Establishment method and application of damp-heat type eczema animal model

Abstract

The invention relates to the technical field of experimental animal disease models, and discloses an establishment method and application of a damp-heat type eczema animal model. The method comprises the following steps: firstly, enabling animals to eat improperly (the content of high-fat feed is increased from 40wt%-50wt% to 100wt% and is kept unchanged) and to feel damp-heat pathogens (the temperature and the humidity are respectively increased from 24-26 DEG C and 70-80% to 28-32 DEG C and 90-100% and are kept unchanged and baijiu is drenched every other day); and then 0.1-7wt% of dinitrochlorobenzene solution is smeared on the abdomen and the back of the animal for sensitization. The eczema animal model and the traditional Chinese medicine damp-heat syndrome animal model are organically combined together, and the ideal damp-heat type eczema animal model is successfully established. The model has high repeatability, is closer to clinical manifestation of patients with damp-heat type eczema, and is beneficial to pathogenesis research of damp-heat type eczema and research and development and efficacy evaluation of new traditional Chinese medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of experimental animal disease models, and in particular to a method for establishing an animal model of damp-heat type eczema and application thereof. Background Art

[0002] Damp-Heat Eczema is a disease caused by a variety of complex internal and external factors. The etiology is complex and the pathogenesis is not yet fully understood. It is divided into acute, subacute and chronic stages, with different pathological manifestations in different stages. A large number of basic and clinical studies have shown that damp-heat eczema is a damp-heat syndrome accompanied by allergic inflammatory diseases, characterized by poor spleen and stomach function, abnormal organ morphology, internal accumulation of dampness and heat, mental depression, body weight loss, and epidermal exudation and erosion, lichenification of skin lesions and scaly pathological changes.

[0003] The Global Burden of Disease study shows that the prevalence of eczema in children is about 20%, and the prevalence in adults is as high as 10%. It ranks 15th among common non-fatal diseases, and the number of patients is increasing year by year. Due to the continuous increase in the number of cases of damp-heat eczema, the socioeconomic burden is becoming increasingly obvious, but the existing drugs for the treatment of damp-heat eczema can only delay the progression of the disease and improve symptoms, but cannot cure it and have obvious side effects. Over the years, global pharmaceutical companies have invested huge amounts of money in the research and development of related drugs, but the efficacy of the drugs developed is not obvious. The main reason is that the commonly used rodent models cannot simulate the real situation of patients, resulting in many candidate drugs failing in clinical trials. Therefore, constructing an animal model that can simulate the real situation of patients has always been a major problem that needs to be solved urgently in the pathogenesis research and drug development in this field.

[0004] Rodents are widely used in research due to their low price, wide resources, high survival rate, clear genetic background, mature genetic manipulation and low breeding cost. However, a single eczema model cannot simultaneously show all the typical symptoms of damp-heat eczema, and a single eczema model does not conform to the pathogenesis and etiology of eczema diseases in the scope of traditional Chinese medicine, and the pathological symptoms are unstable. Therefore, the rodent damp-heat eczema model cannot simulate the real situation of human patients, and the clinical results of new drugs verified by its model are difficult to successfully transform.

[0005] The structure and function of the skin of rodent rats are similar to those of humans. The basic structure of the skin includes the epidermis, dermis and subcutaneous tissue, which can prevent the invasion of foreign substances, prevent excessive loss of water, play a certain protective role for the body, and have the ability to perceive external stimuli. After being damaged, it has a certain self-repair function and restores the integrity of the skin through cell proliferation and differentiation. It can complete the construction of complex skin models commonly used in clinical practice, and the results are highly interoperable. Studies have shown that the selection of a single eczema model cannot obtain an animal model that exhibits both damp-heat syndrome and eczema-like changes. Therefore, the establishment of an animal model of damp-heat eczema with both damp-heat syndrome and eczema-like changes is of great significance for the evaluation of new therapies; among them, damp-heat syndrome is mainly manifested in mental state, emotional changes, drowsiness, hair sheen, perianal condition and defecation; eczema-like is mainly manifested in epidermal changes, body weight, pathological conditions and changes in organ indexes. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems existing in the prior art. On the one hand, the present invention provides a method for establishing an animal model of damp-heat type eczema, which comprises the following steps:

[0007] (1) Normal feeding: 5-6 week old SD rats were fed with water and regular feed for 3-7 days;

[0008] (2) Gradient feeding: Gradient feeding of normally fed SD rats for 2-4 days, wherein the gradient feeding process is carried out in an artificial climate chamber, and the SD rats are placed in the artificial climate chamber for 8-12 hours every day, and the gradient feeding feed contains conventional feed and high-fat feed;

[0009] The high-fat feed content in the gradient feeding feed and the temperature and humidity in the artificial climate box are increased gradually day by day, wherein the high-fat feed content in the gradient feeding feed is increased gradually from 40wt%-50wt% to 100wt%, and the temperature and humidity in the artificial climate box are increased gradually from 24-26°C and 70-80% to 28-32°C and 90-100% respectively;

[0010] (3) High-fat feeding: The SD rats that had undergone gradient feeding were fed with a 100 wt% high-fat diet for 12-19 days, wherein the high-fat feeding process was carried out in an artificial climate chamber, and the SD rats were placed in the artificial climate chamber for 8-12 hours every day, and the temperature and humidity in the artificial climate chamber were 28-32° C. and 90-100%, respectively;

[0011] During the high-fat feeding period, the abdominal region of the SD rats was sensitized with a 0.1-7wt% dinitrochlorobenzene solution for 5-7 days on the 2nd to 4th day, and then the back region of the SD rats was re-sensitized with a 0.1-7wt% dinitrochlorobenzene solution for 3-5 days after 1-2 days;

[0012] During the gradient feeding and high-fat feeding process, the SD rats were gavaged with liquor every other day, and the amount of liquor was 9-11 mL / kg.

[0013] Preferably, in step (1), the ingredients of the conventional feed include corn, bran, flour, soybean meal, fish meal, bone meal and salt.

[0014] Preferably, in step (1), the amount of the conventional feed is 120-15 g / kg.

[0015] Preferably, in step (2), the gradient of increasing the content of high-fat feed in the gradient feeding feed is 20-25wt% / d; and / or

[0016] In step (2), the temperature in the artificial climate box increases at a gradient of 1-2°C / d; and / or

[0017] In step (2), the humidity in the artificial climate box increases at a gradient of 10-15% / d.

[0018] Preferably, in step (2), the gradient of increasing the content of high-fat feed in the gradient feeding feed is 25wt% / d; and / or

[0019] In step (2), the temperature in the artificial climate box increases at a gradient of 2°C / d; and / or

[0020] In step (2), the humidity in the artificial climate box increases at a gradient of 25% / d.

[0021] Preferably, in step (2), the initial content of the high-fat feed in the gradient feeding feed is 50wt%; and / or

[0022] In step (2), the initial temperature in the artificial climate box is 26°C; and / or

[0023] In step (2), the initial humidity in the artificial climate box is 75%.

[0024] Preferably, the high-fat feed comprises lard, egg yolk powder and sugar; and / or

[0025] In step (2), the amount of the gradient feeding feed is 65-80 g / kg.

[0026] Preferably, in step (3), during the sensitization of the abdominal region of the SD rat, the amount of the 0.1-7wt% dinitrochlorobenzene solution is 20-30 μL / cm 2 ; and / or

[0027] In step (3), during the sensitization process of the back region of the SD rat, the dosage of the 0.1-7wt% dinitrochlorobenzene solution is 10-15 μL / cm 2 .

[0028] Preferably, in step (3), the amount of the high-fat feed is 65-80 g / kg.

[0029] The second aspect of the present invention provides an animal model of damp-heat type eczema constructed by the above construction method.

[0030] The third aspect of the present invention provides the application of the damp-heat type eczema animal model in the field of medical technology.

[0031] In the present invention, a damp-heat type eczema SD rat model can be successfully cultivated by combining the traditional eczema model with the damp-heat syndrome model. The damp-heat type eczema rat model is an eczema model with two pathological changes of eczema-like changes and damp-heat symptoms, and both the apparent indicators and organ tissues show damp-heat symptoms and eczema-like changes.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] (1) The animal model established in the present invention is a rat model. The skin structure and functional structure of rats are similar to those of humans. It is the first time that damp-heat syndrome is combined with eczema, which is in line with the pathogenesis and principles of TCM theory. It provides a model for TCM treatment of eczema and can complete the construction of complex skin models commonly used in clinical practice. The results are highly interoperable, and the clinical results of new drugs verified by the model are easy to be successfully transformed;

[0034] (2) The damp-heat type eczema animal model of the present invention enables SD rats to better adapt to the high-fat feeding conditions through the gradient feeding steps, and the model formation rate is high;

[0035] (3) The damp-heat type eczema animal model of the present invention is placed in an artificial climate box and administered with liquor every other day. In the construction of the damp-heat type eczema model, administration of liquor has the following effects:

[0036] 1. Damage to the spleen and stomach: According to TCM theory, excessive drinking can easily damage the spleen and stomach functions. Damage to the spleen and stomach may affect the metabolism and transportation of water, leading to endogenous water and dampness.

[0037] 2. Generates dampness and promotes heat: Liquor is a hot substance. When it enters the body, it may generate internal heat. The stagnation of water and dampness caused by abnormal spleen and stomach transportation and transformation interacts with internal heat, which can easily lead to a damp-heat pathological state.

[0038] 3. Affecting the circulation of Qi and blood: Long-term or heavy drinking may affect the normal circulation of Qi and blood, causing Qi and blood stasis, further aggravating the formation of dampness and heat;

[0039] 4. Simulate bad living habits: In real life, long-term excessive drinking is a bad living habit, which may be one of the factors leading to damp-heat constitution or related diseases. By administering liquor in the model, the effect of this bad factor on the body can be simulated;

[0040] (4) The damp-heat type eczema animal model described in the present invention has a relatively long maintenance period of eczema-like symptoms and damp-heat syndrome. After modeling, the apparent indicators, damp-heat symptoms of organ tissues and eczema-like symptoms change significantly, which is convenient for observation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram of the results of the damp-heat condition (mental state and fur color) of the blank group SD rats and the model group SD rats in Example 1;

[0042] Figure 2 Schematic diagram of the results of damp-heat condition (stool) of SD rats in the blank group and the model group in Example 1;

[0043] Figure 3 Schematic diagram of the eczema-like results of the ventral and dorsal areas of the SD rats in the blank group and the SD rats in the model group in Example 1;

[0044] Figure 4 Schematic diagram of the pathological condition (HE) of the back skin lesions of the SD rats in the blank group and the SD rats in the model group in Example 1;

[0045] Figure 5 Schematic diagram of colon histopathological condition (HE) results of SD rats in the blank group and the model group in Example 1;

[0046] Figure 6 Schematic diagram of the results of gastric lining pathological conditions (HE) of SD rats in the blank group and the model group in Example 1;

[0047] Figure 7 Schematic diagram of the results of body weight, spleen index, stomach index and liver index of SD rats in the blank group and the model group in Example 1. DETAILED DESCRIPTION

[0048] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0049] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0050] In one aspect, the present invention provides a method for establishing an animal model of damp-heat type eczema, the method comprising the following steps:

[0051] (1) Normal feeding: 5-6 week old SD rats were fed with water and regular feed for 3-7 days;

[0052] (2) Gradient feeding: Gradient feeding of normally fed SD rats for 2-4 days, wherein the gradient feeding process is carried out in an artificial climate chamber, and the SD rats are placed in the artificial climate chamber for 8-12 hours every day, and the gradient feeding feed contains conventional feed and high-fat feed;

[0053] The high-fat feed content in the gradient feeding feed and the temperature and humidity in the artificial climate box are increased gradually day by day, wherein the high-fat feed content in the gradient feeding feed is increased gradually from 40wt%-50wt% to 100wt%, and the temperature and humidity in the artificial climate box are increased gradually from 24-26°C and 70-80% to 28-32°C and 90-100% respectively;

[0054] (3) High-fat feeding: The SD rats that had undergone gradient feeding were fed with a 100 wt% high-fat diet for 12-19 days, wherein the high-fat feeding process was carried out in an artificial climate chamber, and the SD rats were placed in the artificial climate chamber for 8-12 hours every day, and the temperature and humidity in the artificial climate chamber were 28-32° C. and 90-100%, respectively;

[0055] During the high-fat feeding period, the abdominal region of the SD rats was sensitized with a 0.1-7wt% dinitrochlorobenzene solution for 5-7 days on the 2nd to 4th day, and then the back region of the SD rats was re-sensitized with a 0.1-7wt% dinitrochlorobenzene solution for 3-5 days after 1-2 days;

[0056] During the gradient feeding and high-fat feeding process, the SD rats were gavaged with liquor every other day, and the amount of liquor was 9-11 mL / kg.

[0057] In the present invention, in step (2), "the content of high-fat feed in the gradient-fed feed and the temperature and humidity in the artificial climate box are both increased gradually day by day, wherein the content of high-fat feed in the gradient-fed feed is increased gradually from 40wt%-50wt% to 100wt%, and the temperature and humidity in the artificial climate box are increased gradually from 24-26°C and 70-80% to 28-32°C and 90-100% respectively" means that the content of high-fat feed in the gradient-fed feed, the temperature and humidity in the artificial climate box are increased gradually from 40wt%-50wt%, 24-26°C and 70-80% to 28-32°C and 90-100% respectively. -26℃ and 70-80% are gradually increased to 100wt%, 28-32℃ and 90%-100% over the same period of time; for example, assuming that the number of days of gradient feeding is 3 days, on the first day, the content of high-fat feed in the feed is 50wt%, and the temperature and humidity in the artificial climate box are 26℃ and 75% respectively; on the second day, the content of high-fat feed in the feed is 75wt%, and the temperature and humidity in the artificial climate box are 28℃ and 85% respectively; on the third day, the content of high-fat feed in the feed is 100wt%, and the temperature and humidity in the artificial climate box are 30℃ and 95% respectively.

[0058] In the present invention, the "humidity" refers to "relative humidity".

[0059] In a preferred embodiment, in step (1), the ingredients of the conventional feed include corn, bran, flour, soybean meal, fish meal, bone meal and salt.

[0060] Further preferably, the nutritional components of the conventional feed are as shown in Table 1.

[0061] Table 1

[0062] project Unit (g / kg) Moisture 99 Crude protein (90% dry matter) 191.8 Crude fat (90% dry matter) 46 Crude fiber (90% dry matter) 49 Crude ash (90% dry matter) 79 Calcium (90% dry matter) 16.3 Total phosphorus (based on 90% dry matter) 8.8

[0063] In a preferred embodiment, in step (1), the amount of the conventional feed is 120-150 g / kg; specifically, the amount of the conventional feed is 120 g / kg, 125 g / kg, 130 g / kg, 135 g / kg, 140 g / kg, 145 g / kg or 150 g / kg.

[0064] In a preferred embodiment, in step (2), the gradient feeding feed consists of conventional feed and high-fat feed.

[0065] In a preferred embodiment, in step (2), the initial content of the high-fat feed in the gradient feeding feed is 50 wt %; the initial temperature in the artificial climate box is 26° C.; and the initial humidity in the artificial climate box is 75%.

[0066] In a preferred embodiment, in step (2), the gradient of increasing the content of high-fat feed in the gradient feeding feed is 20-25wt% / d; more preferably 25wt% / d.

[0067] In a preferred embodiment, in step (2), the temperature in the artificial climate box increases at a gradient of 1-2°C / d; more preferably 2°C / d.

[0068] In a preferred embodiment, in step (2), the humidity in the artificial climate box increases at a gradient of 10-15% / d; more preferably 10% / d.

[0069] In a preferred embodiment, the ingredients of the high-fat feed include lard, egg yolk powder and sugar.

[0070] Further preferably, the calories of the high-fat feed are 4112.2 Kcal / g, the fat mass ratio is 17.09% (energy supply ratio 37.4%), the protein mass ratio is 19.21% (energy supply ratio 18.68%), and the carbohydrate mass ratio is 45.15% (energy supply ratio 43.92%) (all the above are theoretical calculated values).

[0071] In a preferred embodiment, in step (2), the amount of the gradient feeding feed is 65-80 g / kg; specifically, the amount of the gradient feeding feed is 65 g / kg, 68 g / kg, 70 g / kg, 72 g / kg, 75 g / kg, 78 g / kg or 80 g / kg.

[0072] In the present invention, acetone and olive oil are used as matrix solutions to dissolve dinitrochlorobenzene, wherein the volume ratio of acetone to olive oil is 4:1, and the concentration of the prepared dinitrochlorobenzene solution is 0.1-7wt%.

[0073] In the present invention, in step (3), the sensitization and re-sensitization processes are both carried out on the shaved area of ​​the SD rat.

[0074] In a preferred embodiment, in step (3), during the sensitization process of the abdominal region of the SD rat, the amount of the 0.1-7wt% dinitrochlorobenzene solution is 20-30 μL / cm 2 Preferably, the concentration of the dinitrochlorobenzene solution is 6-7wt%, and the amount of the dinitrochlorobenzene solution is 25-30μL / cm 2 .

[0075] In a preferred embodiment, in step (3), during the sensitization process of the back region of the SD rat, the amount of the 0.1-7wt% dinitrochlorobenzene solution is 10-15 μL / cm 2Preferably, the concentration of the dinitrochlorobenzene solution is 2-3wt%, and the amount of the dinitrochlorobenzene solution is 12.5-15μL / cm 2 .

[0076] In a preferred embodiment, in step (3), the amount of the high-fat feed is 65-80 g / kg; specifically, the amount of the high-fat feed is 65 g / kg, 68 g / kg, 70 g / kg, 72 g / kg, 75 g / kg, 78 g / kg or 80 g / kg.

[0077] The second aspect of the present invention provides an animal model of damp-heat type eczema constructed by the above construction method.

[0078] The third aspect of the present invention provides the application of the damp-heat type eczema animal model in the field of medical technology.

[0079] The following examples further illustrate the method for establishing a damp-heat type eczema animal model and its application. The examples are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following examples.

[0080] The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art. The experimental materials used in the following examples, unless otherwise specified, are all commercially available; among them, SD rats were purchased from Henan Sikebeis Biotechnology Co., Ltd., and the procurement procedures were in compliance with the requirements of the laws and regulations of the People's Republic of China. After approval by the Experimental Animal Ethics Committee of Hubei University of Traditional Chinese Medicine, the various indicators of the SD rats met the local inspection and quarantine standards; dinitrochlorobenzene was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the artificial climate box was purchased from Shaoguan Taihong Medical Equipment Co., Ltd.; conventional feed and high-fat feed were purchased from Wuhan Chunyuhong Experimental Animal Feed Co., Ltd.

[0081] Example 1

[0082] (1) Normal feeding: Ten 5-6 week old SD rats were fed with water and regular feed for 7 days. The amount of the regular feed was 130 g / kg. The SD rats were kept in an SPF-grade laboratory with good ventilation, a day and night cycle of 12 h each, a room temperature of 18-22° C., and a relative humidity of 50%-70%;

[0083] (2) Gradient feeding: SD rats that had been fed normally were subjected to gradient feeding for 3 days, wherein the gradient feeding process was carried out in an artificial climate chamber, and the SD rats were placed in the artificial climate chamber for 10 hours every day, the gradient feeding feed contained conventional feed and high-fat feed, and the amount of the gradient feeding feed was 70 g / kg;

[0084] Wherein, on the first day, the content of high-fat feed in the feed was 50wt%, and the temperature and humidity in the artificial climate box were 26°C and 75%, respectively; on the second day, the content of high-fat feed in the feed was 75wt%, and the temperature and humidity in the artificial climate box were 28°C and 85%, respectively; on the third day, the content of high-fat feed in the feed was 100wt%, and the temperature and humidity in the artificial climate box were 30°C and 95%, respectively;

[0085] (3) High-fat feeding: The SD rats that had undergone gradient feeding were fed with 100 wt% high-fat feed for 12 days, the amount of the high-fat feed was 70 g / kg, wherein the high-fat feeding process was carried out in an artificial climate chamber, and the SD rats were placed in the artificial climate chamber for 10 hours every day, and the temperature and humidity in the artificial climate chamber were 30° C. and 95%, respectively;

[0086] During the high-fat feeding period, the abdominal area of ​​the SD rats was shaved with an electric razor on the second day, with an area of ​​2×2 cm. 2 The next day (i.e., the third day of high-fat feeding), the shaved abdominal area of ​​the SD rats was sensitized with 100 μL of 7 wt% dinitrochlorobenzene solution for 5 days, and the back area of ​​the SD rats was shaved with an electric razor every other day, with an area of ​​4×4 cm 2 Then, 1 day later, 200 μL of 2.5 wt% dinitrochlorobenzene solution was used to re-sensitize the shaved back area of ​​the SD rats for 3 days;

[0087] During the gradient feeding and high-fat feeding process, the SD rats were gavaged with liquor every other day, and the amount of liquor was 10 mL / kg.

[0088] Comparative Example 1

[0089] The method of Example 1 is adopted for implementation, except that step (2) is not performed.

[0090] Comparative Example 2

[0091] The embodiment is carried out in the manner of embodiment 1, except that the amount of the liquor used is 15 mL / kg.

[0092] Blank group

[0093] During the entire experiment, 10 SD rats aged 5-6 weeks were fed with water and regular feed at a dosage of 130 g / kg. The SD rats were raised in an SPF-level laboratory with good ventilation, a day and night cycle of 12 h each, a room temperature of 18-22°C, and a relative humidity of 50%-70%. During the sensitization process, the blank group was smeared with the matrix solution.

[0094] Experimental Results

[0095] In the present invention, the 10 SD rats in the blank group were all normal and no death occurred; the 10 SD rats in Example 1 had no death and all were modeled; the modeling rates of Comparative Example 1 and Comparative Example 2 were 70% and 40%, respectively; therefore, in the modeling process, suitable modeling conditions can well guarantee the survival rate and modeling success rate of the model animals.

[0096] Test Case

[0097] The SD rat model of Example 1 was evaluated, and various indicators of the SD rats of Example 1 were compared with various indicators of the SD rats of the blank group;

[0098] (1) The mental state and activity state of SD rats were observed daily, and photos of the SD rats were taken to observe the condition of their fur. The results are as follows: Figure 1 As shown, Figure 1 A is the status diagram of a random SD rat in the blank group. Figure 1 B is the state diagram of a random SD rat in Example 1;

[0099] Depend on Figure 1 It can be seen that compared with the blank group, the SD rats in Example 1 showed symptoms such as mental depression, irritability, strong irritability, drowsiness, laziness and tendency to gather together. At the same time, the hair of the SD rats was loose, rough, dull and matte.

[0100] (2) The feces of SD rats were observed daily, and the damp-heat condition of SD rats was characterized by taking photos of their feces and perianal area. Figure 2 As shown, Figure 2 A is the appearance of feces of a random SD rat in the blank group. Figure 2 B is the appearance of feces of a random SD rat in Example 1;

[0101] Depend on Figure 2 It can be seen that compared with the blank group, the SD rats in Example 1 had loose stools and unclean anus.

[0102] (3) The appearance of the epidermis on the abdomen and back of the SD rats was observed daily, and the eczema condition of the SD rats was characterized by taking photos of the epidermis on the abdomen and back of the SD rats; the results were as follows: Figure 3 As shown, Figure 3 A is a picture of abdominal eczema of a random SD rat in the blank group. Figure 3 C is a picture of the eczema on the back of a random SD rat in the blank group. Figure 3 B is a diagram showing abdominal eczema of a random SD rat in Example 1. Figure 3 D is a picture showing the eczema condition on the back of a random SD rat in Example 1;

[0103] Depend on Figure 3 It can be seen that compared with the blank group, the back and abdomen of the SD rats in Example 1 showed symptoms of erythema, papules, exudation, erosion and lichenification, and thick scaly scabs were generated on the surface of the back and abdomen of the SD rats in Example 1.

[0104] (4) The back skin tissue of SD rats was processed for pathological sectioning and the pathological condition of SD rats was observed under a microscope; the results are as follows: Figure 4 As shown, Figure 4 A and Figure 4 B is the histopathological picture of the back skin lesions of two random SD rats in the blank group (HE). Figure 4 C and Figure 4 D is a picture of the histopathological condition of the back skin lesions of two random SD rats in Example 1 (HE);

[0105] Depend on Figure 4 It can be seen that: compared with the blank group, the SD rats in Example 1 had obvious hyperplasia of the back epidermis accompanied by hyperkeratosis and parakeratosis, acanthosis accompanied by edema, a large number of inflammatory cells were seen in the dermis, and the epidermis was obviously thickened.

[0106] (5) The colon tissue of SD rats was processed for pathological sectioning and the pathological condition of SD rats was observed under a microscope; the results were as follows: Figure 5 As shown, Figure 5 A and Figure 5 B is the colon tissue pathological condition of two random SD rats in the blank group (HE). Figure 5 C and Figure 5 D is a picture of colon pathological condition of two random SD rats in Example 1 (HE);

[0107] Depend on Figure 5 It can be seen that compared with the blank group, the SD rats in Example 1 had more goblet cells missing, different crypt sizes, shapes and directions, and missing crypts, and a large number of inflammatory cell infiltrations in the mucosal muscles.

[0108] (6) The stomach lining tissue of SD rats was pathologically sliced ​​and the pathological conditions of SD rats were observed under a microscope; the results were as follows: Figure 6 As shown, Figure 6 A and Figure 6 B is the pathological condition of the gastric lining tissue of two random SD rats in the blank group (HE). Figure 6 C and Figure 6 D is a picture of the pathological condition of the stomach lining of two random SD rats in Example 1 (HE);

[0109] Depend on Figure 6It can be seen that: compared with the blank group, the gastric mucosa of the SD rats in Example 1 was thinned, defective, and disordered in structure, with obvious aggregation of inflammatory cells, and the structure of the mucosal muscular layer was incomplete.

[0110] (7) Considering that the spleen index can reflect the changes in the immune status of rats, Graph Pad Prism9.5 software was used to process the body weight, spleen index, stomach index and liver index of SD rats. The results Figure 7 As shown, Figure 7 Figure A is a graph showing the significant difference in body weight between the blank group SD rats and the SD rats in Example 1. Figure 7B is a graph showing the significant difference in spleen index between the blank group SD rats and the SD rats in Example 1. Figure 7 C is a graph showing the significant difference in gastric index between the blank group SD rats and the SD rats in Example 1. Figure 7 D is a graph showing significant differences in liver index between SD rats in the blank group and SD rats in Example 1; Figure 7 In the figure, **** represents P<0.0001, indicating that the results of the blank group and the model group (i.e., Example 1) have extremely significant statistical significance; ** represents P<0.01, indicating that the results of the blank group and the model group have a relatively significant statistical significance; * represents P<0.05, indicating that the results of the blank group and the model group have significant statistical significance;

[0111] Depend on Figure 7 It can be seen that: compared with the blank group, the body weight of the SD rats in Example 1 was significantly decreased, and it was extremely statistically significant (P < 0.0001), the spleen index and liver index of the SD rats in Example 1 were both increased, and they were statistically significant (P < 0.05), and the gastric index of the SD rats in Example 1 increased, which was extremely statistically significant (P < 0.01).

[0112] In summary, the construction method described in the present invention can be used to construct a damp-heat type eczema SD rat model, which is mainly manifested in mental depression, irritability, strong irritability, drowsiness, laziness and easy to gather, loose and rough hair, dull and dull, loose stools, unclean perianal area, erythema, papules, exudate, erosion, lichenification, thick scaly crusts on the back and abdominal epidermis, weight loss, and increased spleen index, gastric index and liver index; in addition, the epidermis of the lesion area of ​​SD rats is accompanied by hyperkeratosis and incomplete keratinization, acanthosis with edema, a large number of inflammatory cells can be seen in the dermis, a large number of inflammatory cells infiltrate the mucosal muscle, mast cell infiltration increases, goblet cells and crypts are missing, the gastric mucosa is thinned and deficient, the structure is disordered, obvious inflammatory cells can be seen, and the structure of the mucosal muscle layer is incomplete. The present invention constructs a damp-heat type eczema model that is more in line with the syndromes of traditional Chinese medicine by comparing various indicators of the blank group and the model group constructed in Example 1. The model can provide a powerful tool for exploring the pathogenesis of damp-heat type eczema and finding treatment options for eczema diseases and new drug development. The damp-heat type eczema SD rat model constructed by the construction method described in the present invention has a high model success rate.

[0113] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for establishing an animal model of damp-heat type eczema, characterized in that: The method comprises the following steps: (1) Normal feeding: 5-6 week old SD rats were fed with water and regular feed for 3-7 days; (2) Gradient feeding: Gradient feeding of normally fed SD rats for 2-4 days, wherein the gradient feeding process is carried out in an artificial climate chamber, and the SD rats are placed in the artificial climate chamber for 8-12 hours every day, and the gradient feeding feed contains conventional feed and high-fat feed; The high-fat feed content in the gradient feeding feed and the temperature and humidity in the artificial climate box are increased gradually day by day, wherein the high-fat feed content in the gradient feeding feed is increased gradually from 40wt%-50wt% to 100wt%, and the temperature and humidity in the artificial climate box are increased gradually from 24-26°C and 70-80% to 28-32°C and 90-100% respectively; (3) High-fat feeding: The SD rats that had undergone gradient feeding were fed with a 100 wt% high-fat diet for 12-19 days, wherein the high-fat feeding process was carried out in an artificial climate chamber, and the SD rats were placed in the artificial climate chamber for 8-12 hours every day, and the temperature and humidity in the artificial climate chamber were 28-32° C. and 90-100%, respectively; During the high-fat feeding period, the abdominal region of the SD rats was sensitized with a 0.1-7wt% dinitrochlorobenzene solution for 5-7 days on the 2nd to 4th day, and then the back region of the SD rats was re-sensitized with a 0.1-7wt% dinitrochlorobenzene solution for 3-5 days after 1-2 days; During the gradient feeding and high-fat feeding process, the SD rats were gavaged with liquor every other day, and the amount of liquor was 9-11 mL / kg.

2. The method for establishing an animal model of damp-heat type eczema according to claim 1, characterized in that: In step (1), the ingredients of the conventional feed include corn, bran, flour, soybean meal, fish meal, bone meal and salt; Preferably, in step (1), the amount of the conventional feed is 120-150 g / kg.

3. The method for establishing an animal model of damp-heat type eczema according to claim 1 or 2, characterized in that: In step (2), the gradient of increasing the content of high-fat feed in the gradient feeding feed is 20-25wt% / d; and / or In step (2), the temperature in the artificial climate box increases at a gradient of 1-2°C / d; and / or In step (2), the humidity in the artificial climate box increases at a gradient of 10-15% / d.

4. The method for establishing an animal model of damp-heat type eczema according to claim 3, characterized in that: In step (2), the gradient of increasing the content of high-fat feed in the gradient feeding feed is 25wt% / d; and / or In step (2), the temperature in the artificial climate box increases at a gradient of 2°C / d; and / or In step (2), the humidity in the artificial climate box increases at a gradient of 10% / d.

5. The method for establishing an animal model of damp-heat type eczema according to claim 4, characterized in that: In step (2), the initial content of the high-fat feed in the gradient feeding feed is 50wt%; and / or In step (2), the initial temperature in the artificial climate box is 26°C; and / or In step (2), the initial humidity in the artificial climate box is 75%.

6. The method for establishing an animal model of damp-heat type eczema according to claim 1 or 5, characterized in that: The high-fat feed comprises lard, egg yolk powder and sugar; and / or In step (2), the amount of the gradient feeding feed is 65-80 g / kg.

7. The method for establishing an animal model of damp-heat type eczema according to claim 6, characterized in that: In step (3), during the sensitization process of the abdominal region of the SD rats, the dosage of the 0.1-7wt% dinitrochlorobenzene solution is 20-30 μL / cm 2 ; and / or In step (3), during the sensitization process of the back region of the SD rat, the dosage of the 0.1-7wt% dinitrochlorobenzene solution is 10-15 μL / cm 2 .

8. The method for establishing an animal model of damp-heat type eczema according to claim 1 or 7, characterized in that: In step (3), the amount of the high-fat feed is 65-80 g / kg.

9. An animal model of damp-heat type eczema constructed by the construction method described in any one of claims 1 to 8.

10. Use of the damp-heat type eczema animal model according to claim 9 in the field of medical technology.

Images