A method for constructing a pressure aggravated psoriasis mouse model and application thereof

Abstract

The application belongs to the technical field of animal model construction, and particularly relates to a construction method of a stress-aggravated psoriasis mouse model and application. The construction method comprises the following steps: (1) shaving the back of the mouse, placing the mouse in a 50-milliliter breathable centrifuge tube, and limiting the activity for four hours; (2) taking out the mouse, and waiting for the back skin to dry; (3) uniformly applying imiquimod cream to the back of the mouse, and placing the mouse back into the cage; and (4) repeating the above steps every day until the stress-aggravated psoriasis mouse model is successfully constructed. The construction method can quickly and simply construct the stress-aggravated psoriasis mouse model for subsequent research.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing and applying a stress-induced psoriasis mouse model. Background Technology

[0002] Psoriasis is a chronic, relapsing autoimmune disease clinically characterized by scaly erythematous plaques and patches, itching, and microscopic examination revealing keratinocyte proliferation and inflammatory infiltration in the affected areas. The disease has a long course and can be lifelong. Because the affected areas are often located on the limbs and scalp and are accompanied by itching, patients often experience significant psychological stress, which in turn exacerbates the disease, creating a vicious cycle. The incidence of anxiety, depression, and other mental health issues associated with psoriasis is higher than in the general population. Currently, with the development of targeted therapy using biological agents, psoriasis treatment has achieved remarkable results, but the problems of psoriasis flare-ups and relapses remain unresolved. Psychological stress, as a significant factor in inducing or exacerbating psoriasis flare-ups, will be a key focus of future psoriasis treatment efforts, with an emphasis on understanding its harmful mechanisms and potential therapeutic targets.

[0003] Therefore, it is necessary to have a stress-induced psoriasis mouse model that can be used to study stress as a mechanism for inducing or aggravating psoriasis flare-ups. Summary of the Invention

[0004] To address the above problems, this invention provides a stress-induced psoriasis mouse model, which applies stress to mice by restricting their activity. This model can be used to study the mechanism by which stress induces or aggravates psoriasis flare-ups, and has also identified drugs that can treat stress-induced psoriasis.

[0005] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0006] The present invention provides a method for constructing a stress-induced psoriasis mouse model, comprising: (1) shaving the back of the mouse, placing it in a 50 ml breathable centrifuge tube, and restricting its activity for four hours; (2) releasing the mouse and waiting for the skin on its back to dry; (3) applying imiquimod cream evenly to the back of the mouse and returning it to the cage; (4) repeating the above steps every day until the stress-induced psoriasis mouse model is successfully constructed.

[0007] Another aspect of the present invention provides the use of a glucocorticoid receptor inhibitor in the preparation of a medicament for treating psoriasis accompanied by stress.

[0008] In another aspect, the present invention provides the use of a glucocorticoid receptor inhibitor in the preparation of a medicament for treating stress-induced psoriasis.

[0009] The beneficial effects of this invention include:

[0010] (1) The method for constructing a stress-induced psoriasis mouse model provided in this invention can quickly and easily construct a stress-induced psoriasis mouse model for subsequent research;

[0011] (2) In this invention, glucocorticoid receptor inhibitors are used in a stress-induced psoriasis mouse model, which can effectively reduce skin damage and inflammation in stress-induced psoriasis mice and effectively inhibit macrophage polarization. Attached Figure Description

[0012] Figure 1 The activity trajectory and distance traveled by mice in each group;

[0013] Figure 2 The total distance traveled by mice in each group, the distance traveled in the central region, and the number of times they entered the central region were recorded.

[0014] Figure 3 Microscopic view of the skin of mice in each group;

[0015] Figure 4 The serum levels of common neurotransmitters and hormones in each group of mice were measured.

[0016] Figure 5 The changes in the number of immune cells in the skin of mice in each group;

[0017] Figure 6 The changes in the proportion of immune cells in the skin of mice in each group;

[0018] Figure 7 The average fluorescence intensity of glucocorticoid receptors on the surface of macrophages in each group of mice;

[0019] Figure 8 The average fluorescence of glucocorticoid receptors on the surface of macrophages was detected by immunofluorescence in each group of mice.

[0020] Figure 9 The polarization effect of different concentrations of glucocorticoids on macrophages;

[0021] Figure 10 The effects of different concentrations of glucocorticoids on the expression of phosphorylated protein p-STAT1 in M1 and phosphorylated protein p-STAT6 in M2;

[0022] Figure 11 The effect of the glucocorticoid receptor inhibitor AL082D06 on macrophage polarization;

[0023] Figure 12 The effect of the glucocorticoid receptor inhibitor AL082D06 on the expression of phosphorylated protein p-STAT1 in M1 and phosphorylated protein p-STAT6 in M2;

[0024] Figure 13 The effect of the glucocorticoid receptor inhibitor AL082D06 on the skin of stress-exacerbated psoriasis mice;

[0025] Figure 14 The effect of the glucocorticoid receptor inhibitor AL082D06 on the proportion and number of skin immune cells in stress-exacerbated psoriasis mice;

[0026] Figure 15 The effect of the glucocorticoid receptor inhibitor AL082D06 on M1 and M2 macrophages in the skin of stress-exacerbated psoriasis mice;

[0027] Figure 16 The effect of the glucocorticoid receptor inhibitor AL082D06 on serum cytokines in stress-exacerbated psoriasis mice;

[0028] Figure 17 The back lesions and pathological examination of mice with stress-induced psoriasis after knocking out the glucocorticoid receptor gene;

[0029] Figure 18 The number of macrophages, neutrophils, and dendritic cells in mice with stress-induced psoriasis after knocking out the glucocorticoid receptor gene;

[0030] Figure 19 The proportions of macrophages, neutrophils, and dendritic cells in mice with stress-induced psoriasis after knocking out the glucocorticoid receptor gene;

[0031] Figure 20 The proportions of macrophage subtypes M1 and M2 after knocking out the glucocorticoid receptor gene in mice with stress-induced psoriasis;

[0032] Figure 21 The status of marker genes in M1 macrophages and M2 macrophages after knocking out the glucocorticoid receptor gene in mice with stress-induced psoriasis;

[0033] Figure 22 The skin lesions of mice with psoriasis aggravated by stress after injection of glucocorticoids;

[0034] Figure 23 The number of macrophages, neutrophils, and dendritic cells in mice with psoriasis aggravated by stress after injection of glucocorticoids;

[0035] Figure 24 The proportions of macrophages, neutrophils, and dendritic cells in mice with psoriasis aggravated by stress after injection of glucocorticoids;

[0036] Figure 25The proportions of macrophage subtypes M1 and M2 after injection of glucocorticoids into mice with stress-induced psoriasis;

[0037] Figure 26 The marker genes of M1 macrophages and M2 macrophages after injection of glucocorticoids into mice with stress-induced psoriasis. Detailed Implementation

[0038] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0039] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0040] This invention provides a method for constructing a stress-induced psoriasis mouse model, comprising: (1) shaving the back of a mouse, placing it in a 50 ml breathable centrifuge tube, and restricting its activity for four hours; (2) releasing the mouse and waiting for the skin on its back to dry; (3) applying imiquimod cream evenly to the back of the mouse and returning it to its cage; (4) repeating the above steps daily until the stress-induced psoriasis mouse model is successfully constructed. It should be understood that in the above method for constructing a stress-induced psoriasis mouse model, the breathable centrifuge tube is obtained by drilling a hole in the body of a 50 ml centrifuge tube, and it is used for mouse respiration.

[0041] In some specific embodiments, in the above method for constructing a stress-induced psoriasis mouse model, step (4) is repeated 4 times. It should be noted that repeating 4 times for 5 consecutive days can obtain a stress-induced psoriasis mouse model, which has a short modeling time and high efficiency.

[0042] In some specific embodiments, in the above-described method for constructing a stress-induced psoriasis mouse model, mice are injected daily with 250 μL of 4 μg / μL glucocorticoid. It should be noted that glucocorticoid injection can exacerbate inflammation in the stress-induced psoriasis mouse model and worsen the symptoms.

[0043] In some specific embodiments, in the above-described method for constructing a stress-induced psoriasis mouse model, female 8-week-old mice can be selected.

[0044] Another embodiment of the present invention provides the use of a glucocorticoid receptor inhibitor in the preparation of a medicament for treating psoriasis accompanied by stress. It should be noted that glucocorticoid receptor inhibitors can effectively alleviate the symptoms of psoriasis exacerbated by stress, where stress refers to psychological or mental stress, which can be stress arising from life or work.

[0045] Another embodiment of the present invention provides the use of a glucocorticoid receptor inhibitor in the preparation of a medicament for treating stress-induced psoriasis. It should be noted that the glucocorticoid receptor inhibitor can be used to treat stress-induced psoriasis, especially stress-induced psoriasis relapses.

[0046] In some specific embodiments, the above-mentioned applications include: the use of glucocorticoid receptor inhibitors in the preparation of drugs for inhibiting macrophage polarization. It should be noted that, whether for alleviating symptoms of stress-induced psoriasis or treating stress-induced psoriasis relapses, the goal of alleviating psoriasis symptoms can be achieved by inhibiting macrophage polarization. Furthermore, macrophage polarization can be manifested in: the use of glucocorticoid receptor inhibitors in the preparation of drugs for reducing M1 macrophages and increasing M2 macrophages.

[0047] In some specific embodiments, the glucocorticoid receptor inhibitor AL082D0 is selected in the above applications. It should be noted that the glucocorticoid receptor inhibitor in this invention is known in the art, such as AL082D0.

[0048] In some specific embodiments, the drug is used in the above applications as an ointment or injection. It should be noted that glucocorticoid receptor inhibitors can be prepared in different dosage forms for use, such as ointments or injections, and the specific dosage form can be selected according to clinical needs.

[0049] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0050] Example 1: Psychological stress reduces activity in mice

[0051] Mice were divided into four groups: a Vaseline control group, a Vaseline activity-restricted group, an imiquimod control group, and an imiquimod activity-restricted group. The specific groupings were as follows: Vaseline control group: mice were allowed free movement daily, with Vaseline applied to their backs; Vaseline activity-restricted group: mice were restrained for four hours daily, with Vaseline applied to their backs; Imiquimod control group: mice were allowed free movement daily, with imiquimod cream applied to their backs; Imiquimod activity-restricted group: mice were restrained for four hours daily, with imiquimod cream applied to their backs.

[0052] Five days later, the mice's activity trajectory and distance were examined using a mining experiment, and the results were as follows: Figure 1 As shown, activity restriction reduces the mouse's activity trajectory. Statistical analysis of total activity distance, central region activity distance, and number of entries into the central region confirms the success of the stress model, as shown in the results. Figure 2 As shown.

[0053] Example 2: Psychological stress exacerbates psoriasis-like symptoms in mice

[0054] Five days later, the dorsal skin lesions and pathological examinations of mice in the imiquimod control group (imiquimod) and the imiquimod-restricted activity group (stress / imiquimod) of Example 1 were observed, and the results were as follows: Figure 3 As shown, under a microscope, increased epidermal thickness and increased infiltration of inflammatory cells were observed in restrained mice that restricted their movement, indicating that psychological stress exacerbated imiquimod-induced psoriasis-like symptoms in mice.

[0055] Example 3: Psychological stress activates the hypothalamic-pituitary-adrenal axis in psoriasis-like mice.

[0056] Under stress, the hypothalamic-pituitary-adrenal axis is activated, releasing neurotransmitters into various organs to initiate a response. The levels of common neurotransmitters and hormones (glucocorticoids, adrenocorticotropic hormone, norepinephrine, and serotonin) in the serum of mice in each group were examined in Example 1. The results are as follows: Figure 4 As shown, glucocorticoids and corticotropin-releasing hormone were significantly elevated in the serum of stress / psoriasis mice, indicating that the hypothalamic-pituitary-adrenal axis was activated in anxious psoriasis mice and released corticotropin and glucocorticoids.

[0057] Example 4: Psychological stress increases the number and proportion of macrophages in the skin of psoriasis-like mice.

[0058] The skin of mice from each group in Example 1 was analyzed by flow cytometry to examine changes in the number of immune cells. The results are as follows: Figure 5 The number of macrophages increased in the skin of psoriatic mice under stress. Additionally, the changes in the proportion of immune cells in the skin of mice in each group in Example 1 were analyzed, and the results are as follows: Figure 6As shown, the proportion of macrophages in the skin of psoriatic mice under stress increases.

[0059] Furthermore, the average fluorescence intensity of glucocorticoid receptors in each group of mice in Example 1 was analyzed, and the results are as follows: Figure 7 As shown, the mean fluorescence intensity of glucocorticoid receptors on the surface of macrophages was increased in skin lesions aggravated by psychological stress. Furthermore, immunofluorescence analysis of glucocorticoid receptor fluorescence signals in each group of mice in Example 1 yielded the following results: Figure 8 As shown, an increase in the fluorescence signal of glucocorticoid receptors on the surface of macrophages was found in skin lesions aggravated by psychological stress.

[0060] Example 5: Effects of stress-induced glucocorticoid release on macrophages in vitro

[0061] By simulating the effects of glucocorticoid release on cells in an in vitro monocyte-macrophage cell line, it was found that different concentrations of glucocorticoids (0 μM, 10 μM, 50 μM, 100 μM, 500 μM, and 1000 μM) produced different polarization effects on monocyte-macrophages, such as... Figure 9 As shown, low-concentration glucocorticoid (50 μM) treatment increased the expression of marker genes in M2 macrophages; at high concentrations (1000 μM), the expression of marker genes in M1 macrophages increased.

[0062] In addition, the expression of phosphorylated protein p-STAT1 in M1 and phosphorylated protein p-STAT6 in M2 was also detected. Figure 10 As shown, there were no significant changes when treated with low concentrations of glucocorticoids; however, when treated with high concentrations of glucocorticoids, the expression of the phosphorylated protein p-STAT1 on the surface of M1 macrophages increased.

[0063] Example 6: Validating the effect of blocking glucocorticoid release on macrophages in a monocyte-macrophage cell line.

[0064] When treating monocyte-macrophage cell lines with low (50 μM) and high (1000 μM) concentrations of glucocorticoids, the experimental group received the glucocorticoid receptor inhibitor AL082D06 (10 μM), while the control group received no inhibitor. Macrophage subtype marker genes were detected in each group, and the results are as follows: Figure 11 As shown, the results revealed that macrophage polarization ceased after the use of glucocorticoid receptor inhibitors.

[0065] In addition, by detecting the expression of phosphorylated protein p-STAT1 in M1 and phosphorylated protein p-STAT6 in M2, the results are as follows: Figure 12 As shown, the results showed that the use of the glucocorticoid receptor inhibitor AL082D06 did not significantly alter the phosphorylated protein p-STAT1 on the surface of M1 macrophages.

[0066] Example 7: Observing changes in a stress-induced psoriasis mouse model using glucocorticoid receptor inhibitors.

[0067] In the imiquimod-restricted activity group of mice in Example 1, the experimental group received daily tail vein injections of the glucocorticoid receptor inhibitor AL 082D06, while the control group received daily tail vein injections of the solvent Vehicle without the inhibitor. Skin lesions were observed after 5 days, and the results are as follows: Figure 13 As shown, the application of the glucocorticoid receptor inhibitor AL 082D06 reduced epidermal lesions, decreased inflammatory infiltration, and reduced epidermal thickness in a stress-induced psoriasis mouse model.

[0068] Example 8: Observation of the effects of glucocorticoid receptor inhibitors on immune cells

[0069] The mouse skin from Example 7 was subjected to flow cytometry to observe changes in the proportion and number of immune cells. The results are as follows: Figure 14 As shown, the application of glucocorticoid receptor inhibitors reduced the number of macrophages in mouse skin, and also reduced the number of M1 macrophages.

[0070] RNA was extracted from mouse skin in Example 7, reverse transcribed, and amplified to detect marker expression in M1 and M2 macrophages. The results are as follows: Figure 15 As shown, the glucocorticoid receptor inhibitor AL 082D06 significantly reduced the expression of marker genes in M1 macrophages and significantly increased the expression of marker genes in M2 macrophages.

[0071] The mouse serum from Example 8 was analyzed for cytokines, and the results were... Figure 16 As shown, the levels of psoriasis-related cytokines IL-1β, IL-17A, IL-23, and CCL2 were significantly reduced, indicating that the application of the glucocorticoid receptor inhibitor AL 082D06 significantly alleviated stress-induced psoriasis lesions.

[0072] Example 9: Observation of changes in a stress psoriasis mouse model in mice with conditional knockout of the glucocorticoid receptor gene in macrophages.

[0073] Conditional knockout of the glucocorticoid receptor gene (Nr3c1) using macrophages M KO mice were used as the experimental group, and littermates without knockout background were used as the control group (wild type). The mice underwent activity restriction and were treated with petrolatum and imiquimod cream applied to their backs to establish the skin lesions. Five days later, the skin lesions on the backs of the mice were observed and pathological examinations were performed. The results are as follows: Figure 17 As shown, Nr3c1 was observed under a microscope. MKO mice showed reduced skin thickness and decreased inflammatory infiltration, indicating that Nr3c1 was significantly more effective than littermate controls. M In KO mice, imiquimod-induced psoriasis-like symptoms are reduced under psychological stress.

[0074] In addition, the number of macrophages, neutrophils, and dendritic cells in each group of mice was detected by flow cytometry, and the results were as follows: Figure 18 As shown, there was no significant difference in cell number among the groups.

[0075] Furthermore, the proportions of macrophages, neutrophils, and dendritic cells in each group of mice were measured, and the results are as follows: Figure 19 As shown, there was no significant difference in cell proportion among the groups.

[0076] Furthermore, the proportions of macrophage subtypes M1 and M2 in each group of mice were examined, revealing that Nr3c1 macrophages treated with stress / imiquimod showed improvement. M The reduced percentage of M1 macrophages in KO mice indicates a decrease in the proportion of pro-inflammatory macrophages, as shown in the results. Figure 20 As shown.

[0077] Furthermore, by detecting marker genes of M1 macrophages and M2 macrophages in the skin of mice in each group, it was found that Nr3c1 macrophages treated with stress / imiquimod showed... M KO mice M1 macrophage marker genes Cd80, Nos2, and Il12a were significantly reduced, as shown in the following results. Figure 21 As shown.

[0078] Example 10: Injection of glucocorticoids into stress / imiquimod mice and observation of changes in psoriasis symptoms in the model.

[0079] In experimental mice, 250 μL of 4 μg / μL glucocorticoid (DXMS) was injected via the tail vein, followed by restraint and application of imiquimod cream. On the fifth day, compared with the control group of uninjected stress-induced psoriasis mice, the stress / imiquimod group showed aggravated skin lesions. Figure 22 As shown, microscopic observation revealed that stress / imiquimod mice showed increased skin thickness and inflammatory cell infiltration after tail vein injection of DXMS; while mice treated only with imiquimod topical application showed thinner skin and reduced inflammatory cell infiltration after tail vein injection of DXMS. This suggests that under stress conditions, DXMS administration in psoriatic mice may exacerbate the psoriatic phenotype.

[0080] In addition, the number of macrophages, neutrophils, and dendritic cells in each group of mice was detected by flow cytometry, and the results are as follows: Figure 23 As shown, there was no significant difference in cell number among the groups.

[0081] Furthermore, the proportions of macrophages, neutrophils, and dendritic cells in each group of mice were measured, and the results are as follows: Figure 24 As shown, there was no significant difference in cell proportion among the groups.

[0082] Furthermore, the proportions of macrophage subtypes M1 and M2 in each group of mice were examined. It was found that the percentage of M1 macrophages increased after DXMS injection, indicating an increase in the proportion of pro-inflammatory macrophages. The results are as follows: Figure 25 As shown.

[0083] Furthermore, the marker genes of M1 macrophages and M2 macrophages in the skin of mice in each group were detected. It was found that the marker genes Cd80, Nos2, Tnf, and Il1b of M1 macrophages in DXMS-treated stress / imiquimod mice were significantly increased. The results are as follows: Figure 26 As shown.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for constructing a stress-induced psoriasis mouse model, characterized in that, include: (1) Shave the back of the mouse and put it into a 50 ml breathable centrifuge tube and restrict its activity for four hours; (2) Release the mouse and wait for the skin on its back to dry; (3) Apply imiquimod cream evenly to the back of the mouse and put it back in the cage; (4) Repeat the above steps every day until the stress-induced psoriasis mouse model is successfully constructed; inject the mouse with 250 μl of 4 μg / μl glucocorticoid every day.

2. The method for constructing a stress-induced psoriasis mouse model according to claim 1, characterized in that, In step (4), the number of repetitions is 4.

3. The method for constructing a stress-induced psoriasis mouse model according to claim 1 or 2, characterized in that, Female 8-week-old mice were selected as mice.

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