A preparation method of a dampness syndrome animal model

Abstract

The application provides a preparation method of a dampness syndrome animal model. The method for establishing the dampness syndrome animal model comprises the following steps: injecting pathogenic microorganisms such as Candida albicans and Escherichia coli into the abdominal cavity to establish the dampness syndrome animal model. The typical symptoms of the dampness syndrome, i.e. sticky stool and limb weakness, can appear within 6-12 hours after the animal model is prepared by the above method, and the success rate of model preparation is 100%. The performance of the dampness syndrome animal model prepared by the above method is extremely similar to the performance of the dampness syndrome in clinic. The animal model is simple and effective, has a short preparation time, is highly reproducible, and can effectively represent the symptoms of the dampness syndrome. The dampness syndrome animal model prepared by the above method can be used for the research on the mechanism of the dampness syndrome and intervention treatment. The application creates conditions for further in-depth research on the mechanism of the related TCM syndromes and intervention treatment.

Description

Technical Field

[0001] This invention belongs to the field of animal model technology, specifically relating to a method for establishing a wet syndrome animal model. Background Technology

[0002] Dampness syndrome is a disease name in Traditional Chinese Medicine (TCM). TCM believes that dampness causes a hundred diseases. Because dampness is heavy, sticky, and easily obstructs the flow of Qi, damaging Yang Qi, its symptoms often linger and are not easy to resolve quickly. Its characteristic symptoms include headache, fullness and distension in the epigastrium and abdomen, heaviness and pain in the body, fatigue, a sticky tongue coating, and a slow or weak pulse. Dampness syndrome can be divided into internal dampness and external dampness. Internal dampness is mainly caused by consuming too much rich, sweet, and damp-producing food, exceeding the spleen's digestive capacity; external dampness mainly comes from exposure to dampness pathogens in the external environment. Internal and external dampness can influence each other, indicating that they share a common pathogenesis. According to modern research, both internal and external dampness are inseparable from pathogenic microorganisms. Internal dampness is caused by rich, sweet, and damp-producing foods that promote the colonization and reproduction of pathogenic microorganisms in the intestines. External dampness is caused by pathogenic microorganisms in the air or water invading the mucous membranes of the respiratory and digestive tracts.

[0003] Dampness syndrome is involved in the occurrence and development of almost all diseases. Traditional Chinese medicine (TCM) has a relatively detailed description of the symptoms of dampness syndrome and an analysis of its mechanisms using traditional TCM terminology, and has created many prescriptions and medicines for treating dampness syndrome. However, it still has not clearly elucidated the specific pathophysiological mechanisms of dampness syndrome and has not gained recognition from modern medicine. One important reason is the lack of a convenient animal model to simulate the symptom manifestations of dampness syndrome, which restricts a deeper understanding of the pathological mechanisms of dampness syndrome and the evaluation of the effects and mechanisms of new drugs or interventions related to dampness syndrome.

[0004] Currently, the most commonly used mouse model for dampness syndrome involves using an artificial climate chamber with a high-fat diet, maintaining humidity above 95% for more than 20 days. However, due to significant individual differences among mice, some do not fully develop the model or recover within a few days after leaving the high-humidity environment. Some studies, in addition to a high-humidity environment and a high-fat diet, added a certain amount of alcohol or ethanol by gavage. For the first 10 days, rats were given a high-fat diet, followed by gavage of baijiu (10 mL / kg) every other day, and then placed in an artificial climate chamber simulating a high-temperature and high-humidity environment. Other studies, in addition to a high-humidity environment and a high-fat diet, added E. coli or Salmonella by gavage. Rats were fed a high-fat, high-sugar diet for 10 days, then placed in a high-temperature, high-humidity environment, and gavage with Salmonella typhimurium at 96 and 120 hours, before being removed to a natural environment. The results showed that the main symptoms, signs, and pathogenesis conditions were similar to the damp-heat syndrome in traditional Chinese medicine. The cumbersome and inconsistent methods for preparing dampness syndrome hinder research into its mechanisms and the widespread adoption of animal models. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the existing technology, the main objective of this invention is to provide a method for preparing a wet symptom animal model.

[0006] This invention is achieved through the following technical solution:

[0007] A method for preparing an animal model of wet syndrome includes the following steps:

[0008] (1) Cultivate microorganisms in culture medium, centrifuge to obtain bacterial cells, and resuspend to obtain bacterial cell suspension for later use;

[0009] (2) Prepare healthy experimental animals and inject bacterial suspension into the peritoneum;

[0010] (3) Detect the activity level, stool stickiness, and blood levels of immune inflammatory factors in experimental animals to determine whether the dampness syndrome model has been successfully established.

[0011] The microorganisms mentioned in step (1) are pathogenic or opportunistic pathogenic microorganisms; preferably, they include Candida albicans (… Candida albicans ), Escherichia coli ( Esherichia coli )or K. pintolopesii At least one of the following; more preferably Candida albicans.

[0012] When the microorganism described in step (1) is a fungus, the culture medium is YM medium; when the microorganism is a bacterium, the culture medium is LB medium.

[0013] The cultivation conditions described in step (1) are 35-39℃ for 20-30 hours; preferably 37℃ for 24 hours.

[0014] The centrifugation in step (1) is performed at 3000-7000 r / min for 5-15 min; preferably at 6000 r / min for 10 min.

[0015] The resuspension mentioned in step (1) is to resuspend using sterile PBS.

[0016] The experimental animals mentioned in step (2) are at least one of C57BJ / 6 mice, KM mice and SD rats.

[0017] The intraperitoneal injection mentioned in step (2) is administered at a dose of 0.8–3 × 10⁻⁶ mg / L, depending on the weight of the experimental animal. 6 Inject at a bacterial concentration of CFU / g.

[0018] The experimental animals described in step (2) are divided into experimental group and control group.

[0019] The immune inflammatory factors mentioned in step (3) include at least one of IL-17A and TNF-α; preferably IL-17A and TNF-α.

[0020] The method for determining the stickiness of stool described in step (3) specifically includes the following steps:

[0021] Place the same mass of feces on filter paper, then add another layer of filter paper, place a 50-gram weight on the filter paper, remove the weight after 2 minutes, observe the area of ​​feces residue on the upper filter paper, measure the diameter, and use the diameter value to represent the viscosity of the feces.

[0022] The method for judging the dampness syndrome model described in step (3) is that when the activity level of animals in the experimental group decreases compared to the control group, the diameter of the stool stickiness test exceeds 50% of that in the control group, and the level of immune inflammatory factors in the blood of the animals increases by more than 50% compared to the normal group, then the dampness syndrome model is successfully established.

[0023] The application of the method for preparing the animal model of dampness syndrome in the evaluation and analysis of dampness syndrome.

[0024] The present invention has the following advantages over the prior art:

[0025] The animal model of dampness syndrome created using this invention exhibits symptoms extremely similar to those of clinical dampness syndrome. Based on three recognized criteria—decreased activity, sticky stools, and elevated levels of immune inflammatory factors in the blood—the modeling method of this invention has a 100% success rate.

[0026] Furthermore, existing modeling methods described in the literature are time-consuming, cumbersome, and have poor reproducibility. This invention creates conditions for further in-depth research into the mechanisms and interventional treatments of related TCM diseases.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) The animal model of dampness syndrome prepared by the above method showed symptoms of dampness syndrome such as limb weakness and sticky stool within 6 to 12 hours, with a modeling success rate of 100%.

[0029] (2) The animal model of dampness syndrome prepared by the above method has extremely similar manifestations to the clinical manifestations of dampness syndrome, while the animal model of dampness syndrome itself does not have these manifestations. Therefore, this model can be considered a new invention of a dampness syndrome model.

[0030] (3) The technical feature of the method for establishing the animal model of dampness syndrome in this invention is that it is based on the mechanism of ectopic intestinal bacteria triggering the activation of immune cells and the release of large amounts of mucolytic and inflammatory factors, thereby rapidly preparing the animal model of dampness syndrome. This creates conditions for further in-depth research on the mechanism of related TCM diseases and their intervention and treatment. Attached Figure Description

[0031] Figure 1 This is a photograph of the fecal characteristics of C57BL / 6 mice 8 hours after intraperitoneal injection of the bacterial strain in Example 1.

[0032] Figure 2This is a comparison of the appearance of stool in normal KM mice and mice injected with Candida albicans in Example 2.

[0033] Figure 3 This is a graph showing the results of the injection of Candida albicans into the blood of mice with inflammatory factors TNF-α and IL-17 in Example 2.

[0034] Figure 4 This is a graph showing the effect of Candida albicans on RAW264.7 macrophages in Example 4.

[0035] Figure 5 This is a photograph of the fecal characteristics of SD rats 6 hours after intraperitoneal injection of the bacterial strain in Example 5.

[0036] Figure 6 These are photographs showing the changes in the morphology of mouse feces after modeling in Example 6.

[0037] Figure 7 This is a graph showing the change in IL-17A levels in mouse serum after modeling in Example 6. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0039] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0040] The SD rats and KM mice used in the examples were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. Example

[0041] 1. Evaluation indicators for the wet syndrome model

[0042] Inflammatory factors: All reported models of dampness syndrome showed elevated levels of certain inflammatory factors in the blood, with tumor necrosis factor-α being the most frequent, followed by various interleukins such as IL-β and IL-6.

[0043] General indicators include reduced food intake, decreased activity, and sticky stools. Sticky stools are an important criterion for qualitative analysis.

[0044] Alterations in gut microbiota: Alterations in gut microbiota have been reported in animal models of wet syndrome prepared by different modeling methods. In mice, the Bacteroidetes phylum was significantly reduced and the Firmicutes phylum was significantly increased, reflecting a decrease in the proportion of healthy bacteria in the gut.

[0045] Based on the standards and models disclosed in the existing literature, the present invention mainly uses three evaluation criteria: decreased activity, sticky stools, and the level of immune inflammatory factors in the blood.

[0046] The test method for the degree of sticky stools is as follows: Place the same mass of feces on a filter paper, add another layer of filter paper, and place a 50-gram weight on the filter paper. After 2 minutes, remove the weight and observe the area of the fecal trace adhered to the upper filter paper, and measure the diameter. The diameter value represents the fecal viscosity and humidity.

[0047] When the activity of the animals in the experimental group decreases compared to the control group, the diameter of the sticky stools test exceeds 50% of that of the control group, and the immune inflammatory factors in the animals' blood increase by more than 50% compared to the normal group, it proves that the damp syndrome model is successfully established.

[0048] 2. Establishing a damp syndrome model using C57BJ / 6 mice

[0049] (1) Experimental animals

[0050] C57BL / 6 mice were purchased from the Guangdong Provincial Medical Experimental Animal Center, 10 weeks old. License numbers are SCXK (Guangdong) 2018-0002 and SYXK (Guangdong) 2015-0102, male. They were housed in a common clean environment with a 12 / 12-hour light / dark cycle, and food and water were supplied normally. Ear tags were punched in ascending order of body weight, and the excel random function =RANDBETWEEN(1,4) was used for random grouping, divided into a normal (control) group, an Escherichia coli group, a Candida albicans group, a Klebsiella pneumoniae group, and a Lactobacillus fermentum group.

[0051] (2) Experimental strains

[0052] All experimental strains were purchased from commercial channels. Among them, the ATCC number of Candida albicans ( Candida albicans ) is 10231, the model of Escherichia coli ( Esherichia coli ) is dh5a, K. pintolopesii (Klebsiella pneumoniae) was purchased from CBS strains in the Netherlands, numbered Bio-109890, and Lactobacillus fermentum ( Lactobacillus fermentum ) was purchased from Shandong Jigao Pharmaceutical Co., Ltd., batch number: 2022061608, with a viable count of 10 billion CFU / g.

[0053] (3) Preparation of bacterial cells

[0054] Take 50 - 200 μL of the preserved bacterial liquid into 5 mL of freshly sterilized medium (LB liquid medium for Escherichia coli / Lactobacillus fermentum, and YM liquid medium for Klebsiella pneumoniae / Candida albicans), shake in a 37 °C constant temperature shaker for 24 h to ensure that the bacteria are in the logarithmic growth phase, measure the absorbance OD at 600 nm, and 1OD = 2×10 9CFU / mL, adjust the bacterial concentration to 10. 8 Centrifuge at CFU / mL, 6000 r / min for 10 min, discard the supernatant culture medium, resuspend in sterile PBS to obtain a bacterial suspension, and let stand at 4 ℃ for later use.

[0055] (4) Animal experiments

[0056] Mice (20 g–40 g) were used, with 10 mice per group. The amount of bacteria injected intraperitoneally was determined based on body weight for different bacterial strain injection groups, at a concentration of 0.8 × 10⁻⁶. 6 CFU / g, administered via intraperitoneal injection once. The control group received an equal volume of physiological saline via intraperitoneal injection. Mice were sacrificed 12 hours later, and blood was collected. IL-17A and TNF-α immune inflammatory factors in the mouse blood were detected using an ELISA kit.

[0057] Table 1. Levels of immune factors in mice after intraperitoneal injection

[0058]

[0059] (5) Experimental results

[0060] Experimental results showed that Candida albicans ( Candida albicans Group ) and Escherichia coli ( Esherichia coli Group ) and KP bacteria ( K. pintolopesii C57BL / 6 mice in group () showed symptoms such as fatigue, limb weakness, and sticky stools within 6–12 hours after intraperitoneal injection. Figure 1 As shown, its manifestations are extremely similar to those of "dampness syndrome" in clinical practice, while the group injected with an equal amount of Lactobacillus fermentum (probiotics) did not exhibit these disease characteristics; moreover, the Escherichia coli group, Candida albicans group, and KP group caused a significant increase in TNF levels, proving that the dampness syndrome model was successfully established. Since KP and Candida albicans belong to the same fungus family, the next step is to compare the modeling differences between Escherichia coli and Candida albicans.

[0061] Example 2 Candida albicans ( Candida albicans Intraperitoneal injection of KM mice to establish a dampness syndrome model

[0062] Based on the experiment in Example 1, Candida albicans with good modeling effect was screened out. Further modeling effect verification experiments were conducted on other experimental animals. The specific steps are as follows:

[0063] (1) Laboratory animals

[0064] KM mice, male and 8 weeks old, were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. They were subjected to a 12 / 12-hour light / dark cycle and randomly divided into a control group and an experimental group, with 6 mice in each group.

[0065] (2) Preparation of bacterial cells

[0066] Add 50–200 μL of Candida albicans culture medium to 5 mL of freshly sterilized LB broth and incubate at 37 °C for 24 h to ensure the bacteria are in the logarithmic growth phase. Adjust the culture concentration to 10⁻⁶. 8 Centrifuge at CFU / mL, 6000 r / min for 10 min, discard the supernatant culture medium, resuspend in sterile PBS, and let stand at 4 ℃ until ready for use.

[0067] (3) Animal experiments

[0068] Intraperitoneal injection experiment in mice: Mice (20 g–40 g) were divided into a control group and an experimental injection group, with 6 mice in each group. The amount of bacteria injected intraperitoneally was determined based on body weight, at a concentration of 0.8 × 10⁻⁶. 6 CFU / g was administered via intraperitoneal injection once. Twelve hours after injection, mouse characteristics were observed to determine the modeling status. Tail blood was collected from mice and ELISA kits were used to detect IL-17A and TNF-α immune inflammatory factors in the mouse blood.

[0069] (4) Experimental results

[0070] After 12 hours of observation, and based on the judgment method in Example 1, KM mice exhibited decreased activity, sticky stools, and significantly elevated levels of immune inflammatory factors compared to the normal group. The results are as follows: Figure 2 and 3 As shown, the success rate of wet lesion animal model establishment in the experimental group was 100%, proving that the model has good reproducibility and practicality, and can be used to model different experimental animals.

[0071] Example 3 Modeling a safe dosage experiment

[0072] Experimental methods:

[0073] (1) Laboratory animals

[0074] KM mice were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. They were male and 8 weeks old. They were subjected to a 12 / 12-hour light / dark cycle and randomly divided into four groups: control group, low-dose Cnidium monnieri group, high-dose Cnidium monnieri group, low-dose Escherichia coli group, and high-dose Escherichia coli group, with 10 mice in each group.

[0075] (2) Preparation of bacterial cells

[0076] Add 50–200 μL of the culture medium to 5 mL of freshly sterilized LB liquid medium and incubate at 37 °C for 24 h to ensure the bacteria are in the logarithmic growth phase. Adjust the bacterial concentration to 10⁻⁶. 8 Centrifuge at CFU / mL, 6000 r / min for 10 min, discard the supernatant culture medium, resuspend in sterile PBS, and let stand at 4 ℃ until ready for use.

[0077] (3) Animal experiments

[0078] Intraperitoneal injection experiment in mice (different bacterial species): Mice (20 g–40 g) were divided into a control group and two injection groups: a high / low dose Candida albicans injection group and a high / low dose Escherichia coli injection group, with 10 mice in each group. The amount of bacteria injected intraperitoneally was determined based on body weight for each bacterial species injection group, with the low dose being 1.0 × 10⁻⁶. 6 CFU / g, high dose 3.0×10 6 CFU / g, once intraperitoneally injected; control group injected an equal volume of physiological saline intraperitoneally, and the animals' symptoms and mortality were observed for 3 days.

[0079] (4) Experimental results

[0080] No deaths occurred in either the Candida albicans injection group or the Escherichia coli injection group after low-dose injection. After high-dose injection, one animal died in the Candida albicans injection group and three animals died in the Escherichia coli injection group.

[0081] This indicates that Candida albicans is safer than Escherichia coli, and that using Candida albicans and low concentrations of Escherichia coli to model wet eczema in mice can achieve the desired technical effect quickly and safely. Example

[0082] To verify the modeling mechanism, an in vitro experiment was designed to interact Candida albicans with macrophages and observe whether macrophages were activated to secrete immune inflammatory factors.

[0083] (1) Experimental cells

[0084] RAW264.7 (mouse mononuclear macrophage leukemia cells) cells were purchased from ATCC and cultured in 24-well plates under the following conditions: DMEM + 10% FBS + 1% P / S. The cryopreservation formulation was 55% DMEM + 40% FBS. Cells were cultured at 37 °C with 5% CO2 and passaged at a ratio of 1:3 to 1:6.

[0085] (2) Bacterial culture

[0086] Inoculate *Candida albicans* into 5 mL of freshly sterilized YM liquid medium and incubate at 37 °C for 16 h. For a 1:100 dilution: add 50 μL of the cultured bacterial solution to 5 mL of freshly sterilized YM liquid medium and incubate at 37 °C for 4.5 h to ensure the bacteria are in the logarithmic growth phase.

[0087] (3) Detection of the effect of supernatant from co-culture of Candida albicans and macrophages on the proliferation of different bacterial species

[0088] Take the bacterial culture obtained in step (2), adjust the concentration to OD600=1, add 100μL to the 24-well plate of RAW264.7 cultured in step (1), repeat for 12 wells, and culture for 6h.

[0089] RAW264.7 cell supernatants were collected from 24-well plates after normal culture and after culturing with 100 μL of Candida albicans with OD600=1 for 6 h. The cells were filtered through a 0.22 μm microfiltration filter, and the TNF-α content was detected using an ELISA kit.

[0090] The TNF-α content in the supernatant of cells co-cultured with Candida albicans was detected using an ELISA kit. The experimental results are as follows: Figure 4 As shown, the TNF-α content in the control group was N=41.017 pg / mL, while the TNF-α content in the model group was M=300.992 pg / mL, indicating that Candida albicans can stimulate macrophages to secrete large amounts of the inflammatory factor TNF-α.

[0091] Example 5 Candida albicans ( Candida albicans Intraperitoneal injection of SD rats to establish a dampness syndrome model

[0092] To verify the reproducibility of this modeling method on different experimental animals, a comparative experiment was designed using SD rats under the same conditions, employing existing wet syndrome modeling methods. The experimental steps are as follows:

[0093] (1) Laboratory animals

[0094] SD rats were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. They were normally fed and divided into a control group and an experimental group, with 6 rats in each group.

[0095] (2) Experimental methods

[0096] The preparation method and injection volume of the bacterial solution used were the same as in Example 2. The experimental group was injected intraperitoneally once, and the characterization was observed after injection to determine the modeling situation.

[0097] (3) Experimental results

[0098] Six hours after injection, rats were observed to exhibit symptoms such as fatigue, lethargy, and sticky feces, which closely resembled the clinical manifestations of "dampness syndrome." Fecal samples were tested to determine the degree of stickiness, confirming the presence of these "dampness syndrome" characteristics. Figure 5 As shown. Experimental results demonstrate that the wet lesion model establishment method provided by this invention can quickly model different experimental animals.

[0099] Example 6: Comparison of traditional modeling methods for preparing wet syndrome models

[0100] To compare with existing methods for establishing wet syndrome models, an experiment was designed to model mice using traditional modeling methods. The experimental steps are as follows:

[0101] (1) Laboratory animals

[0102] The experimental animals used were C57BJ / 6 mice, which were housed under the same conditions as the mice in Example 1. They were divided into a normal group (Coltrol) and a traditional model group (Model), with 10 mice in each group.

[0103] (2) Experimental methods

[0104] Normal group: humidity 50%, temperature 25℃.

[0105] Traditional model group: Mice were kept in an artificial climate chamber with a temperature of 25 ℃ and a humidity of 95% from 8:00 to 18:00 every day, with no restrictions on food and water.

[0106] The bottom of the mouse cages was lined with a thick layer of sawdust, which was changed daily. The animals had free access to food and water. The mice's activity level, fur color, and stool consistency were observed daily for 20 days. On day 21, tail blood was collected and an ELISA kit was used to detect the IL-17A immune inflammatory factor in the mouse blood.

[0107] (3) Experimental results

[0108] 1. Changes in activity level and fecal characteristics in mice with "dampness syndrome".

[0109] The experimental results showed that the activity level and fecal characteristics of the model group mice remained essentially unchanged 12 hours after the start of modeling. On day 20, the activity level of the model group mice decreased, and their fecal characteristics changed significantly, appearing stickier than those of the normal group. Figure 6 As shown.

[0110] 2. Changes in the expression of serum and colonic inflammatory factors in mice with "dampness syndrome"

[0111] Experimental results are as follows Figure 7 As shown, the warm and humid model group can lead to an increase in serum IL-17A levels ( P <0.05).

[0112] By combining the characteristics of mouse feces and the level of serum inflammatory factors, it was determined that the traditional model group successfully established the wet syndrome model. However, the changes in mouse traits in the traditional model only appeared after 20 days. It can be seen that the traditional method requires a long time to model and cannot meet the needs of current experiments. The method used in Example 1 of this invention can quickly prepare an animal wet syndrome model, which has significant advantages over the traditional method.

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an animal model of wet syndrome, characterized in that... It consists of the following steps: (1) Cultivate microorganisms in culture medium, centrifuge to obtain bacterial cells, and resuspend to obtain bacterial cell suspension for later use; (2) Prepare healthy experimental animals and inject bacterial suspension into the peritoneum; (3) Detect the activity level, stool stickiness, and blood levels of immune inflammatory factors in experimental animals to determine whether the dampness syndrome model has been successfully established; The culture medium is YM medium or LB medium; The microorganism mentioned in step (1) is Candida albicans; The culture conditions described in step (1) are 35-39℃ for 20-30 hours; The centrifugation mentioned in step (1) is 3000-7000 r / min for 5-15 min; The resuspension mentioned in step (1) refers to resuspension using sterile PBS; The experimental animals mentioned in step (2) are at least one of C57BJ / 6 mice, KM mice, and SD rats; The experimental animals described in step (2) were divided into an experimental group and a control group; The intraperitoneal injection mentioned in step (2) is administered at a dose of 0.8–3 × 10⁻⁶ mg / L, depending on the weight of the experimental animal. 6 Inject at a bacterial count of CFU / g; The immune inflammatory factors mentioned in step (3) include at least one of IL-17A and TNF-α; The method for determining the stickiness of stool described in step (3) specifically includes the following steps: Place the same mass of feces on filter paper, then add another layer of filter paper, place a 50-gram weight on the filter paper, remove the weight after 2 minutes, observe the area of ​​feces residue on the upper filter paper, measure the diameter, and use the diameter value to represent the stickiness and moisture of the feces. The method for determining the wet syndrome model described in step (3) specifically includes the following steps: When the activity level of animals in the experimental group decreases compared to the control group, the diameter of their stool (measured for stickiness) exceeds that of the control group by more than 50%, and the level of immune inflammatory factors in their blood increases by more than 50% compared to the normal group, it proves that the dampness syndrome model has been successfully established.

2. The application of the method for preparing the wet syndrome animal model according to claim 1 in the evaluation and analysis of wet syndrome.

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