Use of VEGFR gene expression inhibitors as or in the preparation of a formulation for treating psoriasis - Patents.com

Abstract

To provide a safe and effective formulation for treating psoriasis.SOLUTION: The present invention relates to the use of a VEGFR gene expression inhibitor as a formulation for treating psoriasis or in its preparation. This invention effectively inhibits an abnormal expression of Rasgrp1 in skin T-cells through NBUVB irradiation, thereby inhibiting IL-17 secretion from the T-cells, thus effectively alleviating IL-17-mediated immune response in skin psoriatic lesions to achieve an effect of treating the psoriasis.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention belongs to the technical field of medicine for skin diseases, and specifically relates to the use of a VEGFR gene expression inhibitor as a preparation or in the preparation of a preparation for treating psoriasis. [Background technology]

[0002] Psoriasis is a chronic immune-inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes, infiltration of lymphocytes (mostly T cells), and changes in dermal blood vessels such as angiogenesis, dilation, tortuosity, and formation of high endothelial venules. Psoriasis is most commonly seen in clinical practice as psoriasis vulgaris. The pathogenesis of psoriasis is broad and the pathogenesis is complex, and immune pathways play an important role in the pathogenesis of psoriasis, which is closely related to chronic inflammation mediated by abnormal activation of T cells. Research has shown that psoriasis is driven by the activation of T cells, which is associated with the secretion of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin IL-17A, and interferon-γ. The presence and accumulation of T cells is one of the early events in the evolution process of psoriatic plaques, and abnormal activation of T cells affects skin immunity and the production of systemic cytokines, thereby stimulating the recruitment of more inflammatory cells and causing psoriasis-specific epidermal changes.

[0003] Currently, the clinical use of biological agents targeting IL17A / F has shown relatively good therapeutic effects on psoriasis, but the related biological agents are expensive and need to be used continuously in the later stages, resulting in high treatment costs and the inability to eradicate the disease.

[0004] Therefore, there is a need for safe and effective formulations for the treatment of psoriasis. Summary of the Invention

[0005] In response to the above problems, one of the objectives of the present invention is to provide the use of VEGFR gene expression inhibitor as a preparation for treating psoriasis or in the preparation of said preparation.Specifically, in the present invention, by inhibiting VEGFR gene expression, IL-17 production by T cells can be effectively inhibited, and psoriasis can be effectively treated.Note that in the present invention, the VEGFR gene expression inhibitor is any reagent known in the art that can inhibit VEGFR gene expression, based on the mechanism of reducing or eliminating psoriasis phenotype by inhibiting VEGFR gene expression.

[0006] Another object of the present invention is to provide the use of Rasgrp1 gene expression inhibitor as a preparation for treating psoriasis or in the preparation of said preparation.Specifically, by inhibiting Rasgrp1 gene, it is possible to effectively inhibit IL-17 production by T cells and effectively treat psoriasis.Similarly, as described above, it is based on the mechanism that by inhibiting Rasgrp1 gene expression, it is possible to reduce or eliminate the psoriasis phenotype.

[0007] Furthermore, in the above use, the preparation includes chemical preparation or biological preparation.For example, the VEGFR gene expression inhibitor can be a chemical preparation such as a VEGFR small molecule inhibitor, or can be a gene editing reagent for knocking down or knocking out VEGFR gene, such as CRISPR-Cas reagent, TALEN reagent, ZFN reagent, etc.; similarly, the Rasgrp1 gene expression inhibitor can be a small molecule chemical preparation for inhibiting Rasgrp1 gene expression, or can be a reagent for knocking down or knocking out Rasgrp1 gene, such as CRISPR-Cas reagent, TALEN reagent, ZFN reagent, etc.

[0008] In addition, in the above-mentioned use, the preparation includes a paste, a powder, or a liquid. Note that the VEGFR gene expression inhibitor or the Rasgrp1 gene expression inhibitor can be prepared into various dosage forms suitable for clinical use by adding an auxiliary agent, and the VEGFR gene expression inhibitor or the Rasgrp1 gene expression inhibitor can be used in combination with other reagents for treating psoriasis to jointly treat psoriasis.

[0009] A further object of the present invention is to provide the use of NBUVB radiation in inhibiting VEGF gene expression.

[0010] A further object of the present invention is to provide the use of NBUVB irradiation in inhibiting Rasgrp1 gene expression.

[0011] A further object of the present invention is to provide the use of NBUVB irradiation in inhibiting IL-17 production by T cells.

[0012] In addition, NBUVB irradiation can inhibit Rasgrp1 gene expression by inhibiting VEGF gene expression in the body, and inhibiting Rasgrp1 gene expression can effectively inhibit IL-17 secreted by downstream T cells, thereby effectively treating psoriasis and achieving the purpose of precise treatment of psoriasis.In addition, the present invention uses the successfully constructed NBUVB-treated psoriasis mouse model to perform RNA-Seq pathway enrichment analysis, and finds that NBUVB can significantly inhibit the RAS pathway, which is helpful in diversifying the pathological mechanism of NBUVB-treated psoriasis treatment, and provides a theoretical basis for the genetic diagnosis, targeted treatment, and research and development of new drugs for psoriasis.

[0013] A further object of the present invention is to provide a method for inhibiting IL-17 production by T cells, which comprises irradiating with NBUVB, knocking out or knocking down the VEGF gene, or knocking out or knocking down the Rasgrp1 gene. Specifically, as described above, in the present invention, IL-17 production by T cells may be effectively inhibited by NBUVB irradiation, IL-17 production by T cells may be inhibited by inhibiting VEGF gene expression, or IL-17 production by T cells may be inhibited by inhibiting the Rasgrp1 gene.

[0014] In addition, psoriasis is a complex disease, and not influenced by a single factor. Finding the intrinsic mechanism of disease occurrence and control and the weight of various influencing factors in the disease is the basis and prerequisite for effectively identifying future disease targets. Vascular endothelial growth factor (VEGF) is an important angiogenesis promoter, and has the effect of promoting increased vascular permeability and inducing angiogenesis. In the present invention, NBUVB can significantly inhibit the expression of VEGF in the skin tissue of psoriasis mice.

[0015] In addition, RasGRP1 is a RAS guanine nucleotide exchange factor and an important regulator of lymphocyte receptor signal transduction. Rasgrp1 is one of the downstream target genes of vascular endothelial growth factor VEGF. Rasgrp1 plays an important role in the growth and development of T cells, and regulates the growth, steady state and differentiation of T cells / B cells by binding T lymphocyte / B lymphocyte antigen receptors with RAS. The signal required for abnormal activation of T cells is the recognition signal of T cell receptor TCR. When T cells are stimulated and activate TCR, Rasgrp1 expression increases and is highly expressed in T cells, playing a very important role in the activation and transmission of TCR signals, acting as a bridge to transmit signals downstream to promote the proliferation and activation of T cells. Activated T cells secrete various cytokines and inflammatory mediators, which mediate the occurrence and development of psoriasis. Effect of the Invention

[0016] The beneficial effects of the present invention are as follows: In the present invention, the abnormal expression of Rasgrp1 in skin T cells can be effectively inhibited by NBUVB irradiation, and the secretion of IL-17 from T cells can be inhibited, so that the immune response mediated by IL-17 in the skin site of psoriasis can be effectively reduced, and the effect of treating psoriasis can be achieved. [Brief description of the drawings]

[0017] [Figure 1] Pathological skin phenotype of NBUVB-irradiated psoriatic mice. [Diagram 2] PASI scoring of NBUVB-irradiated psoriatic mice. [Diagram 3] Pathway enrichment in gene transcriptome sequencing revealed that NBUVB irradiation inhibited the RAS pathway. [Figure 4] 1 shows the results of real-time fluorescent quantitative PCR detection of RAS pathway mRNA in the skin of psoriatic mice irradiated with NBUVB. [Diagram 5] Protein expression of the RAS pathway in the skin of NBUVB-irradiated psoriatic mice. [Figure 6] Flow cytometry results of Rasgrp1+T and IL-17A+T cells in NBUVB-irradiated psoriatic mice. [Figure 7] FIG. 13 is a histogram statistical diagram of flow cytometry results of Rasgrp1+T and IL-17A+T cells in NBUVB-irradiated psoriatic mice. [Figure 8] FIG. 1 shows the skin pathology phenotype of the imiquimod psoriasis mouse model. [Figure 9] Rasgrp1 expression and localization in skin tissue of psoriatic mice. [Figure 10] 1 shows the results of real-time fluorescent quantitative PCR detection of RAS pathway mRNA in the skin of psoriatic mice. [Figure 11] Protein expression of the RAS pathway in psoriatic mouse skin. [Figure 12]These are the results of flow cytometry of Rasgrp1+T, Rasgrp1+Monocytes, and Rasgrp1+Neutrophils cells in the skin tissue of psoriatic mice. [Figure 13] FIG. 13 is a histogram statistical diagram of the flow cytometry results of Rasgrp1+T, Rasgrp1+Monocytes, and Rasgrp1+Neutrophils cells in the skin tissue of psoriatic mice. [Figure 14] Co-labeling of T cells with Rasgrp1 in skin tissue from psoriatic mice was detected by immunofluorescence. [Figure 15] VEGF mRNA in the skin of psoriatic mice exposed to NBUVB was detected by real-time fluorescent quantitative PCR. [Figure 16] The expression and localization of VEGF in skin tissue of NBUVB-irradiated psoriatic mice. [Figure 17] VEGF mRNA in the epidermis of psoriatic mice exposed to NBUVB was detected by real-time fluorescent quantitative PCR. [Figure 18] VEGF protein expression in epidermis of NBUVB-irradiated psoriatic mice. [Figure 19] VEGF mRNA in the dermis of psoriatic mice exposed to NBUVB was detected by real-time fluorescent quantitative PCR. [Figure 20] Protein expression of VEGF in the dermis of NBUVB-irradiated psoriatic mice. [Figure 21] Results of single-cell sequencing of human psoriatic skin lesions reveal that VEGFA is the predominant cell population expressing VEGFA. [Figure 22] HACAT cells were irradiated with NBUVB, and changes in VEGF concentration in the cell supernatant were detected at each irradiation dose gradient and each irradiation time gradient. [Diagram 23] HACAT cells were stimulated with IL-17A and TNF-α cytokines, and the VEGF concentration in the cell supernatant of the NBUVB-unirradiated and NBUVB-irradiated groups was detected by ELISA. [Figure 24] Skin pathology phenotype of psoriatic mice subcutaneously injected with a VEGF stimulator. [Diagram 25]PASI scoring of psoriatic mouse skin following subcutaneous injection of a VEGF stimulator. [Figure 26] Protein expression of the RAS pathway in the skin of psoriatic mice subcutaneously injected with a VEGF stimulator. [Figure 27] Flow cytometry results of Rasgrp1+T and IL-17A+T cells in the skin of psoriatic mice subcutaneously injected with VEGF stimulatory factors. [Figure 28] FIG. 1 shows histogram statistics of flow cytometry results of Rasgrp1+T and IL-17A+T cells in the skin of psoriatic mice subcutaneously injected with a VEGF stimulator. [Figure 29] This shows the skin pathology phenotype of psoriatic mice treated with the VEGFR small molecule inhibitor SU5205. [Diagram 30] PASI scoring of psoriatic mice after applying the VEGFR small molecule inhibitor SU5205 to the skin. [Diagram 31] Protein expression of the RAS pathway in the skin of psoriatic mice after application of the VEGFR small molecule inhibitor SU5205 to the skin. [Diagram 32] These are the results of flow cytometry of Rasgrp1+T and IL-17A+T cells in the skin of psoriatic mice to which the VEGFR small molecule inhibitor SU5205 was applied. [Diagram 33] FIG. 13 is a histogram statistical diagram of the flow cytometry results of Rasgrp1 + T and IL-17A + T cells in the skin of psoriatic mice to which the VEGFR small molecule inhibitor SU5205 was applied. [Diagram 34] This shows the results of real-time fluorescent quantitative PCR detection of IL1b and IL17 mRNA in Jurkat cells overexpressing Rasgrp1 following stimulation with VEGF cytokines. [Diagram 35] This shows the concentrations of IL1b and IL17 in the supernatant of Jurkat cells overexpressing Rasgrp1 following stimulation with VEGF cytokines. [Diagram 36] Protein expression of the RAS pathway in Jurkat cells overexpressing Rasgrp1 following stimulation with VEGF cytokines. [Figure 37] This shows the results of real-time fluorescent quantitative PCR detection of IL1b and IL17 mRNA in Jurkat cells in which Rasgrp1 was knocked down following stimulation with VEGF cytokines. [Figure 38] This shows the concentrations of IL1b and IL17 in the supernatant of Jurkat cells in which Rasgrp1 was knocked down following stimulation with VEGF cytokines. [Figure 39] Protein expression of the RAS pathway in Jurkat cells in which Rasgrp1 was knocked down following stimulation with VEGF cytokines. [Diagram 40] This shows the skin pathology phenotype of psoriatic mice subcutaneously injected with adeno-associated virus overexpressing Rasgrp1. [Diagram 41] PASI scoring of psoriatic mice subcutaneously injected with adeno-associated virus overexpressing Rasgrp1. [Diagram 42] This shows the concentration of IL-1β in the skin tissue of psoriatic mice subcutaneously injected with an adeno-associated virus that overexpresses Rasgrp1. [Diagram 43] This shows the results of real-time fluorescent quantitative PCR detection of RAS pathway mRNA in skin tissue from psoriatic mice subcutaneously injected with an adeno-associated virus that overexpresses Rasgrp1. [Diagram 44] Protein expression of the RAS pathway in skin tissue from psoriatic mice subcutaneously injected with an adeno-associated virus that overexpressed Rasgrp1. [Diagram 45] These are the results of flow cytometry of Rasgrp1+T and IL-17A+T cells in the skin tissue of psoriatic mice that were subcutaneously injected with an adeno-associated virus that overexpresses Rasgrp1. [Figure 46] FIG. 13 is a histogram statistical diagram of the flow cytometry results of Rasgrp1 + T and IL-17A + T cells in the skin tissue of psoriatic mice subcutaneously injected with an adeno-associated virus that overexpresses Rasgrp1. [Figure 47] Skin pathology phenotype of Rasgrp1-overexpressing psoriatic mice irradiated with NBUVB. [Figure 48]PASI scoring of skin in NBUVB-irradiated Rasgrp1-overexpressing psoriatic mice. [Figure 49] IL-1β concentration in skin tissue of N-BUVB-irradiated Rasgrp1-overexpressing psoriatic mice. [Figure 50] 1 shows the results of real-time fluorescent quantitative PCR detection of RAS pathway mRNA in skin tissue of psoriatic mice overexpressing Rasgrp1 irradiated with NBUVB. [Figure 51] NB: Protein expression of the RAS pathway in skin tissue of UVB-irradiated Rasgrp1-overexpressing psoriatic mice. [Figure 52] Flow cytometry results of Rasgrp1+T cells and IL-17A+T cells in skin tissues of NBUVB-irradiated Rasgrp1-overexpressing psoriatic mice. [Figure 53] This is a histogram statistical diagram of the flow cytometry results of Rasgrp1+T and IL-17A+T cells in skin tissue from NBUVB-irradiated Rasgrp1-overexpressing psoriatic mice. [Figure 54] Skin pathology phenotype of Rasgrp1- / - psoriatic mice. [Figure 55] Skin PASI scoring of Rasgrp1− / − psoriatic mice. [Figure 56] IL-1β concentration in skin tissue of Rasgrp1− / − psoriatic mice. [Figure 57] 1 shows the results of real-time fluorescent quantitative PCR detection of RAS pathway mRNA in skin tissues of Rasgrp1− / − psoriatic mice. [Figure 58] Protein expression of the RAS pathway in skin tissue from Rasgrp1- / - psoriatic mice. [Figure 59] Flow cytometry results of IL-17A+T cells in skin tissue of Rasgrp1- / - psoriatic mice. [Figure 60] FIG. 13 is a histogram statistical diagram of flow cytometry results of IL-17A+T cells in skin tissue of Rasgrp1− / − psoriatic mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The illustrated examples are provided to better explain the present invention, but the content of the present invention is not limited to the illustrated examples. Therefore, even if a person skilled in the art makes non-essential improvements and adjustments to the embodiments based on the above content of the invention, they still fall within the scope of protection of the present invention.

[0019] The terms used herein are used only to describe certain embodiments and are not intended to limit the present disclosure. Singular expressions include complex expressions unless the context clearly indicates otherwise. As used herein, terms such as "comprises", "has", "contains" and the like are intended to indicate the presence of features, numbers, operations, assemblies, parts, elements, materials, or combinations thereof, and should be understood to be intended to indicate the presence of features, numbers, operations, assemblies, parts, elements, materials, or combinations thereof. The terms of the present invention are disclosed herein and are not intended to exclude the possibility that one or more other features, numbers, operations, assemblies, parts, elements, materials, or combinations thereof may be present or added. When used herein, " / " can be interpreted as "and" or "or", as the case may be.

[0020] In order to better understand the present invention, the present invention will be further described below using specific examples, but the present invention is not limited to the following examples.

[0021] In the following examples, any experimental procedures not specifically described are routine experimental procedures in the art.

[0022] Example 1 Inhibition of downstream Ras pathway activation in skin tissue of psoriatic mice by NBUVB irradiation and alleviation of mouse phenotype In an embodiment of the present invention, NBUVB irradiation therapy was performed on psoriatic mice. NBUVB irradiation therapy reduced the inflammatory response in the skin of the psoriatic mice, and HE staining revealed significant thinning of the skin epidermis and a reduction in inflammatory cell infiltration, suggesting a reduction in the inflammatory response (Figure 1).

[0023] In addition, the mice's skin was scored using the PASI scale, which revealed a decrease in skin thickness on the back and a reduction in erythema and scale (Figure 2).

[0024] In addition, we also performed RNA-Seq sequencing pathway enrichment analysis, and the results showed that the RAS pathway was significantly inhibited after NBUVB irradiation (Figure 3).

[0025] Furthermore, RT-qPCR detection of Rasgrp1 and its downstream cytokines, IL1b and IL17a, showed that the expression levels of inflammatory genes, such as Rasgrp1 and its downstream cytokines, IL1b and IL17a, were significantly reduced in the skin tissue of psoriatic mice treated with NBUVB irradiation (Figure 4).

[0026] Furthermore, Western blot was used to detect the protein expression of Rasgrp1 downstream pathway genes, and the results showed that the phosphorylated active protein levels of NF-κB and AKT in the skin of psoriatic mice treated with NBUVB irradiation were both lower than those in the control group, and the active protein levels of IL-1β were also lower than those in the control group (Figure 5).

[0027] Furthermore, Rasgrp1+ T cells and IL-17A+ T cells were both significantly decreased in the skin of psoriatic mice after NBUVB irradiation (Figures 6 and 7).

[0028] These results suggest that NBUVB inhibits the Ras pathway, thereby inhibiting IL-17 production by T cells and reducing psoriasis-like skin inflammation.

[0029] Example 2 Rasgrp1 is mainly expressed in skin T cells and the Ras pathway is activated in psoriasis In the examples of the present invention, the skin tissues of psoriasis and normal control mice were stained with HE staining to observe the inflammatory reaction in the skin lesions of the psoriatic mice. HE staining showed dilation of dermal capillaries, thickening of the epidermis, and infiltration of inflammatory cells into the subcutaneous tissue and skin appendages such as hair follicles and hair shafts, suggesting the presence of an inflammatory reaction (Figure 8).

[0030] Furthermore, to detect the expression and localization of Rasgrp1 in psoriasis, immunohistochemical experiments showed that Rasgrp1 was mainly expressed in immune cells, and was strongly positive in the skin tissue of psoriatic mice (Figure 9).

[0031] Furthermore, RT-qPCR detection of Rasgrp1 downstream cytokines such as IL1b and IL17a revealed that the expression levels of inflammatory factors in skin tissue of psoriasis mouse models were significantly higher than those of normal controls (Figure 10).

[0032] Furthermore, Western blot was used to detect the protein expression of Rasgrp1 downstream pathway genes, and the results showed that the total protein levels and phosphorylated active protein levels of NF-κB and AKT in the skin of psoriatic mice were all higher than those in the control group, and the active protein level of IL-1β in the skin of psoriatic mice was higher than those in the control group (Figure 11).

[0033] Furthermore, lymphocytes were extracted from the skin of normal control mice and psoriatic mice, and the isolated mouse skin immune cells were grouped using specific labeled antibodies. When localized to a cell population called T cells, a significant difference was observed in the expression levels of Rasgrp1 in the skin of normal control mice and psoriatic mice, but the difference in monocytes and neutrophils was not statistically significant (Figures 12 and 13).

[0034] Furthermore, co-labeling of T cells with Rasgrp1 in skin tissues from normal control and psoriatic mice was detected by immunofluorescence, revealing Rasgrp1 expression in T cells (Fig. 14).

[0035] Example 3: Inhibition of VEGF secreted mainly from epidermal cells in skin tissue of psoriatic mice by NBUVB irradiation In the examples of the present invention, the gene expression of VEGFA in the dorsal skin of psoriatic mice and NBUVB-irradiated psoriatic mice was detected by RT-qPCR. The results showed that NBUVB irradiation inhibited the expression of VEGF factor in the skin tissue of psoriatic mice (Figure 15). Immunohistochemistry showed that VEGF was mainly expressed in epidermal cells of the skin, and that NBUVB irradiation reduced the expression of epidermal VEGF (Figure 16). The epidermis and dermis of mouse skin were separated, and the expression of VEGF in mouse epidermal tissue was detected by RT-qPCR. It was shown that NBUVB irradiation inhibited the VEGF gene expression in mouse epidermal tissue (Figure 17).

[0036] Furthermore, Western Blot detection showed that NBUVB irradiation inhibited the protein expression of VEGFA in the dorsal epidermal tissue of psoriasis mice (Figure 18). RT-qPCR detection showed that NBUVB irradiation did not affect the VEGF gene expression in the dermis tissue of mice (Figure 19), and Western Blot detection showed that NBUVB irradiation did not affect the protein expression of VEGFA in the dorsal epidermal tissue of psoriasis mice (Figure 20).

[0037] Furthermore, sequencing of single cells from human psoriatic skin lesions showed that VEGFA was mainly secreted from epidermal cells, and in the granular layer of the skin, the expression level of VEGFA in psoriatic skin lesion tissues was significantly higher than that in normal tissues (Figure 21).

[0038] Furthermore, HACAT cells were irradiated with NBUVB, with an irradiation dose gradient of 0mJ / cm2, 200mJ / cm2, 400mJ / cm2, and 800mJ / cm2, and an irradiation time gradient of 8h, 12h, and 24h after irradiation, respectively, and the cell supernatant was collected to detect changes in VEGF concentration.It was shown that when HACAT cells were irradiated with an irradiation dose of 400mJ / cm2 for 8h, the VEGF concentration in the supernatant was significantly decreased (Figure 22).

[0039] Furthermore, HACAT cells were stimulated with IL-17A and TNF-α cytokines, and the cells were irradiated with 400 mJ / cm2 of NBUVB. The VEGFA concentration in the supernatant of cells that were not irradiated with NBUVB and cells in the NBUVB-irradiated group was detected by ELISA. The VEGFA secretion concentration from cells in the NBUVB-irradiated group was significantly lower than that of the unirradiated group (Figure 23).

[0040] Example 4: Subcutaneous injection of VEGF aggravates phenotype of psoriasis mice, and VEGFR inhibitor alleviates phenotype of psoriasis mice In the present embodiment, the VEGFR inhibitor SU5205 was dissolved in DMSO solution to prepare a stock solution, and the stock solution was diluted with cyclodextrin. The back hair of 7-8 week old wild type mice was shaved to expose the skin area of ​​2cm x 2cm, and 150μl of the freshly prepared VEGF inhibitor solution was applied to the skin surface at a mouse application concentration of 10mg / kg / d, thus establishing a mouse psoriasis model with VEGFR inhibitor application, and the control group animals were applied with the control agent (DMSO + cyclodextrin 150μl). After 4h of application, IMQ cream or the control agent VAS was applied evenly to the exposed skin of the mice.

[0041] In the present embodiment, VEGF165 protein was dissolved in DMSO solution to prepare a stock solution, and the stock solution was diluted with cyclodextrin. The back hair of 7-8 week old wild type mice was shaved to expose the skin area of ​​2cm×2cm, and 100μl of freshly prepared VEGF165 solution with a mouse injection concentration of 10mg / kg / d was subcutaneously injected, thus establishing a VEGF subcutaneous injection psoriasis model, and the control group animals were subcutaneously injected with the control agent (DMSO+cyclodextrin 100μl). After 4h of application, IMQ cream or the control agent VAS was evenly applied to the exposed skin of the mice.

[0042] In the present invention, an IMQ-induced psoriasis mouse model based on VEGF overexpression was created according to the above method. The results of H&E staining and PASI scoring of skin sections showed that the psoriasis-like phenotype of VEGF165 subcutaneously injected mice was significantly worse than that of IMQ-induced control mice, and the thickness of the back skin was increased, and the erythema and scale were worsened (Figures 24 and 25).

[0043] Furthermore, Western blot was used to detect the protein expression of Rasgrp1 downstream pathway genes, and the results showed that the phosphorylation activity protein levels of NF-κB and AKT were all significantly increased in the skin of VEGF-overexpressing psoriatic mice, among which the activity protein level of IL-1β was also significantly higher than that of the control group (Figure 26).

[0044] Furthermore, in VEGF-overexpressing psoriatic mice, Rasgrp1+ T cells and IL-17A+ T cells in the skin were significantly increased (Figures 27 and 28), suggesting that overexpression of VEGF in skin tissue activates the secretion of Rasgrp1 and downstream inflammatory factors, thereby regulating IL-17 production by T cells and affecting psoriasis-like skin inflammation.

[0045] To further clarify the mechanism of action of VEGF in psoriasis, we created a mouse model of psoriasis by applying the VEGF receptor inhibitor SU5205 to the skin of mice to inhibit VEGFR in the skin. H&E staining and PASI scoring of skin sections showed that the psoriasis-like phenotype of the SU5205-treated mice was significantly reduced compared to the IMQ-induced control mice, with a reduction in the thickness of the skin on the back and a reduction in erythema and scale (Figures 29 and 30).

[0046] Furthermore, Western blot analysis to detect protein expression of Rasgrp1 downstream pathway genes showed that the phosphorylation activity protein levels of both NF-κB and AKT in the skin of psoriatic mice treated with SU5205 were significantly reduced, and the activity protein level of IL-1β was also significantly lower than that of the control group (Figure 31).

[0047] Furthermore, in psoriatic mice treated with SU5205, Rasgrp1 + T cells and IL-17A + T cells in the skin were significantly reduced (FIGS. 32 and 33).

[0048] These findings suggest that inhibition of VEGFR in skin tissue reduces the secretion of Rasgrp1 and downstream inflammatory factors, regulates IL-17 production by T cells, and alleviates psoriasis-like skin inflammation.

[0049] Example 5 Promotion of Jurkat cell activation by VEGF, and changes in secretion of inflammatory factors downstream of the Ras pathway by overexpression and knockdown of Rasgrp1 In the examples of the present invention, Jurkat cells were transfected with Rasgrp1 lentivirus to establish stable overexpression and knockdown cell models, and Jurkat cells were stimulated with VEGF cytokines to detect whether VEGF can promote the activation of Jurkat cells, and whether overexpression and knockdown of Rasgrp1 can cause changes in the secretion of inflammatory factors downstream of the Ras pathway.

[0050] Furthermore, RT-qPCR experiments showed that in VEGF-stimulated Jurkat cells, the secretion of Rasgrp1 and its downstream inflammatory factors was significantly increased, and under VEGF stimulation, overexpression of Rasgrp1 significantly increased the secretion of inflammatory factors downstream of the RAS pathway (Figure 34).

[0051] Furthermore, ELISA results showed that the secretion levels of IL-1β and IL-17 in the supernatants of VEGF-stimulated Rasgrp1-overexpressing Jurkat cells were significantly higher than in the control group (Figure 35). Western blot results showed that VEGF stimulation significantly increased the phosphorylation activity protein levels of Rasgrp1 and its downstream pathways, NFκb and AKT, in Jurkat cells, and VEGF stimulation significantly increased the phosphorylation activity protein levels of NF-κB and AKT in Rasgrp1-overexpressing cells, among which the active protein level of IL-1β was also significantly higher than in the control group (Figure 36).

[0052] Furthermore, RT-qPCR showed that IL1b and IL17 transcriptome levels were significantly downregulated in VEGF-stimulated Rasgrp1 knockdown Jurkat cells compared to non-knockdown cells (Figure 37).

[0053] Furthermore, ELISA results showed that the secretion levels of IL-1β and IL-17 in the supernatants of VEGF-stimulated Rasgrp1 knockdown cells were significantly lower than those of the control group (Figure 38). Western blot results showed that VEGF stimulation significantly reduced the phosphorylation activity protein levels of NF-κB and AKT in Rasgrp1 knockdown cells, and the IL-1β activity protein levels were also significantly lower than those of the control group (Figure 39).

[0054] These findings suggest that VEGF promotes the activation of Jurkat cells, and that overexpression and knockdown of Rasgrp1 induce changes in the secretion of inflammatory factors downstream of the RAS pathway.

[0055] Example 6 Overexpression of Rasgrp1 in the skin aggravates the skin phenotype of psoriatic mice In the embodiment of the present invention, a Rasgrp1 adeno-associated virus subcutaneous injection psoriasis model was established by subcutaneously injecting mice with Rasgrp1 adenovirus, the viral dose per mouse was 5 x 1011 VG / mouse, the adenovirus was diluted with PBS, and the mice were divided into 8 groups, including 4 control groups and 4 injection groups at different time points. The control group was injected with PBS, and the mice were killed on days 7, 14, 21, and 28 after injection. Fluorescence quantitative QPCR experiments were performed using the skin tissue to detect the concentration of Rasgrp1 in the mouse skin tissue.

[0056] In the examples of the present invention, in order to further clarify the mechanism of action of Rasgrp1 in psoriasis, an IMQ-induced psoriasis mouse model based on Rasgrp1 overexpression was created according to the above-mentioned method, and H&E staining and PASI scoring of skin sections showed that Rasgrp1-overexpressing mice had a significantly worse psoriasis-like phenotype compared to IMQ-induced wild-type (WT) mice, as observed by increased skin thickness on the back and worsening erythema and scales (Figures 40 and 41).

[0057] Furthermore, ELISA results showed that IL-1β concentrations were significantly increased in the skin tissues of Rasgrp1-overexpressing psoriatic mice compared to the control group (FIG. 42).

[0058] Furthermore, detection of Rasgrp1 and its downstream cytokines by RT-qPCR showed that the expression levels of Rasgrp1, IL1b, IL17a and related inflammatory genes were significantly increased in the skin tissue of Rasgrp1-overexpressing psoriatic mice (Figure 43). Furthermore, detection of protein expression of Rasgrp1 downstream pathway genes by Western blot showed that the phosphorylation activity protein levels of NF-κB and AKT were all significantly increased in the skin of Rasgrp1-overexpressing psoriatic mice, and among them, the activity protein level of IL-1β was also significantly higher than that of the control group (Figure 44).

[0059] Furthermore, IL-17A+ T cells were significantly increased in the skin of Rasgrp1-overexpressing psoriatic mice (FIGS. 45 and 46).

[0060] These findings suggest that overexpression of Rasgrp1 in skin tissue activates the secretion of downstream inflammatory factors, thereby regulating IL-17 production by T cells and affecting psoriasis-like skin inflammation.

[0061] Example 7. Attenuation of the skin phenotype of Rasgrp1-overexpressing psoriatic mice by NBUVB irradiation In the examples of the present invention, in order to further clarify the mechanism of action of NBUVB in psoriasis, a Rasgrp1-overexpressing psoriasis mouse model treated with NBUVB irradiation was created, and H&E staining and PASI scoring of skin sections showed that the psoriasis-like phenotype was clearly alleviated in the Rasgrp1-overexpressing psoriasis mice after NBUVB irradiation treatment compared to the Rasgrp1-overexpressing psoriasis mice, as observed by reduced skin thickness on the back and reduced erythema and scale (Figures 47 and 48).

[0062] Furthermore, the results of the ELISA showed that IL-1β concentration was significantly reduced in the skin tissue of Rasgrp1-overexpressing psoriatic mice by NBUVB irradiation compared to the control group (Figure 49).

[0063] Furthermore, RT-qPCR detection of Rasgrp1 and its downstream cytokines IL1b and IL17a showed that the gene expression levels of Rasgrp1, IL1b, and IL17a were all significantly decreased in the skin tissue of Rasgrp1-overexpressing psoriatic mice after NBUVB irradiation (Figure 50).

[0064] Furthermore, Western blot was used to detect protein expression of Rasgrp1 downstream pathway genes. The results showed that in the skin of Rasgrp1-overexpressing psoriatic mice after NBUVB irradiation, the total protein levels and phosphorylated active protein levels of NF-κB and AKT were all significantly decreased, and the active protein level of IL-1β was also significantly lower than that of the control group (Figure 51).

[0065] Furthermore, NBUVB irradiation significantly reduced IL-17A+ T cells in the skin of Rasgrp1-overexpressing psoriatic mice (Figures 52 and 53).

[0066] These results suggest that NBUVB irradiation inhibits the Ras pathway overexpressed in skin tissue, inhibits downstream inflammatory factors, inhibits IL-17 production by T cells, and reduces psoriasis-like skin inflammation.

[0067] Example 8 Knocking out Rasgrp1 alleviates the phenotype of psoriatic mice In the examples of the present invention, in order to further clarify the mechanism of action of Rasgrp1 in psoriasis, Rasgrp1 knockout mice were constructed and it was observed whether knockout of Rasgrp1 affected the phenotype of psoriatic mice. As a result of H&E staining and PASI scoring of skin sections, the psoriasis-like phenotype of Rasgrp1 knockout mice was clearly alleviated compared to IMQ-induced wild-type (WT) mice, as observed in the form of reduced thickness of the back skin and reduced erythema and scale (Figures 54 and 55).

[0068] Furthermore, the results of ELISA showed that the concentration of IL-1β was significantly reduced in the skin tissue of Rasgrp1 knockout psoriatic mice compared to the control group (Figure 56). The results of detecting Rasgrp1 downstream cytokines by RT-qPCR showed that the expression levels of IL1b, IL17a and related inflammatory genes were all significantly reduced in the skin tissue of Rasgrp1 knockout psoriatic mice (Figure 57). Furthermore, the results of detecting the protein expression of Rasgrp1 downstream pathway genes using Western blot showed that the phosphorylation activity protein levels of NF-κB and AKT were all significantly reduced in the skin of Rasgrp1 knockout psoriatic mice, and the activity protein level of IL-1β was also significantly lower than that of the control group (Figure 58).

[0069] Furthermore, IL-17A+ T cells were significantly reduced in the skin of Rasgrp1 knockout psoriatic mice (Figures 59 and 60).

[0070] These findings suggest that knockout of Rasgrp1 in skin tissue inhibits the secretion of downstream inflammatory factors, inhibits IL-17 production by T cells, and reduces psoriasis-like skin inflammation.

[0071] It should be noted that the above embodiments are only used to explain the technical solutions of the present invention, and are not limited thereto. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art can understand that the technical solutions of the present invention can be modified or substituted with equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these are included in the scope of the claims of the present invention.

Claims

1. Use of a VEGFR inhibitor in the preparation of a formulation for treating psoriasis, wherein the VEGFR inhibitor is SU5205; The use of the preparation is characterized in that it reduces Rasgrp1+ T cells and IL-17A+ T cells by application.

2. The use according to claim 1, characterized in that the formulation comprises a chemical formulation or a biological formulation.

3. The use according to claim 2, characterized in that the formulation comprises a paste, a powder or a solution.

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