Establishment method and application of damp-heat intestine accumulation type ulcerative colitis animal model

Abstract

The invention discloses an establishment method and application of a damp-heat intestine accumulation type ulcerative colitis animal model, and belongs to the field of animal models.The establishment method comprises the steps that S1, high-fat feed treatment is conducted, specifically, experimental animals freely eat high-fat feed for 6-10 weeks from the first day; s2, DSS treatment: feeding 1.5% (w / v) DSS solution to the experimental animal for 2 weeks; and S3, observing the damp-heat syndrome type and ulcerative colitis related indications of the experimental animals, and selecting the experimental animals meeting the scoring standard, thereby establishing the ulcerative colitis mouse model. Aiming at the situation that most patients induce ulcerative colitis due to excess energy due to the change of dietary structures and lifestyles at present, the invention constructs and evaluates the damp-heat accumulation type ulcerative colitis mouse model by inducing internal dampness by a direct pathogenesis (high fat diet), which is closer to the actual situation of clinical patients. Therefore, a model and reference are provided for research and treatment of pathology of the damp-heat accumulation type ulcerative colitis and pharmacological mechanisms of traditional Chinese medicines.

Description

Technical Field

[0001] The present invention relates to a method for establishing an animal model and its application, and in particular to a method for establishing an animal model of ulcerative colitis of the damp-heat accumulation in the intestine type and its application. Background Art

[0002] Research on animal models of ulcerative colitis (UC) primarily focuses on simulating the pathological characteristics and mechanisms of the human disease. These models are generally divided into genetically engineered models and inducible models. Genetically engineered models study the immunopathological processes of UC by knocking out or knocking in specific genes, such as IL-10 knockout mice. Inducible models induce disease through chemical substances or antigen stimulation, such as chemical methods, immunological methods, and combined methods. Chemical methods include induction with chemical substances such as acetic acid, trinitrobenzenesulfonic acid (TNBS), oxazolidinone, and dextran sodium sulfate (DSS). The DSS-induced model is of great value in studying disease pathogenesis and drug screening because of its similar clinical and histological manifestations to human ulcerative colitis and its simple and easy-to-replicate operation.

[0003] Currently, most animal models of ulcerative colitis use DSS alone to induce UC symptoms in mice and rats, ignoring the Traditional Chinese Medicine (TCM) principle of syndrome differentiation and treatment based on individual disease symptoms. This has hindered in-depth research into the efficacy and mechanisms of Chinese medicine, particularly prescriptions, tailored to specific syndromes. Some studies have used multiple factors, including a high-sugar, high-fat diet, a high-temperature, high-humidity environment, and baijiu (white liquor), to establish animal models of large intestine damp-heat ulcerative colitis. However, these studies overlook the fact that people are not exposed to such complex, multi-factorial environments of dampness and heat in their daily lives. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for establishing and applying an animal model of ulcerative colitis of the damp-heat accumulation type in the intestine, so as to solve the problem that most current UC animal models use dextran sulfate sodium (DSS) as a single factor to induce UC symptoms in mice and rats, while ignoring the need for syndrome differentiation and treatment based on different disease symptoms.

[0005] To achieve the above objectives, one aspect of the present invention provides a method for establishing an animal model of ulcerative colitis of the damp-heat accumulation type in the intestine, comprising the steps of:

[0006] S1, high-fat diet treatment: experimental animals were fed a high-fat diet ad libitum for 6–10 weeks starting from day 1;

[0007] S2, DSS treatment: experimental animals were fed with 1.5% (w / v) DSS solution for 2 weeks;

[0008] S3, observe the damp-heat syndrome and ulcerative colitis-related signs of experimental animals, select experimental animals that meet the scoring criteria, and thus establish an ulcerative colitis mouse model.

[0009] Furthermore, in step S1, the composition of the high-fat feed is: 60% fat calories, 20% protein calories, 20% carbohydrate calories, 5.24kcal﹒ g -1 .

[0010] Furthermore, in step S2, during the above-mentioned animal model construction process, the experimental animals used are mice.

[0011] Furthermore, in step S3, the method for selecting animals that meet the scoring criteria includes: verifying the damp-heat accumulation in the intestine type ulcerative colitis animal model through the damp-heat accumulation in the intestine syndrome symptom score and the ulcerative colitis disease activity index (DAI); verifying the damp-heat accumulation in the intestine type ulcerative colitis animal model through the "clearing heat and drying dampness" prescription Gegenqinlian Decoction "using prescription to test syndrome".

[0012] Furthermore, the related indicators of damp-heat accumulation in the intestines include: mental state, body shape, hair coat, perianal condition and bowel movements.

[0013] Furthermore, the ulcerative colitis disease activity index (DAI)-related indicators include: the percentage of weight loss of mice, stool texture, and blood in stool.

[0014] Another aspect of the present invention provides an application of an animal model of ulcerative colitis of the damp-heat accumulation in the intestine type, comprising applying the animal model obtained by the above method to the treatment of chronic nonspecific inflammatory bowel disease.

[0015] In summary, the present invention has the following beneficial effects compared to the prior art:

[0016] (1) The present invention successfully replicated the mouse model of ulcerative colitis of damp-heat accumulation in the intestine by only using a long-term high-fat diet combined with DSS. The method is simple and can reflect the pathological characteristics of ulcerative colitis of damp-heat accumulation in the intestine in clinical and real-life situations, in which improper diet induces internal dampness, which turns into heat over time and then spreads to the intestine.

[0017] (2) Diversity of time windows for drug intervention

[0018] Therapeutic Window: Medication (especially traditional Chinese medicine) treatment should focus on the 14-day acute phase of DSS-induced disease. During this period, mice develop the foundation of damp-heat accumulation in the intestines and the acute lesions of ulcerative colitis, effectively simulating the pathological state of the clinical acute phase.

[0019] Prevention window: Feasibility of drug preventive intervention: Starting from the high-fat induction period, a group design can be used to observe the improvement effects of drugs, especially prescriptions, on damp-heat syndrome and colitis symptoms.

[0020] (3) The present invention aims to address the current changes in dietary structure and lifestyle. Most patients develop ulcerative colitis due to excess energy. Therefore, the direct cause (high-fat diet) is used to induce internal dampness to construct and evaluate a mouse model of damp-heat lumbar ulcerative colitis, which is closer to the actual situation of clinical patients, thereby providing a model and reference for the study and treatment of the pathology and pharmacological mechanism of traditional Chinese medicine for damp-heat lumbar ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 This is the animal experimental grouping situation of Experiment 1;

[0023] Figure 2 This is the establishment of the damp-heat accumulation in the intestines syndrome model in Experiment 1;

[0024] Figure 3 This is the establishment of the ulcerative colitis model in Experiment 1;

[0025] Figure 4 The spleen mass, spleen index, and colon length of mice in each group in Experiment 1;

[0026] Figure 5 HE staining of the colon tissues of mice in each group in Experiment 1;

[0027] Figure 6 This is the HE histological score of Experiment 1;

[0028] Figure 7 This is the status of pro-inflammatory and anti-inflammatory factors in the colon tissue of mice in Experiment 1;

[0029] Figure 8 This is the grouping of animals in Experiment 2;

[0030] Figure 9 This is the general condition of mice in each group in Experiment 2;

[0031] Figure 10 The spleen weight, spleen index, and colon length of mice in each group in Experiment 2;

[0032] Figure 11 This is the HE staining of mouse colon tissue in Experiment 2;

[0033] Figure 12 This is the HE histological score in Experiment 2;

[0034] Figure 13 This is the expression of pro-inflammatory and anti-inflammatory factors in the colon tissue of mice in Experiment 2. DETAILED DESCRIPTION

[0035] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form can also include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0037] Unless otherwise specifically stated, the relative arrangement of the parts and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention. At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to actual proportional relationships. The techniques, methods and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the techniques, methods and equipment should be considered as part of the authorized specification. In all examples shown and discussed herein, any specific values should be interpreted as being merely exemplary and not as limitations. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following figures, and therefore, once an item is defined in one figure, it does not need to be further discussed in subsequent figures.

[0038] See also Figures 1 to 13 As shown, one aspect of the present invention provides a method for establishing an animal model of ulcerative colitis of damp-heat accumulation in the intestine, comprising the steps of:

[0039] S1, high-fat diet treatment: experimental animals were fed a high-fat diet ad libitum for 6–10 weeks starting from day 1;

[0040] In other words, the time design of this model has a certain degree of flexibility, and researchers can adjust the ratio of high-fat diet and DSS induction according to specific experimental needs. For example, shortening the high-fat diet period (e.g., 6-8 weeks) while maintaining the 14-day (2-week) acute phase of DSS induction can better meet the needs of drug intervention studies.

[0041] S2, DSS treatment: experimental animals were fed with 1.5% (w / v) DSS solution for 2 weeks;

[0042] S3, observe the damp-heat syndrome and ulcerative colitis-related signs of experimental animals, select experimental animals that meet the scoring criteria, and thus establish an ulcerative colitis mouse model.

[0043] As a preferred method, the selection of animals that meet the scoring criteria includes: verifying the animal model of damp-heat accumulation in the intestine type ulcerative colitis through the damp-heat accumulation in the intestine syndrome symptom score and the ulcerative colitis disease activity index (DAI); verifying the animal model of damp-heat accumulation in the intestine type ulcerative colitis through the "clearing heat and drying dampness" prescription Gegenqinlian Decoction "using prescription to test syndrome".

[0044] As a preferred embodiment, the composition of high-fat feed is: 60% fat calories, 20% protein calories, 20% carbohydrate calories, 5.24kcal﹒ g -1 .

[0045] As a preference, in the process of constructing the above-mentioned animal model, the experimental animals used are usually mice.

[0046] As a preferred method, the related indicators of damp-heat intestinal syndrome include: mental state, body shape, hair coat, perianal condition, bowel movements, etc.

[0047] As a preferred embodiment, the ulcerative colitis disease activity index (DAI)-related indicators include: percentage of mouse weight loss, stool texture, and blood in stool.

[0048] In order to verify the reliability of the method for establishing an animal model of ulcerative colitis of the damp-heat accumulation type provided by the present invention, the present invention conducted the following related experiments:

[0049] 1. Controlled Experiment on Mouse Model of Damp-Heat Intestinal Ulcerative Colitis (Experiment 1)

[0050] (1) Mice were selected and adaptively cultured for 3 days, then randomly divided into a normal group (Control) and a damp-heat intestinal syndrome group (HFD);

[0051] (2) The damp-heat intestinal accumulating syndrome group was fed a high-fat diet, while the normal group was fed a control diet for 10 weeks. This single diet was used to induce a damp-heat intestinal accumulating syndrome mouse model. During the model establishment period, the mice were observed for general indicators of damp-heat intestinal accumulating syndrome, such as hair condition, curling, anal cleanliness, and bowel movements. The mice's weight and length were recorded weekly, and the obesity index (Lee's index) was calculated.

[0052] That is to say, in S2, the mice in the damp-heat intestinal syndrome group were fed with high-fat feed for 10 weeks, which caused the mice in this group to have an irregular diet and eventually develop damp-heat intestinal syndrome. In the present invention, the mice that successfully develop damp-heat intestinal syndrome are referred to as.

[0053] High-fat diet (60% fat calories, 20% protein calories, 20% carbohydrate calories, 5.24 kcal﹒g-1);

[0054] Control diet (10% fat calories, 20% protein calories, 70% carbohydrate calories, 3.85 kcal﹒g-1)

[0055] (3) The mice in the normal group were randomly divided into the normal group (Control) and the ulcerative colitis group (UC); the mice in which the damp-heat syndrome model was successfully established were randomly divided into the damp-heat accumulation in the intestine group (HFD) and the damp-heat accumulation in the intestine type ulcerative colitis group (HUC).

[0056] The mice in the control group and the HFD group were fed with sterile water for 14 days; the mice in the UC group and the HUC group were fed with 1.5% (w / v) DSS solution for 14 days to establish the ulcerative colitis mouse model. Figure 1 shown.

[0057] (4) Macroscopic index evaluation: The damp-heat intestinal syndrome and ulcerative colitis disease activity index (DAI) of mice were scored, and the fluid volume (fat content) in the mice was detected using Minispec LF90 II body composition analyzer every week.

[0058] Symptom scoring for damp-heat intestinal syndrome: The relevant symptoms of damp-heat syndrome in humans and animals were converted, and the general conditions of each group of mice were observed and recorded daily, including mental state, body shape, hair coat, perianal condition, and bowel movements (Table 1). The average of the six scores was the damp-heat intestinal syndrome score, and the symptom scores of the mice were calculated weekly.

[0059] Table 1 Syndrome scoring criteria for the mouse model of damp-heat accumulation in the intestines syndrome

[0060]

[0061] Ulcerative colitis disease activity index (DAI) score: The scoring criteria are shown in Table 2. The model mice are comprehensively evaluated based on the severity of three aspects: percentage of mouse weight loss, stool texture, and blood in stool. The average of the three scores is the DAI score.

[0062] Table 2 Disease Activity Index Scoring Criteria

[0063]

[0064] (5) After the experiment, the mice were injected with sodium pentobarbital (10 mL·kg -1) were anesthetized, the eyeballs were removed and blood was collected. After standing for 4 hours, the blood was centrifuged at 500×g for 10 minutes to collect the serum. The colon was separated, and the colon tissue about 1.5 cm away from the anus and 1 cm away was selected and fixed in 4% paraformaldehyde solution for at least 48 hours. The spleen was separated, washed, and the excess water was absorbed before weighing its mass.

[0065] (6) Comparison of colon length: After separating the colon, place it on a blank paper and measure the colon length with the lower end of the ileocecal region as the starting point using a ruler and take a photo to record it.

[0066] (7) Comparison of spleen index: After isolating the spleen, clean the blood stains with PBS, dry the water with filter paper, and weigh the spleen mass (mg) using an electronic balance. The spleen index was calculated using the following formula: spleen index (%) = spleen mass (mg) / mouse body weight (g).

[0067] (8) Colon tissue preparation and staining: dehydrate, wax-immerse, embed, slice, dewax, stain, dehydrate, and seal.

[0068] (9) Tissue damage score: The colon tissue structure was observed and photographed under an optical microscope to evaluate the histopathological damage score. The scoring criteria are shown in Table 3.

[0069] Table 3 Scoring criteria for colon tissue injury

[0070]

[0071] (10) Serum biochemical index detection: The mouse serum was taken out from the -80°C refrigerator, dissolved in a 4°C refrigerator, and allowed to stand at room temperature for 30 min. The TG, TC, HDL-C, and LDL-C in the serum were measured using an automatic biochemical analyzer according to the kit instructions.

[0072] (11) Method for detecting the expression of inflammatory factors in mouse colon tissue. Preparation of mouse colon tissue ELISA samples: weigh 30 mg of colon tissue and put it into a sterile EP tube. Add 3 steel balls and 300 μL of RIPA high-efficiency lysis buffer containing 1 mM PMSF to each tube. Use a high-throughput tissue homogenizer at 1000 rpm min. -1 After grinding for 4 minutes, place it on ice for 3 minutes, grind it again for 4 minutes, grind the colon tissue into a homogenate, place it in a 4°C refrigerator and let it stand for 30 minutes, use a refrigerated centrifuge to centrifuge at 4°C and 12,000×g for 10 minutes, aspirate the supernatant, and detect according to the specific steps of the ELISA kit.

[0073] 2. Results

[0074] (1) Establishment of a mouse model of damp-heat accumulation in the intestines

[0075] During the establishment of the damp-heat intestinal syndrome model, the mice in the Control group had smooth fur, good mental state, and oval feces; while the mice in the HFD group were obese, had greasy fur, curled up, had soft or loose stools, and had moist perianal area.

[0076] Compared with the Control group, the body weight and Lee's index of mice in the HFD group increased significantly (P < 0.01), the rectal temperature and damp-heat syndrome symptom score increased significantly at week 5 (P < 0.05), and the fluid volume increased significantly. At week 10, compared with the Control group, the body weight, Lee's index, rectal temperature, damp-heat syndrome symptom score and serum biochemical indicators of the HFD group increased significantly (P < 0.01, P < 0.001). Figure 2 (Compared with the Control group, * P<0.05, ** P<0.01, *** P<0.001), indicating that the mouse model of damp-heat intestinal syndrome was successfully established.

[0077] (2) Establishment of mouse ulcerative colitis and damp-heat intestinal ulcerative colitis models

[0078] The weight and DAI scores of mice showed that during the establishment of the ulcerative colitis mouse model, compared with the control group, the weight of mice in the UC and HUC groups decreased significantly (P < 0.01), and the DAI scores increased significantly. Both groups showed symptoms of ulcerative colitis model, such as emaciation, diarrhea, mucus, pus and blood in stool, hunched back, and hair loss. Compared with the UC group, the weight of mice in the HUC group decreased significantly (P < 0.05), and the DAI scores increased (P < 0.05). The symptoms related to ulcerative colitis appeared earlier and were more severe. Figure 3 (Compared with the Control group, ** P<0.01, *** P<0.001; compared with UC group, # P<0.05), indicating that the mouse models of ulcerative colitis and damp-heat accumulation in the intestine type ulcerative colitis were successfully established.

[0079] (3) Spleen mass, spleen index, and colon length of mice

[0080] Compared with the Control group, the spleen weight and spleen index of the mice in the UC and HUC groups were significantly increased (P < 0.001), and the colon length was significantly shortened (P < 0.001). The colon showed obvious swelling, bleeding and ulcers. Compared with the UC group, the spleen weight and spleen index of the mice in the HUC group were increased (P < 0.05, P < 0.01), and the colon length was shortened (P < 0.05). Figure 4 (Compared with the Control group, *** P<0.001; compared with UC group,# P<0.05, ## P < 0.01), indicating that damp-heat syndrome exacerbated spleen inflammation in UC mice and led to colon shortening, bleeding, ulceration, and erosion due to intestinal inflammation.

[0081] (5) Pathological changes in mouse colon tissue

[0082] In order to observe the pathological changes of the lesion sites in each group of mice, the present invention performed HE staining on the colon of each group of mice. The results are shown in Figure 5 (Scale bar length is 100 μm) and Figure 6 (Compared with the Control group, * P<0.05, *** P<0.001; compared with UC group, ## The colons of mice in the control and HFD groups were structurally intact, with normal, neatly arranged crypts. Compared with the control group, the HFD group showed localized inflammatory cell infiltration and an increased histological score (P < 0.05). The UC and HUC groups showed significant signs of colonic inflammation, including structural destruction, irregular crypt surfaces, upward displacement of crypts, and inflammatory cell infiltration, with increased histological scores (P < 0.001). Compared with the UC group, the HUC group showed more pronounced inflammatory manifestations of colonic tissue, with larger and deeper ulcers and inflammatory infiltration, a large number of plasma cells aggregated in the basal layer, and a significantly increased histological score (P < 0.01). This suggests that damp-heat syndrome exacerbates the structural and pathological changes in the colonic tissue of UC model mice.

[0083] (6) Expression of pro-inflammatory and anti-inflammatory factors in the colon tissues of mice in each group

[0084] Compared with the Control group, the expression level of inflammatory factor TNF-α in the HFD group, UC group, and HUC group was significantly increased (P < 0.001); the expression levels of IL-1β and IL-17A in the HFD group were increased (P > 0.05), and the difference was not statistically significant. The expression levels of IL-1β and IL-17A in the UC group and HUC group were significantly increased (P < 0.001). Compared with the UC group, the expression levels of TNF-α, IL-1β, and IL-17A in the HUC group were increased (P < 0.05, P < 0.01). Compared with the Control group, the expression levels of anti-inflammatory factor IL-10 in the HFD group, UC group, and HUC group were decreased (P < 0.05, P < 0.001); compared with the UC group, the expression level of IL-10 in the HUC group was significantly decreased (P < 0.01). Figure 7 (Compared with the Control group, * P<0.05, ** P<0.01, *** P<0.001; compared with UC group,# P<0.05).

[0085] The expression levels of pro-inflammatory and anti-inflammatory factors in the colon tissues of the above groups of mice showed that damp-heat syndrome promoted the release of pro-inflammatory factors TNF-α, IL-1β and IL-17A in the colon tissues of UC mice, inhibited the release and expression of anti-inflammatory factor IL-10, and further aggravated the intestinal and systemic inflammatory response in HUC model mice.

[0086] Conclusion: A combined HUC disease and syndrome mouse model was constructed and evaluated using various research indicators. The results showed that this model exhibited characteristics of UC and distinct TCM syndromes of damp-heat intestinal tract infection, making it a reliable HUC mouse model.

[0087] 3. The "heat-clearing and dampness-drying" prescription Gegenqinlian Decoction was used to verify the animal model of ulcerative colitis with damp-heat accumulation in the intestines (Experiment 2).

[0088] The experimental animals and experimental procedures were the same as above. The preparation steps of Gegenqinlian decoction in this experiment were as follows:

[0089] The herbs required for Gegenqinlian decoction (Kuanggen: Huangqin: Huanglian: Zhigancao 8:3:3:2) were soaked in 10-fold water for 30 minutes, brought to a boil over high heat, and then simmered over low heat for 30 minutes. The liquid was poured out, and 8-fold water was added to the herbs and boiled again. The liquid was combined and filtered through gauze. The crude drug was concentrated to 1.5 g mL using a large rotary evaporator. -1 .

[0090] (1) Reproduction and administration of damp-heat ulcerative colitis model

[0091] The damp-heat intestinal syndrome animal model was established in C57BL / 6 mice using the above method. The mice with successful damp-heat syndrome model were randomly divided into 6 groups, namely damp-heat syndrome group (HFD), damp-heat intestinal ulcerative colitis group (HUC), Gegenqinlian decoction low-dose group (GQD-L) (0.75g﹒Kg-1), Gegenqinlian decoction medium-dose group (GQD-M) (1.5g﹒Kg -1 ), Gegenqinlian decoction high-dose group (GQD-H) (3g﹒Kg -1 ), mesalazine group (MESA) (310 mg﹒Kg -1 The dosage of mesalazine for adults is 1.5g﹒d. -1 According to the calculation conversion, the mouse dose is 12.3 times that of human. Except for the UC and HUC groups, the other treatment groups were given drugs by gavage. The experimental groups are shown in Figure 8 .

[0092] IV. Results

[0093] (1) Gegenqinlian decoction improves the general symptoms of mice with ulcerative colitis of the damp-heat type

[0094] Compared with the HFD group, the body weight and Lee's index of the mice in the HUC group were significantly reduced, and the DAI score and damp-heat syndrome score were significantly increased (P < 0.001). After drug treatment, the weight loss trend of the mice was slowed down. Compared with the HUC group, the body weight and Lee's index of the mice in the GQD-H group were significantly increased, and the DAI score and damp-heat syndrome score were significantly decreased (P < 0.05, P < 0.01, P < 0.001). Figure 9 .

[0095] Compared with the HFD group, the spleen weight and spleen index of the mice in the HUC group were significantly increased (P < 0.001), and the colon length was significantly shortened (P < 0.001). Compared with the HUC group, the spleen weight and spleen index of the mice after GQD treatment were significantly decreased (P < 0.01, P < 0.001), and the colon length was significantly increased (P < 0.05, P < 0.01). Figure 10 .

[0096] (2) Gegenqinlian decoction inhibits pathological damage in the colon of mice with ulcerative colitis of the damp-heat type

[0097] Compared with the HFD group, the HUC group showed significantly disordered colon structure, with essentially destroyed crypts and a large number of inflammatory cells infiltrating, with the infiltration reaching the basal layer. The histological score was significantly increased (P < 0.001). Compared with the HUC group, the GQD-M and MESA groups showed significantly improved colon conditions, with only the intestinal epithelium damaged, and the histological score was significantly reduced (P < 0.01, P < 0.001). The GQD-H group showed essentially intact colon structure, with neatly arranged crypts and a significantly reduced histological score (P < 0.001). Figure 11 (Scale bar length is 100 μm) and Figure 12 (Compared with the HFD group, *** P<0.001; compared with the HUC group, # P<0.05,##P<0.01, ### P<0.001).

[0098] (3) Gegenqinlian decoction alleviates colonic inflammation in mice with ulcerative colitis of the damp-heat type

[0099] Compared with the HFD group, the expression levels of proinflammatory factors TNF-α, IL-1β, and IL-17A in the HUC group were significantly increased (P < 0.001), and the expression level of anti-inflammatory factor IL-10 was significantly decreased (P < 0.001). Compared with the HUC group, the expression levels of proinflammatory factors TNF-α, IL-1β, and IL-17A in the GQD-L group, GQD-M group, GQD-H group, and MESA group were significantly decreased (P < 0.05, P < 0.01, P < 0.001), and the expression level of anti-inflammatory factor IL-10 was increased (P < 0.05, P < 0.001). Figure 13 (Compared with the HFD group, ***P<0.001; compared with the HUC group, #P<0.05, ##P<0.01, ###P<0.001).

[0100] Conclusion: Based on the efficacy of Gegenqinlian decoction in clearing away heat and drying dampness, the damp-heat pathological state of the HUC model was significantly improved through multiple indexes, verifying the syndrome type of damp-heat accumulation in the intestines in this model.

[0101] Another aspect of the present invention provides an application of an animal model of ulcerative colitis of the damp-heat accumulation in the intestine type, comprising applying the animal model obtained by the above method to the treatment of chronic nonspecific inflammatory bowel disease.

[0102] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for establishing an animal model of ulcerative colitis of the damp-heat accumulation type in the intestine, characterized in that: Including steps: S1, high-fat diet treatment: experimental animals were fed a high-fat diet ad libitum for 6–10 weeks starting from day 1; S2, DSS treatment: experimental animals were fed with 1.5% (w / v) DSS solution for 2 weeks; S3, observe the damp-heat syndrome and ulcerative colitis of experimental animals, select experimental animals that meet the scoring criteria, and then establish an ulcerative colitis mouse model.

2. The method for establishing an animal model of ulcerative colitis of damp-heat accumulation in the intestine according to claim 1, characterized in that: In step S1, the composition of high-fat feed is: 60% fat calories, 20% protein calories, 20% carbohydrate calories, 5.24kcal﹒ g -1 .

3. The method for establishing an animal model of ulcerative colitis of damp-heat accumulation in the intestine according to claim 1, characterized in that: In step S2, during the above-mentioned animal model construction process, the experimental animals used are mice.

4. The method for establishing an animal model of ulcerative colitis of damp-heat accumulation in the intestine according to claim 1, characterized in that: In step S3, the method for selecting animals that meet the scoring criteria includes: verifying the damp-heat accumulation in the intestine type ulcerative colitis animal model through the damp-heat accumulation in the intestine syndrome symptom score and the ulcerative colitis disease activity index (DAI); verifying the damp-heat accumulation in the intestine type ulcerative colitis animal model through the "clearing heat and drying dampness" prescription Gegenqinlian Decoction "using prescription to test syndrome".

5. The method for establishing an animal model of ulcerative colitis of damp-heat accumulation in the intestine according to claim 4, characterized in that: The related indicators of damp-heat accumulation in the intestines include: mental state, body shape, hair coat, perianal condition and bowel movements.

6. The method for establishing an animal model of ulcerative colitis of damp-heat accumulation in the intestine according to claim 4, characterized in that: Indicators related to the ulcerative colitis disease activity index (DAI) include: percentage of mouse body weight loss, stool texture, and blood in stool.

7. An application of an animal model of ulcerative colitis of damp-heat accumulation in the intestine, characterized in that: The method comprises applying the animal model obtained by the method for establishing an animal model of damp-heat accumulation in the intestine type ulcerative colitis according to any one of claims 1 to 6 to the treatment of chronic nonspecific inflammatory bowel disease.

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