A method for establishing an animal model of damp-heat type eczema

Abstract

This invention discloses a method for establishing an animal model of damp-heat type eczema. First, animals are subjected to an irregular diet (45% high-fat, high-sugar feed) and exposed to damp-heat pathogens (placed in an artificial climate chamber at 35°C and 95% humidity for 8 hours daily). Then, sensitization and stimulation are achieved by applying 1% and 1.5% DNCB solutions to the animals' backs, respectively. The damp-heat type eczema animal model of this invention is characterized by increased body weight, water intake, and rectal temperature; decreased food intake and gripping strength; increased skin lesions and transepidermal water loss; increased serum IgE levels, spleen index, and scratching frequency; thickening of the epidermis and dermis; and increased mast cell infiltration. This invention organically combines an eczema animal model with a traditional Chinese medicine damp-heat syndrome animal model, successfully establishing a relatively ideal damp-heat type eczema animal model. The model has high reproducibility and more closely resembles the clinical manifestations of damp-heat type eczema patients, which is beneficial for research on the pathogenesis of damp-heat type eczema and for the development and efficacy evaluation of new traditional Chinese medicine drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically to the field of basic research on damp-heat type eczema in traditional Chinese medicine and experimental animal science. It relates to a method for establishing an animal model, specifically a method for establishing an animal model of damp-heat type eczema. Background Technology

[0002] Eczema is an inflammatory skin disease affecting the superficial dermis and epidermis, caused by a variety of internal and external factors. Clinically, in the acute phase, the lesions are mainly papules and vesicles with a tendency to ooze; in the chronic phase, the lesions are mainly lichenification and are prone to recurrence. [1,2] .

[0003] Traditional Chinese medicine believes that the pathogenesis of eczema is due to inherent intolerance, improper diet, or excessive consumption of spicy, stimulating, and greasy foods, which damages the spleen and stomach, impairs their function, and leads to the internal generation of damp-heat. This is compounded by external wind pathogens, resulting in the interaction of these two pathogens and the invasion of wind, dampness, and heat pathogens into the skin. [6] Traditional Chinese medicine diagnostic results show that damp-heat type accounts for about 60% of eczema cases in clinical practice. [7] It is the most frequently occurring type of certificate. [8] The causes of damp-heat type eczema are complex and varied, the course of the disease is prolonged and difficult to cure, and it is prone to recurrence. There is an urgent need to conduct in-depth research on the pathogenesis of damp-heat type eczema in order to develop drugs that can prevent, alleviate or treat patients with damp-heat type eczema.

[0004] Animal models are an effective means of in-depth research into the pathogenesis of diseases and the exploration and discovery of new treatment methods. Currently, common animal models of eczema include rabbits, rats, mice, guinea pigs, and dogs. Among them, mice are favored due to their low cost, ease of model establishment, and ease of gene manipulation. According to the establishment method and inducing factors, these eczema animal models can be roughly divided into three categories: (1) spontaneous animal models: Nc / Nga mice; (2) sensitizer-induced models: ovalbumin, dust mites, Staphylococcus aureus, haptens [such as oxazolone (Ox), trinitrochlorobenzene (TNCB), 2,4-dinitrofluorobenzene (DNFB), 2,4-dinitrochlorobenzene (DNCB), etc.]; (3) gene-based models, such as transgenic mice and gene knockout mice. [9] Damp-heat type eczema is a major syndrome in TCM clinical practice. However, most existing studies have focused on constructing animal models of eczema, and there are no reports on animal models of damp-heat type eczema. This has severely limited the research progress on the pathogenesis of the disease and the treatment of damp-heat type eczema with TCM. Therefore, it is imperative to establish a stable animal model of damp-heat type eczema.

[0005] References:

[0006] [1] Zhang Xuejun, Zheng Jie. Dermatology and Venereology. 9th ed. [M]. Beijing: People's Medical Publishing House, 2018: 108.

[0007] [2]Endre KMA, L,LeBlanc M,et al.Eczema distribution in girls andboys during infancy:A cohort study on atopic dermatitis[J].J Allergy ClinImmunol Pract.2021;9(9):3513-3516.e2.

[0008] [3] Li Linfeng, Li Yuanwen. Clinical application guidelines for the treatment of eczema with traditional Chinese medicine (2020) [J]. Chinese Journal of Integrated Traditional and Western Medicine, 2021, 41(2):133-142.

[0009] [4]Silverberg JI, Gelfand JM, Margolis DJ, et al.Symptoms and diagnosis of anxiety and depression in atopic dermatitis in USadults[J].Br JDermatol.2019,181(3):554-565.

[0010] [5]Tsai TF,Rajagopalan M,Chu CY,et al.Burden of atopic dermatitis inAsia[J].J Dermatol.2019,46(10):825-834.

[0011] [6] Chen Hongfeng. Traditional Chinese Medicine Surgery. 5th ed. [M]. Beijing: China Traditional Chinese Medicine Press, 2021: 171.

[0012] [7] Shang Qian. A study on the treatment patterns of eczema by contemporary famous dermatologists based on the TCM inheritance auxiliary platform [D]. Beijing University of Chinese Medicine, 2016.

[0013] [8] Liu Qi, Guo Jie, Huang Zhibin, et al. Analysis of the distribution of eczema symptoms in literature from 1990 to 2019 [J]. Chinese Journal of Medical Science, 2021, 11(7):36-39.

[0014] [9] Li Yunzhu, Li Linfeng. Research progress on animal models of atopic dermatitis [J]. Chinese Medical Abstracts (Dermatology), 2016, 33(2):138-143+90. Summary of the Invention

[0015] To address the problems of existing technologies, this invention provides a method for establishing an animal model of damp-heat type eczema, overcoming the lack of unified standards and instability of existing animal models of damp-heat type eczema that combine traditional Chinese medicine syndromes.

[0016] The objective of this invention is achieved through the following technical solutions:

[0017] A method for establishing a damp-heat type eczema animal model is described, which uses DNCB (2,4-dinitrochlorobenzene), a high-fat, high-sugar diet, and an artificial climate chamber to induce a damp-heat type eczema model, as detailed below:

[0018] ① High-fat and high-sugar diet treatment: Animals were allowed free access to a high-fat and high-sugar diet from day 1 until the end of the experiment;

[0019] ②DNCB treatment: Use a mixture of acetone and olive oil in a volume ratio of 4:1 to 3:1 to dissolve DNCB.

[0020] Sensitization: Before sensitization, the animals were anesthetized by inhalation of isoflurane and their hair was removed. The next day, a 0.1-7% DNCB solution was applied to the back of the animals to induce sensitization, and the application was repeated for 3-5 days.

[0021] Stimulation: Apply 0.1-7% DNCB solution to the back of the animal to stimulate it, and repeat the stimulation after one day.

[0022] ③ Artificial climate chamber treatment: Starting from day 1, place the animals in an artificial climate chamber with a temperature of 30-40℃ and a humidity of 80-95% for 1-12 hours each day; do not place the mice in the artificial climate chamber during the sensitization period, and place them in the artificial climate chamber after the mice have recovered their weight; during the challenge period, place the mice in the artificial climate chamber on the day of challenge, take them out and challenge them, and place them in the artificial climate chamber one day apart, for a total of 7-10 challenges; the interval between sensitization and challenge is 3-14 days.

[0023] Preferably, in step ②, the volume ratio of acetone to olive oil is 3:1; the sensitization concentration of the DNCB solution is 1%, and the activation concentration of the DNCB solution is 1.5%; in step ③, the temperature is 35°C, the humidity is 95%, and the time is 8 hours; the interval between sensitization and activation is 7 days, and a total of 8 activations are performed.

[0024] The high-fat, high-sugar feed mentioned in step ① is a purified feed with more than 30% high fat and sugar content. It should be given in a fixed amount every day to avoid softening after being placed in a hot and humid environment, which would lead to loss and affect the accurate recording of feed intake.

[0025] The acetone and olive oil base solutions in ② must be prepared and used immediately.

[0026] The process after modeling also includes testing of the model animals. The testing methods include the detection of signs of damp-heat type, signs related to damp-heat type eczema, key indicators of damp-heat type eczema, and immunological and pathological signs.

[0027] The indicators of damp-heat type eczema in the animal model include the detection of animal weight, food intake, water intake, anal temperature, and grip strength.

[0028] The aforementioned indicators related to hot and humid eczema include transepidermal water loss, morphology, photographs and scoring of skin lesions, and detection of scratching.

[0029] The key indicator for the aforementioned damp-heat type eczema is the determination of serum immunoglobulin E levels.

[0030] The immunological and pathological indicators of the aforementioned damp-heat type eczema include spleen index, skin pathological changes at the lesion site, and mast cell infiltration.

[0031] The model animal used can be a rat or a mouse, with mice being preferred, and the preferred mouse strain being BALB / c.

[0032] The purpose of an animal model of damp-heat type eczema prepared by the above method in screening or developing drugs that can prevent, alleviate or treat damp-heat type eczema.

[0033] Mice with damp-heat type eczema induced by the method of this invention exhibited symptoms of damp-heat type in traditional Chinese medicine, such as poor appetite, excessive thirst, fatigue, and fever.

[0034] The severity of skin lesions and scores, transepidermal water loss, scratching frequency, spleen index, skin pathological changes, and mast cell infiltration in the eczema mice with damp-heat type eczema were all more severe than those in the normal group and DNCB group.

[0035] The beneficial effects of this invention are:

[0036] 1. This invention provides a method for establishing a damp-heat type eczema animal model. First, a model animal was designed to simulate the damp-heat environment of dietary imbalance and high temperature and humidity in traditional Chinese medicine by feeding it a high-fat, high-sugar diet and placing it in a high-temperature, high-humidity artificial climate chamber. Second, mice were sensitized and challenged by applying different concentrations of DNCB solution to their backs to establish an eczema mouse model. By organically combining the eczema animal model with the traditional Chinese medicine damp-heat type animal model, a relatively ideal damp-heat type eczema animal model was successfully established. Results show that the animal model constructed by this method has good stability, high reproducibility, and is easy to operate.

[0037] 2. This invention addresses the current lack of animal models for eczema caused by damp heat. The results of the embodiments show that a damp heat environment exacerbates the severity of eczema in model animals. Initially, the animals were subjected to an irregular diet (45% high-fat, high-sugar feed) and exposed to damp heat (placed in an artificial climate chamber at 35°C and 95% humidity for 8 hours daily). Then, sensitization and stimulation were achieved by applying 1% and 1.5% DNCB solutions to the animals' backs, respectively. This invention optimizes the modeling method, resulting in a high success rate and low mortality.

[0038] 3. Traditional Chinese medicine (TCM) treatment for eczema is more flexible than Western medicine, emphasizing "syndrome differentiation and treatment, and individualized treatment." Currently, there are no reported animal models of damp-heat type eczema. Damp-heat syndrome is located on the skin, with internal and external damp-heat interfering and infiltrating the skin. The animal model of damp-heat type eczema established in this invention effectively simulates the occurrence and development of damp-heat type eczema, more closely resembling the clinical manifestations of patients with this condition. This is beneficial for research on the pathogenesis of damp-heat type eczema and for the development and efficacy evaluation of new TCM drugs.

[0039] 4. This invention evaluates the established damp-heat eczema model from multiple aspects, including damp-heat type indicators, damp-heat type eczema-related indicators, key indicators of damp-heat type eczema, and immunological and pathological indicators. This avoids the problem of strong subjectivity in the evaluation indicators of traditional animal models, and uses data to evaluate the model intuitively, making it more scientific and standardized. Attached Figure Description

[0040] Figure 1 Figure A shows the change in mouse body weight and the change in body weight before sampling; Figure B shows the weight of the mice on the day of sampling; *P < 0.05 indicates that the difference is statistically significant.

[0041] Figure 2 Figure A shows the changes in food intake of mice and the changes in food intake before sampling; Figure B shows the changes in food intake of mice before sampling; *P < 0.05 indicates that the difference is statistically significant.

[0042] Figure 3 Figure A shows the changes in water intake of mice before and after sample collection; Figure B shows the changes in water intake of mice before sample collection; *P < 0.05 indicates that the difference is statistically significant.

[0043] Figure 4 The values ​​represent changes in rectal temperature in mice; *P < 0.05 indicates that the difference was statistically significant.

[0044] Figure 5 The changes in grip strength in mice are shown; *P < 0.05 indicates that the difference is statistically significant.

[0045] Figure 6This describes the morphological changes in mice;

[0046] Figure 7 This represents the change in transdermal water loss in mice; *P < 0.05 indicates that the difference is statistically significant.

[0047] Figure 8 The figures show the degree of skin lesions and changes in scores in mice; Figure A shows the degree of skin lesions on the back of the mice; Figure B shows the skin lesion scores; *P < 0.05 indicates that the difference is statistically significant.

[0048] Figure 9 The changes in the number of scratches by mice; *P<0.05 indicates that the difference is statistically significant;

[0049] Figure 10 The expression of IgE in mouse serum; *P<0.05 indicates that the difference is statistically significant;

[0050] Figure 11 The changes in the spleen index of mice were shown; *P<0.05 indicates that the difference was statistically significant.

[0051] Figure 12 HE staining of mouse skin tissue was used to observe pathological changes; Figure A shows the pathological changes in mouse skin; Figure B shows the changes in mouse epidermal thickness; Figure C shows the changes in mouse dermal thickness; *P < 0.05 indicates that the differences are statistically significant;

[0052] Figure 13 Toluidine blue staining was used to observe mast cell infiltration in mouse skin; Figure A shows the changes in mast cell infiltration in mouse skin; Figure B shows the changes in the number of mast cells in mouse skin; *P<0.05 indicates that the difference is statistically significant. Detailed Implementation

[0053] To facilitate a better understanding of the present invention, the present invention will be described in more detail and comprehensively below with reference to the embodiments, and the scope of protection of the present invention is not limited to the embodiments described below.

[0054] Unless otherwise defined, the technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention.

[0055] Unless otherwise specified, all reagents and consumables used in this invention are commercially available products.

[0056] This invention discloses a method for establishing and identifying a damp-heat type of eczema combined with an animal model, as follows:

[0057] 1. Experimental materials and reagents are shown in the table below.

[0058]

[0059]

[0060] Experimental mice: Thirty 4-week-old SPF-grade female BALB / c mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were housed in an environment with a temperature of 22±1℃ and a humidity of 50% to 60%, with a 12-hour dark / light cycle, free access to water, and normal feed for one week for acclimatization before the formal experiment began.

[0061] 2. Model building methods

[0062] Mice were randomly divided into three groups based on their body weight: a normal group (10 mice), a DNCB group (10 mice), and a moist heat + DNCB group (10 mice), and were housed separately.

[0063] (1) Normal group and DNCB group: fed with normal feed;

[0064] (2) Humid heat + DNCB group: fed with 45% high fat and high sugar purified feed and placed in an artificial climate chamber. This operation continued until the end of the experiment.

[0065] ① High-fat, high-sugar diet treatment: Mice were given a high-fat, high-sugar diet freely from day 1 until the end of the experiment;

[0066] ②DNCB treatment: DNCB was dissolved in a mixture of acetone and olive oil in a specific ratio. Sensitization: Mice were anesthetized with isoflurane inhalation before the experiment, and their hair was removed. The following day, DNCB solution was applied to the backs of the mice for sensitization, and this was repeated for three consecutive days. Challenge: DNCB solution was applied to the backs of the mice for challenge, and this was repeated after a one-day interval. During the sensitization and challenge processes, normal mice were treated with an equal volume of a matrix solution made of acetone and olive oil in a ratio of 4:1 to 3:1.

[0067] ③ Artificial climate chamber treatment: Starting from day 1, mice were placed in a high-temperature and high-humidity artificial climate chamber every day. During the sensitization period, mice were not placed in the artificial climate chamber. They were placed in the artificial climate chamber after their weight recovered. During the stimulation period, mice were placed in the artificial climate chamber on the day of stimulation, and then stimulated after being taken out. They were placed in the artificial climate chamber every other day. This stimulation was repeated several times. This operation continued until the end of the experiment.

[0068] ④ During the rearing period, provide the mice with feed and drinking water, and change the feed and drinking water daily.

[0069]

[0070]

[0071] 3. Model Evaluation Method (Based on Example 1)

[0072] (1) Based on the TCM diagnostic method of damp-heat type, various characteristics of mice, such as body weight, loss of appetite (food intake), thirst (water intake), body temperature (rectal temperature), and weakness (gripping strength), were recorded to evaluate the damp-heat model of mice.

[0073] (2) Observe the mice’s mental state and activity status daily, and take photos of the mice to observe the color of their fur.

[0074] (3) After DNCB stimulation, the mice were tested for moisture loss on the skin lesions on their backs using a transdermal moisture loss meter. The shorter end of the cylindrical hollow cavity of the moisture loss test probe was pressed vertically against the surface of the skin to be tested, and then the start button was pressed to record the skin moisture loss results.

[0075] (4) On the day of collection, photographs were taken of the skin lesions on the back of the mice to observe the condition of the skin lesions and to score the skin lesions on the back of the mice to quantify the severity of the skin lesions.

[0076] The rating table is shown below:

[0077]

[0078] (5) Number of scratches: The scratching action is defined as the mouse's hind paw leaving the bottom of the cage and scratching the skin of the ear, head and other drug application sites until the hind paw touches the ground or licks the hind paw. This is counted as one continuous scratch. If the scratching time is more than 3 seconds, it is counted as 2 scratches. A video is recorded using a digital camera, and 20 minutes are extracted for statistical analysis.

[0079] (6) Serum IgE content: Eczema is an allergic and hypersensitivity skin disease, and elevated serum IgE levels are a characteristic change in eczema. Therefore, after enucleating the eyeballs of mice, the blood was allowed to stand at room temperature for 2 hours, then centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and stored at -20℃ for later use. The expression level of serum IgE was detected by ELISA.

[0080] (7) Changes in spleen index: Immune dysfunction plays a very important role in the pathogenesis of eczema, and the spleen index is an indicator of changes in the immune status of mice. The spleen of the mouse was removed under sterile conditions: The mouse was placed in a supine position, and the abdominal cavity was opened along the midline of the abdomen below the ribs to fully expose the abdominal organs. The spleen was extracted and separated using ophthalmic forceps, and the fascia and adipose tissue were removed. The spleen was weighed and the value was recorded. The spleen index was calculated as follows: Spleen index = Spleen mass / Mouse body mass × 100%.

[0081] (8) Skin embedding and sectioning: After the experiment, skin samples from each group of mice (sensitized sites, preferably 2cm × 2cm) were taken. A portion of the tissue was dehydrated, fixed, and then embedded in paraffin to prepare paraffin sections. Mouse skin tissue was taken and thoroughly fixed with 4% paraformaldehyde. The tissue material was cut flat, and the mouse skin tissue was dehydrated, cleared, and paraffin-impregnated in the following order: Dehydration: 70% alcohol, 30 min; 80% alcohol, 30 min; 90% alcohol, 30 min; 95% alcohol, 30 min; 100% I alcohol, 10 min; 100% II alcohol, 10 min; 100% III alcohol, 30 min. Clearing: Xylene I, 5 min; Xylene II, 15 min. Paraffin impregnation: Paraffin (soft paraffin, 52℃~54℃), 10 min; Paraffin (hard paraffin, 56~58℃ or 60~62℃), 30 min (time depends on the clearing effect). Embedding: Place the tissue in the center of a metal mold, inject molten wax, and embed. After embedding, the wax block can gradually solidify at room temperature, or it can be placed on an embedding cooling stage to accelerate solidification. Sectioning: Use a paraffin microtome to section the tissue, adjust the thickness to 4μm, place it in water at 40-45℃ for spreading, then retrieve the section, attach it to a glass slide, and number it promptly.

[0082] (9) Skin hematoxylin-eosin (HE) staining: Place glass slides at intervals in the slide holder and place them in a 60℃ oven for 1 hour before staining. Dewaxing paraffin sections to water: ① Xylene I 10 min ② Xylene II 10 min ③ 100% alcohol I 5 min ④ 100% alcohol II 5 min ⑤ 95% alcohol 5 min ⑥ 80% alcohol 5 min ⑦ 70% alcohol 5 min ⑧ Distilled water I 5 min ⑨ Distilled water II 5 min; HE staining: ① Hematoxylin staining solution 2 min ② Rinse slightly with running water to remove hematoxylin solution ③ Differentiate with differentiation solution for 10-60 s ④ Rinse with distilled water ⑤ Re-blue with tap water for 10 min ⑥ Stain with eosin solution for 20 s. Dehydration: 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, 100% I alcohol, 3s each, 100% II alcohol, 1min; Clearing: Xylene I, 1min; Xylene II, 1min; Mounting: Neutral resin adhesive for mounting, observe the changes in pathological tissue under a microscope.

[0083] (10) Toluidine blue staining of skin: Place glass slides at intervals in the slide holder and place them in a 60℃ oven for 1 hour before staining. Dewax paraffin sections to water: ① Xylene I 10 min ② Xylene II 10 min ③ 100% alcohol I 5 min ④ 100% alcohol II 5 min ⑤ 95% alcohol 5 min ⑥ 80% alcohol 5 min ⑦ 70% alcohol 5 min ⑧ Distilled water I 2 min (9) Distilled water II 2 min. Immerse in mast cell staining solution (toluidine blue method) for 10 min. Wash with tap water for 2 min and blot dry with filter paper. Separate the colors with 80% alcohol for 5 min until the mast cells are purple-blue and the background is light blue. 95% alcohol 1 min, 100% alcohol I 2 min, 100% alcohol II 2 min, clear: Xylene I, 1 min; Xylene II, 1 min. Mount with neutral resin and observe the infiltration of mast cells under a microscope.

[0084] (11) Statistical methods: Experimental data were plotted using GraphPad Prism 9.0 software. When the data met the normal distribution, one-way ANOVA was used. P < 0.05 indicated statistical difference.

[0085] 4. Experimental Results

[0086] In this invention, the normal control group used a matrix solution applied as a blank control; all 10 mice were normal and there were no deaths. The DNCB group used DNCB applied to construct an eczema mouse model; all 10 mice successfully developed the model and there were no deaths. The damp-heat + DNCB group used a high-fat, high-sugar purified diet combined with an artificial climate chamber and DNCB application to construct a damp-heat eczema mouse model. In the preliminary experiment (comparative example), the artificial climate chamber treatment involved placing mice in a high-temperature, high-humidity artificial climate chamber daily from day 1, and placing them in the climate chamber every other day after stimulation, with more than 10 stimulations. This operation continued until the end of the experiment. However, after sensitization, the damp-heat caused a significant decrease in mouse weight, resulting in the death of 3 mice, with only 7 mice successfully developing the model, indicating a high mortality rate. After optimizing the conditions, mice were not placed in the artificial climate chamber during the sensitization period. They were placed in the artificial climate chamber only after their weight recovered, and the number of stimulations was reduced. As a result, all 10 mice successfully developed the model and there were no deaths. Therefore, during the modeling process, the model animals should not be placed in an artificial climate chamber during the sensitization period. Exploring an appropriate number of stimulations can effectively ensure the survival rate and modeling success rate of the model animals.

[0087] In the first two weeks, the weight of mice fed a high-fat, high-sugar purified diet combined with an artificial climate chamber showed a trend of increasing weight compared to both the normal and DNCB groups. After stimulation with 1% DNCB solution, the weight of the mice in this group decreased significantly compared to the normal group. Once the weight of the mice returned to a constant level, stimulation with 1.5% DNCB solution was administered to induce eczema symptoms. Until the end of the experiment, the weight of the mice in the humid heat + DNCB group was higher than that of the mice in the normal and DNCB groups. Figure 1 A). The body weight of the mice on the day of sampling was collected and analyzed, such as... Figure 1 Results B showed that, compared with the control group and the DNCB group, the body weight of mice in the humid heat + DNCB group was significantly increased, and the difference was statistically significant (P < 0.05).

[0088] In the first two weeks, a comparison of food intake in mice revealed that the food intake of the moist heat + DNCB group gradually decreased compared to the other two groups. After challenge and sensitization with different concentrations of DNCB, the food intake of mice in the DNCB group increased, while the food intake of mice in the moist heat + DNCB group showed a gradual decreasing trend compared to the other two groups. Figure 2 A). Simultaneously, this invention collects data on the food intake of mice before sample collection, such as... Figure 2 Results B showed that the food intake of mice in the DNCB group was significantly higher than that of mice in the normal group, but the food intake of mice in the damp-heat + DNCB group was significantly lower, indicating that the damp-heat type caused a decrease in food intake in mice, and this was statistically significant (P < 0.05), which is consistent with the poor appetite characteristics of damp-heat type in traditional Chinese medicine.

[0089] After treatment with high-fat, high-sugar purified feed and an artificial climate chamber, the water intake of mice showed an increasing trend. Following challenge and sensitization with different concentrations of DNCB, the water intake of mice in the DNCB group increased. The water intake of mice in the humid heat + DNCB group showed a decreasing trend compared to the DNCB group, but still showed an increasing trend compared to the normal group. Figure 3 A). Simultaneously, this invention collects the water intake of mice before sampling, such as... Figure 3 Results B showed that the water intake of mice in the DNCB group was significantly higher than that of mice in the normal group, but the water intake of mice in the damp-heat + DNCB group was significantly lower, indicating that the damp-heat type caused a decrease in water intake in mice, and this was statistically significant (P < 0.05), which is consistent with the thirst symptoms of damp-heat type in traditional Chinese medicine.

[0090] Rectal temperature monitoring results showed that the average rectal temperature of mice in the DNCB group was 36.61℃, significantly higher than that of the normal group, and the difference was statistically significant (P < 0.05). The average rectal temperature of mice in the damp-heat + DNCB group was 37.19℃, which also showed an increasing trend compared to the normal group and the DNCB group, and the difference was statistically significant (P < 0.05), consistent with the fever characteristics of damp-heat type in traditional Chinese medicine. (See attached image) Figure 4 .

[0091] The results of grip strength tests in mice showed that the grip strength of mice in the DNCB group was significantly weaker than that of mice in the normal group (P < 0.05). The grip strength of mice in the damp-heat + DNCB group was significantly lower than that in the DNCB group, indicating that the damp-heat condition exacerbated the decrease in grip strength in mice, and this was statistically significant (P < 0.05), consistent with the weakness characteristics of damp-heat type in Traditional Chinese Medicine. (See Appendix) Figure 5 .

[0092] Meanwhile, during the experiment, morphological data of the mice were collected. Comparison revealed no difference in fur color or mental state between the DNCB group and the normal group. However, the mice in the humid heat + DNCB group exhibited more bushy fur and lethargy compared to the other two groups. (See attached image) Figure 6 .

[0093] The results of transdermal water loss assays in mice showed that the moist heat + DNCB group experienced the most severe water loss, which was significantly higher than that in the normal group and the DNCB group (P < 0.05). This is consistent with the fact that after the onset of eczema, the skin barrier is damaged, and transdermal water loss is aggravated. (See attached image) Figure 7 .

[0094] In addition, before sampling, photographs were taken of the skin lesions on the backs of the mice for analysis. Figure 8 A) The results showed that mice in both the DNCB group and the damp-heat + DNCB group exhibited symptoms consistent with eczema, showing a certain degree of skin lesions and significantly increased skin lesion scores compared to the normal group (P < 0.05). Compared to the DNCB group, the damp-heat + DNCB group showed more severe skin lesions and significantly increased skin lesion scores (P < 0.05). See [link to article]. Figure 8 B.

[0095] Given that mice with damp-heat type eczema also exhibit scratching, video recordings of the scratching activity were made, and the number of scratches was counted. The results showed that mice in the DNCB group and the damp-heat + DNCB group exhibited itchy skin and increased scratching frequency, which was statistically significant compared to the normal group (P < 0.05). The damp-heat + DNCB group showed an even greater trend in scratching frequency than the DNCB group, which was statistically significant (P < 0.05). (See attached image). Figure 9 .

[0096] Given that damp-heat type eczema can also cause changes in serum IgE levels in mice, serum IgE levels in mice were measured and statistically analyzed. The results showed that serum IgE levels were increased in the DNCB group and the damp-heat + DNCB group, with statistically significant differences compared to the normal group (P < 0.05); the serum IgE levels in the damp-heat + DNCB group showed an increasing trend compared to the DNCB group, with statistically significant differences (P < 0.05). (See Appendix) Figure 10 .

[0097] Given that the spleen index can reflect changes in the immune status of mice, the spleen weight of mice was measured after sampling. The results showed that the spleen index of mice in the DNCB group and the damp-heat + DNCB group increased significantly (P < 0.05). Furthermore, compared with the DNCB group, the spleen index of mice in the damp-heat + DNCB group was significantly higher (P < 0.05). (See Appendix) Figure 11 .

[0098] Given that damp-heat type eczema presents with pathological changes in mouse skin, microscopic observation and photographic analysis of mouse skin pathology results (see...) Figure 12 A) In the normal group, the skin structure of the mice was clear and intact, with no thickening observed. Compared with the normal group, the DNCB group showed increased skin lesion thickness, manifested as increased thickness of both the epidermis and dermis, which was statistically significant (P < 0.05). The thickness of both the epidermis and dermis in the damp-heat + DNCB group was significantly greater than that in the DNCB group (P < 0.05). See Appendix. Figure 12 B and 12C.

[0099] Given that hot and humid eczema can also present with mast cell infiltration in mouse skin, microscopic observation and photographic analysis of mast cell infiltration in mouse skin showed the following results (see...). Figure 13 A) Mice in the DNCB group and the damp-heat + DNCB group showed extensive mast cell infiltration, with a significantly higher number than the normal group (P < 0.05). The damp-heat + DNCB group showed an increasing trend in mast cell infiltration compared to the DNCB group (P < 0.05). (See Appendix) Figure 13 B.

[0100] In summary, the conditions used in this invention can construct a mouse model of damp-heat type eczema, mainly characterized by increased body weight, decreased food intake, increased water intake, elevated rectal temperature, weakened gripping strength, increased severity and score of skin lesions, aggravated transepidermal water loss, increased serum IgE levels, spleen index, and increased scratching frequency. Additionally, the epidermis and dermis at the lesion sites show thickening, and mast cell infiltration is increased. This embodiment, by comparing various indicators between the damp-heat + DNCB group and the DNCB group, constructs a damp-heat type eczema model that better conforms to the TCM syndrome differentiation. This model can provide a powerful tool for exploring the pathogenesis of damp-heat type eczema and for finding treatment options and developing new drugs for eczema.

Claims

1. A method for establishing an animal model of damp-heat type eczema, characterized in that, A humid-heat type eczema model was induced using DNCB, a high-fat, high-sugar diet, and an artificial climate chamber. The specific steps are as follows: ① High-fat and high-sugar diet treatment: Animals were allowed free access to a high-fat and high-sugar diet from day 1 until the end of the experiment; ② DNCB treatment: Use a mixture of acetone and olive oil in a volume ratio of 4:1 to 3:1 to dissolve DNCB. Sensitization: Before sensitization, the animals were anesthetized by inhalation of isoflurane and their hair was removed. The next day, a 0.1-7% DNCB solution was applied to the back of the animals to induce sensitization, and the application was repeated for 3-5 days. Stimulation: Apply 0.1-7% DNCB solution to the back of the animal to stimulate it, and repeat the stimulation after one day. ③ Artificial climate chamber treatment: Starting from day 1, place the animals in an artificial climate chamber with a temperature of 30-40℃ and a humidity of 80-95% for 1-12 hours each day; do not place the mice in the artificial climate chamber during the sensitization period, and place them in the artificial climate chamber after the mice have recovered their weight; during the challenge period, place the mice in the artificial climate chamber on the day of challenge, take them out and challenge them, and place them in the artificial climate chamber one day apart, for a total of 7-10 challenges; the interval between sensitization and challenge is 3-14 days.

2. The method for establishing an animal model of damp-heat eczema according to claim 1, characterized in that, In step ②, the volume ratio of acetone to olive oil is 3:1; the sensitization concentration of the DNCB solution is 1%, and the activation concentration of the DNCB solution is 1.5%; in step ③, the temperature is 35℃, the humidity is 95%, and the time is 8 hours; the interval between sensitization and activation is 7 days; and a total of 8 activations are performed.

3. The method for establishing an animal model of damp-heat type eczema according to claim 1, characterized in that: The high-fat, high-sugar feed mentioned in step ① is a purified feed containing more than 30% high fat and high sugar, and is given in fixed quantities every day; The acetone and olive oil base solution in step ② should be prepared and used immediately.

4. The method for establishing an animal model of damp-heat type eczema according to claim 1, characterized in that: The process after modeling also includes testing the model animals. The testing methods include the detection of damp-heat type indicators, damp-heat type eczema-related indicators, key indicators of damp-heat type eczema, and immunological and pathological indicators in the damp-heat type eczema animal model.

5. The method for establishing an animal model of damp-heat type eczema according to claim 4, characterized in that, The indicators of damp-heat type eczema in the animal model include the detection of animal weight, food intake, water intake, anal temperature, and grip strength.

6. The method for establishing an animal model of damp-heat type eczema according to claim 4, characterized in that, The aforementioned indicators related to hot and humid eczema include transepidermal water loss, morphology, photographs and scoring of skin lesions, and detection of scratching.

7. The method for establishing an animal model of damp-heat type eczema according to claim 4, characterized in that, The key indicator for the aforementioned damp-heat type eczema is the determination of serum immunoglobulin E levels.

8. The method for establishing an animal model of damp-heat type eczema according to claim 4, characterized in that, The immunological and pathological indicators of the aforementioned damp-heat type eczema include spleen index, skin pathological changes at the lesion site, and mast cell infiltration.

9. The method for establishing an animal model of damp-heat type eczema according to claim 1, characterized in that, The animal in question is a rat or a mouse.

10. The use of an animal model of damp-heat type eczema prepared by the method of claim 1 in screening or developing drugs that can prevent, alleviate or treat damp-heat type eczema.

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