present invention first discovered that Tripterygium wilfordii polysaccharide can prevent and treat inflammatory bowel disease, and play the role of reducing the infiltration of inflammatory cells in the colon mucosa, repairing the intestinal mucosal barrier, and alleviating intestinal wall edema, which broadens the scope of application of Tripterygium wilfordii polysaccharide and also provides a new approach for the treatment of inflammatory bowel disease. In addition, the present invention first discovered that Tripterygium wilfordii polysaccharide can increase the expression levels of tight junction proteins such as Claudin‑1, Occludin, ZO‑1 and G protein-coupled receptors such as GPR41 and GPR43 in the colon in vivo and in vitro, which can not only play a role in the prevention and treatment of inflammatory bowel disease, but also is expected to provide a new approach for the treatment of its related diseases and the study of its physiological functions.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of Tripterygium wilfordii polysaccharide in preparing medicines for preventing and treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, excessive inflammation-induced intestinal immune dysfunction disease characterized by repeated intestinal inflammation and intestinal mucosal damage, mainly including ulcerative colitis (UC) and Crohn's disease (CD). IBD has a low mortality rate but a high disability rate, is difficult to cure, and may even cause cancer in severe cases, posing a serious threat to human health. For the treatment of IBD, clinical drugs are mainly salicylic acid (ASA), glucocorticoids (GC), and immunosuppressive drugs (ISD), but they can only relieve symptoms and cannot have a radical effect, and long-term medication has many adverse reactions. To date, a large proportion of patients still do not respond to first-line and second-line drugs for the treatment of IBD. Therefore, it is of great clinical significance to prepare drugs that can deeply relieve or even cure IBD.

[0003] Studies have confirmed that intestinal mucosal barrier function plays an important role in the development of IBD. Clinical experiments have shown that increased intestinal permeability is an early symptom of IBD, which allows allergenic substances (microorganisms, protein particles) to enter the intestinal tissue, followed by the destruction of intestinal IECs tight junctions and the infiltration of immune cells, which causes the immune system to over-respond, leading to repeated inflammation and inducing the further development of IBD. Intestinal mucosal barrier dysfunction has become an important predictor of recurrence and serious complications in IBD patients. In addition, intestinal flora plays a key regulatory role in intestinal repair and regeneration. Probiotics can promote the expression of tight junction proteins in intestinal epithelial cells, reduce epithelial cell apoptosis, regulate the thickness of the intestinal mucus layer, and enhance intestinal barrier function. Intestinal pathogens directly invade intestinal epithelial cells, affect the energy metabolism of intestinal epithelial cells, lead to epithelial cell damage, secrete enterotoxins that increase intestinal epithelial permeability, and secrete immunosuppressive proteins that lead to mucosal immune disorders, inducing the occurrence of intestinal inflammation. These studies have shown that regulating intestinal flora, correcting immune dysfunction, improving inflammatory responses, and repairing intestinal mucosal barriers provide new therapeutic targets for the treatment of IBD.

[0004] Traditional Chinese medicine has the advantages of stable efficacy, low adverse reactions and recurrence rate in the clinical treatment of IBD. The scientific name of Tetrastigma hemsleyanum Diels et Gilg is a rare medicinal plant of the genus Tetrastigma in the family Vitaceae, which is unique to my country. It is mainly distributed in a few provinces such as Zhejiang, Guangxi, and Jiangxi. Its underground roots or whole plants are used as medicine. At present, the research focus on the medicinal value of Tripterygium wilfordii is mostly on flavonoids and ethyl acetate extracts, and less attention is paid to the polysaccharide components. In addition, the research on Tripterygium wilfordii polysaccharides is mostly focused on anti-tumor and anti-liver damage, and it has not been reported that it has the effect of preventing and treating inflammatory bowel disease. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the use of Tripterygium wilfordii polysaccharide in the preparation of drugs for preventing and treating inflammatory bowel disease. The present invention first discovered that Tripterygium wilfordii polysaccharide has the effect of preventing and treating inflammatory bowel disease, is safe and effective, broadens the application scope of Tripterygium wilfordii polysaccharide, and also provides a new way for the treatment of inflammatory bowel disease.

[0006] The specific technical scheme of the present invention is:

[0007] In a first aspect, the present invention provides the use of Tripterygium wilfordii polysaccharide in the preparation of a drug for preventing and treating inflammatory bowel disease.

[0008] The present invention team has found that Tripterygium wilfordii polysaccharide can prevent and treat inflammatory bowel disease (IBD). The experimental evidence and specific manifestations are as follows:

[0009] (1) The results of in vivo experiments showed that Tripterygium wilfordii polysaccharide can alleviate colon damage in IBD and reduce symptoms such as weight loss and shortened colon length. Further pathological examinations found that Tripterygium wilfordii polysaccharide can reduce the degree of intestinal wall edema, intestinal wall inflammatory infiltration, and mucosal epithelial loss in IBD.

[0010] (2) The results of in vivo experiments showed that Tripterygium wilfordii polysaccharide could affect the levels of inflammatory cytokines in the colon and inhibit the expression levels of pro-inflammatory factors (such as IL-6, TNF-α, and IFN-γ).

[0011] (3) In vivo test results show that Tripterygium wilfordii polysaccharide can increase the levels of mucin 2 (MUC-2), β-defensin and secretory immunoglobulin A in colon tissue. Previous studies have shown that MUC-2, β-defensin and SIgA can reflect the intestinal chemical barrier function; among them, MUC-2 is a highly glycosylated macromolecular mucus-like glycoprotein substance that mainly attaches to the surface of the intestinal mucus layer. The protein body of MUC-2 can not only lubricate and protect the intestinal mucosal epithelium, but the sugar chains attached to the mucin surface can also participate in intercellular signal transduction and the regulation of intestinal mucosa-related immune factors.

[0012] (4) In vivo test results show that Tripterygium wilfordii polysaccharide can reduce the changes in the intestinal flora structure in IBD, increase the abundance of Firmicutes and Bacteroidetes (such as Lactobacillus, Ruminococcus, Eubacterium, Lachnospiraceae, etc.), and restore the intestinal flora to normal. The intestinal flora plays a key regulatory role in the process of intestinal repair and regeneration. Therefore, by regulating the structure of the intestinal flora, Tripterygium wilfordii polysaccharide can play a role in preventing and treating IBD.

[0013] (5) The results of in vivo and in vitro experiments showed that Tripterygium wilfordii polysaccharide can increase the expression level of colon tight junction proteins (such as Claudin-1, Occludin, and ZO-1); and in vitro experiments on Caco-2 cells showed that Tripterygium wilfordii polysaccharide has little toxic side effects on Caco-2 cells. Intercellular tight junction proteins (such as Claudin-1, Occludin, and ZO-1) can reflect the integrity of the mouse intestinal barrier. They are expressed on the cell membrane and are closely related to tight junctions. Tight junctions are located at the top of epithelial cells and are an important component for maintaining mucosal permeability. Therefore, by increasing the expression level of colon tight junction proteins, Tripterygium wilfordii polysaccharide can alleviate intestinal mucosal barrier damage in IBD.

[0014] (6) The results of in vivo and in vitro experiments showed that Tripterygium wilfordii polysaccharide can activate the expression of G protein-coupled receptors (such as GPR41 and GPR43) in colon tissue; and in vitro experiments on Caco-2 cells showed that Tripterygium wilfordii polysaccharide had little toxicity to Caco-2 cells. This suggests that Tripterygium wilfordii polysaccharide may produce anti-inflammatory effects through the GPR41 / 43 signaling pathway to respond to changes in intestinal flora composition and SCFAs, and play a role in repairing tight junctions of colon cells.

[0015] Preferably, the inflammatory bowel disease is acute inflammatory bowel disease.

[0016] Preferably, the drug is used to protect the integrity of the intestinal mucosal barrier or repair intestinal mucosal barrier damage, or to relieve intestinal wall edema caused by inflammatory bowel disease, or to relieve shortening of colon length caused by inflammatory bowel disease, or to reduce weight loss caused by inflammatory bowel disease, or to reduce changes in intestinal flora caused by inflammatory bowel disease.

[0017] Furthermore, the drug is used to increase the abundance of one or more bacteria in the genera Lactobacillus, Ruminococcus, Eubacterium and Lachnospira in the intestinal flora.

[0018] Preferably, the drug is used to reduce the level of proinflammatory factors in colon tissue, or to increase the level of G protein coupled receptor, mucin 2, secretory immunoglobulin A, β-defensin or tight junction protein in colon tissue.

[0019] Furthermore, the pro-inflammatory factors include one or more of IL-6, TNF-α and IFN-γ.

[0020] Furthermore, the G protein coupled receptors include GPR41 and / or GPR43.

[0021] Furthermore, the tight junction proteins include one or more of Claudin-1, Occludin and ZO-1.

[0022] Preferably, the preparation method of the Tripterygium wilfordii polysaccharide comprises the following steps: crushing the Tripterygium wilfordii medicinal material, extracting it with ethanol-water solution, taking the residue, extracting it with water, collecting the extract obtained by the water extraction, concentrating it, separating the supernatant, removing free protein, adding a precipitant to precipitate the polysaccharide, separating the precipitate and drying it to obtain Tripterygium wilfordii polysaccharide.

[0023] The clover polysaccharide prepared by the method of the present invention has a higher content of sugar and uronic acid, which is conducive to its better role in preventing and treating inflammatory bowel disease.

[0024] Furthermore, the extraction temperature is 75-85°C.

[0025] Furthermore, the water extraction temperature is 95-100°C.

[0026] In a second aspect, the present invention provides the use of Tripterygium wilfordii polysaccharide in the preparation of a drug for regulating the levels of G protein coupled receptors, mucin 2, secretory immunoglobulin A, β-defensin or tight junction proteins in colon tissue in vivo.

[0027] The present invention found that the polysaccharide of Tripterygium wilfordii can increase the expression of G protein-coupled receptors (such as GPR41 and GPR43) in colon tissue. Studies have shown that G protein-coupled receptors GPR41 and GPR43 can play a regulatory role in leptin levels, intestinal absorption of nutrients, body fat formation, and growth and development of vertebrates. Therefore, in addition to preventing and treating inflammatory bowel disease, Tripterygium wilfordii polysaccharide is also expected to be used in the fields of improving obesity, animal breed improvement and seed selection by regulating the level of G protein-coupled receptors in colon tissue.

[0028] Preferably, the pro-inflammatory factors include one or more of IL-6, TNF-α and IFN-γ.

[0029] Preferably, the G protein coupled receptors include GPR41 and / or GPR43.

[0030] Preferably, the tight junction proteins include one or more of Claudin-1, Occludin and ZO-1.

[0031] In a third aspect, the present invention provides the use of Tripterygium wilfordii polysaccharide in regulating the level of G protein coupled receptors or tight junction proteins in colon tissue in vitro.

[0032] In in vitro cytological experiments, it was found that Tripterygium wilfordii polysaccharide could increase the expression levels of G protein-coupled receptors (such as GPR41, GPR43) and tight junction proteins (such as Claudin-1, Occludin, ZO-1) in colon tissue, which can provide a new tool for the study of the physiological functions of G protein-coupled receptors and tight junction proteins in colon tissue.

[0033] Preferably, the G protein coupled receptors include GPR41 and / or GPR43.

[0034] Preferably, the tight junction proteins include one or more of Claudin-1, Occludin and ZO-1.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] (1) The present invention first discovered that Tripterygium wilfordii polysaccharide can prevent and treat inflammatory bowel disease, play a role in reducing the infiltration of inflammatory cells in the colon mucosa, repairing the intestinal mucosal barrier, and relieving intestinal wall edema, which broadens the application scope of Tripterygium wilfordii polysaccharide and also provides a new approach for the treatment of inflammatory bowel disease;

[0037] (2) The present invention discovers for the first time that Tripterygium wilfordii polysaccharide can increase the expression levels of tight junction proteins (such as Claudin-1, Occludin, ZO-1) and G protein-coupled receptors (such as GPR41, GPR43) in the colon both in vivo and in vitro, which is expected to provide a new approach for the treatment of related diseases and the study of its physiological functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The effect of Tripterygium wilfordii polysaccharide on the pathological changes of colon tissue HE staining in DSS-induced IBD mice (40×).

[0039] Figure 2 The effect of Tripterygium wilfordii polysaccharide on the body weight of IBD mice induced by DSS, where * indicates p<0.05, and ** indicates p<0.01.

[0040] Figure 3 The effect of Tripterygium wilfordii polysaccharide on the colon length of DSS-induced IBD mice. Among them, * indicates p<0.05, and ** indicates p<0.01.

[0041] Figure 4 The effect of Tripterygium wilfordii polysaccharide on PAS staining of colon tissue in DSS-induced IBD mice (40×).

[0042] Figure 5 The effect of Tripterygium wilfordii polysaccharide on the expression of MUC-2, SIgA, and β-defensin in DSS-induced IBD mice. Figure 5 (A) is the effect of Tripterygium wilfordii polysaccharide on the expression of MUC-2 in DSS-induced IBD mice; Figure 5 (B) is the effect of Tripterygium wilfordii polysaccharide on SIgA expression in DSS-induced IBD mice; Figure 5 (C) Effect of Tripterygium wilfordii polysaccharide on the expression of β-defensins in DSS-induced IBD mice. Figure 5 (A)~ Figure 5 In (C), * indicates p < 0.05, and ** indicates p < 0.01.

[0043] Figure 6 The effect of Tripterygium wilfordii polysaccharide on the cytokine content in the serum of IBD mice induced by DSS. Figure 6 (A) is the effect of Tripterygium wilfordii polysaccharide on serum TNF-α content in DSS-induced IBD mice; Figure 6 (B) is the effect of Tripterygium wilfordii polysaccharide on serum IL-6 content in DSS-induced IBD mice; Figure 6 (C) Effect of Tripterygium wilfordii polysaccharide on serum INF-γ content in DSS-induced IBD mice. Figure 6 (A)~ Figure 6 In (C), * indicates p < 0.05, and ** indicates p < 0.01.

[0044] Figure 7 The effect of Tripterygium wilfordii polysaccharide on the expression of tight junction proteins (Claudin-1, Occludin, ZO-1) in the colon of IBD mice induced by DSS. Among them, * indicates p<0.05, ** indicates p<0.01.

[0045] Figure 8 The effect of Tripterygium wilfordii polysaccharide on the expression of Claudin-1 in the colon tissue of DSS-induced IBD mice (immunofluorescence).

[0046] Fig. 9 The effect of Tripterygium wilfordii polysaccharide on the expression of Occludin in the colon tissue of DSS-induced IBD mice (immunofluorescence).

[0047] Fig.10 The effect of Tripterygium wilfordii polysaccharide on the expression of ZO-1 in the colon tissue of DSS-induced IBD mice (immunofluorescence).

[0048] Fig.11 The effect of Tripterygium wilfordii polysaccharide on the fecal flora composition (species composition) of DSS-induced IBD mice. Fig.11 (A) is the species composition of fecal microbiota at the phylum level; Fig.11(B) is the species composition of fecal microbiota at the genus level.

[0049] Fig.12 This is the effect of Tripterygium wilfordii polysaccharide on the fecal flora composition of DSS-induced IBD mice (statistical charts at the phylum and genus levels).

[0050] Fig.13 The effect of Tripterygium wilfordii polysaccharide on the content of short-chain fatty acids in feces of DSS-induced IBD mice. Fig.13 (A) is the effect of Tripterygium wilfordii polysaccharide on fecal acetic acid content in DSS-induced IBD mice; Fig.13 (B) is the effect of Tripterygium wilfordii polysaccharide on the fecal propionic acid content of DSS-induced IBD mice; Fig.13 (C) is the effect of Tripterygium wilfordii polysaccharide on fecal butyric acid content in DSS-induced IBD mice; Fig.13 (D) is the effect of Tripterygium wilfordii polysaccharide on fecal isobutyric acid content in DSS-induced IBD mice; Fig.13 (E) is the effect of Tripterygium wilfordii polysaccharide on fecal valeric acid content in DSS-induced IBD mice; Fig.13 (F) Effect of Tripterygium wilfordii polysaccharide on fecal isovaleric acid content in DSS-induced IBD mice. Fig.13 In (A) to 13(F), * indicates p<0.05, and ** indicates p<0.01.

[0051] Fig.14 It is the effect of Tripterygium wilfordii polysaccharide on the expression levels of G protein-coupled receptors (GPR41, GPR43) in colon tissue of DSS-induced IBD mice.

[0052] Fig.15 This is the effect of the fermentation broth of Tripterygium wilfordii polysaccharide on the viability of Caco-2 cells, where * indicates p<0.05, and ** indicates p<0.01.

[0053] Fig.16 The effect of the fermentation broth of Tripterygium wilfordii polysaccharide on the expression of tight junction proteins and G protein-coupled receptors in Caco-2 cells stimulated by LPS. Fig.16 (A) is the effect of the fermentation broth of Tripterygium wilfordii polysaccharide on the level of claudin-1 mRNA in Caco-2 cells stimulated by LPS; Fig.16 (B) is the effect of Tripterygium wilfordii polysaccharide fermentation broth on the occludin mRNA level in Caco-2 cells stimulated by LPS; Fig.16 (C) is the effect of the fermentation broth of Tripterygium wilfordii polysaccharide on the ZO-1 mRNA level in Caco-2 cells stimulated by LPS; Fig.16 (D) is the effect of Tripterygium wilfordii polysaccharide fermentation broth on the GPR41 mRNA level in Caco-2 cells stimulated by LPS; Fig.16(E) is the effect of Tripterygium wilfordii polysaccharide fermentation broth on the GPR43 mRNA level in Caco-2 cells stimulated by LPS; Fig.16 (F) is the effect of the fermentation broth of Tripterygium wilfordii polysaccharide on the GPR41 mRNA level in Caco-2 cells stimulated by GPRs inhibitors; Fig.16 (G) is the effect of Tripterygium wilfordii polysaccharide fermentation broth on the GPR43 mRNA level in Caco-2 cells stimulated by GPRs inhibitors. Fig.16 In (A) to 16(G), * indicates p<0.05, and ** indicates p<0.01. DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with the embodiments.

[0055] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0056] Unless otherwise described, the implementation of the present invention will employ conventional techniques such as cell biology, molecular biology, and genetic engineering techniques, which are known to those skilled in the art.

[0057] Overall embodiment

[0058] Application of Tripterygium wilfordii polysaccharide in the preparation of drugs for preventing and treating inflammatory bowel disease.

[0059] As a specific embodiment, the inflammatory bowel disease is acute inflammatory bowel disease.

[0060] As a specific embodiment, the drug is used to protect the integrity of the intestinal mucosal barrier or repair intestinal mucosal barrier damage, or to relieve intestinal wall edema caused by inflammatory bowel disease, or to relieve shortening of colon length caused by inflammatory bowel disease, or to reduce weight loss caused by inflammatory bowel disease, or to reduce changes in intestinal flora caused by inflammatory bowel disease.

[0061] Furthermore, the drug is used to increase the abundance of one or more bacteria in the genera Lactobacillus, Ruminococcus, Eubacterium and Lachnospira in the intestinal flora.

[0062] As a specific embodiment, the drug is used to reduce the level of proinflammatory factors in colon tissue, or to increase the level of G protein coupled receptor, mucin 2, secretory immunoglobulin A, β-defensin or tight junction protein in colon tissue.

[0063] Furthermore, the pro-inflammatory factors include one or more of IL-6, TNF-α and IFN-γ.

[0064] Furthermore, the G protein coupled receptors include GPR41 and / or GPR43.

[0065] Furthermore, the tight junction proteins include one or more of Claudin-1, Occludin and ZO-1.

[0066] As a specific embodiment, the preparation method of the Tripterygium wilfordii polysaccharide comprises the following steps: crushing the Tripterygium wilfordii medicinal material, extracting it with ethanol aqueous solution at 75-85°C, taking the residue, extracting it with water at 95-100°C, collecting the extract obtained by the water extraction, concentrating it, separating the supernatant, removing free protein, adding a precipitant to precipitate the polysaccharide, separating the precipitate and drying it to obtain Tripterygium wilfordii polysaccharide.

[0067] Application of Tripterygium wilfordii polysaccharide in the preparation of drugs for regulating the levels of G protein coupled receptors, mucin 2, secretory immunoglobulin A, beta-defensins or tight junction proteins in colon tissue in vivo.

[0068] As a specific embodiment, the pro-inflammatory factors include one or more of IL-6, TNF-α and IFN-γ.

[0069] As a specific embodiment, the G protein coupled receptor includes GPR41 and / or GPR43.

[0070] As a specific embodiment, the tight junction proteins include one or more of Claudin-1, Occludin and ZO-1.

[0071] Application of Tripterygium wilfordii polysaccharide in regulating the levels of G protein-coupled receptors or tight junction proteins in colon tissue in vitro.

[0072] As a specific embodiment, the G protein coupled receptor includes GPR41 and / or GPR43.

[0073] As a specific embodiment, the tight junction proteins include one or more of Claudin-1, Occludin and ZO-1.

[0074] Example 1: Preparation of Tripterygium wilfordii polysaccharide

[0075] Take 100g of Sanyeqing medicinal material, crush it, extract it twice with 95% (v / v) ethanol aqueous solution by cold soaking (temperature is 80℃), dry the residue in a ventilated place at room temperature, then extract it twice with hot water (temperature is 100℃), filter, combine the extracts, concentrate, centrifuge, remove free protein from the supernatant by sevage method, centrifuge, concentrate the supernatant to a small volume, add 95% (v / v) ethanol aqueous solution, centrifuge, and precipitate and freeze-dry to obtain total polysaccharide, which is Sanyeqing polysaccharide.

[0076] The sugar content in the polysaccharide of Tripterygium wilfordii was measured by sulfuric acid-phenol method, which was 39.6%; the uronic acid content in the polysaccharide of Tripterygium wilfordii was 51.6%; and the protein content in the polysaccharide of Tripterygium wilfordii was 0.93%.

[0077] Example 2: Pharmacological efficacy test of Tripterygium wilfordii polysaccharide in relieving inflammatory bowel disease

[0078] 2.1 Experimental Materials

[0079] 2.1.1 Experimental animals and drugs

[0080] 40 C57BL / 6 male mice, weighing 16-18 g, were purchased from the Animal Experiment Center of Zhejiang Chinese Medical University, with animal qualification certificate number: SYXK(Zhe)2018-0012. The experimental animals were housed in separate cages, and the bedding was changed every 3 days. They were adaptively fed for 2 days and had free access to water and food during the experiment. The temperature of the breeding environment was 20±2℃, the relative humidity was 60%, and the light and dark cycles were 12 hours per day.

[0081] The trifoliate green polysaccharide is the polysaccharide extracted from the trifoliate green in Example 1.

[0082] 2.1.2 Experimental reagents and instruments Chemical reagents: DSS (dextran sulfate sodium, MP Biomedicals, LOT NO: 116531050), PBS buffer, analytical grade formaldehyde solution, 10% formalin. Sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate (provided by Sinopharm Chemical Reagent Co., Ltd.).

[0083] Kits: BCA protein concentration detection kit (Biyuntian Biotechnology Co., Ltd., batch number P0012), mouse MUC-2 enzyme-linked immunosorbent assay kit (Wuhan Enzyme Immunoassay Biotechnology Co., Ltd., MM-44508M1), mouse SIgA enzyme-linked immunosorbent assay kit (Wuhan Enzyme Immunoassay Biotechnology Co., Ltd., MM-0040M1), mouse β-defensin enzyme-linked immunosorbent assay kit (Wuhan Enzyme Immunoassay Biotechnology Co., Ltd., MM-1116M1).

[0084] Experimental instruments: FA2204 electronic balance (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.), microplate spectrophotometer (bio-tek), Allegra64R desktop high-speed refrigerated centrifuge (Beckman Coulter Trading China Co., Ltd.), pipette, vortexer, mini microcentrifuge.

[0085] Experimental equipment: elbow forceps, surgical scissors, beaker, measuring cylinder, EP tube, centrifuge tube.

[0086] 2.1.3 Drug configuration

[0087] 2.1.3.1 Preparation of Tripterygium wilfordii polysaccharide suspension

[0088] Use an electronic balance to weigh 10, 20, and 30 mg of Tripterygium wilfordii polysaccharide powder and pour it into a 15 mL centrifuge tube. Add double distilled water and make the volume to 10 mL. Dissolve it by ultrasonic and prepare working solutions with final concentrations of 10, 20, and 30 mg / mL. Place it in a 4°C refrigerator, return to room temperature before each use, and vortex to mix.

[0089] 2.1.3.2DSS solution preparation

[0090] Weigh 12.5 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 2.5%. Re-prepare the solution every two days and feed the animals with the new solution.

[0091] 2.1.3.3 Preparation of tissue fixative

[0092] Use a measuring cylinder to measure 50 mL of analytical grade formaldehyde solution into a wide-mouth bottle, and then add 450 mL of prepared PBS buffer to obtain 500 mL of 4% formaldehyde solution.

[0093] 2.2 Experimental methods

[0094] 2.2.1 Animal grouping and model establishment

[0095] 40 C57BL / 6 mice were randomly divided into 6 groups: CON group was the normal control group; DSS group was the DSS model group; DSS+5-ASA group was the 5-aminosalicylic acid positive control group; DSS+THP-L group was the low-dose group of Tripterygium wilfordii polysaccharide (100 mg / kg); DSS+THP-M group was the medium-dose group of Tripterygium wilfordii polysaccharide (200 mg / kg); DSS+THP-M group was the high-dose group of Tripterygium wilfordii polysaccharide (300 mg / kg). Except for 10 mice in the DSS group, there were 6 mice in each group; the drinking water of all groups except the CON group was replaced with an aqueous solution containing 2.5% DSS, and the DSS+5-ASA and DSS+THP-L / M / H groups were intragastrically administered with doses of 300 mg / kg, 100 mg / kg, 200 mg / kg, and 300 mg / kg, respectively; at the same time, the CON group and the DSS group were intragastrically administered with equal amounts of double distilled water, once a day, for 7 consecutive days.

[0096] 2.2.2 Experimental process

[0097] The activities, eating, mental state, body weight, fecal status and death of mice were observed and recorded every day. Seven days after the model was established, the mice were weighed, their eyes were removed to collect blood, the cecum and colon were cut off, the length of the colon was recorded and photographed for preservation, and the part of the colon connected to one side of the cecum was cut off and placed in 10% formalin; the remaining colon was stored at -80°C.

[0098] 2.2.3 Animal grouping and model establishment

[0099] After the mouse was dissected, the colon near the cecum was selected, and the tissue of about 0.5 cm in length was cut off and fixed in 10% formalin. The fixed tissue sections were dehydrated and embedded in paraffin. HE staining: the samples were hydrated first, then stained with hematoxylin and eosin, and finally dehydrated; PAS staining: the sections were hydrated, stained with periodic acid and Schiff's solution, and finally dehydrated; Immunofluorescence: after extracting the antigen, the intestinal sections were incubated with Claudin-1, Occludn, and ZO-1 at 4°C, and incubated with secondary antibody-bound antibodies in a 37°C water bath for 30 minutes. The sections were observed under an inverted microscope and photographed.

[0100] 2.2.4 Preparation of intestinal tissue homogenate and determination of MUC-2, SIgA, and β-defensin

[0101] Weigh about 50 mg of colon tissue into a 2 mL EP tube, add 500 μL PBS buffer, homogenize with a homogenizer, aspirate the homogenate into a new 1.5 mL EP tube, centrifuge at 3500 rpm for 10 min, divide the supernatant after centrifugation into 0.6 mL EP tubes, 100 μL per tube, mark them, and store them at -80°C; take out one tube of intestinal homogenate for each sample, and use ELISA (refer to the instructions of the MUC-2, SIgA, and β-defensin kits) to determine the contents of MUC-2, SIgA, and β-defensin in the intestinal homogenate supernatant.

[0102] 2.2.5 WB detection of Claudin-1, Occludin and ZO-1 proteins in intestinal tissue The mouse colon tissue was lysed with RIPA lysis buffer to extract proteins, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (PVDF membrane); blocked with Beyotime blocking solution for 1 hour, incubated with corresponding antibodies at room temperature overnight, incubated with secondary antibodies for 1 hour, and developed with color developer on a Bio-Rad gel imager.

[0103] 2.2.6 Detection of intestinal flora in mouse feces using high-throughput sequencing technology

[0104] The feces of each mouse were collected aseptically, frozen in liquid nitrogen, and then sent to Shanghai Meiji Biological Co., Ltd. for examination.

[0105] 2.2.7 Detection of short-chain fatty acids in mouse feces by high performance gas chromatography

[0106] 100 mg of fecal sample was placed in a 10 mL centrifuge tube, 1.6 mL of deionized water was added to dissolve, vortexed for 2 min, 0.4 mL of sulfuric acid (50% sulfuric acid solution) and 1.5 mL of ether were added, shaken at 250 r / min for 45 min, and centrifuged at 3000 r / min for 5 min. The supernatant was placed in a sterile test tube, anhydrous calcium chloride was added, and dehydrated in a 0.22 μm filter membrane, and the volume was adjusted to 1 mL with ether before injection, and SCFAs were detected by gas chromatography.

[0107] 2.2.8 WB detection of G protein-coupled receptors GPR41 and GPR43 proteins in intestinal tissue The mouse colon tissue was lysed with RIPA lysis buffer to extract proteins, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (PVDF membrane); blocked with Beyotime blocking solution for 1 hour, incubated with corresponding antibodies at room temperature overnight, incubated with secondary antibodies for 1 hour, and developed with a color developer on a Bio-Rad gel imager.

[0108] 2.2.9 Statistics

[0109] All measurement data were expressed as mean ± standard deviation (mean ± SD). The differences among the groups were compared with analysis of variance, and pairwise comparisons were performed with one-way ANOVA. P < 0.05 was used to judge statistical significance. "#" indicated p < 0.05 compared with the normal control group, "##" indicated p < 0.01 compared with the normal control group, "*" indicated p < 0.05 compared with the model group; "**" indicated p < 0.01 compared with the model group. Statistics were performed using Graphpad Prism 9.0.

[0110] 2.3 Experimental Results

[0111] 2.3.1 Effect of Tripterygium wilfordii polysaccharide on the body weight of DSS-induced IBD mice

[0112] The research results are as follows Figure 2 As shown: the weight of the normal control group increased by an average of 5.35% from the 1st day to the 8th day, while the weight of the mice that continuously drank 2.5% DSS decreased by an average of 8.6% from the 1st day to the 8th day, indicating that the health of the mice in the model group declined sharply, which is one of the signs of successful modeling. The weight loss rate of the mice in the THP intervention group was relatively slow compared with the model group, and the weight of the mice in the THP medium and high dose groups and the positive control group all recovered until the end of the experiment. The positive control drug (mesalazine) was not effective in improving weight loss.

[0113] 2.3.2 Effect of Tripterygium wilfordii polysaccharide on colon length in DSS-induced IBD mice

[0114] The research results are as follows Figure 3As shown: The experimental results showed that the colon length of the model group mice was relatively shorter than that of the normal group; after THP intervention, the colon length changed, among which the length of the THP high-dose group was closest to that of the normal group, the THP medium-dose group became longer than the normal group, and the low-dose group became shorter than the normal group.

[0115] 2.3.3 Effect of Tripterygium wilfordii polysaccharide on colon tissue morphology in DSS-induced IBD mice

[0116] HE staining results showed that ( Figure 1 ): By observing the colon sections of mice in the normal control group, it was found that the colon tissue was intact, with clear layers, the mucosal layer, submucosal layer, serosal layer and muscular layer of the colon tissue were intact, the colon mucosal epithelial cells were neat and orderly, and there was no edema and inflammatory cell infiltration in the submucosal layer. The integrity of the colon tissue structure of the model group mice was destroyed, the colon mucosal epithelial cells were disordered, the mucosal layer cells were necrotic and detached, inflammatory cells infiltrated the submucosal layer, and the glandular structure disappeared. However, after THP intervention, the integrity of the colon structure was enhanced to varying degrees, and the inflammatory response was alleviated, which was manifested in the basic integrity of the mucosal layer and submucosal layer of the colon tissue, the reduction of inflammatory cell infiltration, and the integrity of its tissue structure tended to the normal control group.

[0117] The results of PAS staining were Figure 4 As shown: the goblet cells in the colon of the normal group were purple-red in color and had a high expression level; while in the model group, the colon structure was destroyed, the goblet cell expression was low, and the number of goblet cells was significantly reduced compared with the normal group; the colon structure of the drug-treated group was protected, the number of goblet cells was close to that of the normal group, and significantly increased compared with the model group.

[0118] 2.3.4 Effects of Tripterygium wilfordii polysaccharide on MUC-2, SIgA, and β-defensins in colon tissues of DSS-induced IBD mice

[0119] MUC-2, β-defensin and SIgA can reflect the intestinal chemical barrier function. After DSS modeling, the level of SIgA in the intestine was significantly reduced, and the content of β-defensin was at a high level, indicating that the model was successfully established. Figure 5 As shown: After THP administration, the secretion levels of SIgA and β-defensin in the intestine of mice were higher than those in the model group; compared with the normal group, the MUC-2 content in the intestine of the model group mice decreased; compared with the model group, the MUC-2 content in the low-, medium-, and high-dose THP groups and the mesalazine group increased.

[0120] 2.3.5 Effect of Tripterygium wilfordii polysaccharide on the levels of IL-6, TNF-α, and IFN-γ in the serum of DSS-induced IBD mice Figure 6): Compared with the model group, THP could significantly reduce the increased levels of TNF-α(p<0.01), IL-6(p<0.01), and IFN-γ(p<0.01) in the intestine of mice.

[0121] 2.3.6 Effect of Tripterygium wilfordii polysaccharide on the expression levels of tight junction proteins (Claudin-1, Occludin, and ZO-1) in the colon of DSS-induced IBD mice

[0122] Intercellular tight junction proteins (Claudin-1, Occludin, ZO-1) can reflect the integrity of the mouse intestinal barrier. They are expressed and play a role on the cell membrane and are closely related to tight junctions. Tight junctions are located at the top of epithelial cells and are an important component for maintaining mucosal permeability. Protein expression results are shown in Figure 2. Figure 7 The immunofluorescence results are shown in Figures 8 to 10 As shown: tight junction proteins were highly expressed in normal group mice; the protein expression levels of the three tight junction proteins in model group mice decreased, and the immunofluorescence intensity decreased; the fluorescence intensity of the drug-treated group was close to that of the normal group, showing a high expression level, which was higher than that of the model group.

[0123] 2.3.7 The results showed that the effect of Tripterygium wilfordii polysaccharide on the fecal flora composition of DSS-induced IBD mice ( Fig.11 and 12 ): THP treatment significantly changed the intestinal flora to the normal group, while DSS treatment significantly changed the overall structure of the intestinal flora. It is worth noting that the THP-H group was also shown to be different from the THP-L and THP-M groups, indicating that 300 mg / kg THP may greatly change the composition of the intestinal flora. Firmicutes and Bacteroidetes were the two most common phyla, but the proportions of these phyla were much lower in the DSS group, while the species composition of the drug-treated group was comparable to that of the normal control group. The results of our genus-level analysis of the species composition map showed that the proportions of Lactobacillus, Ruminococcus, Eubacterium, Lachnospira, etc. were significantly downregulated in the DSS group, and their abundance increased after THP treatment, with significant differences in the THP-H group.

[0124] 2.3.8 Effect of Tripterygium wilfordii polysaccharide on fecal short-chain fatty acids (SCFAs) content in DSS-induced IBD mice Microorganisms and their metabolites such as SCFAs (acetic acid, propionic acid, butyric acid, etc.) play an important role in maintaining intestinal homeostasis. Acetic acid is the short-chain fatty acid with the highest content in feces. Fig.13 As shown: the contents of acetic acid, isobutyric acid, valeric acid and isovaleric acid in feces of the model group decreased, while THP treatment significantly increased the contents of acetic acid and isovaleric acid in feces, but only slightly increased the contents of propionic acid, butyric acid and valeric acid in feces.

[0125] 2.3.9 Effect of Tripterygium wilfordii polysaccharide on the expression level of G protein-coupled receptors (GPR41, GPR43) in colon tissue of DSS-induced IBD mice We studied the expression pattern of G protein-coupled receptors (including GPR41 and GPR43) in colon tissue by Western blotting. Fig.14 As shown: At the protein level, the expression of GPR41 / 43 in the model group was significantly lower than that in the control group, while its expression was significantly increased after THP administration. The above results suggest that THP may produce anti-inflammatory effects by activating GPR41 / 43 in colon cells in response to changes in intestinal flora composition and increase in SCFAs.

[0126] Example 3: In vitro study of signaling pathways in the treatment of inflammatory bowel disease with Tripterygium wilfordii polysaccharide

[0127] 3.1 Experimental Materials

[0128] 3.1.1 Experimental cells and drug preparation

[0129] Caco-2 cells are a human colon cancer cell line cultured at 37°C, 5% CO2 in DMEM (Gibco, USA) supplemented with 20% heat-inactivated fetal bovine serum and 1% penicillin and streptomycin.

[0130] The culture supernatant (CS) was derived from the intestinal bacteria in the feces of mice in each group of Experiment 2. To obtain CS, the intestinal bacteria were cultured anaerobically in anaerobic medium at 37°C for 24 h, then centrifuged at 3000 rpm for 10 min and filtered through a 0.22 μm filter.

[0131] 3.1.2 Experimental reagents Chemical reagents: CCK8 (Biosharp, cat. no. BS350A), lipopolysaccharide (Sigma-Aldrich, cat. no. l2880), pertussis toxin (Glpbio, cat. no. 70323-44-3), U71322 (Macklin, cat. no. 112648-68-7), cell RNA extraction kit (ES Science), high capacity cDNA reverse transcription kit (Biosystems), SYBR premix (Thermo Fisher, LOT, 91231261)

[0132] 3.2 Experimental methods

[0133] 3.2.1 Caco-2 cell viability assay

[0134] Cell viability analysis was performed using CCK-8 according to the manufacturer's instructions. Cells were plated at 1 × 10 4The cells / well were seeded in 96-well plates and cultured with CS of intestinal bacteria from different THP groups (THP-CS) for 24 h. Incubated at 37°C for 1-2 h. Finally, the optical density (OD) was measured at 450 nm.

[0135] 3.2.2 RT-qPCR detection of tight junction proteins (Claudin-1, Occludin, ZO-1) and GPR41, GPR43 mRNA expression levels in Caco-2 cells

[0136] Caco-2 cells were cultured in 6-well plates. After Caco-2 cells were stimulated with LPS (2 μg / mL) for 6 h, they were incubated with DSS-CS and THP-CS (10 μL / mL) for 24 h, and cell samples were collected for subsequent detection. We incubated Caco-2 cells with pertussis toxin (200 ng / mL) and U73122 (10 μM) for 24 h to inhibit the GPRs signaling pathway, and then collected cell samples for subsequent detection after incubation in THP-CS for 24 h. RNA was extracted according to the instructions of the cell RNA extraction kit. cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit. Quantitative real-time fluorescence quantitative PCR (qRT-PCR) was performed using SYBR green premix (using Biosystems).

[0137] 3.3 Experimental Results

[0138] 3.3.1 Effect of THP-CS on Caco-2 cell viability

[0139] like Fig.15 As shown: When the concentration of the intestinal flora culture supernatant of the mice in the Tripterygium wilfordii polysaccharide group was 0-62.5 μL / mL, there was no significant difference in the cell viability value compared with the normal control cells (p>0.05), indicating that within the concentration range of 0-62.5 μL / mL, the fecal fermentation liquid had no toxic side effects on Caco-2 cells. The mouse fecal fermentation liquid significantly reduced the viability of Caco-2 cells at 125-500 μL / mL (p<0.05), indicating that the growth of Caco-2 cells would be inhibited within this dose range, resulting in toxic side effects. Within the safety range, the maximum dose selected was used to treat Caco-2 cells, so a concentration of 100 μL / mL was selected for subsequent experiments.

[0140] 3.3.2 Effects of THP-CS on tight junctions and expression of GPR41 and GPR43 in Caco-2 cells stimulated by LPS In order to confirm whether the regulatory effect of THP on intestinal flora is mainly through the activation of SCFAs-GPRs to improve the intestinal barrier, we incubated Caco-2 cell monolayers with CS of intestinal bacteria in the control and treatment groups for 24 h. Then, we detected the effects of intestinal bacteria collected from different groups on the expression levels of ZO-1, occludin and claudin-2 in Caco-2 cell monolayers stimulated by LPS. Fig.16 As shown in (A) to 16 (E): After 24 h of DSS-CS treatment, the expression of ZO-1, occludin and claudin-1 mRNA in Caco-2 cells was significantly reduced; THP-CS treatment can regulate the expression of these genes, and after Caco-2 cells were treated with THP-CS, the expression of GPR41 / 43 was significantly increased, indicating that GPRs have been activated by THP-CS. In addition, THP-CS can significantly upregulate the level of GPR41 / 43 mRNA in Caco-2 treated with GPRs inhibitors, as shown in Figure 1. Fig.16 (F) and 16(G).

[0141] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the experimental methods described herein are conventional methods unless otherwise specified; the reagents, biological materials and equipment described herein are commercially available unless otherwise specified.

[0142] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. The use of Tripterygium wilfordii polysaccharide in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The preparation steps of the trifoliate green polysaccharide include: taking trifoliate green medicinal materials and crushing them, cold-immersing and extracting them twice with 95% ethanol aqueous solution at 75-85° C., air-drying the residues in a ventilated place at room temperature, extracting them twice with hot water at 95-100° C., filtering, combining the extracts, concentrating, centrifuging, removing free proteins from the supernatant by sevage method, centrifuging, concentrating the supernatant, adding 95% ethanol aqueous solution to precipitate polysaccharides, centrifuging, and freeze-drying the precipitate to obtain total polysaccharides, namely, trifoliate green polysaccharide.

2. The use according to claim 1, characterized in that The drug is used for protecting the integrity of the intestinal mucosal barrier or repairing the damage of the intestinal mucosal barrier.

3. The use according to claim 1, characterized in that The drug is used to relieve intestinal wall edema caused by ulcerative colitis, or to relieve shortening of colon length caused by ulcerative colitis, or to reduce changes in intestinal flora caused by ulcerative colitis.

4. The use according to claim 1, characterized in that The drug is used to reduce weight loss caused by ulcerative colitis.

5. The use according to claim 1, characterized in that The drug is used to reduce the level of pro-inflammatory factors in colon tissue.

6. The use according to claim 1, characterized in that Used to increase the levels of G protein-coupled receptors, mucin 2, secretory immunoglobulin A, beta-defensins, or tight junction proteins in colon tissue.

7. The use according to claim 5, characterized in that The pro-inflammatory factors include one or more of IL-6, TNF-α and IFN-γ.

8. The use according to claim 6, characterized in that The G protein coupled receptors include GPR41 and / or GPR43.

9. The use according to claim 6, characterized in that The tight junction proteins include one or more of Claudin-1, Occludin and ZO-1.

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