Construction method and application of inflammatory bowel disease combined atopic dermatitis animal model

Abstract

The invention relates to a construction method and application of an inflammatory bowel disease combined atopic dermatitis animal model, and relates to the technical field of animal experiment models. The construction method comprises the following steps: applying a dinitrochlorobenzene (DNCB) solution with a first concentration to animal skin for AD sensitization, and after the AD sensitization is finished, continuously applying a dinitrochlorobenzene solution with a second concentration for AD excitation; an animal orally takes a dextran sodium sulfate (DSS) solution with a set concentration for continuous induction to obtain the inflammatory bowel disease combined atopic dermatitis animal model. By taking the animal model as a research object, a concurrent mechanism, early prevention and simultaneous treatment of the inflammatory bowel disease and the atopic dermatitis can be explored, the animal model can also be used for finding and screening multiple-effect drugs, and the dual-effect drug dendrobium officinale with both the inflammatory bowel disease resistance and the atopic dermatitis resistance is finally found.

Description

Technical Field

[0001] This invention relates to the field of animal experimental model technology, and in particular to a method for constructing an animal model of inflammatory bowel disease combined with atopic dermatitis and its application. Background Technology

[0002] Inflammatory Bowel Disease (IBD) is a chronic, relapsing disease. Its pathogenesis mainly involves impaired intestinal mucosal barrier function, intestinal flora imbalance, and dysregulation of the immune cell response to the intestinal flora. Typical clinical manifestations of IBD primarily involve the gastrointestinal tract, including recurrent abdominal pain, mucus or bloody stools, and diarrhea, often accompanied by weight loss, anemia, and fatigue. Its lesions can affect not only the digestive tract but also various extraintestinal organs, including the skin, with related skin diseases including atopic dermatitis (AD). AD, as a systemic inflammatory skin disease, presents with clinical symptoms including intense itching, dryness and desquamation, eczematous erythema, papules, vesicles, exudation, and crusting; prolonged scratching can lead to lichenification of the skin. The close relationship between the etiologies of IBD and AD suggests the possibility of treating different diseases with the same approach.

[0003] Currently, animal models for Alzheimer's disease (AD) and intradermal brain injury (IBD) are relatively well-established. Commonly used inducers for AD animal models include 2,4-dinitrochlorobenzene (DNCB) and oxazolone. Both compounds induce typical AD pathological features such as erythema, edema, and epidermal thickening. Their standard induction protocols typically include sensitization and challenge phases. On the other hand, for IBD animal models, direct ingestion of dextran sulfate sodium (DSS) solution and rectal instillation of trinitrobenzenesulfonic acid (TNBS) are widely used induction methods.

[0004] However, existing studies usually focus on a single disease. Compared to single-disease models, comorbidity models are often difficult to construct while simultaneously and accurately simulating the natural pathological processes of two diseases and ensuring their interaction is similar to human clinical conditions, while also ensuring model reproducibility. All of these factors increase the difficulty of constructing comorbidity models. The comorbidity modeling path is complex, the interaction mechanism is difficult to control, different diseases often involve multiple pathogenic factors, and the superposition or interaction of these factors may lead to superimposed toxicity. Moreover, the induction methods of the two diseases may affect or even exclude each other, and the timing of the occurrence of different diseases is difficult to grasp, all of which may lead to a decrease in the success rate of modeling. When constructing a comorbidity animal model of AD and IBD, we found that simply superimposing the two modeling methods has the following significant drawbacks: (1) the superposition effect of two strong stressors may lead to excessively high animal mortality and the inability to establish a stable model; (2) simple superposition may not be able to simulate the real clinical comorbidity state. Therefore, there is an urgent need for a stable, reliable animal model construction method that can realistically simulate the complex characteristics of clinical comorbidity. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an animal model of inflammatory bowel disease complicated with atopic dermatitis that exhibits low mortality and good reproducibility by screening inducing drug combinations and optimizing dosage ratios. This overcomes the shortcomings of existing technologies, such as simple superposition modeling methods leading to high animal mortality and the inability to simulate real clinical comorbidity. This animal model of inflammatory bowel disease complicated with atopic dermatitis can be used to verify the efficacy of drugs related to the comorbidity.

[0006] The first objective of this invention is to provide a method for constructing an animal model of inflammatory bowel disease (IBD) combined with atopic dermatitis (AD), comprising the following steps:

[0007] S1. Apply a first concentration of dinitrochlorobenzene (DNCB) solution to the skin of animals to induce AD ​​sensitization, and then apply a second concentration of dinitrochlorobenzene solution to stimulate AD stimulation.

[0008] S2. Animals were orally administered a set concentration of dextran sulfate sodium (DSS) solution for further induction to obtain an animal model of inflammatory bowel disease combined with atopic dermatitis.

[0009] Furthermore, the animal in question is a mouse.

[0010] Further, in step S1, the first concentration is 0.8% to 1.2% (w / v); and / or the second concentration is 0.2% to 0.4% (w / v).

[0011] Further, in step S2, the set concentration is 1.5% to 2.5% (w / v).

[0012] Further, in step S1, the AD sensitization includes the following steps: applying a first concentration of dinitrochlorobenzene solution to the skin of the animal 1-3 times within one week, with each application being 100-300 μL.

[0013] Further, in step S1, the AD stimulation includes the following steps: within two weeks after the completion of AD sensitization, applying a second concentration of dinitrochlorobenzene solution once every 2-4 days, with each application being 100-300 μL.

[0014] Furthermore, in step S2, the animals are orally administered a pre-set concentration of sodium dextran sulfate solution for one week.

[0015] Furthermore, the specific steps are as follows:

[0016] (1) AD sensitization stage: The skin of mice was shaved, and the area was 2cm×3cm. From day 1 to day 7, 1.0% DNCB acetone solution was applied to the skin of mice to induce skin lesions, twice a week.

[0017] (2) AD stimulation phase: From day 8 to 21, mice were stimulated by applying 0.4% DNCB solution to their skin every other day.

[0018] (3) IBD induction phase: On days 15-21, the drinking water was replaced with a solution containing 2.5% (w / v) DSS and the mice were allowed to drink freely.

[0019] This invention first explored inducing drugs for constructing an animal model of inflammatory bowel disease (IBD) combined with atopic dermatitis (AD). Through screening various drug combinations that can induce IBD and AD, the optimal drug combination was determined to be DNCB+DSS. Subsequently, the dosages of DNCB and DSS were explored separately. It was found that only 0.2%–0.4% DNCB stimulation combined with 1.5%–2.5% DSS could simultaneously induce AD ​​and IBD and significantly reduce mortality. In particular, using 0.4% DNCB stimulation combined with 2.5% DSS could induce effective disease and reduce inter-group differences in the model. Finally, the model was validated, and the effectiveness of the model was demonstrated through the therapeutic effect of Dendrobium officinale, confirming its application in the screening of drugs for treating IBD combined with atopic dermatitis. Furthermore, we found that the order in which drugs for inducing different diseases are used significantly affects the success of the model and phenotypic stability when constructing an animal model of the two combined diseases. The first disease fundamentally alters the animal's internal physiological environment, providing a different "soil" for the occurrence and development of the second disease. Furthermore, the first drug may alter the animal's metabolism, absorption, distribution, and excretion of the second inducing drug, producing accelerating or inhibiting effects, thereby changing its toxicity or efficacy. Therefore, in the preliminary experimental stage, we designed experimental groups using DNCB followed by DSS, DSS followed by DNCB, and DNCB and DSS simultaneously. We found that the mortality rate in the simultaneous use group was abnormally high, and the DSS followed by DNCB group could not achieve synergy between the two. Only the DNCB followed by DSS approach of this invention can establish an effective disease comorbidity model.

[0020] The second objective of this invention is to provide an animal model of inflammatory bowel disease complicated with atopic dermatitis constructed by the above-described method.

[0021] A third objective of this invention is to provide the application of the animal model of inflammatory bowel disease combined with atopic dermatitis in screening therapeutic drugs for inflammatory bowel disease combined with atopic dermatitis.

[0022] A fourth objective of this invention is to provide the application of the animal model of inflammatory bowel disease combined with atopic dermatitis in the preparation of a drug screening model for the treatment of inflammatory bowel disease combined with atopic dermatitis.

[0023] The fifth objective of this invention is to provide the application of Dendrobium officinale in the preparation of a treatment for inflammatory bowel disease complicated with atopic dermatitis.

[0024] By means of the above-described solution, the present invention has at least the following advantages:

[0025] 1. This invention effectively overcomes the core obstacle of high animal mortality caused by simple model stacking by screening drug combinations and optimizing stimulation dosages, making the successful construction of this comorbidity model possible. Subsequently, a systematic evaluation was conducted using multiple dimensions of indicators, including mouse body weight change, disease activity index (DAI), and AD skin lesion score. The results show that this model has a short experimental cycle, high success rate, strong reproducibility, and is closer to the actual situation of clinical patients, providing an easily scalable animal model for subsequent research on complex comorbidity mechanisms and drug evaluation.

[0026] 2. This invention evaluates the animal model of inflammatory bowel disease combined with atopic dermatitis constructed in this invention, and uses this model as a platform to discover and screen dual-effect drugs that simultaneously combat inflammatory bowel disease and atopic dermatitis. This provides a research foundation and data support for studying the pleiotropic activity of traditional Chinese medicine and exploring its "treating different diseases with the same method" mechanism. This invention confirms that the traditional Chinese medicine Dendrobium officinale can effectively alleviate both DNCB-induced skin inflammation and DSS-induced colitis, manifested as improved body weight, decreased DAI (diabetic arterial inflammation), reduced skin lesion score, and decreased IgE level.

[0027] 3. This invention simultaneously meets the relevant requirements for animal models of inflammatory bowel disease and atopic dermatitis. It can not only be used as a research subject to explore the concurrent mechanism, early prevention and simultaneous treatment of inflammatory bowel disease and atopic dermatitis, but also can be used for the discovery and screening of drugs with multiple effects.

[0028] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 This invention provides a schematic diagram of the method for establishing an animal model of inflammatory bowel disease complicated with atopic dermatitis and its drug intervention.

[0031] Figure 2 The change in body weight of mice in each group as described in Example 2 of this invention, where n = 6.

[0032] Figure 3 The results are the DAI scores of each group of mice described in Example 2 of this invention, where n = 6.

[0033] Figure 4 The skin lesion scores of each group of mice described in Example 2 of the present invention are given, where n = 6.

[0034] Figure 5 The results of IgE concentration detection in the serum of mice in each group described in Example 2 of the present invention are shown, where n = 6. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0036] The technical solution involved in this invention is as follows:

[0037] This invention provides a method for establishing an animal model of inflammatory bowel disease combined with atopic dermatitis, comprising the following steps: (1) Preparation of experimental animals: Six-week-old male C57BL / 6 mice were selected and acclimatized for one week. (2) AD sensitization stage: The skin on the back of the mice was shaved, covering an area of ​​about 2cm×3cm. From day 1 to day 7, 1.0% DNCB acetone solution (DNCB dissolved in a mixture of acetone and olive oil, with a volume ratio of 3:1) was applied to the back (200μL) and ears (15μL) of the mice to induce skin lesions, twice a week. (3) AD stimulation stage: From day 8 to day 21, 0.4% DNCB solution was applied to the back (200μL) and ears (15μL) of the mice every other day to stimulate stimulation. (4) IBD induction stage: From day 15 to day 21, the drinking water was replaced with a solution containing 2.5% (w / v) DSS, and the mice were allowed to drink freely.

[0038] During this period, the mice's body weight, skin condition, diarrhea, and bloody stools were observed and recorded daily. After the model was established, serum, skin tissue, and colon tissue were collected for further analysis.

[0039] The evaluation criteria for the successful establishment of the comorbidity model were as follows: compared with the single-disease model group, the mice in the comorbidity model group showed the following synergistic effects: the rate of weight loss and DAI were significantly greater than those in the IBD-only group. The AD skin lesion score was significantly higher than that in the AD-only group.

[0040] Example 1

[0041] This embodiment provides a method for establishing an animal model of inflammatory bowel disease combined with atopic dermatitis, including the following steps: (1) AD sensitization: The skin on the back of the mouse was shaved, covering an area of ​​about 2cm × 3cm. From day 1 to day 7, 1.0% DNCB acetone solution (w / v, DNCB dissolved in a mixture of acetone and olive oil, with a volume ratio of 3:1) was applied to the back (200μL) and ears (15μL) of the mouse to induce skin lesions, twice (days 1 and 4, the same below); (2) AD challenge: From day 8 to day 21, 0.4% (w / v) DNCB solution was applied to the back (200μL) and ears (15μL) of the mouse to challenge the disease at a frequency of once every other day; (3) The inflammatory bowel disease model was established from day 15 to day 21. The mice were allowed free access to 2.5% DSS solution (w / v, 40kDa) to induce inflammatory bowel disease.

[0042] Example 2

[0043] This embodiment provides a method for establishing an animal model of inflammatory bowel disease combined with atopic dermatitis, and its application in verifying drug efficacy. The specific steps are as follows: Six-week-old healthy male SPF-grade C57BL / 6 mice were randomly divided into 5 groups (n=6) after one week of adaptive feeding: blank control group (CTL), DNCB-induced AD model group (DNCB), DSS-induced IBD model group (DSS), DNCB combined with DSS-induced atopic dermatitis-inflammatory bowel disease comorbidity model group (DD), and the traditional Chinese medicine Dendrobium officinale solution treatment group (DOE). The mouse animal model of inflammatory bowel disease combined with atopic dermatitis was established according to the method described in Example 1. From day 15 to 21, the candidate traditional Chinese medicine Dendrobium officinale (DOE) was administered to the animal model of inflammatory bowel disease combined with atopic dermatitis of this invention. The drug was administered by gavage at a dose of 250 mg / kg / day from day 8 to 21 of the experiment.

[0044] The method for establishing the DNCB group includes the following steps: (1) AD sensitization: Shave the skin on the back of the mouse, with an area of ​​about 2cm×3cm. On days 1-7, apply 1.0% DNCB acetone solution (w / v, DNCB is dissolved in a mixture of acetone and olive oil, with a volume ratio of 3:1) to the back (200μL) and ears (15μL) of the mouse to induce skin lesions, for a total of 2 times; (2) AD challenge: On days 8-21, challenge the mouse by applying 0.4% (w / v) DNCB solution to the back (200μL) and ears (15μL) at a frequency of once every other day.

[0045] The DSS group establishment method includes the following steps: (1) On days 1-7, apply a mixture of acetone and olive oil (volume ratio 3:1) to the back (200 μL) and ears (15 μL) of mice twice; (2) On days 8-21, apply a mixture of acetone and olive oil to the back (200 μL) and ears (15 μL) of mice every other day; (3) Establish the inflammatory bowel disease model from days 15-21. Mice were allowed free access to 2.5% DSS solution (w / v, 40 kDa) to induce inflammatory bowel disease.

[0046] Comparative Example 1

[0047] The difference between this comparative example and Example 1 is that the method for inducing IBD is rectal instillation of TNBS. The steps are as follows: (1) AD sensitization: The skin on the back of the mouse was shaved, covering an area of ​​about 2cm × 3cm. From day 1 to day 7, 1.0% DNCB acetone solution (w / v, DNCB dissolved in a mixture of acetone and olive oil, with a volume ratio of 3:1) was applied to the back (200μL) and ears (15μL) of the mouse to induce skin lesions, for a total of 2 times; (2) AD challenge: From day 8 to day 21, 0.4% (w / v) DNCB solution was applied to the back (200μL) and ears (15μL) of the mouse for challenge at a frequency of once every other day; (3) On day 15, the mouse was fasted for 4 hours. Subsequently, 1.5mg of TNBS (dissolved in 100μL of 50% (v / v) ethanol aqueous solution) was instilled into the rectum via the anus. During infusion, the animal was lightly anesthetized, and a round-tipped gavage needle or catheter was gently inserted into the rectum about 3-4 cm. The solution was slowly injected, and the mouse was kept in an inverted position for 60 seconds to ensure that the solution was fully retained. The animal was then observed until day 21.

[0048] Comparative Example 2

[0049] The only difference between this comparative example and Example 1 is that the drug used to induce AD ​​is Oxazolone. The steps are as follows: (1) AD sensitization: The skin on the back of the mice was shaved, covering an area of ​​about 2cm × 3cm. From day 1 to day 7, 1.0% Oxazolone acetone solution (w / v, Oxazolone dissolved in a mixture of acetone and olive oil, with a volume ratio of acetone to olive oil of 3:1) was applied to the back (200μL) and ears (15μL) of the mice to induce skin lesions, twice in total; (2) AD challenge: From day 8 to day 21, 0.4% (w / v) Oxazolone solution was applied to the back (200μL) and ears (15μL) of the mice every other day to challenge them; (3) The inflammatory bowel disease model was established from day 15 to day 21. Mice were allowed free access to 2.5% DSS solution (w / v, 40kDa) to induce inflammatory bowel disease.

[0050] Comparative Example 3

[0051] The difference between this comparative example and Example 1 is that the drug used to induce AD ​​is Oxazolone, and the method used to induce IBD is rectal instillation of TNBS. The steps are as follows: (1) AD sensitization: The skin on the back of the mouse was shaved, covering an area of ​​about 2cm × 3cm. From day 1 to day 7, 1.0% Oxazolone acetone solution (w / v, Oxazolone dissolved in a mixture of acetone and olive oil, with a volume ratio of 3:1) was applied to the back (200μL) and ears (15μL) of the mouse to induce skin lesions, twice in total; (2) AD challenge: From day 8 to day 21, 0.4% (w / v) Oxazolone solution was applied to the back (200μL) and ears (15μL) of the mouse to challenge the AD, once every other day; (3) On day 15, the mouse was fasted for 4 hours. Subsequently, 1.5mg of TNBS (dissolved in 100μL of 50% (v / v) ethanol aqueous solution) was instilled into the rectum. During infusion, the animal was lightly anesthetized, and a round-tipped gavage needle or catheter was gently inserted into the rectum about 3-4 cm. The solution was slowly injected, and the mouse was kept in an inverted position for 60 seconds to ensure that the solution was fully retained. The animal was then observed until day 21.

[0052] Examples 3-6

[0053] The steps are the same as in Example 1, except that the dosage of the inducing drug is different, as detailed in Table 1 below:

[0054] Table 1 shows the difference in induction drug dosage between Example 1 and Example 1.

[0055] Group DNCB excitation concentration DSS-induced concentration Example 3 1％ 2.5％ Example 4 0.4％ 3.5％ Example 5 0.2％ 2.5％ Example 6 0.4％ 1.5％

[0056] Test case

[0057] (1) Mortality rate

[0058] The survival status and number of deaths of mice were observed and recorded daily. The cumulative mortality rate was calculated at the end of the experiment, using the initial number of animals in each group as the denominator. The formula was: Mortality rate (%) = (Number of dead mice / Initial number of mice) × 100%.

[0059] (2) Mouse body weight change rate and decrease rate

[0060] Record the mice's weight daily. Using the weight before IBD modeling as a baseline (W0), calculate the weight loss rate (%) using the following formula: [(W0 - W0)] t [W0]×100%, weight change rate (%) = W t / W0×100%, where W t Weight at the end of IBD modeling.

[0061] (3) Disease Activity Index (DAI) score

[0062] According to the DAI scoring criteria, mouse body weight, fecal viscosity, and fecal occult blood were observed and scored daily to assess the severity of colitis in mice. The mouse disease activity index scoring criteria described in the embodiments and comparative examples of this invention are shown in Table 2 below:

[0063] Table 2 Disease Activity Index Scoring Criteria

[0064]

[0065]

[0066] (4) Assessment of the severity of skin lesions in mice with atopic dermatitis

[0067] The severity of dermatitis in mice was assessed on day 21. The specific criteria were based on four clinical symptoms of Alzheimer's disease (AD): erythema, edema, erosion, and scaling. The scoring system was 0 (none), 1 (mild), 2 (moderate), and 3 (severe). The final dermatitis score was the sum of all scores.

[0068] (5) Serum IgE concentration detection

[0069] Blood samples from mice in each group were obtained using the enucleation method in Example 2 and placed in EP tubes, then incubated at room temperature for 2 hours. The EP tubes were then centrifuged at 4°C (1000 g / min × 15 min). The supernatant was collected and stored at -80°C for later testing. Before testing, the samples needed to be thawed, mixed, and centrifuged at 3000 rpm for a total of 10 min. Serum immunoglobulin E (IgE) levels were detected using enzyme-linked immunosorbent assay (ELISA). The specific procedure was performed according to the kit instructions. After incubation, the reaction was terminated, and the optical density (OD) was measured using a microplate reader. A standard curve was constructed, an equation was fitted, and the sample concentration was calculated based on the OD values.

[0070] Statistical analysis

[0071] Experimental data are expressed as mean ± standard deviation (SEM). The T-test was used for comparisons between two groups. For comparisons involving only a single factor, one-way ANOVA was used; for comparisons involving two independent factors, two-way ANOVA was used. Tukey's test was used to determine significance. *p < 0.05 and **p < 0.01 were considered statistically significant. Data were analyzed using GraphPad Prism 9.5.

[0072] The results are as follows:

[0073] The mortality rate, weight loss rate, DAI score, and AD score on the last day of the experiment are shown in Table 3 below:

[0074] Table 3 Test Results

[0075]

[0076] Compared with the DD group of Example 2, *p<0.05 and **p<0.01 were the criteria for statistical significance.

[0077] As shown in Table 3, the DD group in Example 2 showed the best overall effect, with a mortality rate of 0%, indicating model stability. In addition, the weight loss rate (18.2±1.1%) was significantly higher than that in the DSS group (12.4±1.5%, p<0.05); the DAI score (9.7±0.2) was significantly higher than that in the DSS group (7.8±0.4, p<0.01); and the AD score (9.8±0.5) was significantly higher than that in the DNCB group (7.7±0.4, p<0.05), demonstrating that the two diseases exacerbate each other when they coexist, rather than simply adding to each other.

[0078] In contrast, Comparative Examples 1 and 3, due to the strong stimulation of rectal instillation of TNBS, showed significantly higher rates of weight loss (22.8±1.6% and 24.8±0.8%, respectively, p<0.01), significantly higher DAI scores (11.2±0.2, p<0.05 and 11.5±0.2, respectively), and significantly higher mortality rates (33.3%, p<0.01 and 50.0%, respectively). This is likely due to the additive effect of the strong intestinal inflammatory response induced by TNBS and the skin inflammation, leading to severe systemic reactions in the mice. This excessively strong stimulation results in poor model stability and large individual variability, failing to meet the basic requirements of reproducibility and operability for comorbidity studies. Therefore, TNBS is not suitable for establishing animal models of comorbidity of inflammatory bowel disease and atopic dermatitis. Although Comparative Example 2 had a higher AD score (11.3±0.2), its mortality rate was 16.7% (p<0.01). Therefore, oxazolone is not suitable for establishing an animal model of comorbid inflammatory bowel disease and atopic dermatitis.

[0079] After determining that DNCB and DSS were the optimal combination, the dosage was further optimized. The results are shown in Table 3. The dosage (0.4% DNCB stimulation + 2.5% DSS) used in the DD group of Example 2 was the optimal dosage combination. Example 3 (1.0% DNCB provocation + 2.5% DSS) resulted in an increased AD score of 11.2 ± 0.4, but the mortality rate reached 16.7%; Example 4 (0.4% DNCB provocation + 3.5% DSS) caused severe colitis, with the DAI score rising to 11.2 ± 0.3 (p<0.05), but the mortality rate was as high as 50%; Example 5 (0.2% DNCB provocation + 2.5% DSS) significantly reduced the AD score to 6.8 ± 0.5 (p<0.01), lower than the single AD model group, and was insufficient to induce effective skin inflammation; Example 6 (0.4% DNCB provocation + 1.5% DSS) significantly reduced the DAI score to 7.2 ± 0.4 (p<0.01), lower than the single IBD model group, indicating insufficient intestinal inflammation induction. These results demonstrate that the dosage combination in Example 1 is key to achieving balanced disease induction and ensuring animal survival.

[0080] Based on the established optimization model, the efficacy of Dendrobium officinale extract was verified. The efficacy experiment results are as follows: Figures 2-5As shown, the combination of DNCB-induced atopic dermatitis and DSS-induced inflammatory bowel disease caused significant skin and intestinal damage in mice, including weight loss, elevated DAI (diabetic arterial blood lipids), and increased skin lesion scores. However, after two weeks of continuous intervention with DOE (densitosterol), the weight loss and elevated DAI in mice were significantly alleviated (P<0.05), indicating that DOE has a mitigating effect on inflammatory bowel disease. Simultaneously, the AD symptoms in mice also improved, as evidenced by a significant decrease in skin lesion scores and serum IgE concentrations (P<0.05). This demonstrates that the model can effectively evaluate the efficacy of drug treatment, and that Dendrobium officinale has a significant therapeutic effect on the comorbidity model.

[0081] In summary, this embodiment verifies the feasibility of the animal model constructed in this invention, which can be used to screen and discover dual-effect drugs that can simultaneously improve inflammatory bowel disease and atopic dermatitis. The candidate drug DOE showed varying degrees of improvement in the relevant quantitative indicators of this model, demonstrating that DOE has dual effects against inflammatory bowel disease and atopic dermatitis.

[0082] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing an animal model of inflammatory bowel disease complicated with atopic dermatitis, characterized in that, Includes the following steps: S1. Apply a first concentration of dinitrochlorobenzene solution to the skin of animals to induce AD ​​sensitization, and then apply a second concentration of dinitrochlorobenzene solution to stimulate AD stimulation. S2. Animals were orally administered a pre-set concentration of dextran sulfate sodium solution for further induction to obtain an animal model of inflammatory bowel disease combined with atopic dermatitis.

2. The construction method according to claim 1, characterized in that, In step S1, the second concentration is 0.2% to 0.4% (w / v); And / or, in step S2, the set concentration is 1.5% to 2.5% (w / v).

3. The construction method according to claim 1 or 2, characterized in that, In step S1, the first concentration is 0.8% to 1.2% (w / v).

4. The construction method according to any one of claims 1-3, characterized in that, In step S1, the AD sensitization includes the following steps: applying a first concentration of dinitrochlorobenzene solution to the skin of the animal 1-3 times within one week, with each application being 100-300 μL; And / or, in step S1, the AD stimulation includes the step of: applying a second concentration of dinitrochlorobenzene solution once every 2-4 days within two weeks after the end of AD sensitization, with each application being 100-300 μL; And / or, in step S2, the animal is orally administered a set concentration of sodium dextran sulfate solution for one week.

5. The construction method according to claim 1, characterized in that, The specific steps are as follows: (1) AD sensitization stage: The skin of mice was shaved, and the area was 2cm×3cm. From day 1 to day 7, 1.0% DNCB acetone solution was applied to the skin of mice to induce skin lesions, twice a week. (2) AD stimulation phase: From day 8 to 21, mice were stimulated by applying 0.4% DNCB solution to their skin every other day. (3) IBD induction phase: On days 15-21, the drinking water was replaced with a solution containing 2.5% (w / v) DSS and the mice were allowed to drink freely.

6. The construction method according to claim 1, characterized in that, The animal in question is a mouse.

7. An animal model of inflammatory bowel disease combined with atopic dermatitis constructed by the construction method according to any one of claims 1-6.

8. The use of the animal model of inflammatory bowel disease complicated with atopic dermatitis as described in claim 7 in screening drugs for the treatment of inflammatory bowel disease complicated with atopic dermatitis.

9. The application of the animal model of inflammatory bowel disease combined with atopic dermatitis as described in claim 7 in the preparation of a drug screening model for the treatment of inflammatory bowel disease combined with atopic dermatitis.

10. Application of Dendrobium officinale in the preparation of drugs for the treatment of inflammatory bowel disease complicated with atopic dermatitis.

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