A method for establishing an animal model of psoriasis

Abstract

The application discloses a method for establishing a psoriasis animal model. The method for establishing the psoriasis animal model is realized by injecting cytokines and applying imiquimod cream externally. The model is characterized by the severity of skin lesions, histopathological changes of skin lesions, expression of CD4 + and CD8 + T cells in immunohistochemistry of skin lesions, and the level of serum inflammatory factors, and conforms to the characteristics of psoriasis. Compared with the model induced by imiquimod cream alone, the model has the advantages of faster occurrence and development of psoriasis, more obvious characteristics of the model, higher stability of the model, longer maintenance time of the model, more diversified mechanisms of the model, and reduced systemic immune response. The model established by the application is simple and easy to operate, and can be widely popularized and applied, and provides an experimental tool for the research and development of the pathogenesis and drugs of psoriasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal models, in particular to a method for establishing a psoriasis animal model. BACKGROUND

[0002] Psoriasis is a chronic, recurrent, inflammatory and systemic skin disease induced by the interaction of genetics and environment, and is an immune-mediated disease. The typical clinical manifestations of skin lesions are scaly erythema or plaques. Psoriasis cannot be cured at present, and patients are prone to relapse after treatment, so they often need lifelong treatment, which causes serious psychological burden to patients. Psoriasis is caused by a variety of pathogenic factors stimulating the immune system, causing dysfunction of dendritic cells, T cells, macrophages and other cells, and abnormal intracellular signal transduction, inducing immune system disorder and inflammation, leading to epidermal keratinocyte hyperplasia, abnormal differentiation, and massive inflammatory cell infiltration. The pathogenesis of psoriasis has not been clearly elucidated.

[0003] The establishment of animal models plays an important role in the pathogenesis of psoriasis and drug development. However, there is no animal model that matches the pathogenesis and therapeutic drug response of human psoriasis. Psoriasis animal models such as spontaneous mouse models, genetically engineered mouse models and xenotransplantation models each have advantages and disadvantages, but are relatively expensive or complex to operate, and have poor applicability and generalizability. Drug-induced animal models are inexpensive and easy to use, among which the most widely used is the psoriasis animal model induced by imiquimod. The length of the modeling time is similar to the early and late histopathology of human psoriasis, and is considered to be caused by the synergistic effect of multiple mechanisms. However, there are also some shortcomings such as unstable model, short maintenance time after modeling, and long-term use causing systemic inflammation and immune response. Therefore, the establishment of a new psoriasis animal model or the optimization of the existing model is of great significance for the discovery of the etiology and therapeutic drugs of psoriasis.

[0004] Cytokines such as interleukin-2 (IL-2) are involved in the pathogenesis of psoriasis, which induces or aggravates the occurrence of psoriasis by activating T cells, stimulating dendritic cells, macrophages and other cells to secrete inflammatory factors. It was found that the level of IL-2 in the body of psoriasis patients increased significantly, and interleukin 2 monoclonal antibody had a certain therapeutic effect on psoriasis by selectively targeting the blockage of IL-2 in the body and inhibiting its function, which confirmed that IL-2 accompanied the occurrence and development of psoriasis. Therefore, in the establishment of psoriasis animal model, giving IL-2 cytokine can promote the occurrence of psoriasis, make the model more stable, and also can reduce the systemic side effects and other advantages. In addition, the animal model induced by multiple mechanisms is more consistent with the pathogenesis of psoriasis.

[0005] Therefore, the IL-2 cytokine is combined with imiquimod to induce a psoriasis animal model, which can promote the occurrence of psoriasis, aggravate the development of psoriasis, improve the stability of the model induced by imiquimod alone, the maintenance time of the model, and the systemic immune response, and can be widely promoted and applied, thereby providing an experimental tool for the pathogenesis of psoriasis and the research and development of drugs. SUMMARY

[0006] The application provides a method for establishing a novel psoriasis animal model, which adopts injection of IL-2 cytokine combined with external use of imiquimod cream to establish the psoriasis animal model. + The model is consistent with the characteristics of psoriasis in terms of the severity of skin lesions, histopathological changes of skin lesions, expression of CD4 + T cells in immunohistochemistry of skin lesions, and the level of serum inflammatory factors.

[0007] To achieve the above object, the application adopts the following technical scheme.

[0008] In a first aspect, the application provides a method for establishing a psoriasis animal model, which comprises injection of a cytokine combined with external application of imiquimod to induce rats or mice to establish a psoriasis animal model.

[0009] Preferably, the cytokine is interleukin-2 (IL-2), and the model induced by the interleukin-2 is more stable.

[0010] In an embodiment of the application, the imiquimod is applied externally to the modeling site of the rats or mice in the form of imiquimod cream. Further, the mass content of imiquimod in the imiquimod cream is 5%, and the dosage is 20 mg of imiquimod cream per cm 2 of the area of the modeling site. Further, the frequency of external application is once a day, and the total time is 7-14 days.

[0011] Further, the modeling site is the skin of the back, the ear or other parts of the animal after hair removal.

[0012] Further, the administration mode of the cytokine is intraperitoneal injection, intravenous injection or subcutaneous injection. The injection dose is 0.5-180,000 IU / kg of the body weight of the rats or mice.

[0013] In an embodiment of the application, the administration mode of the cytokine is intraperitoneal injection. According to the conversion of the clinical dose, the application recommends that the injection dose of interleukin-2 is 1-90,000 IU / kg of the body weight of the rats or mice, and the injection dose of TNF-α is 0.5-45,000 IU / kg of the body weight of the rats or mice.

[0014] In one embodiment of the present application, the injection time of the cytokine is within 30 minutes before and after the daily application of imiquimod cream.

[0015] Specifically, the method is:

[0016] After the rats or mice are adaptively fed, the back or ear is depilated, and then the following treatment is performed once a day for 7-14 days: 20 mg / cm 2 The imiquimod cream (Sichuan Mingxin Pharmaceutical Co., Ltd., batch number: 39220301) is evenly applied, and the cytokine injection solution is intraperitoneally injected at 0.5-180,000 IU / kg of the body weight of the rats or mice within 30 minutes to obtain the psoriasis animal model.

[0017] In a second aspect, the present application further provides a psoriasis animal model established by the above method.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] ①Compared with the psoriasis animal model established by using imiquimod alone, the psoriasis animal model established by the present method has a shorter time of occurrence of psoriasis-like skin lesions, a more severe degree of skin lesions, a longer and more stable model maintenance time;

[0020] ②Compared with the psoriasis animal model established by using imiquimod alone, the psoriasis animal model established by the present method has more obvious histopathological features of skin lesions, higher expression levels of CD4 + and CD8 + T cells in the skin lesion tissues;

[0021] ③Compared with the psoriasis animal model established by using imiquimod alone, the psoriasis animal model established by the present method has more induced inflammatory factors in the serum and higher expression levels thereof. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Skin lesion conditions of the psoriasis rats in each group at different modeling days.

[0023] Figure 2 Psoriasis lesion area and severity index (PASI) scores of the rats in each group.

[0024] Figure 3Skin histopathology observation (HE, ×200) of each group of rats. A: normal control group, skin structure is complete and clear, granular layer is clear; B: IMQ group, pathological features such as incomplete keratinization, acanthosis, inflammatory cell infiltration can be seen; C-E: TNF-α (45, 15, 0.5 million IU / kg) + IMQ group, pathological features such as hyperkeratosis (blue arrow), incomplete keratinization (orange arrow), inflammatory cell infiltration (red arrow) can be seen; F-H: IL-2 (90, 30, 10 million IU / kg) + IMQ group, pathological features such as Munro microabscess (black arrow), capillary dilation (purple arrow), acanthosis, granular layer disappearance, skin fluctuation (cyan arrow), papillary up (yellow arrow) can be seen.

[0025] Figure 4 Skin histopathology observation score (Baker score) of each group of rats.

[0026] Figure 5 TNF-α, IL-1β and IFN-α levels in serum of each group of rats.

[0027] Figure 6 IL-17A and IL-23 levels in serum of each group of rats.

[0028] Figure 7 CD4 + and CD8 + T cell expression (IHC, ×200) in skin tissue of each group of rats.

[0029] Figure 8 Average optical density value of CD4 + and CD8 + T cell expression in skin tissue of each group of rats. DETAILED DESCRIPTION

[0030] The application will be further described below in conjunction with specific examples. The following examples are only specific embodiments of the application, and the protection scope of the application is not limited to this.

[0031] Example 1

[0032] I. Materials and methods

[0033] 1. Experimental animals: 80 SPF Wistar rats, male, body weight (200 ± 20) g, purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Jing) 2016-0006, bred by Zhejiang Province Traditional Chinese Medicine Research Institute Experimental Animal Center, use license number: SYXK (Zhejiang) 2019-0010. The feeding environment: the room temperature is (20 ± 2) ℃, the humidity is (60 ± 10) %, and the standard feed is kept for 12 h light and 12 h dark alternation, free drinking water. The animal experiment content is approved by Zhejiang Province Traditional Chinese Medicine Research Institute Ethics Committee (Zhejiang Medical Ethics Review No.

[2021] 020).

[0034] 2. Main reagents and instruments: 5% imiquimod cream (Sichuan Mingxin Pharmaceutical Co., Ltd., batch number: 39220301); human interleukin-2 (IL-2, Shandong Quan'gang Pharmaceutical Co., Ltd., batch number: 202107020); recombinant modified human tumor necrosis factor (TNF-α, Shenyang Sansheng Pharmaceutical Co., Ltd., batch number: 20211234); dextran (National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number: 30191228); interleukin-17A (IL-17A), interleukin-1β (IL-1β), interleukin-23 (IL-23), tumor necrosis factor (TNF-α) and interferon-α (IFN-α) enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., item numbers are m1037365, m1003057, m1003152, m1002859, m1003203, respectively); CD4 + and CD8 + T cell antibodies (Abcam, UK, batch numbers are 1002220-1, 1015767-1, respectively). Multifunctional enzyme marker (Thermo, USA); Nikon eclipse 80i microscope (Nikon, Japan).

[0035] 3. Grouping and modeling: 80 SPF male Wistar rats were randomly divided into normal control group, imiquimod alone group, IL-2 (90,000, 30,000, 10,000 IU / kg) combined with imiquimod group, TNF-α (45,000, 15,000, 5,000 IU / kg) combined with imiquimod group, a total of 8 groups, 10 rats in each group. After adaptive feeding of rats for several days, the hair on the central area (2 cm × 2 cm) of the back of each rat was shaved off, and the surface short hair was removed with a mild depilatory cream, washed and dried for use. If the back hair grows during the experiment, the hair is removed again with a depilatory cream. The imiquimod alone group rats were given 20 mg / cm 2Uniformly apply 5% imiquimod cream (Sichuan Mingxin Pharmaceutical Co., Ltd., batch number: 39220301); IL-2 combined with imiquimod group: apply 5% imiquimod cream on the back of the rats at the same dose, and 15 min after application, inject IL-2 cytokine injection at doses of 90,000, 30,000 and 10,000 IU / kg intraperitoneally; TNF-α combined with imiquimod group: apply 5% imiquimod cream on the back of the rats at the same dose, and 15 min after application, inject TNF-α cytokine injection at doses of 45,000, 15,000 and 5,000 IU / kg intraperitoneally; the blank control group: apply the same amount of vaseline on the back of the rats, once a day, for 10 consecutive days.

[0036] 4. Psoriasis Area and Severity Index (PASI) score: According to the PASI score standard, give the rats a score of 0-4 for the degree of erythema, scaling and epidermal infiltration thickening in the lesion. The scoring standard is as follows: 0, none; 1, mild; 2, moderate; 3, severe; 4, extremely severe. Take the average of the scores of each group of rats, and observe the changes in the lesions of each group of rats daily.

[0037] 5. ELISA detection of IL-17A, TNF-α, IL-23, IFN-α and IL-1β levels: After the modeling is completed, the rats are anesthetized with 20% urethane, blood is collected from the abdominal aorta, centrifuged at 3500 r / min for 15 min, and serum is separated. The levels of IL-17A, TNF-α, IL-23, IFN-α and IL-1β in the serum are detected by enzyme-linked immunoassay (ELISA) according to the instructions of the kit.

[0038] 6. Histopathological observation of skin: Take the skin lesion tissue from the back of the rats, fix it with 4% neutral formaldehyde, embed it in paraffin, section it, stain it with HE, observe the morphological changes of the skin tissue under a microscope, and perform Baker scoring.

[0039] The specific standard of Baker scoring is as follows: 2.0 points for finding Munro abscess in the epidermal layer; 0.5 points for hyperkeratosis; 1.0 points for parakeratosis; 1.0 points for thinning or disappearance of the granular layer; 1.0 points for thickening of the spinous layer; 0.5 points, 1.0 points and 1.5 points for elongation and undulation of the dermal papilla according to mild, moderate and severe; 0.5 points, 1.0 points and 1.5 points for single or multinucleated cell infiltration in the dermal layer according to mild, moderate and severe; 0.5 points for papillary apophysis; 0.5 points for capillary dilation.

[0040] 7. Immunohistochemical detection of CD4 + and CD8 +T cell expression level: staining was performed according to the two-step immunohistochemical procedure in the product manual. That is, paraffin-embedded skin lesion tissue was cut into sections and baked in a 60°C oven for 2 h; the sections were deparaffinized to water; distilled water was washed for 2 min; high-pressure thermal repair was performed; 3% H2O2 solution was used to block peroxidase for 10 min; PBS was washed for 5 min x 3 times; the primary antibody was added and incubated at 37°C for 1 h; PBS was washed for 5 min x 3 times; the secondary antibody was added and incubated at 37°C for 60 min; PBS was washed for 5 min x 3 times; DAB was developed for 2 min; after staining and transparency, the sections were mounted. The parts to be analyzed of each section were photographed under a microscope, and image analysis was performed to determine the average optical density of CD4 + and CD8 + T cells in the tissue.

[0041] 8. Statistical method: SPSS 20.0 was used for statistical analysis of the data. The data are expressed as mean ± standard deviation, and one-way ANOVA was used for comparison among multiple groups, and LSD-t test was used for comparison between two groups. For data with unequal variance, Kruskal-Wallis H test was used. P < 0.05 was considered statistically significant.

[0042] II. Experimental results

[0043] 1. Establishment of psoriasis rat model and PASI score

[0044] On the 3rd day after modeling, the epidermis of the skin of the model group rats was slightly thickened, and the erythema was pale red; on the 5th day, the epidermis was covered with a layer of scales, and the elevation was moderate; on the 7th day, the skin was significantly thickened, the scales were thick and layered, and the erythema was red; on the 9th day, some scales began to fall off, with bleeding and scabbing symptoms, and the erythema was dark red or purple red, with Figure 1 . After the 10th day, the skin of the normal control group rats was smooth, tender, flat and undamaged; the skin of the rats in the imiquimod alone group was mostly covered with scales, the skin lesions were thickened, the elevation was obvious, and the erythema was pale red or red; and the severity of the skin scale and erythema damage in the TNF-α or IL-2 combined with imiquimod group was more obvious than that in the imiquimod alone group, and Figure 1 .

[0045] ​The PASI score of rats in the imiquimod alone group gradually increased and reached the peak on day 7, then showed a downward trend. The PASI score of rats in the TNF-α or IL-2 combined with imiquimod group reached the peak on day 6, then remained stable or decreased slowly. Compared with the imiquimod alone group, the PASI score of rats in the TNF-α combined with imiquimod group was significantly increased on day 3 (P<0.01), and the PASI score of rats in the TNF-α (150,000 IU / kg) combined with imiquimod group was significantly increased on day 5 (P<0.05); the PASI score of rats in the IL-2 combined with imiquimod group was significantly increased on days 3 and 5 (P<0.01, 0.05), and the PASI score of rats in the IL-2 (900,000 IU / kg) combined with imiquimod group was significantly increased on day 10 (P<0.05), as shown in Table 1. Figure 2 .

[0046] 2. Histopathological changes in the skin of rats in each group: The skin structure of the normal control group was complete and clear, the granular layer was clearly visible, the epidermis was slightly keratinized, and no acanthosis, parakeratosis, capillary dilation, or dermal layer infiltration of inflammatory cells was observed. The skin of the imiquimod alone group and the imiquimod combined with cytokine group showed different degrees of psoriasis pathological features, such as obvious hyperkeratosis, parakeratosis, acanthosis, thinning or disappearance of the granular layer, skin protrusion, inflammatory cell infiltration, and capillary dilation, as shown in Table 2. Figure 3 Compared with the imiquimod alone group, the Baker score of rats in the TNF-α (450,000 IU / kg) and IL-2 (900,000 IU / kg) combined with imiquimod group was significantly increased (P<0.01), as shown in Table 3. Figure 4 .

[0047] 3. Changes in the levels of TNF-α, IL-17A, IL-1β, IFN-α, and IL-23 in the serum of rats in each group: Compared with the normal control group, the levels of TNF-α, IL-1β, and IL-17A in the imiquimod alone group and the TNF-α and IL-2 combined with imiquimod group were significantly increased (P<0.01); the levels of IL-23 and IFN-α in the TNF-α and IL-2 combined with imiquimod group were significantly increased (P<0.01, 0.05), while the imiquimod alone group had no significant effect (P>0.05). Compared with the imiquimod alone group, the level of IL-17A in the IL-2 (300,000 IU / kg) combined with imiquimod group was significantly increased (P<0.05), and the levels of IL-23 and IFN-α in the IL-2 (900,000 IU / kg) combined with imiquimod group were significantly increased (P<0.05), as shown in Table 4. Figure 5 , Figure 6 .

[0048] 4. Changes in the levels of CD4 + and CD8 +T cell expression difference: compared with normal control group, CD4 + and CD8 + T cell expression of imiquimod alone group, TNF-α (45,000 IU / kg) and IL-2 (90,000 IU / kg) combined with imiquimod group were significantly increased (P<0.05, 0.01). Compared with imiquimod alone group, CD4 + and CD8 + T cell expression of imiquimod combined with IL-2 (90,000 IU / kg) were significantly increased (P<0.05), see Figure 7 , Figure 8 .

[0049] In summary, IL-2 (90,000 IU / kg) combined with imiquimod to establish psoriasis animal model, from the clinical lesion apparent score, lesion histopathological features, inflammatory cytokine levels and T cell expression in lesion tissue, etc. are in line with the physiological and pathological characteristics of psoriasis. Compared with imiquimod alone group, it forms psoriasis-like skin lesions faster, the severity of skin lesions is more serious, the pathological features are more obvious, the model maintains for a longer time and is stable, the serum inflammatory factor level is higher, the CD4 + and CD8 + T cell expression level in lesion tissue is higher, which is more in line with the characteristics of psoriasis, and can be widely promoted and applied.

Claims

1. A method for establishing an animal model of psoriasis, characterized by The method is: cytokine combined with imiquimod to induce rats to establish psoriasis animal model; the cytokine is interleukin-2 or TNF-alpha, the mass content of imiquimod in the imiquimod cream is 5%, and the dosage is 20 mg imiquimod cream / cm 2 The modeling site area; the frequency of external coating is 1 time / day, and the total is 7-14 days; the injection dose of interleukin-2 is 1-90,000 IU / kg rat body weight, and the injection dose of TNF-alpha is 0.5-4.5,000 IU / kg rat body weight; the injection time of the cytokine is within 30 min before and after the daily application of imiquimod cream.

2. The method for preparing a psoriasis animal model according to claim 1, wherein the mouse is a BALB / c mouse. The molding site is the skin of the back, the skin of the ear or other parts of the animal after depilation. The molding site is the skin of the back, the skin of the ear or other parts of the animal after depilation.

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