Use of a traditional Chinese medicine compound composition in preparing an anti-inflammatory colon cancer drug

Abstract

The present invention belongs to the field of medical technology, and specifically relates to the use of a Chinese medicine compound composition in the preparation of a drug for resisting ulcerative colitis and intestinal fibrosis and colon cancer caused by colitis. The Chinese medicine compound composition of the present invention has significant effects on resisting acute ulcerative colitis, improving the degree of intestinal fibrosis caused by colitis, and resisting colon cancer, and can be used to improve and treat ulcerative colitis and intestinal fibrosis and colon cancer caused by colitis. The Chinese medicine compound composition resists acute ulcerative colitis by reducing the level of oxidative stress, inhibiting intestinal inflammation, restoring intestinal barrier function, and regulating immunity, and improves colon fibrosis and colon cancer caused by colitis. A new drug with significant efficacy and few adverse reactions is provided for patients with ulcerative colitis and intestinal fibrosis and colon cancer caused by colitis to relieve the suffering of patients.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a new use of a traditional Chinese medicine compound composition in preparing drugs for resisting ulcerative colitis, colitis-induced intestinal fibrosis and colon cancer. Background Art

[0002] Tangcao Tablets is a pure Chinese medicine preparation, composed of 20 kinds of Chinese medicines, including Geranium, Honeysuckle, Bupleurum, Elsholtzia, Trichosanthes peel, Pomegranate peel, Astragalus, Licorice, Kapok, etc. It has the effects of clearing away heat and detoxifying, promoting blood circulation and invigorating Qi. It is currently used clinically to treat HIV-infected people and AIDS patients, and has the effect of increasing CD4 lymphocyte counts, and can improve symptoms such as fatigue, hair loss, loss of appetite and diarrhea.

[0003] Ulcerative colitis (UC) is a chronic recurrent inflammatory disease, which is mainly manifested by abdominal pain, diarrhea, mucous and bloody stools, etc. It has the characteristics of repeated attacks, poor prognosis and incurability. Recurrent inflammatory bowel disease has been proven to be the main cause of colitis-associated colorectal cancer (CAC). However, existing therapeutic drugs have problems such as easy recurrence after discontinuation of medication, reduced efficacy of long-term medication, and many adverse reactions. Therefore, it is urgent to develop more ideal drugs for the prevention and treatment of UC and CAC. According to statistics, the longer the course of inflammatory bowel disease patients, the higher the incidence of colon cancer. The overall prevalence of colon cancer in UC patients is 3.2%, and the cumulative risk of cancer after 10, 20 and 30 years of illness is 2%, 8% and 18%, respectively. In addition, current treatments cannot cure inflammatory bowel disease, but can only relieve symptoms to a certain extent, which greatly increases the risk of colorectal cancer. A large number of studies have shown that inhibiting intestinal inflammation and restoring intestinal barrier function can effectively fight colitis-associated colon cancer.

[0004] Repeated stimulation of the mouse intestine with dextran sulfate sodium (DSS) simulates the changes in the acute and remission phases of human ulcerative colitis, which is an ideal model for studying the pathogenesis of UC and evaluating the efficacy of drugs. The azoxymethane / dextran sulfate sodium (AOM / DSS) model is an effective and reproducible experimental model that uses the chemical substance AOM to induce DNA damage, which is converted into highly active alkylated products through metabolism by the CYP2E1 enzyme, and then forms O6 methylguanine adducts in DNA, leading to gene mutations. AOM is absorbed after entering the colon epithelial cells and exerts a carcinogenic effect. Combined with DSS, it provides a two-step method for establishing a tumor model. This model has the characteristics of a relatively short timeline and accurate modeling of colorectal cancer. Tumors can be formed in as little as 10 weeks. In addition, AOM / DSS-induced tumors present key features of human CAC in histopathology, such as distal tumors and invasive adenocarcinomas. It is widely used to study the pathogenesis of inflammatory colorectal cancer and is a powerful research tool.

[0005] Tangcao tablets are a pure Chinese medicine preparation for the auxiliary treatment of AIDS. They can improve symptoms such as anorexia and diarrhea in patients. Many of the ingredients in them have been proven to have multiple functions such as anti-inflammatory, antioxidant, immune function promotion, and intestinal protection, and have potential therapeutic effects on the treatment of UC. Many Chinese medicinal ingredients in Tangcao tablets have been proven to have significant anti-inflammatory effects. Geranium is a perennial herb of the Geranium family, mainly used to dispel wind and dredge meridians; promote blood circulation; clear heat and remove dampness. In recent years, more than 90 chemical components such as flavonoids, organic acids and volatile oils in geranium have been isolated and proven to have anti-inflammatory, antibacterial, antiviral, antioxidant and immunomodulatory effects. Existing studies have shown that geranium has a significant therapeutic effect on inflammatory diseases. For example, the ethanol extract of geranium significantly reduced the swelling of rat ears and feet induced by xylene or carrageenan, and reduced capillary permeability. In addition, in the rat adjuvant arthritis model, geranium ointment can significantly relieve allergic inflammation caused by Freund's complete adjuvant and reduce joint swelling. Honeysuckle has multiple pharmacological effects, involving effects on inflammation, fever, viruses, bacteria, immune response, liver, nervous system and heart. The active ingredients in Bupleurum, saikosaponin A and saikosaponin D, have multiple pharmacological effects such as antiviral, anti-inflammatory, anti-tumor, and liver and kidney protection. In the ulcerative colitis model induced by sodium dextran sulfate, saikosaponin D can effectively reduce the disease activity index, increase colon length and improve pathological characteristics, significantly inhibit the mRNA levels of pro-inflammatory cytokines such as tumor necrosis factor α, interleukin 6 and interleukin 1β, and increase the mRNA level of anti-inflammatory cytokine interleukin 10. In addition, the ingredients in Tangcao tablets such as Astragalus, Elsholtzia, and Licorice also have significant antibacterial and anti-inflammatory pharmacological effects. The compounds such as flavonoids and phenolic acids in Tangcao tablets have the effects of improving immunity, antiviral, antioxidant, and antibacterial and anti-inflammatory.

[0006] Oxidative stress plays a vital role in the pathogenesis of inflammatory bowel disease. It has the dual effects of generating free radicals and enhancing lipid peroxidation, and is the main mechanism of disease evolution. When inflammatory bowel disease occurs, the expression of NOX1 and NOX2 increases abnormally, the generation of oxygen free radicals such as malondialdehyde (MDA) increases, and the level of glutathione (GSH) decreases, leading to an imbalance of oxidative stress in the intestinal mucosa. Nitric oxide (NO) has the effect of promoting neutrophil infiltration in the distal colon, thereby causing tissue damage. In the colonic mucosa, myeloperoxidase (MPO) is an indicator used to measure the degree of neutrophil infiltration, and its activity level can reflect the number of neutrophils.

[0007] The pathogenesis of inflammatory bowel disease is closely related to the loss of intestinal epithelial barrier function, leading to excessive bacterial translocation, which may be the initiation or early event of the disease. Restoring the integrity of the mucosal barrier is expected to become a future treatment for UC. When UC occurs, a large number of inflammatory cytokines accumulate in the intestinal mucosa. At this time, the permeability of the intestinal mucosa is significantly increased, the expression of intestinal epithelial cell proteins is reduced, and intestinal mucosal barrier dysfunction occurs. Tight junctions are destroyed, and the permeability of the intestinal mucosa to harmful substances increases. At the same time, various inflammatory factors and endotoxins can further increase the permeability of the intestinal mucosa by affecting the expression of various proteins. In addition, the content of diamine oxidase (DAO) in colonic tissue goblet cells and serum also reflects the integrity of the colonic mucosal barrier.

[0008] Nuclear factor κB (NF-κB) is a core regulator of inflammatory response and is involved in the pathogenesis of many inflammatory diseases, including inflammatory bowel disease. This pathway induces the expression of proinflammatory cytokines and leads to inflammation-related tissue damage. In the mouse colitis model, NF-κB can participate in regulating the transcription of multiple inflammatory factors, induce the production of multiple proinflammatory cytokines, and cause abnormal immune responses. During the activation of NF-κB, the inhibitory protein IκB is degraded, p65-NF-κB is released and phosphorylated, and then transported into the nucleus to regulate the transcription of target genes. The phosphorylation level of NF-κB p65 is positively correlated with the pathological grade of UC, and plays a key role in the occurrence and development of UC. IL-6 is a 26KD glycoprotein that can be synthesized by a variety of cells, including monocytes, macrophages, lymphocytes, fibroblasts, and some tumor cells. The IL-6 receptor exists in two forms: IL-6R and CD126, one is membrane-bound and the other is soluble. After the IL-6 / IL-6R complex binds to IL-6, it recruits another transmembrane glycoprotein, gp130, also known as the IL-6 signal converter or CD130. The IL-6 signal activates the tyrosine kinases JAK1, JAK2, and TYK2 through IL-6R and gp130, causing the phosphorylation of STAT1 and STAT3. STAT3 activation can be caused by a variety of cytokines and growth factors, including the use of IL-6 signals to transfer to the receptor chain gp130. In addition, STAT3 mediates important signal transduction cascades triggered by intracellular proteins such as tyrosine kinase oncoproteins, and plays an important role in inflammatory bowel disease.

[0009] Inflammatory bowel disease is also believed to be caused by an excessive immune response of a genetically susceptible host to intestinal flora and environmental factors. Among the various immune cells that overreact in the intestinal mucosa, CD4+ helper T cells play an important role in acquired immunity, among which the subtypes believed to be involved in inflammatory diseases include effector T cells: Th1, Th2, Th17 and Treg. The pathogenesis of UC involves changes in the balance between effector T cells and inhibitory T cells. Studies have shown that IL-12-induced Th1 cells express the transcription factor T-bet and secrete interferon IFN-γ; Th2 cells express the transcription factor Gata3 and secrete IL-4 and IL-13 under the stimulation of IL-4 and IL-33; Th17 mainly produces cytokines such as IL-17 to play a pro-inflammatory role in pro-inflammatory reactions; Treg cells have immunosuppressive functions in autoimmune diseases, which can inhibit the activation of helper T cells and maintain the body's homeostasis. Excessive activation of Th17 cells and lack of Treg cells will lead to intestinal inflammation. Studies have found that the proportion of Th17 cells is upregulated and Treg is downregulated. The imbalance between the two plays an important role in the pathogenesis of autoimmune diseases. Therefore, inhibiting the differentiation of Th17 cells and promoting the differentiation of Treg cells may be an effective way to treat UC.

[0010] Relevant studies have demonstrated the relationship between the balance of Th1 / Th2 cells and fibrosis in various organs, and found that the fibrosis process of various organs can be inhibited or promoted by regulating the secretion status of Th1 and Th2 cells. Th2 cells secrete IL-4 to activate B cell proliferation, promote B cell isotype conversion, and cause a series of pathological changes, including autoantibody secretion and collagen deposition, and recruit mast cells, macrophages, eosinophils and mesenchymal cells to directly induce fibroblast activation. As a negative immune regulatory cell, Treg secretes inhibitory cytokines IL-10 and TGF-β, and has the function of protecting the body from excessive immune activation and tissue damage. At the same time, it can promote epithelial cell repair, inhibit fibroblast aggregation, and regulate the balance of pro-inflammatory and anti-inflammatory factors to inhibit the occurrence of fibrosis. This part of the experiment detected a significant increase in the expression of RORγt and IL-17 in colon tissue, indicating that DSS-induced chronic UC may promote the differentiation of Th17 cells by inducing the expression of RORγt. Th17 cells promote the proliferation and migration of neutrophils, the activation of endothelial cells and the proliferation of fibroblasts by secreting IL-17, and recruit macrophages and produce high levels of profibrotic TGF-β, which is necessary for fibrosis. Tregs inhibit fibrosis by inhibiting the production and function of pro-inflammatory factors and antagonizing Th17 and its cytokines.

[0011] Tangcao tablets have a preventive and therapeutic effect on ulcerative colitis, which is mainly characterized by inflammatory response, as well as intestinal fibrosis and colon cancer in the later stages of inflammation. Summary of the invention

[0012] In order to better treat ulcerative colitis and colon fibrosis and colon cancer caused by colitis, and relieve the pain of patients. The present invention provides a new use of a Chinese medicine compound composition, specifically, a new use in the preparation of drugs for ulcerative colitis, intestinal fibrosis and colon cancer caused by colitis. The Chinese medicine compound composition is produced by Shanghai Baisuixing Pharmaceutical Co., Ltd., and the product name is Tangcao Tablets, and the approval number is a pure Chinese medicine preparation of National Medicine Standard Z20050291. The present invention is a secondary research and development of the existing product, and unexpectedly found that Tangcao Tablets have a certain effect of resisting ulcerative colitis and intestinal fibrosis and colon cancer caused by colitis.

[0013] The specific technical scheme of the present invention is:

[0014] The invention provides a use of a Chinese medicine compound composition in preparing a drug for resisting ulcerative colitis and intestinal fibrosis and colon cancer caused by colitis. The Chinese medicine compound composition is composed of the following components in parts by weight: 120-150 parts of geranium, 120-150 parts of astragalus, 120-200 parts of nightshade, 120-200 parts of honeysuckle, 60-100 parts of kapok, 40-60 parts of terminalia chebula, and 120-200 parts of scutellaria baicalensis. 80-150 parts of grass, 40-60 parts of pomegranate peel, 150-200 parts of glutinous rice roots, 80-150 parts of water chestnuts, 40-60 parts of trichosanthes peels, 60-100 parts of bupleurum, 40-60 parts of elsholtzia, 60-80 parts of liquorice, 120-150 parts of millettia reticulata, 40-60 parts of safflower, 40-60 parts of ginkgo leaves, 60-120 parts of purslane, 40-60 parts of picrophora, and 28-40 parts of scorpion.

[0015] Furthermore, the Chinese medicine compound composition is prepared into conventional oral dosage forms including tablets, capsules, powders and granules.

[0016] Furthermore, the Chinese medicine compound composition is made into film-coated tablets.

[0017] Furthermore, the dosage of the Chinese medicine compound composition is adjusted based on the severity of the disease and the presence or absence of previous medical history.

[0018] The tangcao tablets of the present invention have significant anti-acute ulcerative colitis effect, can significantly reduce MPO activity and MDA and NO contents in colon tissue, and increase GSH level; reduce the phosphorylation levels of NF-κB-p65 and STAT3 in colon tissue; significantly improve mouse mucosal barrier function, which is mainly manifested in increasing the number of epithelial goblet cells in colon tissue and reducing Evans blue dye exudation, etc.; the expression of specific transcription factors Tbet and RoRγt and related cytokines of Th1 and Th17 in colon tissue is decreased; the expression of specific transcription factors Gata3 and Foxp3 and related cytokines of Th2 and Treg is increased, and the balance of Th1 / Th2 and Th17 / Treg biased towards Th1 and Th17 is significantly corrected.

[0019] The tangerine tablets of the present invention have the effect of significantly inhibiting intestinal fibrosis caused by chronic colitis, significantly reducing pathological damage to colon tissue, and reducing collagen deposition and HYP content. The pathological characteristics of mice with chronic colitis, such as weight loss, increased DAI level, shortened colon, enlarged spleen, thymus atrophy, and colon damage, are significantly improved; the structural integrity of the intestinal epithelial mucosa is maintained by increasing the protein expression of Occludin. The drift toward Th2 and Th17 in the Th1 / Th2 and Th17 / Treg balance systems in colon tissue is significantly corrected.

[0020] The tangerine tablets of the present invention have the effects of significantly reducing the size of colon tumors caused by colitis, the maximum diameter of tumors, and the proportion of tumors with a diameter > 4 mm. Improve the symptoms related to colitis in mice with colon cancer; increase the expression level of Occludin in the colon tissue of mice with colon cancer. Reduce the expression levels of Gata3, RoRγt, Foxp3, IL-4, IL-6, IL-1β, TNF-α, IL-17, IL-10, and TGF-β in intestinal tissues; increase the expression levels of T-bet and IFN-γ, that is, continuously correct the drift of Th cells to Th2 and Th17 in the Th1 / Th2 and Th17 / Treg systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are HE staining images of colon tissue pathology sections of mice with acute ulcerative colitis, where A is the colon tissue pathology section of mice in the normal group, B is the colon tissue pathology section of mice in the model group, C is the colon tissue pathology section of mice in the positive group, D is the colon tissue pathology section of mice in the low-dose Tangcao tablets group, and E is the colon tissue pathology section of mice in the high-dose Tangcao tablets group. Figure 2 The figures are comparative diagrams of the expression of inflammatory signaling pathway proteins in the colon tissue of mice with acute ulcerative colitis, wherein A is a protein immunoblotting map of phosphorylated NF-κB-p65, IL-6, and P-STAT3 / STAT3 in the colon tissue of mice with acute ulcerative colitis, B is a comparative bar graph of P-STAT3 / STAT3 protein expression, C is a comparative bar graph of NF-κB-p65 protein expression, and D is a comparative bar graph of IL-6 protein expression.

[0022] Figure 3 The figures are comparative graphs of the expression levels of Th1 / Th2 specific markers and cytokines in the colon tissue of mice with acute ulcerative colitis, wherein A is a protein immunoblotting graph of T-bet and Gata3, B is a comparative bar graph of T-bet protein expression, C is a comparative bar graph of Gata3 protein expression, D is a comparative bar graph of T-bet / Gata3 ratio, E is a protein immunoblotting graph of cytokines IFN-γ and IL-4, F is a comparative bar graph of IFN-γ protein expression, and G is a comparative bar graph of IL-4 protein expression.

[0023] Figure 4The figures are comparative graphs of the expression levels of Th17 / Treg-specific transcription factors and related cytokines in the colon tissue of mice with acute ulcerative colitis, wherein A is a protein immunoblotting atlas of RoRγt and Foxp3, B is a comparative bar graph of RoRγt protein expression, C is a comparative bar graph of Foxp3 protein expression, D is a comparative bar graph of RoRγt / Foxp3 ratio, E is a protein immunoblotting atlas of cytokines IL-6, IL-1β, TNF-α, IL-17, TGF-β, and IL-10, F is a comparative bar graph of IL-6 protein expression, G is a comparative bar graph of IL-1β protein expression, H is a comparative bar graph of TNF-α protein expression, I is a comparative bar graph of IL-17 protein expression, J is a comparative bar graph of TGF-β protein expression, and K is a comparative bar graph of IL-10 protein expression.

[0024] Figure 5 These are pathological sections of goblet cells in colon tissue of mice with acute ulcerative colitis, where A is the pathological section of mice in the normal group, B is the pathological section of mice in the model group, C is the pathological section of mice in the positive group, D is the pathological section of mice in the low-dose group of Tangcao tablets, and E is the pathological section of mice in the high-dose group of Tangcao tablets.

[0025] Figure 6 The figure is a comparative bar graph of the Evans blue exudation content of colon tissues of mice with acute ulcerative colitis.

[0026] Figure 7 The figures are comparative diagrams of the expression levels of tight junction protein Occludin in colon tissue of mice with acute ulcerative colitis, wherein A is a protein immunoblot atlas of tight junction protein Occludin, and B is a bar graph comparing the expression of tight junction protein Occludin.

[0027] Figure 8 This is a comparison of the survival time of mice with acute ulcerative colitis.

[0028] Fig. 9 This is a comparison chart of body weight changes in mice with chronic ulcerative colitis.

[0029] Fig.10 Comparison of disease activity index scores in mice with chronic ulcerative colitis

[0030] Fig.11 These are HE staining images of colon tissue pathology sections of mice with chronic ulcerative colitis, where A is the colon tissue pathology section of mice in the normal group, B is the colon tissue pathology section of mice in the model group, C is the colon tissue pathology section of mice in the positive group, D is the colon tissue pathology section of mice in the low-dose group of Tangcao tablets, and E is the colon tissue pathology section of mice in the high-dose group of Tangcao tablets.

[0031] Fig.12The figure is a comparison of α-SMA expression levels in colon tissues of mice with chronic ulcerative colitis, wherein A is an α-SMA protein immunoblotting map, and B is a bar graph of α-SMA expression comparison.

[0032] Fig.13 The figures are comparative diagrams of the expression levels of tight junction protein Occludin in colon tissue of mice with chronic ulcerative colitis, wherein A is a protein immunoblotting map of tight junction protein Occludin, and B is a bar graph comparing the expression of tight junction protein Occludin.

[0033] Fig.14 The figures are comparative graphs of the expression levels of Th1 / Th2 specific markers and related cytokines in the colon tissue of mice with chronic ulcerative colitis, wherein A is a protein immunoblotting graph of T-bet and Gata3, B is a comparative bar graph of T-bet protein expression, C is a comparative bar graph of Gata3 protein expression, D is a comparative bar graph of T-bet / Gata3 ratio, E is a protein immunoblotting graph of cytokines IFN-γ and IL-4, F is a comparative bar graph of IFN-γ protein expression, and G is a comparative bar graph of IL-4 protein expression.

[0034] Fig.15 The figures are comparative graphs of the expression levels of Th17 / Treg-specific transcription factors and related cytokines in the colon tissue of mice with chronic ulcerative colitis, wherein A is a protein immunoblotting atlas of Foxp3 and RoRγt, B is a comparative bar graph of Foxp3 protein expression, C is a comparative bar graph of RoRγt protein expression, D is a comparative bar graph of RoRγt / Foxp3 ratio, E is a protein immunoblotting atlas of cytokines IL-1β, TNF-α, IL-6, IL-17, IL-10, and TGF-β, F is a comparative bar graph of IL-1β protein expression, G is a comparative bar graph of TNF-α protein expression, H is a comparative bar graph of IL-6 protein expression, I is a comparative bar graph of IL-17 protein expression, J is a comparative bar graph of IL-10 protein expression, and K is a comparative bar graph of TGF-β protein expression.

[0035] Fig.16 Comparison of body weight changes in CAC mice induced by AOM / DSS.

[0036] Fig.17 Comparison of the changes in disease activity index scores in AOM / DSS-induced CAC mice.

[0037] Fig.18These are HE staining images of colon tissue pathology sections of AOM / DSS-induced CAC mice, where A is the colon tissue pathology section of normal group mice, B is the colon tissue pathology section of model group mice, C is the colon tissue pathology section of positive group mice, D is the colon tissue pathology section of Tangcao tablets low-dose group mice, and E is the colon tissue pathology section of Tangcao tablets high-dose group mice.

[0038] Fig.19 The figure is a comparison of the occludin expression levels in the colon tissue of AOM / DSS-induced CAC mice, wherein A is a protein immunoblot atlas of tight junction protein occludin, and B is a bar graph comparing the expression of tight junction protein occludin.

[0039] Fig. 20 The figures are comparative graphs of the expression levels of Th1 / Th2 specific markers and related cytokines in the colon tissue of AOM / DSS-induced CAC mice, wherein A is the immunoblotting graph of Gata3 and T-bet proteins, B is the comparative bar graph of Gata3 protein expression, C is the comparative bar graph of T-bet protein expression, D is the immunoblotting graph of cytokines IL-4 and IFN-γ proteins, E is the comparative bar graph of IL-4 protein expression, and F is the comparative bar graph of IFN-γ protein expression.

[0040] Fig.21 Figure 3 is a comparison of the expression levels of Th17 / Treg-specific transcription factors and related cytokines in the colon tissue of AOM / DSS-induced CAC mice, where A is the protein immunoblotting atlas of Foxp3 and RoRγt, B is the comparative bar graph of Foxp3 protein expression, C is the comparative bar graph of RoRγt protein expression, D is the protein immunoblotting atlas of cytokines TNF-α, IL-6, IL-1β, IL-17, IL-10, and TGF-β, E is the comparative bar graph of TNF-α protein expression, F is the comparative bar graph of IL-6 protein expression, G is the comparative bar graph of IL-1β protein expression, H is the comparative bar graph of IL-17 protein expression, I is the comparative bar graph of TGF-β protein expression, and J is the comparative bar graph of IL-10 protein expression. DETAILED DESCRIPTION

[0041] The present invention is further illustrated below through the description of specific implementation modes, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0042] Example 1: The preventive and therapeutic effects of Tangcao tablets on DSS-induced acute ulcerative colitis in mice

[0043] 1 Main experimental materials

[0044] SPF male BALB / c mice, about 6-8 weeks old, weighing about 18-20 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Animal Certificate No.: No. 210726221101241328). Tangcao tablets, batch number: 190308, approval number: National Medicine Standard Z20050291, Shanghai Baisuixing Pharmaceutical Co., Ltd. Dextran sulfate sodium salt, Shanghai Aladdin Biochemical Technology Co., Ltd.; mesalazine sustained-release granules, Shanghai Aifa Pharmaceutical Co., Ltd.; 4% paraformaldehyde tissue fixative, Wuhan Sewell Biotechnology Co., Ltd.; sodium carboxymethylcellulose, Shanghai Linen Technology Development Co., Ltd.; No. 12 mouse gavage needle, Henan Zhike Hongrun Environmental Protection Technology Co., Ltd.; 1mL sterile syringe, Shanghai Mishawa Medical Industry Co., Ltd.; methanol, Komiou Chemical Reagent Co., Ltd. (Tianjin); 1× PBS buffer, Beijing Solebao Technology Co., Ltd.; 0.5% Evans blue dye, Beijing Solebao Technology Co., Ltd.; formamide, Tianjin Damao Chemical Reagent Factory; GSH assay kit, Nanjing Jiancheng Biological Institute of Engineering; MDA assay kit, Nanjing Jiancheng Bioengineering Institute; MPO assay kit, Nanjing Jiancheng Bioengineering Institute; NO assay kit, Yacoin (Wuhan) Biotechnology Co., Ltd.; DAO assay kit, Nanjing Jiancheng Bioengineering Institute; 5× protein loading buffer, Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.; protein phosphatase inhibitor cocktail (100×), Beijing Solebow Technology Co., Ltd.; BCA protein concentration assay kit, GLPBIO; 96-well plate, Wuxi Nice Biotechnology Co., Ltd.; skim milk powder, Beijing Solebow Technology Co., Ltd.; 10× electrotransfer solution, Beijing Solebow Technology Co., Ltd.; PVDF membrane (pore size 0.45μm), Merck Millipore, Germany; prestained protein molecular weight standard (marker), CISTRO; SDS-PAGE gel preparation kit, Beijing Solebao Technology Co., Ltd.; ultrasensitive ECL chemiluminescence detection kit, Shanghai Yazyme Biotechnology Co., Ltd.; Tris, Beijing Solebao Technology Co., Ltd.; glycine, Beijing Solebao Technology Co., Ltd.; SDS, Beijing Solebao Technology Co., Ltd.; GAPDH antibody, Wuhan Sewell Biotechnology Co., Ltd.; goat anti-rabbit IgG-HRP SAB, Shanghai Sabo Biotechnology Co., Ltd.; goat anti-mouse IgG-HRP, SAB Shanghai Sabo Biotechnology Co., Ltd.; Occludin antibody, Abcam; IL-6 antibody, Signal wayAntibody, USA; IL-1β antibody, SAB Shanghai Sabo Biotechnology Co., Ltd.; TNF-α antibody, SAB Shanghai Sabo Biotechnology Co., Ltd.; IL-4 antibody, Protein tech, USA; IL-10 antibody, Protein tech, USA; TGF-β antibody, Protein tech, USA tech; IFN-γ antibody, Beijing Bioson Biotechnology Co., Ltd.; Tbet antibody, Beijing Bioson Biotechnology Co., Ltd.; Gata3 antibody, Wuhan Fine Biotechnology Co., Ltd.; RoRγt antibody, Beijing Bioson Biotechnology Co., Ltd.; Foxp3 antibody, Abcam; IL-17 antibody, Wuhan Fine Biotechnology Co., Ltd.; STAT3 antibody, Signal Way Antibody, USA; P-STAT3 antibody, Signal Way Antibody, USA Antibody; IVC feeding system in animal room, Beijing Zhongshi Technology Co., Ltd.; -80℃ low temperature refrigerator, Suzhou Antai Air Technology Co., Ltd.; -20℃ low temperature refrigerator, Sanyo Co., Ltd. (Japan); ice maker, Panasonic Electric Industrial Co., Ltd. (Japan); horizontal centrifuge, Thermo Fisher Scientific; electric blast drying oven, Shanghai Senxin Co., Ltd. (DGG-9140); Milli-Q ultrapure water manufacturing system, Millipore Co., Ltd. (USA); analytical balance Quintix type, Sartorius Group (Germany); pipette, Eppendorf Life Sciences, Germany; water bath, Shanghai Jinghong Experimental Equipment Co., Ltd.; low temperature high speed centrifuge, Eppendorf, Germany; full wavelength microplate reader Multiskan GO type, Thermo Fisher Scientific; horizontal shaker, Sartorius Group (Germany); high throughput tissue grinder, Ningbo Xinzhi Biotechnology Co., Ltd.; Western Blot device, Bio-Rad, USA; WB chemiluminescence imager, Shanghai Peiqing Technology Co., Ltd. .

[0045] 2 Experimental methods

[0046] 2.1 Drug preparation: Tangcao tablets are divided into two dosage groups of 0.5g / kg and 1g / kg, and 0.5% sodium carboxymethylcellulose solution is used to prepare Tangcao tablet suspensions with concentrations of 0.05g / ml and 0.1g / ml, respectively. Mesalazine sustained-release granules (National Medicine Standard H20143164) are converted according to the equivalent dose conversion formula for mice and humans. After the granules are ground into powder, the drug is weighed on a balance and dissolved in 0.5% sodium carboxymethylcellulose solution to prepare a 52mg / ml mesalazine suspension as a positive drug. Weigh 35g of sodium dextran sulfate solid, add ultrapure water and stir to completely dissolve it, then dilute to 1000ml, prepare a 3.5% DSS solution and dispense it into the drinking water bottles of mice.

[0047] 2.2 Establishment and treatment of acute ulcerative colitis model: After one week of adaptive feeding of mice, they were randomly divided into blank control group, model group, positive drug mesalazine group, low-dose group of Tangcao tablets (0.5g / kg), and high-dose group of Tangcao tablets (1g / kg). Five days before modeling, the positive drug mesalazine group, low-dose group of Tangcao tablets, and high-dose group of Tangcao tablets were gavaged with mesalazine suspension and corresponding dose of Tangcao tablets suspension at a fixed time every morning, and the drugs were concomitantly administered during the modeling period until the end of the experiment. The mice in the model group were gavaged with an equal amount of 0.5% sodium carboxymethyl cellulose solution once a day. After five days, except for the blank group, the mice in other groups drank 3.5% DSS solution freely for 7 consecutive days, and the blank control group drank ordinary drinking water, and the first day of the mice drinking DSS solution was counted as d1. The weight of the mice and the diarrhea and bloody stools were recorded daily during the modeling period.

[0048] 2.3 Specimen collection: The grouping model was established according to the method in 2.2. After the experiment ended on d7, the mice were fasted for 24 hours and had free access to water. On d8, blood was collected from the mouse orbits, and part of it was added to an EP tube containing an anticoagulant for routine blood tests. The remaining blood was allowed to stand for 1 hour and then centrifuged to obtain the supernatant, which was frozen at -80°C for later use. After the mice were killed, the thymus and colon tissues were collected and the organ weights were weighed. The distance between the rectum and the cecum, i.e., the colon length, was measured with a ruler, and the cecum tissue was then removed. About 1 cm of the distal colon was taken, its mass was weighed, and it was fixed in a 4% paraformaldehyde solution. The remaining colon tissue was longitudinally cut open, and the mass was weighed after the feces in the intestinal cavity was removed. The mass of the two parts was added together and recorded as the colon mass of the mouse. Colon mass per unit length = colon mass (mg) / colon length (cm); organ index = organ mass (mg) / body weight (g). After washing the tissue with PBS, place it on filter paper to absorb moisture, and then divide the tissue into smaller packages for subsequent experiments. The divided tissues are quickly frozen in liquid nitrogen and then transferred to -80°C for frozen storage.

[0049] 2.4 Weight change and calculation of disease activity index: The model was built according to the method in 2.2, and the percentage of weight change of mice from d1 to d7 was recorded and calculated. The initial weight of the mice was recorded, and the percentage of weight change was calculated = (weight on the day - initial weight) / initial weight × 100%. The weight and fecal status of the mice were recorded, and fecal occult blood test kit was performed. The average of the three scores of weight loss percentage, fecal characteristics, and occult blood test kit results (benzidine method) was calculated as the disease activity index (DAI).

[0050] 2.5 HE staining to observe pathological changes of colon tissue: The colon tissue retained in 2.3 was fixed, dehydrated, waxed, embedded, paraffin-sectioned (4 μm), dewaxed to water, HE stained, dehydrated, and mounted for microscopic examination and image acquisition and analysis.

[0051] 2.6 Colon histopathological scoring: The pathological sections in 2.5 were observed under an optical microscope for colon tissue morphology and inflammatory infiltration, and histological scoring was performed.

[0052] 2.7 PAS staining to observe goblet cells in colon tissue: The pathological sections of 2.5 were dewaxed, soaked in gradient ethanol solutions, rinsed with running water, incubated with oxidants, rinsed with running water, dripped with Schiff reagent, rinsed with running water, stained with hematoxylin staining solution for nuclei, differentiated with acidic differentiation solution, rinsed with running water, blued with warm water, dehydrated with gradient ethanol, transparentized with xylene, sealed with neutral gum, dried and stored in the dark. Microscopic examination and image analysis were performed.

[0053] 2.8 Determination of oxidative stress level in colon tissue: Prepare 10% mouse colon tissue homogenate with the colon tissue retained in 2.3 according to the ratio of colon tissue mass (mg): physiological saline (μL) = 1:9, centrifuge for 10 min (4500r / min), and keep the supernatant. Detect the protein concentration of the homogenate supernatant with a BCA kit. Strictly follow the instructions, standardize the preparation of the required reagents, detect MDA, GSH, MPO, and NO according to the operating procedures, and calculate the content.

[0054] 2.9 Protein immunoblotting analysis was used to detect the expression levels of tight junction proteins, inflammatory signaling pathway proteins, Th1 / Th2 specific marker factors and related cytokines, Th17 / Treg specific transcription factors and related cytokines in colon tissue: the colon tissue retained in 2.3 was cut into pieces for protein sample preparation, protein quantification and denaturation, and SDS-PAGE gel electrophoresis was used to quantitatively analyze the protein expression of tight junction protein Occludin protein, inflammatory signaling pathway proteins NF-κB-p65, IL-6, P-STAT3, Th1 / Th2 specific marker factors Tbet, Gata3 and related cytokines IFN-γ, IL-4, Th17 / Treg cell specific marker factors RoRγt, Foxp3 and their related cytokines IL-6, TNF-α, IL-17, TGF-β, IL-10.

[0055] 2.10 Determination of serum diamine oxidase (DAO) content: According to the procedure in the instruction manual, the blood sample retained in 2.3 is measured and calculated at a wavelength of 340 nm using an ultraviolet spectrophotometer.

[0056] 2.11 Routine blood test: The blood samples in the anticoagulation tubes reserved in 2.3 were subjected to routine blood test using a fully automatic blood cell analyzer to detect the white blood cell count (10 9 / L), red blood cell count (10 12 / L), hemoglobin concentration (g / L), platelet count (10 9 / L) and other related indicators.

[0057] 2.12 Observation of survival period of acute ulcerative colitis model: The model was established by grouping according to the method in 2.2. The DSS solution was stopped on d7 and replaced with ordinary drinking water for continued feeding. The survival period of mice in each group was observed and recorded within 14 days.

[0058] 2.13 Evans blue staining to detect endothelial permeability: The grouping model was established according to the method in 2.2, and the following operations were performed on d7: 200 μL of 0.5% Evans blue dye solution was injected into the tail vein of the mouse using a tail vein injector. After 60 minutes, the mouse was killed by cervical dislocation. After removing the colon tissue, it was cut open longitudinally to remove the feces. Then 100 mg of tissue was placed in an EP tube containing 1 mL of formamide solution. The EP tube was placed in a 55°C water bath and incubated for 24 hours. Centrifuge at 2000 rpm for 10 minutes at room temperature, and the supernatant was taken for subsequent determination. Use formamide as a solvent to prepare a standard curve of Evans blue solution with a suitable concentration gradient. Use an enzyme marker to measure the absorbance value at a wavelength of 620 nm.

[0059] 2.14 Statistical analysis: Data were expressed as mean ± standard deviation (Mean ± SD). Graphpad Prism 8.4.2 software was used to plot the data, and SPSS 27.0 software was used for statistical analysis. For multiple groups of data that followed a normal distribution, one-way analysis of variance was used to compare the mean differences between groups. If the variances of the data groups were equal, the LSD method was used for pairwise comparisons; if the variances were unequal, the Games-Howell method was used for pairwise comparisons. For multiple groups of data that did not follow a normal distribution, the Kruskal-Wallis method was used for mean comparisons, and the Bonferroni method was used for pairwise comparisons between groups. For two groups of data that followed a normal distribution, the T test was used for mean comparisons. For two groups of data that did not follow a normal distribution, the Mann-Whitney U method was used for mean comparisons. For two groups of data that followed a normal distribution, the Person method was used for correlation analysis. For two groups of data that did not follow a normal distribution, the Spearman method was used for correlation analysis. If the calculated P < 0.05, it was considered to be statistically significant.

[0060] 3 Experimental results

[0061] 3.1 Effects of Tangcao Tablets on Colon Length and Organ Index in Mice with Acute Ulcerative Colitis

[0062] In the colitis model experiment, as the disease worsens, the colon gradually shortens. Compared with the blank group, the colon length of mice in the model group was significantly shortened, and the colon mass coefficient was significantly increased. Compared with the model group, Tangcao tablets significantly inhibited colon shortening and reduced the colon mass coefficient. Compared with the blank group, the thymus tissue of mice in the model group showed obvious atrophy, and the thymus index of the high-dose group of Tangcao tablets (1g / kg) was significantly higher than that of the model group, indicating that Tangcao tablets significantly improved the thymus atrophy symptoms of mice with DSS-induced acute ulcerative colitis.

[0063] Table 1 Effects of Tangcao tablets on colon length and organ index in DSS-induced acute ulcerative colitis in mice

[0064]

[0065] ***P<0.001vs.Control,#P<0.05,##P<0.01vs.Model.

[0066] 3.2 Effects of Tangcao Tablets on Body Weight Change and Disease Activity Index in Mice with Acute Ulcerative Colitis:

[0067] Disorders in colon function can affect absorption and even cause symptoms such as diarrhea, abdominal distension, and abdominal pain. Therefore, weight changes and disease activity index (DAI) are often used to assess the severity of the disease. The weight change curve showed that the mice in the model group lost weight on d2, which was significantly different from the blank group (P<0.05). The DAI curve showed that the mice in the model group developed diarrhea from d3, and the DAI curve increased. The low-dose group (0.5g / kg) and the high-dose group (1g / kg) of Tangcao tablets can significantly improve the weight loss of mice and reduce symptoms such as diarrhea and bloody stools in mice. This suggests that Tangcao tablets can effectively relieve the symptoms of acute ulcerative colitis in mice

[0068] Table 2 Effect of Tangcao tablets on body weight of mice with acute ulcerative colitis induced by 3.5% DSS

[0069]

[0070] *P<0.05,**P<0.01,***P<0.001vs.Control,#P<0.05,##P<0.01,###P<0.001vs.Model.

[0071] Table 3 Effect of Tangcao tablets on DAI in mice with acute ulcerative colitis induced by 3.5% DSS

[0072]

[0073] *P<0.05,**P<0.01,***P<0.001vs.Control,#P<0.05,##P<0.01,###P<0.001vs.Model.

[0074] 3.3 Effect of Tangcao Tablets on Colonic Histopathology in Mice with Acute Ulcerative Colitis

[0075] Microscopic observation revealed that the colon mucosal epithelial cells of the normal group mice were continuous and intact, with no structural changes or inflammatory infiltration, neatly arranged glands, and normal crypt structure. However, microscopic observation of the colon mucosa of the model group mice showed obvious structural destruction, a decrease in the number of goblet cells, and the disappearance of the crypt structure, with more severe inflammatory infiltration in the mucosa and submucosa. Compared with the model group, the range of colon mucosal lesions in the Tangcao Tablets group mice was reduced, and microscopic observation showed that mucosal edema was reduced, crypt structure was repaired, and mucosal damage was reduced. Among them, the mucosal inflammatory infiltration in the high-dose Tangcao Tablets group (1g / kg) was milder, the mucosal epithelium was basically intact, the crypt structure was restored, and the colon structure basically returned to normal. See for details. Figure 1 .

[0076] Table 4 Effect of Tangcao tablets on histopathological scores of mice with DSS-induced acute ulcerative colitis

[0077] Group N Histopathological scoring Blank Group 3 / Model Group 3 <![CDATA[3.93±0.15 *** ]]> Positive group 3 <![CDATA[1.00±0.33 ### ]]> Tangcao tablets (0.5g / kg) 3 3.00±1.45 Tangcao tablets (1g / kg) 3 <![CDATA[1.00±0.58 ### ]]>

[0078] ***P<0.001vs.Control,###P<0.001vs.Model.

[0079] 3.4 Effect of Tangcao Tablets on Oxidative Stress Levels in Colonic Tissue of Mice with Acute Ulcerative Colitis

[0080] Compared with the model group, the low-dose group (0.5g / kg) and high-dose group (1g / kg) of Tangcao tablets increased the GSH content, and the GSH content of the colon tissue of mice in the high-dose group (1g / kg) of Tangcao tablets was significantly higher than that in the model group (P<0.05). Compared with the model group, the low-dose group (0.5g / kg) and high-dose group (1g / kg) of Tangcao tablets significantly reduced the levels of MDA, MPO, and NO. The above results suggest that Tangcao tablets can regulate the oxidative stress level of colon tissue in mice with acute ulcerative colitis and improve the balance of oxidation and antioxidant systems in the body.

[0081] Table 5 Effects of Tangcao tablets on oxidative stress level in colon tissue of mice with acute ulcerative colitis

[0082] Group GSH (μmol / gprot) MDA (μmol / gprot) MPO(U / g) NO(μM) Blank Group 27.42±10.23 1.52±0.73 0.72±0.29 4.37±2.16 Model Group <![CDATA[14.35±6.62 * ]]> <![CDATA[2.43±1.41 ** ]]> <![CDATA[3.08±0.55 *** ]]> <![CDATA[10.38±8.19 * ]]> Positive group <![CDATA[29.25±2.94 # ]]> <![CDATA[1.46±0.68 # ]]> <![CDATA[1.25±1.00 ## ]]> <![CDATA[3.69±0.99 ## ]]> Tangcao tablets (0.5g / kg) 13.26±7.17 <![CDATA[0.91±0.37 ### ]]> <![CDATA[1.79±0.77 # ]]> 6.25±3.89 Tangcao tablets (1g / kg) <![CDATA[28.62±12.85 # ]]> <![CDATA[1.15±0.44 ## ]]> <![CDATA[1.23±0.39 ## ]]> <![CDATA[4.26±1.47 # ]]>

[0083] *P<0.05,**P<0.01,***P<0.001vs.Control,#P<0.05,##P<0.01,###P<0.001vs.Model.

[0084] 3.5 Effect of Tangcao Tablets on the Expression of Inflammatory Signaling Pathway Proteins in Colonic Tissue of Mice with Acute Ulcerative Colitis

[0085] Western Blot technology was used to detect the expression levels of inflammatory signaling pathway proteins NF-κB-p65, IL-6, and P-STAT3 in the colon tissues of mice with acute ulcerative colitis. Figure 2 As shown, compared with the model group, the expressions of phosphorylated NF-κB-p65, IL-6, and P-STAT3 / STAT3 in the colon tissue of mice in the Tangcao tablets group were significantly reduced, suggesting that Tangcao tablets inhibited the activation of the inflammatory signaling pathway NF-κB / IL-6 / STAT3 in the colon tissue of mice with DSS-induced acute ulcerative colitis.

[0086] 3.6 Effect of Tangcao Tablets on the Expression Levels of Th1 / Th2 Specific Markers and Cytokines in Colonic Tissue of Mice with Acute Ulcerative Colitis

[0087] T-bet and Gata3 are Th1 and Th2 cell-specific transcription factors, and their expression levels reflect the number of Th1 and Th2 cells. Figure 3 As shown in the results, compared with the blank control group, the expression of T-bet protein in the colon tissue of the model group mice increased, the expression of Gata3 protein decreased, the T-bet / Gata3 ratio increased, and the balance was biased towards Th1. Tangcao tablets reduced the expression level of IFN-γ in the colon tissue of UC mice, increased the level of IL-4, and reduced the T-bet / Gata3 ratio by regulating the expression of T-bet and Gata3. This suggests that Tangcao tablets correct the imbalance of Th1 / Th2 in mice with acute ulcerative colitis induced by DSS.

[0088] 3.7 Effects of Tangcao Tablets on Th17 / Treg-specific transcription factors and related cytokines in colon tissue of mice with acute ulcerative colitis

[0089] RoRγt and Foxp3 are specific markers for Th17 and Treg cells, and their expression levels reflect the number of Th17 and Treg cells. Figure 4 As shown in the figure, compared with the control group, the expression of RoRγt protein in the colon tissue of the model group mice was significantly increased, the expression of Foxp3 protein was decreased, the RoRγt / Foxp3 ratio was increased, and the balance was biased towards Th17. Tangcao tablets reduce the expression levels of IL-6, TNF-α, and IL-17 in the colon tissue of mice, increase the expression of TGF-β and IL-10, regulate the expression levels of RoRγt and Foxp3, and reduce the RoRγt / Foxp3 ratio. This suggests that Tangcao tablets correct the imbalance of Th17 / Treg in the colon tissue of mice with acute ulcerative colitis. For details, see Figure 4 .

[0090] 3.8 Effects of Tangcao Tablets on Hematological Parameters in Mice with Acute Ulcerative Colitis

[0091] During the course of ulcerative colitis, the ulcers are located in the mucosa and submucosa, and after invading the blood vessels, mucous purulent stools appear, causing anemia, and anemia may be the most common systemic complication in patients with inflammatory bowel disease. Therefore, we performed a routine blood test on mice with acute ulcerative colitis and found that compared with the blank group, the red blood cell and hemoglobin counts in the model group were significantly reduced, indicating that DSS-induced acute UC mice showed obvious anemia symptoms. The red blood cell count and hemoglobin count of mice in the Tangcao tablet group increased significantly, indicating that Tangcao tablets significantly improved the symptoms of anemia in mice. In addition, the white blood cell count of mice in the model group increased, which may be due to the stress effect of inflammation. After treatment with Tangcao tablets, the white blood cell count decreased significantly, indicating that Tangcao tablets can improve the inflammatory response of mice caused by DSS and relieve anemia symptoms.

[0092] Table 6 Effects of Tangcao tablets on hematological parameters of mice with acute ulcerative colitis

[0093]

[0094]

[0095] *P<0.05,**P<0.01,***P<0.001vs.Control,#P<0.05,##P<0.01,###P<0.001vs.Model.

[0096] 3.9 Effect of Tangcao Tablets on Colonic Mucosal Barrier Function in Mice with Acute Ulcerative Colitis

[0097] Effect of Tangcao Tablets on Goblet Cells in Colonic Tissue of Mice with Acute Ulcerative Colitis: The main component of the intestinal mucosal epithelial villus structure is goblet cells, a type of mucus-secreting cell distributed between the mucosal columnar epithelium. Its main function is to synthesize and secrete mucin, which has the function of protecting the intestinal mucosa. As shown in the figure, the number of red-stained goblet cells in the colonic tissue of mice in the blank control group was relatively large and their morphological structure was normal; while the number of goblet cells in the model group was extremely small and their morphology changed; compared with the model group, the number of goblet cells in the low-dose Tangcao Tablets group (0.5g / kg) was relatively small and irregularly distributed; the number of goblet cells in the high-dose Tangcao Tablets group (1g / kg) was relatively large and evenly distributed, and the morphological structure was improved and close to that of the control group. This suggests that Tangcao Tablets can restore the self-protection function of the intestinal mucosal barrier by increasing the number of goblet cells. See for details. Figure 5 .

[0098] Evans blue staining to detect the permeability of endothelial cells in mouse colon tissue: Evans blue is a dye that can bind to albumin. Under normal physiological conditions, endothelial cells have no permeability to albumin, and Evans blue dye only remains inside blood vessels. When the colon tissue of mice is stimulated by DSS, the tight junctions between endothelial cells are lost, resulting in increased permeability to small molecules such as albumin, thereby causing Evans blue dye to seep out of the tissue. The permeability of endothelial cells can be quantitatively evaluated through the Evans blue dye extravasation experiment. 0.5% Evans blue dye was injected into the tail vein of mice, and the mice were killed by dislocation after 60 minutes, and the colon tissue was removed. It was observed that the colon tissue of mice in the blank control group was basically not stained with blue, and the colon tissue of mice in the model group was deeply stained due to the large amount of Evans blue dye that leaked out (P<0.001). The colon tissue of the Tangcao tablets group (1g / kg) showed light blue, and the exudation of Evans blue dye was significantly reduced (P<0.05). This suggests that Tangcao tablets can significantly reduce the permeability of endothelial cells in mice with DSS-induced acute ulcerative colitis. Figure 6 .

[0099] Detection of diamine oxidase (DAO) levels in mouse serum: DAO is located in the cytoplasm of the chorionic epithelial cells in the upper layer of the intestinal mucosa. When the intestinal mucosal function is impaired, it is released into the blood. Therefore, the increase in DAO content in the blood is considered to be a potential indicator of intestinal mucosal barrier damage in inflammatory bowel disease. The serum DAO content of mice in the normal group was 38.03±13.17U / L. Compared with the normal group, the serum DAO content of the model group was 62.72±15.85U / L, which was significantly different (P<0.01). Compared with the model group, the low-dose group (0.5g / kg) and high-dose group (1g / kg) of Tangcao tablets could reduce the DAO content to 54.90±17.55U / L and 41.00±16.07U / L, respectively. Among them, the high-dose group (1g / kg) of Tangcao tablets had a significant statistical difference compared with the model group (P<0.01). It is suggested that Tangcao tablets reduce the permeability of colonic mucosa in mice with acute ulcerative colitis.

[0100] Table 7 Effect of Tangcao tablets on DAO levels in mice with acute ulcerative colitis induced by 3.5% DSS

[0101] Group N DAO(U / L) Blank Group 10 38.03±13.17 Model Group 10 <![CDATA[62.72±15.85 ** ]]> Positive group 10 <![CDATA[49.38±20.95 # ]]> Tangcao tablets (0.5g / kg) 10 54.90±17.55 Tangcao tablets (1g / kg) 10 <![CDATA[41.00±16.07 ## ]]>

[0102] ***P<0.001vs.Control,#P<0.05,##P<0.01vs.Model.

[0103] Detection of the expression level of tight junction protein Occludin in mouse colon tissue: The integrity of the intestinal barrier is mainly supported by tight junctions of epithelial cells, which effectively prevent harmful substances (such as pathogens and endotoxins) from entering the blood through the intestinal mucosa. Down-regulation of tight junction protein expression can lead to increased intestinal permeability in inflammatory bowel disease. Compared with the normal group, the expression of tight junction protein Occludin in the colon mucosa of mice in the model group was significantly reduced, and the expression of tight junction protein in the Tangcao tablets group was significantly increased. This suggests that Tangcao tablets can increase the expression level of tight junction proteins and improve the mechanical barrier function of the intestinal mucosa. See details Figure 7 .

[0104] 3.10 Effect of Tangcao Tablets on the General Condition and Survival Period of Mice with Acute Ulcerative Colitis

[0105] The mice in the model group showed obvious weight loss, diarrhea, and bloody stools on d4, which led to reduced activity, poor condition, rough fur color, and reduced food intake. However, compared with the model group, the mice in the Tangcao tablet group had increased activity, good mental state, mild diarrhea and bloody stools, and whiter fur. This suggests that Tangcao tablets can significantly improve the general condition of mice with acute ulcerative colitis. The survival results of mice showed that there were no deaths in the blank group, while the mice in the model group died on d8 due to severe diarrhea and bloody stools, and the survival rate of mice was only 60% on d14 (P<0.05). The mice in the low-dose group (0.5g / kg) of Tangcao tablets died on d13, but the overall number of deaths was lower than that in the model group, and the survival rate on d14 reached 90% (P=0.0891). There was no death in the high-dose group (1g / kg) of Tangcao tablets, and the survival rate of mice reached 100%, which was significantly higher than that in the model group (P<0.05). This suggests that Tangcao tablets can prolong the survival time of acute UC mice and reduce the mortality rate. Figure 8 .

[0106] Example 2: Therapeutic effect of Tangcao tablets on DSS-induced chronic colitis and colonic fibrosis

[0107] 1 Main experimental materials

[0108] HYP assay kit, Nanjing Jiancheng Bioengineering Institute; α-SMA antibody, Abcam (Shanghai) Trading Co., Ltd.; Masson staining kit, Shanghai ELISA Biological. The rest of the main experimental materials are the same as those in Example 1.

[0109] 2 Experimental methods

[0110] 2.1 Drug preparation: Weigh 20 g of DSS solid, add it to ultrapure water and stir until fully dissolved, dilute to 1000 ml, prepare a 2% DSS solution, and dispense it into the drinking water bottles of mice. The rest of the operation is the same as the drug preparation in Example 1.

[0111] 2.2 Establishment of a mouse model of chronic ulcerative colitis and drug intervention: After one week of adaptive feeding, 92 male BALB / c mice were randomly divided into five groups, namely, a blank group, a model group, a positive drug mesalazine group, a low-dose group of Tangcao tablets (0.5 g / kg), and a high-dose group of Tangcao tablets (1 g / kg). Except for the blank control group with 12 mice, the remaining groups had 20 mice each. The blank group was given normal diet and drinking water. The model group and the drug group were used to establish a chronic enteritis and intestinal fibrosis model for three cycles: 2% DSS solution was given for 5 days and ordinary drinking water for 10 days in the first cycle; the second and third cycles were treated in the same way as the first cycle. After the third cycle, the mice were killed, and the first day of DSS solution placement was counted as d1. The entire modeling process lasted for 45 days. The drug intervention group was given oral administration starting from d6 after the end of the first cycle of 2% DSS solution, once a day, and the model group was given an equal amount of sodium carboxymethyl cellulose solution.

[0112] 2.3 Specimen collection: Modeling and drug administration were performed according to 2.2. After modeling, specimens were collected according to the specimen collection method in Example 1, and the colon length and organ coefficient were calculated.

[0113] 2.4 DAI scoring: Modeling and drug administration were performed according to 2.2, and the DAI calculation method in Example 1 was used for scoring.

[0114] 2.5 HE staining to observe pathological changes in colon tissue: The colon tissue retained in 2.3 was subjected to HE staining according to the method in Example 1 to observe pathological changes in colon tissue.

[0115] 2.6 Masson staining to detect fibrosis in mouse colon tissue: The colon tissue retained in 2.3 was fixed in a wax block using paraffin embedding technology, and then 4 μm thick sections were prepared from the tissue samples. The sections were soaked in a dewaxing agent, and then the paraffin on the sections was gradually removed using an ethanol gradient. Acid fecal protein staining, acid fuchsin staining, reddish VB staining, green color developing solution staining, and blue color developing solution staining were performed in sequence, and the sections were sealed after dehydration and fixation, and observed under a microscope.

[0116] 2.7 Determination of the hydroxyproline (HYP) content in colon tissue: The colon tissue retained in 2.3 was used to detect the HYP content in the tissue according to the instructions of the HYP determination kit (using the alkaline hydrolysis method).

[0117] 2.8 Protein immunoblotting analysis to detect α-smooth muscle actin, Th1 / Th2, Th17 / Treg related proteins: The colon tissue retained in 2.3 was detected according to the method of protein immunoblotting analysis in Example 1 to detect colon tissue specific transcription factors and related cytokines.

[0118] 2.9 Statistical method: Same as the statistical method in Example 1.

[0119] 3 Experimental results

[0120] 3.1 Effect of Tangcao Tablets on the General Condition of Mice with Chronic Ulcerative Colitis

[0121] During the first cycle of drinking DSS, mice developed erect hair, dull luster, curled up bodies, reduced activity, reduced water intake and food intake, and a significant decrease in weight, accompanied by diarrhea and bloody stools. After changing to ordinary drinking water, recovery was slow. After drug treatment, the above symptoms of mice in the low-dose group (0.5g / kg) and high-dose group (1g / kg) of Tangcao tablets improved to varying degrees compared with the model group, and their mental state was relatively good. Diarrhea and bloody stools were significantly lighter, which preliminarily showed the efficacy of Tangcao tablets on mice with chronic ulcerative colitis. On the 45th day of establishing the chronic ulcerative colitis model, no mice in the blank control group died, while 3 mice in the model group died, and no mice in the low-dose group (0.5g / kg) and high-dose group (1g / kg) of Tangcao tablets died. It suggests that Tangcao tablets can improve the general condition of mice with chronic ulcerative colitis.

[0122] 3.2 Effect of Tangcao Tablets on Body Weight of Mice with Chronic Ulcerative Colitis

[0123] Compared with the body weight of mice in the blank group, the body weight of mice in the model group began to decrease in the first cycle, and the body weight dropped to the lowest on d7, and began to increase from d8; at the beginning of the second cycle, the body weight of mice in the model group remained basically unchanged, and after the discontinuation of the DSS solution (starting from the 21st day), the body weight began to gradually increase; in the third cycle, the body weight of mice in the model group showed a downward trend. The Tangcao Tablets group began treatment at the end of the first cycle of 2% DSS solution, that is, on d6, so the overall trend of weight change in the first cycle was consistent with that of the mice in the model group, but starting from the second cycle, the body weight of mice in the Tangcao Tablets group was on an upward trend, and the weight of mice was always higher than that of the model group until the end of the experiment. See for details. Fig. 9 .

[0124] 3.3 Effect of Tangcao Tablets on DAI Changes in Mice with Chronic Ulcerative Colitis

[0125] The mice in the blank group were in good condition, without diarrhea or bloody stools, and the DAI score was low and basically unchanged. Compared with the blank group, the DAI score of the mice in the model group gradually increased during the period of drinking 2% DSS solution. After the Tangcao tablets group began treatment, the change trend of the DAI score of the mice was consistent with that of the model group, and the DAI score was lower than that of the model group, and the symptoms of the mice were improved. Fig.10 .

[0126] 3.4 Effects of Tangcao Tablets on Colon Length and Organ Index in Mice with Chronic Ulcerative Colitis

[0127] Compared with the colon length of mice in the blank group, the colon length of mice in the model group was significantly shortened. The colon length of mice increased significantly after treatment with Tangcao tablets, indicating that Tangcao tablets can effectively improve the shortening of the colon in mice with chronic ulcerative colitis induced by DSS. The spleen and thymus are important immune organs in the human body. The thymus and spleen coefficients are important indicators reflecting the body's immune function. Clinical reports show that UC patients will have symptoms such as splenomegaly and thymus atrophy. The thymus coefficient of mice in the model group tended to atrophy compared with that of mice in the blank group, but there was no statistical difference. The spleen of mice in the model group showed splenomegaly compared with that in the blank group. The high-dose group of Tangcao tablets (1g / kg) can significantly reduce the spleen coefficient of mice. It is suggested that Tangcao tablets play a role by protecting the immune organs of mice with chronic ulcerative colitis.

[0128] Table 8 Effects of Tangcao tablets on colon length and organ coefficient in DSS-induced chronic ulcerative colitis mice

[0129]

[0130] *P<0.05,**P<0.01,***P<0.001vs.Control,#P<0.05,##P<0.01,###P<0.001vs.Model.

[0131] 3.5 Effect of Tangcao Tablets on Colonic Histopathology in Mice with Chronic Ulcerative Colitis

[0132] Microscopic observation of mouse colon tissue sections revealed no abnormalities in the colon mucosa, submucosa, and muscularis structure of mice in the blank group. The glandular structure was clear and neatly arranged, the goblet cells were filled, and there was less inflammatory cell infiltration. A large number of inflammatory cell infiltrations were found in the colon tissues of mice in the model group, and erosions and ulcers appeared. The glands were destroyed, the structure was irregular, the crypt structure disappeared, and the goblet cells decreased. After treatment with Tangcao tablets, the range of mucosal lesions in the colon tissues of mice was reduced, the crypt structure was repaired, and the mucosal damage was alleviated. Among them, the mucosal inflammatory infiltration in the high-dose group of Tangcao tablets (1g / kg) was milder, the mucosal epithelium was basically intact, the crypt structure was restored, and the colon structure basically returned to normal integrity. See for details. Fig.11 .

[0133] 3.6 Effect of Tangcao Tablets on Colonic Tissue Fibrosis in Mice with Chronic Ulcerative Colitis

[0134] Effect of Tangcao Tablets on Colonic Tissue Fibrosis in Chronic UC Mice: Chronic inflammation in inflammatory bowel disease induces reconstruction of intestinal wall tissue, produces excessive extracellular matrix, causes intestinal cavity diameter to shrink and stenosis to occur, and leads to intestinal fibrosis. In the process of intestinal fibrosis formation, collagen fibers in colon tissue are excessively deposited, mainly distributed in the mucosa and submucosa, especially in the submucosa. Masson staining results of colon tissues of mice in each group showed that only a small amount of collagen fibers were deposited in the colon tissues of blank group mice, while fibroblasts in colon tissues of model group mice proliferated a lot, blue collagen fibers increased in the submucosa, and collagen deposition increased significantly; compared with the model group, the blue collagen fibers in the submucosa of colon tissues of mice in the Tangcao Tablets group decreased, and the percentage of collagen-positive areas was significantly reduced, indicating that Tangcao Tablets can inhibit the deposition of collagen fibers in colon tissues of mice with chronic ulcerative colitis induced by DSS.

[0135] Table 9 Effect of Tangcao tablets on collagen deposition in colonic tissue of mice with chronic ulcerative colitis induced by DSS

[0136] Group N Positive area (%) Blank Group 3 3.43±0.36 Model Group 3 24.03±0.91 Positive group 3 9.18±7.28 Tangcao Tablets (0.5g / kg) 3 9.36±1.21 Tangcao Tablets (1g / kg) 3 5.4±1.86

[0137] ***P<0.001vs.Control; ###P<0.001vs.Model.

[0138] Effect of Tangcao Tablets on Hydroxyproline Content in Colon Tissue of Chronic UC Mice: During the development of the disease, increased collagen synthesis leads to the occurrence of fibrosis, and the accelerated rate of collagen biosynthesis in fibroblasts depends on changes in proline hydroxylase activity. During the collagen synthesis process, proline hydroxylase activity usually increases, and the hydroxyproline content increases, so hydroxyproline can be used as an important biomarker for quantitative collagen content. The results showed that compared with the blank group, the HYP content in the colon tissue of the model group mice was significantly increased, and the high-dose group of Tangcao Tablets (1g / kg) could significantly reduce its content. It is suggested that Tangcao Tablets can inhibit the increase of HYP content in colon tissue and reduce collagen synthesis.

[0139] Table 10 Effect of Tangcao tablets on HYP content in DSS-induced chronic ulcerative colitis in mice

[0140] Group N HYP content (μg / mg) Blank Group 10 0.69±0.11 Model Group 10 <![CDATA[0.95±0.20 *** ]]> Positive group 10 <![CDATA[0.77±0.10 # ]]> Tangcao Tablets (0.5g / kg) 10 0.87±0.10 Tangcao Tablets (1g / kg) 10 <![CDATA[0.78±0.14 # ]]>

[0141] ***P<0.001vs.Control, #P<0.05vs.Model.

[0142] Effect of Tangcao Tablets on the Content of α-Smooth Muscle Actin (α-SMA) in Colonic Tissue of Mice with Chronic Ulcerative Colitis: α-SMA exists in smooth muscle cells and myofibroblasts, and its upregulation is an important indicator of cell fibrosis. The expression level of α-SMA in colonic tissue of each group of mice was detected by Western Blot. The results showed that the expression level of α-SMA in colonic tissue of mice with chronic ulcerative colitis induced by DSS was significantly increased, and the expression level of α-SMA in colonic tissue was significantly decreased after treatment with Tangcao Tablets, indicating that Tangcao Tablets can reduce the expression of α-SMA in colonic tissue of mice with chronic enteritis and improve tissue fibrosis. See for details. Fig.12 .

[0143] 3.7 Effect of Tangcao Tablets on Occludin Content in Colonic Tissue of Mice with Chronic Ulcerative Colitis

[0144] The expression of Occludin protein in the colon tissue of mice in the normal group was higher, while the expression of tight junction protein Occludin in the colon tissue of mice in the model group was significantly lower than that in the normal group. After treatment with Tangcao tablets, the content of Occludin protein in the colon tissue was significantly higher than that in the model group, indicating that Tangcao tablets can significantly increase the expression of Occludin, a tight junction protein between intestinal epithelial cells in mice with chronic enteritis, and protect the intestinal epithelial barrier function. Fig.13 .

[0145] 3.8 Effect of Tangcao Tablets on Th1 / Th2 Balance in Colonic Tissue of Mice with Chronic Ulcerative Colitis

[0146] Compared with the control group, the IFN-γ in the colon tissue of the model group mice was significantly decreased, and the IL-4 level was increased. Tangcao tablets increased the expression level of IFN-γ in the colon tissue of UC mice and reduced the IL-4 level. The expression of T-bet and Gata3 in the model group mice decreased, but the decrease in T-bet was the most significant. Therefore, the Gata3 / T-bet ratio was significantly increased compared with the blank group, showing that the balance was biased towards Th2. The high-dose group of Tangcao tablets (1g / kg) can significantly reduce the Gata3 / T-bet ratio by regulating cytokines and regulate the imbalance of Th1 / Th2 in the colon tissue of mice with chronic ulcerative colitis. See details. Fig.14 .

[0147] 3.9 Effect of Tangcao Tablets on the Expression Levels of Th17 / Treg-specific Transcription Factors and Related Cytokines in Colonic Tissue of Mice with Chronic Ulcerative Colitis

[0148] Compared with the control group, the expression of RoRγt protein in the colon tissue of mice in the model group was increased, the expression of Foxp3 was decreased, the RoRγt / Foxp3 was increased, and the balance was biased towards Th17. The expression level of RoRγt in the mice in the Tangcao tablets group was decreased, the expression level of Foxp3 was increased, and the ratio of RoRγt to Foxp3 was significantly reduced. Compared with the blank control group, the levels of IL-6, IL-1β, TNF-α, IL-17 and other cytokines secreted by Th17 cells of mice in the model group were significantly increased, and the expression of TGF-β, IL-10 and other cytokines secreted by Treg was decreased. Tangcao tablets can significantly reduce the expression level of IL-17 and other cytokines, increase TGF-β, etc., and correct the balance of Th17 / Treg. See for details. Fig.15 .

[0149] Example 3: The preventive and therapeutic effects of Tangcao tablets on AOM / DSS-induced colitis-related colon cancer

[0150] 1 Main experimental materials

[0151] Azoxymethane, Sigma, USA; Enteric-coated aspirin tablets, Bayer HealthCare Manufacturing. Other main experimental materials are the same as those in Example 1.

[0152] 2 Experimental methods

[0153] 2.1 Drug preparation: AOM preparation method: Accurately weigh AOM, add appropriate amount of physiological saline, prepare AOM into a liquid with a concentration of 10mg / kg, store in a dark place, and prepare and use immediately. Positive drug aspirin preparation method: Grind enteric-coated aspirin tablets into powder, weigh an appropriate amount of powdered aspirin on a balance and dissolve it in 0.5% CMC-Na solution to prepare a 3mg / ml solution, and gavage each mouse at 0.1ml / 10g body weight. The metering setting and preparation method of Tangcao tablets, and the preparation method of 2% DSS solution are the same as in Example 2.

[0154] 2.2 Establishment of AOM / DSS-induced CAC mouse model and drug intervention: After one week of adaptive feeding, 95 male Balb / c mice were randomly divided into five groups, namely blank control group, model group, positive drug aspirin group, low-dose group of Tangcao tablets (0.5g / kg), and high-dose group of Tangcao tablets (1g / kg). Among them, there were 11 mice in the blank control group, 24 mice in the model group, and 20 mice in the other groups. The normal control group was intraperitoneally injected with normal saline, and the other groups were intraperitoneally injected with 10mg / kg AOM. After one week, mice with obvious weight loss were eliminated, and the remaining mice drank 2% DSS solution for 7 days and ordinary drinking water for 14 days. The above 21 days were regarded as a cycle, and repeated three cycles to establish a mouse colitis-related colon cancer model. The first day of DSS solution placement was counted as d1. After the end of the third cycle, the mice were fed with ordinary drinking water and feed for 7 days, that is, d71, and the mice were killed. The whole modeling process lasted 77 days. The drug intervention group was given oral administration once a day when the mice began to drink 2% DSS solution in the first cycle, i.e., on d1. The mice in the model group were given an equal amount of sodium carboxymethylcellulose solution.

[0155] 2.3 Changes in mouse body weight and disease activity index score: Modeling and drug administration were performed according to 2.2, and the disease activity index was scored using the same method as in Example 1.

[0156] 2.4 Sample collection: Modeling and drug administration were performed according to 2.2. After modeling, samples were collected according to the specimen collection method in Example 1, and the colon length and organ coefficient were calculated.

[0157] 2.5 Counting the occurrence of colon tumors: The colon tissue retained in 2.4 was cut open longitudinally and the feces in the intestinal cavity was removed. The colon tissue was placed on a ruler and the occurrence of tumors was counted under a dissecting microscope. The tumor incidence, number of tumors, tumor diameter, tumor load, maximum tumor diameter, etc. were counted, and the number of tumors with diameters of <1 mm, 1-2 mm, 2-3 mm, and >4 mm were recorded respectively.

[0158] 2.6 HE staining to observe pathological changes in colon tissue: The colon tissue retained in 2.4 was subjected to HE staining according to the method in Example 1 to observe pathological changes in colon tissue.

[0159] 2.7 Investigation of hematological parameters of CAC mice: The blood samples retained in 2.4 were subjected to routine blood testing according to the method in Example 1.

[0160] 2.9 Western blot detection of Th1 / Th2, Th17 / Treg related proteins in non-tumor areas of colon tissue: The colon tissue retained in 2.4 was detected according to the protein immunoblotting analysis method in Example 1 to detect Th1 / Th2, Th17 / Treg related specific transcription factors and related cytokines in colon tissue.

[0161] 2.10 Statistical analysis: The statistical method is the same as that in Example 1.

[0162] 3 Experimental results

[0163] 3.1 Effects of Tangcao Tablets on the General Condition of AOM / DSS-Induced CAC Mice

[0164] During the experiment, the mice in the blank group had smooth fur, good mental state, agile movements, weight gain, and no diarrhea or blood in the stool. During the first cycle of drinking 2% DSS, the model group had dull fur, reduced activity and food intake, weight loss, and severe diarrhea and blood in the stool. Tangcao tablets improved the above symptoms to varying degrees. At the end of the experiment, on d71, most mice in the model group had anal prolapse, while only a few animals in the Tangcao tablets group had this phenomenon.

[0165] 3.2 Effect of Tangcao Tablets on Body Weight of AOM / DSS-Induced CAC Mice

[0166] After the administration of 2% DSS in the first cycle, the model group had a significant weight loss, while the weight of the other groups decreased slowly relative to the model group. When the DSS solution was discontinued on d7 and replaced with normal drinking water, the weight of each group began to recover. The model group mice recovered the slowest, while the drug-treated group recovered faster and was higher than the model group. Long-term observations have found that the weight of the model group is always at the lowest level, indicating that AOM / DSS will significantly affect the weight changes of animals, and drug intervention can improve this situation. See for details. Fig.16 .

[0167] 3.3 Effect of Tangcao Tablets on DAI Levels in AOM / DSS-Induced CAC Mice

[0168] After drinking 2% DSS solution in the first cycle, the model group developed severe diarrhea and bloody stool symptoms on d5. With the use of DSS solution for multiple cycles, the disease activity index of the mice in the model group continued to increase compared with the blank group, and the diarrhea and bloody stool symptoms worsened. After the intervention treatment with Tangcao tablets, the disease activity index of the mice increased slowly compared with the model group, and the diarrhea and bloody stool symptoms were milder than those in the model group, indicating that Tangcao tablets can improve the clinical manifestations of the disease in AOM / DSS colon cancer mice. See details. Fig.17 .

[0169] 3.4 Effect of Tangcao Tablets on Blood Routine of AOM / DSS-induced CAC Mice

[0170] We performed hematological analysis on mice. As shown in the figure, compared with the blank group, the RBC (P<0.01) and HGB (P<0.001) of the model group mice were extremely low, indicating that the mice had anemia symptoms. The intervention of Tangcao tablets significantly increased the counts of red blood cells (P<0.05) and hemoglobin (P<0.05), and significantly improved the anemia symptoms of mice.

[0171] Table 11 Effects of Tangcao tablets on hematological parameters of mice with colitis-associated colon cancer

[0172]

[0173] *P<0.05,**P<0.01,***P<0.001vs.Control,#P<0.05,##P<0.01,###P<0.001vs.Model.

[0174] 3.5 Effect of Tangcao Tablets on Colon Length and Colon Mass Coefficient in AOM / DSS Colon Cancer Mice

[0175] As shown in the table, the colon length of mice in the model group was significantly shorter than that in the blank group, while the colon length of mice increased significantly after treatment with Tangcao tablets, and the difference was statistically significant. This shows that Tangcao tablets can significantly inhibit the shortening of colon length in CAC mice induced by AOM / DSS. In addition, the colon mass per unit length of mice in the model group increased significantly due to excessive adenoma load, and the colon mass in the Tangcao tablet group was lighter than that in the model group. The above results show that the intervention treatment of Tangcao tablets improves the shortening of colon length and the increase of colon mass per unit length in CAC mice induced by AOM / DSS.

[0176] Table 12 Effects of Tangcao tablets on colon length and mass coefficient of AOM / DSS-induced CAC mice

[0177]

[0178] **P<0.01vs.Control,###P<0.001vs.Model.

[0179] 3.6 Effects of Tangcao Tablets on Organ Coefficients of AOM / DSS-Induced CAC Mice

[0180] The spleen coefficient showed that the AOM / DSS model group was significantly larger than the blank group, and the Tangcao tablet group could significantly improve the spleen enlargement of mice caused by AOM / DSS. The thymus coefficient showed that the model group mice had thymus atrophy, and Tangcao tablets had a tendency to improve thymus atrophy, but the results did not constitute a statistical difference. This suggests that Tangcao tablets play a role by protecting the immune organs of AOM / DSS-induced CAC model mice.

[0181] Table 13 Effects of Tangcao Tablets on the organ coefficients of immune organs in AOM / DSS-induced CAC mice

[0182] Group N Thymus index (mg / g) Spleen index (mg / g) Blank Group 11 0.80±0.19 4.15±0.66 Model Group 21 0.59±0.30 <![CDATA[6.73±3.14 ** ]]> Positive group 16 0.71±0.26 5.10±1.53 Tangcao tablets (0.5g / kg) 18 0.62±0.30 <![CDATA[4.13±0.94 ### ]]> Tangcao tablets (1g / kg) 20 0.68±0.21 <![CDATA[4.15±0.78 ### ]]>

[0183] **P<0.01vs.Control,###P<0.001vs.Model.

[0184] 3.7 Effects of Tangcao Tablets on the Number and Size of Tumors in AOM / DSS-Induced CAC Mice

[0185] Observation and recording of colon sections revealed that only one animal in the model group did not form a tumor, with a tumor formation rate of 91.67%, while eight animals in the high-dose group of Tangcao tablets (1g / kg) did not form a tumor, with a tumor formation rate of 60%. This suggests that Tangcao tablets reduce the incidence of tumors after AOM / DSS modeling. In order to better evaluate the status of tumors in different groups, we examined the following indicators: tumor load (sum of diameters), number of tumors, tumor size (average diameter), maximum diameter of tumors, proportion of tumors with a diameter greater than 4mm, and distribution of tumors of different sizes. According to the results, the average number of tumors in the AOM / DSS model group was 7.04±5.10, the average tumor diameter was 2.45mm, the average tumor load was about 16mm, the maximum tumor diameter was about 3mm, and tumors greater than 4mm accounted for 16%. In the high-dose group of Tangcao tablets (1g / kg), the number of tumors was about 2.75±3.21, the average tumor diameter was 1.26mm, the average tumor load was 5.95mm, and the number of tumors greater than 4mm accounted for 6%. This suggests that Tangcao tablets can alleviate the severity of tumors in AOM / DSS-induced CAC mice to a certain extent. Although the average values ​​of various indicators are lower than those of the model group, due to the large differences within each group, some indicators have no statistical differences. The above results suggest that although Tangcao tablets intervention cannot reduce the proportion of tumors larger than 4mm in AOM / DSS-induced CAC mice, it can significantly reduce indicators such as the number, load, and size of tumors.

[0186] Table 14 Effect of Tangcao tablets on colon tumors in CAC mice induced by AOM / DSS

[0187]

[0188] ***P<0.001vs.Control,#P<0.05,##P<0.01vs.Model.

[0189] 3.8 Effect of Tangcao Tablets on Colonic Histopathology in AOM / DSS-Induced CAC Mice

[0190] like Fig.18As shown in the figure, the size and shape of the glands in the colon tissue of the blank group mice were normal, neatly arranged, and the crypt structure, goblet cells and epithelial mucus layer were clearly visible. The glandular duct structure of the colon tissue of the AOM / DSS model group mice was back-to-back and sieve-like, and adenoma-like; the nuclei were large and darkly stained, and some showed nuclear polarity disappearance and multinucleation, involving the mucosal layer and submucosal layer; goblet cells basically disappeared, and inflammatory cell infiltration was severe. The colon structure of the positive drug group was slightly improved compared with the AOM / DSS model group, but the glandular hyperplasia was still severe; the inflammatory cell infiltration of the colon tissue of the Tangcao tablet group mice was less, the nuclear polarity changed slightly, and the crypt structure and epithelial mucus layer partially disappeared. This suggests that Tangcao tablets can protect the integrity of the colon mucosa and reduce the occurrence of tissue atypia.

[0191] 3.9 Effect of Tangcao Tablets on Occludin Expression in Colonic Tissue of AOM / DSS-Induced CAC Mice

[0192] The expression of Occludin protein in the non-tumor area of ​​the colon tissue of mice in the AOM / DSS model group decreased significantly (P<0.001), and the expression of Occludin in the colon tissue of the Tangcao tablet group increased, among which the high-dose Tangcao tablet group (1g / kg) increased most significantly, and the difference was significant (P<0.001). This suggests that Tangcao tablets can increase the expression of tight junction proteins in the intestinal tissue of the non-tumor area. Fig.19 .

[0193] 3.10 Effect of Tangcao Tablets on Th1 / Th2 Balance in Non-tumor Colon Tissue of CAC Mice

[0194] Western Blot was used to detect the expression levels of Th1 / Th2 specific functional proteins and cytokines in the colon tissue of non-tumor areas of CAC mice induced by AOM / DSS. As shown in the figure, the expression of Th1 specific functional protein T-bet in the model group mice was significantly decreased, the expression of Th2 transcription factor Gata3 was increased, the Gata3 / T-bet ratio was increased, and the balance was biased towards Th2. Compared with the model group, the Tangcao tablets group had increased levels of T-bet and IFN-γ, decreased levels of Gata3 and IL-4, and decreased Gata3 / T-bet ratio. This suggests that Tangcao tablets regulate the balance of Th1 / Th2 in the colon tissue of non-tumor areas to prevent and treat colitis-related colon cancer. For details, see Fig. 20 .

[0195] 3.11 Effect of Tangcao Tablets on Th17 / Treg Balance in Colonic Tissue of CAC Mice

[0196] Western Blot was used to detect the expression levels of Th17 / Treg-specific functional proteins and cytokines in the colon tissue of non-tumor areas of CAC mice induced by AOM / DSS. The results are shown in the figure. Compared with the blank control group, the expression levels of RoRγt and Foxp3 in the model group mice increased, but the increase in RoRγt was more significant, and the balance was biased towards Th17. Tangcao tablets reduced the ratio of RoRγt and Foxp3 by reducing cytokines such as IL-6, TNF-α, and IL-1β, indicating that Tangcao tablets can regulate the levels of Th17 and Treg cytokines in colon tissues to affect the balance of Th17 / Treg and play a preventive and therapeutic role in CAC mice. See details. Fig.21 .

Claims

1. Use of a Chinese medicine compound composition in the preparation of a drug for treating ulcerative colitis, intestinal fibrosis caused by colitis, and colon cancer caused by colitis, the Chinese medicine compound composition comprising the following components in parts by weight: 120-150 parts of geranium, 120-150 parts of astragalus, 120-200 parts of nightshade, 120-200 parts of honeysuckle, 60-100 parts of kapok, 40-60 parts of terminalia chebula, 80-150 parts of oldenlandia diffusa, 40-60 parts of pomegranate peel, 150-200 parts of glutinous rice roots, 80-150 parts of water chestnuts, 40-60 parts of trichosanthes peel, 60-100 parts of bupleurum, 40-60 parts of elsholtzia, 60-80 parts of liquorice, 120-150 parts of millettia, 40-60 parts of safflower, 40-60 parts of ginkgo leaves, 60-120 parts of purslane, 40-60 parts of picrorhizome, and 28-40 parts of scorpion.

2. The use according to claim 1, characterized in that The Chinese medicine compound composition is prepared into conventional oral dosage forms including tablets, capsules, powders and granules.

3. The use according to claim 1, characterized in that The Chinese medicinal compound composition is made into film-coated tablets.

4. The use according to claim 1, characterized in that The dosage of the Chinese medicine compound composition is adjusted based on the severity of the disease and whether or not there is a previous medical history.

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