Use of lupine ketone in the preparation of a medicament for treating psoriasis

Abstract

This invention discloses the application of lupinone in the preparation of drugs for treating psoriasis, belonging to the field of pharmaceutical technology. In this invention, lupinone is used to treat psoriasis by regulating CD4 levels in psoriasis patients. + FoxP3 + The number of Tregs, regulatory T lymphocytes, reduces the expression of CD3-positive cells and the level of inflammatory factors in the skin lesions of psoriasis patients, thereby regulating the immune response of psoriasis patients and thus achieving the treatment of psoriasis. Specifically, it can reduce the severity of psoriasis, improve the degree of erythema, scaling and infiltration of skin lesions, significantly reduce the PASI (Psoriasis Area and Severity Index) score of skin lesions, reduce the thickness of the epidermis at the skin lesion site and inhibit epidermal proliferation, improve hyperkeratosis and parakeratosis, and improve acanthosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of lupinone in the preparation of drugs for treating psoriasis. Background Technology

[0002] Psoriasis is an immune-mediated chronic inflammatory disease, linked to T-cell-mediated immune and inflammatory responses, and also influencing the pathogenesis of many major diseases, including arthritis, obesity-metabolic syndrome, cardiovascular disease, and malignancies (including kidney cancer, breast cancer, lung cancer, and colon cancer). Therefore, psoriasis imposes a significant psychological and physiological burden on patients. Approximately 2% of the world's population suffers from psoriasis, making it the most common autoimmune skin disease in adults.

[0003] Current research has found that Foxp3 levels in the peripheral blood of psoriasis patients are high. + The number of Treg cells is increased, and this increase is positively correlated with the Psoriasis Activity and Severity Index (PASI). CD4 + CD25 + FoxP3 + Treg cells were also present in psoriatic lesions, and similar to peripheral blood, their levels were higher in lesion biopsies than in control or healthy skin biopsies. These findings suggest that regulatory T cells (Tregs) play a role in the pathogenesis of psoriasis. However, how to suppress psoriasis development by regulating Treg cells remains unexplored. Summary of the Invention

[0004] According to one aspect of the invention, the use of lupinone in the preparation of a medicament for treating psoriasis is provided.

[0005] The applicant of this invention has surprisingly discovered that lupinone has a significant therapeutic effect on psoriasis, significantly reducing the severity of psoriasis, improving the degree of erythema, scaling, and infiltration of skin lesions, significantly reducing the PASI (Psoriasis Area and Severity Index) score of skin lesions, reducing epidermal thickness at the lesion site and inhibiting epidermal proliferation, improving hyperkeratosis and parakeratosis, improving acanthosis, and improving weight loss. This is because lupinone can significantly induce regulatory T cells (Tregs, specifically CD4+) in the spleen and lymph nodes. + FoxP3 + An increase in the number of Treg cells can regulate the immune response in psoriasis patients, thereby achieving the treatment of immune-mediated psoriasis.

[0006] In some embodiments, the above application refers to the use of lupinone in the preparation of psoriasis drugs having one or more of the following effects: improving the degree of erythema, scaling and infiltration thickness of skin lesions; reducing the PASI score of skin lesions; reducing the thickness of the epidermis at the skin lesion and inhibiting epidermal proliferation; improving hyperkeratosis, parakeratosis, and acanthosis.

[0007] In some embodiments, the dosage of lupinone in the psoriasis treatment drug is 100 mg / kg body weight / day.

[0008] According to another aspect of the invention, the use of lupinone in the preparation of a medicament for increasing the number of regulatory T lymphocytes in vivo is provided.

[0009] In some embodiments, the above application is the use of lupinone in the preparation of a drug that increases the number of regulatory T lymphocytes in the spleen and / or lymph nodes.

[0010] In some embodiments, the aforementioned regulatory T lymphocytes are CD4 cells. + FoxP3 + Treg cells. Among them, CD4 + FoxP3 + Treg cells are generated by CD4 + CD25 - It originates from the differentiation of T cells.

[0011] According to another aspect of the present invention, the use of lupinone in the preparation of a medicament that reduces CD3-positive expression in psoriatic lesions is provided.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This invention is the first to discover that lupinone has a significant therapeutic effect on imiquimod-induced psoriasis in mice. This invention utilizes lupinone to treat psoriasis by upregulating CD4 levels in psoriasis patients. + FoxP3 + The number of Tregs, a type of regulatory T lymphocyte, reduces the expression of CD3 and the level of inflammatory factors in the skin lesions of psoriasis patients, improves the infiltration of inflammatory cells in the skin tissue, regulates the immune response of psoriasis patients, and thus achieves the treatment of psoriasis. It significantly reduces the severity of psoriasis, reduces the PASI score of the skin lesions and the thickness of the epidermis at the lesion site, inhibits epidermal proliferation, and lupinone has the characteristics of low side effects and good safety. Attached Figure Description

[0014] Figure 1 These are photographs of the skin lesions on the backs of mice in different groups during the experiments of this invention.

[0015] Figure 2 These are the PASI scores of mice in different groups during the experiments of this invention.

[0016] Figure 3 HE staining of skin tissue from different groups of mice in the experiment of this invention.

[0017] Figure 4 These are immunohistochemical stainings of skin tissues from different groups of mice used in the experiments of this invention.

[0018] Figure 5 In the experiment of this invention, ELISA was used to measure the release of cytokines IL-6, TNF-α, and IL-17A from the skin tissue of mice in different groups.

[0019] Figure 6 These are regulatory T lymphocytes (CD4+) from the spleen and lymph nodes of mice in different groups during the experiments of this invention. + FoxP3 + Flow cytometry density plot of Tregs.

[0020] Figure 7 These are regulatory T lymphocytes (CD4+) from the spleen (A) and lymph nodes (B) of different groups of mice in the experiment of this invention. + FoxP3 + A bar chart of Tregs. Detailed Implementation

[0021] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0022] Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. The lupenone (CAS: 1617-70-5) used in this invention has the molecular formula C0.05. 30 H 48 O, molecular weight: 424.7, structural formula:

[0023]

[0024] To verify the therapeutic effect of lupinone on psoriasis, the applicant constructed an imiquimod-induced mouse psoriasis model and conducted the following experiments. Cytokine assays were performed using the Mouse IL-6 ELISA Kit, Mouse TNF-α ELISA Kit, and Mouse IL-17A ELISA Kit (Wuhan Boster Biological Engineering Co., Ltd.). Immunohistochemical staining was performed using standard methods; specifically, the primary antibody was Anti-CD3 (1:200, Abcam), and the secondary antibody was enzyme-labeled goat anti-rabbit IgG polymer (Wuhan Boster Biological Engineering Co., Ltd.).

[0025] I. Animal Modeling and Drug Administration

[0026] 1. Construction of a mouse model of psoriasis

[0027] Healthy male Balb / c SPF mice aged 6-8 weeks were used. The mice were dehaired and randomly divided into a normal control group (Ctrl), a model group (Vehicle), and a lupenone group. Both the model and lupenone groups received topical application of 5% (w / w) imiquimod (62.5 mg) ointment (IMQ, Sichuan Mingxin Pharmaceutical Co., Ltd.) to the dehaired areas for 7 days, covering an area of ​​approximately 3cm × 3cm, once daily. On day 3 after application, the mice began to show skin thickening and scaling. By day 7, increased scaling, significant skin thickening, and pronounced erythema were observed, indicating successful establishment of the psoriasis model.

[0028] 2. Animal dosage

[0029] The lupinone group was given lupinone (100 mg / kg / day) by gavage for 7 consecutive days starting from day 1, while the normal control group and the model group were given the same amount of physiological saline for 7 consecutive days.

[0030] After the psoriasis mice treated with the above drugs were raised to the corresponding event, various indicators of psoriasis were tested.

[0031] II. Experimental Methods and Results

[0032] 1. Symptoms of skin lesions on the back

[0033] Daily digital photography was used to record and observe the condition of the skin lesions on the backs of mice treated with different drugs, and the lesions were scored (erythema, scaling, and infiltration / thickness) (scored from mild to severe on a scale of 0-4). The scores for all three factors were added together to obtain the total score. The PASI scoring criteria are as follows: 0, none; 1, mild; 2, moderate; 3, severe; 4, very severe. Figure 1 , Figure 2 It can be seen from this:

[0034] Compared to the normal group, the psoriasis model group showed significantly thickened dorsal skin with symptoms such as erythema and scaling, resembling human psoriasis-like lesions, and an increased PASI score. Compared to the model group, the lupin ketone group showed reduced scaling, erythema, and infiltration symptoms, and a lower PASI score after lupin ketone administration.

[0035] 2. Inflammatory cell infiltration

[0036] Skin samples (0.5cm x 0.5cm) were excised from the mouse skin lesions to observe the extent of the damage. Histological staining with hematoxylin and eosin (HE) was performed on the skin lesions. Figure 3 It can be seen from this:

[0037] In HE staining, compared with the normal control group mice, the psoriasis model group mice showed significantly thickened epidermal spinous cells, parakeratosis in the stratum corneum with residual shrunken cell nuclei, and abundant inflammatory cell infiltration in the dermis. These symptoms are consistent with the histological characteristics of psoriasis-like lesions. Compared with the model group mice, the lupinone group mice showed significantly reduced epidermal spinous cells and decreased inflammatory cell infiltration in the dermis.

[0038] 3. CD3 positive expression in psoriatic lesions

[0039] Skin samples of the mouse lesion, each measuring 0.5cm × 0.5cm, were excised to observe the extent of skin damage. Histological immunohistochemical staining of the skin lesion was performed. Figure 4 It can be seen from this:

[0040] In immunohistochemical staining, the positive expression of CD3 in the epidermis and dermis of the psoriasis model group was significantly increased. In the lupin group, the positive expression of CD3 in the epidermis and dermis of the psoriasis model group was significantly decreased.

[0041] 4. Inflammatory factor levels

[0042] Skin damage can also be identified by the production and secretion of cytokines IL-6, TNF-α, and IL-17A. These cytokines released from the skin tissue of mice in each group were detected using ELISA. Figure 5 It can be seen from this:

[0043] Compared with the control group, the levels of IL-6, TNF-α, and IL-17A in the skin lesions of mice in the model group were significantly upregulated (P < 0.01). Compared with the model group, the levels of IL-6, TNF-α, and IL-17A in the skin lesions of mice in the lupinone group were significantly downregulated (P < 0.01).

[0044] 5. CD4 in psoriatic mice + FoxP3 + Number of Treg cells

[0045] Mice were treated with different drugs and fed for one week. The proportion of regulatory T cells in the spleen and lymph nodes of the mice was then quantitatively analyzed using flow cytometry. Figures 6-7 It can be seen from this:

[0046] CD4 in spleen and lymph nodes in the lupinone group + FoxP3 + The proportions of Treg cells were 6.43% ± 0.87% and 7.63% ± 0.97%, respectively, which were higher than those of the control group by 2.87% ± 0.95% (P < 0.01) and 3.10% ± 0.76% (P < 0.01), respectively.

[0047] The above results indicate that lupinone can significantly induce an increase in the number of regulatory T cells in the spleen and lymph nodes of mice, thereby regulating the immune response in psoriatic mice.

[0048] The above five experiments and their results indicate that lupinone can significantly improve psoriasis symptoms, reduce PASI scores of skin lesions, improve the infiltration of inflammatory cells in the skin, reduce CD3-positive expression and inflammatory factor levels in psoriasis model skin lesions, and upregulate regulatory T lymphocytes (CD4+) in vivo. + FoxP3 + The proportion of Tregs.

[0049] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. Application of lupinone in the preparation of drugs for treating psoriasis.

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