A method for constructing an ulcerative dermatitis mouse model

Abstract

The application relates to the technical field of experimental animal model construction, and particularly discloses a construction method of an ulcerative dermatitis mouse model, which comprises the following steps: first, feeding mice with high-fat feed A for 4 weeks; in the fifth week, feeding the mice with mixed feed of the high-fat feed A and high-fat feed B lacking vitamin B7; in the sixth to twelfth week, feeding the mice with the high-fat feed B lacking vitamin B7; in the sixth to tenth week, smoking the mice in a smoking device for 2-3 hours every day; in the tenth to twelfth week, injecting vancomycin with a concentration of 100-120 mg / ml into the tail veins of the mice, the injection frequency being 2-3 times per week, and the injection amount being 40-60 mg / kg. The construction method of the ulcerative dermatitis mouse model can accurately simulate the pathogenesis of human ulcerative dermatitis, and provides a reliable experimental basis for researching the pathogenesis and treatment methods of the ulcerative dermatitis.

Description

Technical Field

[0001] This application relates to the field of experimental animal model construction technology, specifically to a method for constructing a mouse model of ulcerative dermatitis. Background Technology

[0002] In the field of biomedical research, the construction of animal models is crucial for a deeper understanding of disease pathogenesis and the exploration of effective treatments. Animal models can simulate the occurrence and development of human diseases, providing researchers with a controlled experimental environment that helps accelerate drug development and the innovation of treatment methods. This is especially true for diseases that are difficult to study directly on humans; reliable animal models are indispensable tools. In the field of dermatology research, ulcerative dermatitis is a common and complex disease with multiple factors involved in its pathogenesis, impacting the health of both humans and animals. Establishing animal models of ulcerative dermatitis can provide an important foundation for further research into the pathogenesis and treatment of this disease.

[0003] Currently, the main methods for constructing animal models of ulcerative dermatitis are physical injury, chemical stimulation, and biological infection. Physical injury involves using tools such as scalpels and lasers to cut or burn the animal's skin to create damage, thus simulating ulcerative dermatitis. Chemical stimulation involves applying strong acids or alkalis to the animal's skin to induce inflammation and ulceration. Biological infection involves inoculating the animal with specific pathogens, such as bacteria or fungi, to induce symptoms of ulcerative dermatitis. However, while these methods can create models resembling ulcerative dermatitis to some extent, they cannot accurately simulate the natural pathogenesis and pathological characteristics of human ulcerative dermatitis. This leads to discrepancies between research results and actual conditions, and fails to provide reliable model support for further research into the pathogenesis and treatment of this type of ulcerative dermatitis.

[0004] Therefore, there is an urgent need to establish a reliable animal model of ulcerative dermatitis in order to further study the pathogenesis and treatment of this type of ulcerative dermatitis. Summary of the Invention

[0005] To overcome the shortcomings of existing methods for constructing mouse models of ulcerative dermatitis, which fail to accurately simulate the natural pathogenesis and pathological characteristics of human ulcerative dermatitis and lead to discrepancies between research results and actual situations, this application provides a method for constructing a mouse model of ulcerative dermatitis.

[0006] In one aspect, this application provides a method for constructing a mouse model of ulcerative dermatitis, which adopts the following technical solution: A method for constructing a mouse model of ulcerative dermatitis includes the following steps: First, feed mice with high-fat diet A for 4 weeks; in the 5th week, switch to a mixed diet of high-fat diet A and high-fat diet B lacking vitamin B7; from the 6th to the 12th week, switch to high-fat diet B lacking vitamin B7. During weeks 6-10, mice were placed in a fumigation device for 2-3 hours daily; during weeks 10-12, mice were injected intravenously with vancomycin at a concentration of 100-120 mg / ml, 2-3 times per week, at a dose of 40-60 mg / kg.

[0007] This application provides a method for constructing a mouse model of ulcerative dermatitis. The method begins by feeding mice a high-fat diet (A) for four weeks. This induces rapid weight gain, laying a foundation for the mice's physical condition, and exposes them to a high-fat diet, affecting their metabolic balance. Then, in the fifth week, the diet is changed to a mixture of high-fat diet A and a vitamin B7-deficient high-fat diet (B). This transitional feeding method allows the mice to gradually adapt to the vitamin B7-deficient diet, avoiding excessive stress from sudden dietary changes. From weeks 6 to 12, the mice are fed exclusively with vitamin B7-deficient high-fat diet B. This prolonged vitamin B7 deficiency more effectively induces normal skin metabolism and function, leading to impaired skin barrier function and subsequently triggering inflammation and ulceration. Furthermore, during weeks 6-10, mice were exposed to smoke. Particulate matter from the smoke adhered to the mouse's skin surface. When the mice scratched or their skin was moist, this particulate matter rubbed against broken skin, causing physical abrasions. Simultaneously, tar and polycyclic aromatic hydrocarbons in the smoke penetrated the damaged stratum corneum, directly stimulating blood vessels and nerve endings in the dermis, leading to local redness, increased exudation, and ultimately, skin ulcers. In summary, this application constructs a more realistic mouse model of ulcerative dermatitis using the above methods. This model can more accurately simulate the pathogenesis of human ulcerative dermatitis, providing a reliable experimental basis for further research on the pathogenesis and treatment of this type of ulcerative dermatitis. It helps researchers gain a deeper understanding of the nature of the disease and develop more effective treatment methods.

[0008] Optionally, the high-fat feed B lacking vitamin B7 is made from the following raw materials in parts by weight: 20-23 parts corn starch, 18-22 parts casein, 14-17 parts cocoa butter, 8-12 parts lard, 10-13 parts sucrose, 5-10 parts plant extracts, and 0.8-1.2 parts of a vitamin B7-deficient mixture. The plant extract is selected from one or more of allicin, bromelain, and eugenol.

[0009] The high-fat diet B used in this application lacks vitamin B7. Feeding mice with this diet leads to disruption of normal skin metabolism and function, impaired skin barrier function, and consequently, inflammation and ulceration. Furthermore, the high-fat diet B used in this application also contains a plant extract made from a mixture of allicin, bromelain, and / or eugenol. Allicin, with its strong volatility and fat solubility, can rapidly penetrate the stratum corneum, stimulating vasodilation and causing local redness, swelling, and increased exudation. It can also directly irritate the gastrointestinal mucosa, causing gastrointestinal inflammation. This inflammation releases inflammatory factors through blood circulation, which reach the skin, exacerbating the inflammatory state and inducing or worsening skin ulcers. Bromelain can reach the damaged skin through blood circulation, damaging the protein structure of blood vessel walls, leading to increased local vascular permeability, plasma leakage, and inducing local erythema and blisters. Ruptured blisters form ulcers. Excessive eugenol is converted into quinone toxic metabolites in the body. On the one hand, it can deplete the skin's antioxidants, weakening the skin's own antioxidant defense capabilities and making the skin more susceptible to damage from internal inflammatory factors and external stimuli such as minor friction and bacteria, laying the foundation for ulcer formation. On the other hand, quinone toxic metabolites can induce the massive release of systemic pro-inflammatory factors such as TNF-α and IL-6. These factors act on the skin's microvessels, increasing vascular permeability and causing plasma components to seep into the subcutaneous tissue, leading to skin edema and redness. At the same time, inflammatory factors inhibit the proliferation and migration of keratinocytes, damaging the skin barrier integrity. Under scratching and other actions, this can gradually develop into superficial erosions. The erosions are continuously attacked by inflammation, and the repair ability is inhibited, gradually deepening into the dermis and eventually forming skin ulcers.

[0010] Optionally, the plant extract is a mixture of allicin, bromelain, and eugenol; the weight ratio of allicin, bromelain, and eugenol is 1:(1.5-4):(3-6).

[0011] In some embodiments, the weight ratio of allicin, bromelain, and eugenol is 1:(1.5-2):5, 1:(1.5-2.5):5, 1:(1.5-3):5, 1:(1.5-4):5, 1:(2-2.5):5, 1:(2-3):5, 1:(2-4):5, 1:(2.5-3):5, 1:(2.5-4):5, 1:(3-4):5, 1:2.5:(3-4), 1:2.5:(3-5), 1:2.5:(3-6), 1:2.5:(4-5), 1:2.5:(4-6), or 1:2.5:(5-6).

[0012] In one specific implementation, the weight ratio of allicin, bromelain, and eugenol can also be 1:1.5:5, 1:2:5, 1:2.5:5, 1:3:5, 1:4:5, 1:2.5:3, 1:2.5:4, or 1:2.5:6.

[0013] Optionally, the high-fat feed B lacking vitamin B7 further includes the following ingredients: 6-8 parts maltodextrin, 4-7 parts cellulose, 2-3 parts soybean oil, 1-2 parts cholesterol, 1-2.5 parts calcium carbonate-dicalcium phosphate, 1-2 parts potassium citrate, 0.5-1.5 parts mineral mixture, 0.2-0.4 parts L-cysteine, and 0.1-0.3 parts choline bitartrate.

[0014] Optionally, the high-fat feed A is D12492 high-fat feed.

[0015] Optionally, during the 7 days of the 5th week, the weight ratio of A:B in the mixed feed fed each day is 90:10, 70:30, 50:50, 40:60, 30:70, 20:80, and 10:90, respectively.

[0016] Optionally, the mice in question are 6-week-old APOE mice.

[0017] Secondly, this application provides a mouse model of ulcerative dermatitis, which is obtained using the method for constructing a mouse model of ulcerative dermatitis.

[0018] In summary, this application has the following beneficial effects: 1. This application provides a method for constructing a mouse model of ulcerative dermatitis. The method uses three factors—mouse diet, environmental fumigation, and vancomycin injection—to stimulate mice to develop ulcerative dermatitis. This application can accurately simulate the pathogenesis of human ulcerative dermatitis through the above method. The obtained mouse model of ulcerative dermatitis provides a reliable experimental basis for researchers to deeply understand and study the pathogenesis and treatment of this type of ulcerative dermatitis.

[0019] 2. The high-fat diet B used in this application contains a plant extract made from a mixture of allicin, bromelain, and / or eugenol. These plant extracts can interfere with normal skin metabolism and function, impairing the skin barrier function and thus causing inflammation and ulcers. Experimental studies in this application have shown that further use of a mixture of allicin, bromelain, and eugenol as a plant extract resulted in a better mouse model of ulcerative dermatitis, with an average skin ulcer area reaching 600 mm². 2 above.

[0020] 3. This application further controls the weight ratio of allicin, bromelain, and eugenol within the range of 1:(2-3):(4-5), resulting in an average skin ulcer area of ​​800 mm² in the obtained ulcerative dermatitis mouse model. 2 above. Attached Figure Description

[0021] Figure 1 This is the ulcerative dermatitis mouse model obtained in Example 1 of this application; Figure 2 This is the mouse model of ulcerative dermatitis obtained in Comparative Example 1 of this application; Figure 3 This is the mouse model of ulcerative dermatitis obtained in Comparative Example 2 of this application. Detailed Implementation

[0022] This application provides a method for constructing a mouse model of ulcerative dermatitis, comprising the following steps: (1) Select male 6-week-old APOE mice and feed them with high-fat diet A for 4 weeks to induce rapid weight gain. (2) In the 5th week, the diet was changed to a mixture of high-fat diet A and high-fat diet B lacking vitamin B7. (3) During weeks 6-12, mice were fed a high-fat diet (B) lacking vitamin B7 to induce fat metabolism disorder. During weeks 6-10, mice were placed in a fumigation device for 2-3 hours daily. During weeks 10-12, mice were injected with vancomycin at a concentration of 100-120 mg / ml via the tail vein, 2-3 times per week, at a dose of 40-60 mg / kg. By week 12, ulcerative dermatitis appeared on the ears, head and neck, back, and upper tail root of the mice, thus establishing the ulcerative dermatitis mouse model.

[0023] The high-fat feed A is D12492 high-fat feed; the vitamin B7-deficient high-fat feed B is made from the following ingredients in parts by weight: 20-23 parts corn starch, 18-22 parts casein, 14-17 parts cocoa butter, 8-12 parts lard, 10-13 parts sucrose, 5-10 parts plant extract, 6-8 parts maltodextrin, 4-7 parts cellulose, 2-3 parts soybean oil, 1-2 parts cholesterol, 1-2.5 parts calcium carbonate-dicalcium phosphate, 1-2 parts potassium citrate, 0.5-1.5 parts mineral mixture, 0.8-1.2 parts vitamin B7-deficient mixture, 0.2-0.4 parts L-cysteine, and 0.1-0.3 parts choline bitartrate.

[0024] The plant extract is selected from one or more of allicin, bromelain, and eugenol; further, the plant extract is a mixture of allicin, bromelain, and eugenol; the weight ratio of allicin, bromelain, and eugenol is 1:(1.5-4):(3-6).

[0025] In the embodiments of this application, D12492 high-fat feed was purchased from Speford (Beijing) Biotechnology Co., Ltd.; allicin was garlic extract, purchased from Shaanxi Muling Biotechnology Co., Ltd., catalog number MLSW-DS; bromelain was pineapple extract, purchased from Xi'an Zhongyan Kangze Biotechnology Co., Ltd., CAS number 9001-00-7, 50,000 IU; eugenol was purchased from Xiamen Zhongnongke Chemical New Materials Co., Ltd., CAS number 97-53-0. All other raw materials, reagents, solvents, etc. used in this application can be obtained commercially.

[0026] The present application will be further described in detail below with reference to preparation examples, embodiments, performance testing tests and accompanying drawings.

[0027] Preparation Examples 1-11 Preparation Examples 1-11 provide a high-fat diet B that is deficient in vitamin B7.

[0028] The difference between the above preparation examples lies in the types and weight ratios of the plant extracts, as shown in Table 1 below.

[0029] The preparation method of the above-mentioned high-fat feed B lacking vitamin B7 is as follows: Weigh out 21g of corn starch, 20g of casein, 16g of cocoa butter, 10g of lard, 11g of sucrose, 8g of plant extract, 1g of vitamin B7-deficient mixture (containing vitamin A and vitamin E in a 1:1 weight ratio), 7g of maltodextrin, 5g of cellulose, 2.5g of soybean oil, 1.5g of cholesterol, 2g of calcium carbonate-dicalcium phosphate, 1.5g of potassium citrate, 1g of mineral mixture, 0.3g of L-cysteine, and 0.2g of choline bitartrate; mix the above components evenly to obtain high-fat feed B lacking vitamin B7.

[0030] Table 1. Types and weight ratios of plant extracts used in Preparation Examples 1-11 Examples 1-11 Examples 1-11 provide a method for constructing a mouse model of ulcerative dermatitis.

[0031] The difference between the above embodiments is that the high-fat diet B lacking vitamin B7 used in Examples 1-11 was derived from Preparation Examples 1-11.

[0032] The method for constructing the above-mentioned mouse model of ulcerative dermatitis includes the following steps: (1) Male APOE mice of 6 weeks were selected. The mice were first fed with D12492 high-fat diet for 4 weeks to induce rapid weight gain. The feeding amount was 10g / mouse / day. (2) In the 5th week, the diet was changed to a mixture of D12492 high-fat feed and vitamin B7-deficient high-fat feed B, with a feeding amount of 10g / animal / day; during the 7 days, the weight ratio of A:B in the mixed feed was 90:10, 70:30, 50:50, 40:60, 30:70, 20:80, and 10:90 respectively.

[0033] (3) From week 6 to week 12, mice were fed a high-fat diet (B) lacking vitamin B7 to induce fat metabolism disorder. Meanwhile, from week 6 to week 10, mice were placed in a fumigation device for 2 hours every day. From week 10 to week 12, mice were injected with vancomycin at a concentration of 100 mg / ml via the tail vein twice a week at a dose of 50 mg / kg. In week 12, ulcerative dermatitis appeared on the ears, head and neck, back, and upper tail root of the mice, and the ulcerative dermatitis mouse model was completed.

[0034] Comparative Example 1 Example 1 provides a method for constructing a mouse model of ulcerative dermatitis.

[0035] The difference between Comparative Example 1 and Example 6 is that the mice were not subjected to smoke treatment during weeks 6-10.

[0036] Comparative Example 2 Example 2 provides a method for constructing a mouse model of ulcerative dermatitis.

[0037] The difference between Comparative Example 1 and Example 6 is that the vitamin mixture lacking vitamin B7 (containing vitamin A and vitamin E in a weight ratio of 1:1) was replaced in equal amounts with a vitamin mixture containing vitamin B7 (containing vitamin A, vitamin B and vitamin E in a weight ratio of 1:1:1).

[0038] Performance testing (1) Experimental animals: 39 male 6-week-old APOE mice were randomly divided into 13 groups, with 3 mice in each group.

[0039] (2) Experimental methods: Ulcerative dermatitis mouse models were induced and constructed using the construction methods provided in Examples 1-11 and Comparative Examples 1-2, respectively, with 3 mice induced in parallel for each method; then the skin ulcer area of ​​the mice was measured by ImageJ, and the average value of the 3 mice was taken.

[0040] Table 2. Construction effects of various mouse models of ulcerative dermatitis According to the test results in Table 2, the average skin ulcer area of ​​the mouse models of ulcerative dermatitis constructed in Examples 1-11 was 334.6-837.5 mm². 2 In contrast, the average skin ulcer area of ​​the mouse models of ulcerative dermatitis constructed in Comparative Examples 1-2 was only 32.5-48.1 mm². 2 Therefore, this application demonstrates that feeding mice with a specific high-fat diet and subjecting them to fumigation for a specific period of time can stimulate mice to develop ulcerative dermatitis, thus obtaining a mouse model of ulcerative dermatitis with a pathogenesis essentially consistent with that of human ulcerative dermatitis.

[0041] The test results of Examples 1-11 show that the average skin ulcer area of ​​the mouse model of ulcerative dermatitis constructed using a mixture of allicin, bromelain, and eugenol as plant extracts in high-fat diet B in Examples 4-11 was 664.9-937.5 mm². 2 (≥600mm 2 In contrast, Examples 1-3, which used allicin, bromelain, or eugenol as plant extracts in high-fat diet B, resulted in mouse models of ulcerative dermatitis with an average skin ulcer area of ​​only 334.6-467.1 mm². 2 Therefore, this application demonstrates that using a mixture of allicin, bromelain, and eugenol as plant extracts in high-fat diet B results in a better mouse model of ulcerative dermatitis, with an average skin ulcer area of ​​500 mm². 2 above.

[0042] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for constructing a mouse model of ulcerative dermatitis, characterized in that, Includes the following steps: Mice were first fed a high-fat diet A for 4 weeks; in the 5th week, they were fed a mixture of high-fat diet A and high-fat diet B lacking vitamin B7; from the 6th to the 12th week, they were fed a high-fat diet B lacking vitamin B7. During weeks 6-10, mice were placed in a fumigation device for 2-3 hours daily; during weeks 10-12, mice were injected via tail vein with vancomycin at a concentration of 100-120 mg / ml, 2-3 times per week, at a dose of 40-60 mg / kg. The high-fat feed A is D12492 high-fat feed; The vitamin B7-deficient high-fat feed B is made from the following ingredients in parts by weight: 20-23 parts corn starch, 18-22 parts casein, 14-17 parts cocoa butter, 8-12 parts lard, 10-13 parts sucrose, 5-10 parts plant extract, 0.8-1.2 parts vitamin B7-deficient mixture, 6-8 parts maltodextrin, 4-7 parts cellulose, 2-3 parts soybean oil, 1-2 parts cholesterol, 1-2.5 parts calcium carbonate-dicalcium phosphate, 1-2 parts potassium citrate, 0.5-1.5 parts mineral mixture, 0.2-0.4 parts L-cysteine, and 0.1-0.3 parts choline bitartrate. The plant extract is selected from one or more of allicin, bromelain, and eugenol.

2. The method for constructing a mouse model of ulcerative dermatitis according to claim 1, characterized in that, The plant extract is a mixture of allicin, bromelain, and eugenol; the weight ratio of allicin, bromelain, and eugenol is 1:(1.5-4):(3-6).

3. The method for constructing a mouse model of ulcerative dermatitis according to claim 1, characterized in that, During the 7 days of the 5th week, the weight ratio of A:B in the mixed feed was 90:10, 70:30, 50:50, 40:60, 30:70, 20:80, and 10:90, respectively.

4. The method for constructing a mouse model of ulcerative dermatitis according to any one of claims 1-3, characterized in that, The mice were 6-week-old APOE mice.

Images