Application of PRMT6 gene as target spot in preparation of medicine for treating psoriasis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a PRMT6 gene as a target spot in preparation of a medicine for treating psoriasis. It is confirmed for the first time that PRMT6 is highly expressed in skin lesion tissue of a psoriasis patient, expression in a psoriasis keratinocyte model is increased, psoriasis keratinocyte proliferation is slowed down due to PRMT6 deficiency, and generation of inflammatory factors and chemotactic factors is reduced. In animal experiments, the PRMT6 inhibitor can relieve psoriasis mouse skin inflammation induced by imiquimod, including improvement of scale and skin lesion thickness, and reduction of generation of inflammatory factors and chemotactic factors. The invention provides a new thought and means for treating and preventing psoriasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving the application of the PRMT6 gene as a target in the preparation of drugs for treating psoriasis. Background Technology

[0002] Psoriasis, commonly known as "cowhide rash," is a common chronic inflammatory skin disease characterized by erythema and scaling. It is accompanied by hyperkeratosis and various inflammatory reactions. The condition is stubborn and prone to recurrence, causing a heavy burden on patients' lives and mental distress.

[0003] Psoriasis has a complex pathogenesis and currently lacks a cure. For many years, traditional treatments such as topical therapies and phototherapy have improved symptoms to some extent, but their efficacy has been limited. Biologics targeting psoriasis use single receptors or cytokines as intermediate targets, neutralizing, blocking, and modulating various psoriatic immune abnormalities, thus improving clinical efficacy to some extent. However, because their targets are primarily concentrated on the immune pathways of psoriasis, they may interfere with the patient's immune system during use. For patients with moderate to severe psoriasis, this can lead to numerous adverse reactions and poor tolerance, limiting the use of biologics. Therefore, finding new treatment strategies for psoriasis remains imperative. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of protein arginine methyltransferase 6 or its encoding gene as a target in the preparation of drugs for treating psoriasis.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the invention provides the use of the PRMT6 gene as a target in screening drugs for the treatment or inhibition of psoriasis.

[0006] A second aspect of the invention provides a pharmaceutical composition comprising an agent that inhibits or silences PRMT6 gene expression and pharmaceutically acceptable excipients.

[0007] A third aspect of the invention provides the use of the pharmaceutical composition described herein in the preparation of a product for treating psoriasis.

[0008] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention discovers a correlation between psoriasis and the expression of the PRMT6 gene. By inhibiting or silencing the expression of the PRMT6 gene, the treatment or prevention of related psoriasis can be achieved. Based on this, the PRMT6 gene can serve as a novel target for screening drugs to treat or inhibit psoriasis. This invention provides a novel application of the PRMT6 gene as a target in psoriasis, offering a new approach and method for the treatment and prevention of the disease. Attached Figure Description

[0009] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and the description thereof are used to explain the invention and do not constitute an improper limitation of the invention.

[0010] Figure 1 This illustrates the relative expression of the PRMT6 gene in the skin lesions of psoriasis patients and the normal skin of healthy controls in this embodiment of the invention. Figure 2 This diagram illustrates the process of stimulating keratinocytes (HEKa cells) with M5 for 24 h in an embodiment of the present invention, extracting total RNA and protein from the cells, and detecting the mRNA and protein expression of PRMT6 using real-time quantitative PCR and Western blotting methods, respectively; where A represents the relative mRNA expression level of the PRMT6 gene, and B represents the PRMT6 protein expression diagram. Figure 3 The figures shown are from an embodiment of the present invention. After M5 stimulation was added to HEKa cells and PRMT6 was knocked down with PRMT6 siRNA for 48 h, the expression of PRMT6 mRNA and protein was detected by real-time quantitative PCR and Western blotting, respectively. In the figure, A is the relative expression level of PRMT6 mRNA, B is the protein expression level, and *** indicates that the difference is significant at the p < 0.001 level. Figure 4 In this embodiment of the invention, after adding M5 stimulation to HEKa cells and knocking down PRMT6 with PRMT6 siRNA for 48 h, the cell proliferation activity was detected by the CCK-8 method. *** indicates that the difference is significant at the p < 0.001 level. Figure 5This is a graph showing the mRNA expression levels of psoriasis-related inflammatory factors in HEKa cells after M5 stimulation and PRMT6 knockdown with siRNA for 48 h, as determined in this embodiment of the invention. A represents the relative mRNA expression level of IL1β, B represents the relative mRNA expression level of IL6, C represents the relative mRNA expression level of IL8, D represents the relative mRNA expression level of IL-17A, E represents the relative mRNA expression level of IL-22, and F represents the relative mRNA expression level of IL-23A. * indicates a significant difference at p < 0.05, ** indicates a significant difference at p < 0.01, and *** indicates a significant difference at p < 0.001. Figure 6 These are photographs illustrating the efficacy of topical PRMT6 inhibitor EPZ020411 in treating imiquimod-induced psoriasis mice in this embodiment of the invention; wherein, A is the base cream group, B is the base cream + 30mg / kg EPZ020411 group, C is the imiquimod group, and D is the imiquimod + 30mg / kg EPZ020411 group; Figure 7 The images show the histopathological changes in skin lesions in mice treated with topical PRMT6 inhibitor imiquimod after HE staining. A represents the base cream group, B represents the base cream + 30 mg / kg EPZ020411 group, C represents the imiquimod group, and D represents the imiquimod + 30 mg / kg EPZ020411 group. The scale bar is 500 μm. Figure 8 This is a graph showing the mRNA expression levels of inflammatory factors in skin lesions of mice with imiquimod psoriasis after treatment with the topical PRMT6 inhibitor EPZ020411 using RT-qPCR in an embodiment of the present invention. In the graph, A represents the relative mRNA expression level of IL-1β, B represents the relative mRNA expression level of IL-6, C represents the relative mRNA expression level of IL-8, D represents the relative mRNA expression level of IL-22, E represents the relative mRNA expression level of IL-17A, and F represents the relative mRNA expression level of IL-23A. ** indicates a significant difference at p < 0.01, *** indicates a significant difference at p < 0.001, and ns indicates no statistically significant difference. Detailed Implementation

[0011] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0012] Arginine methyltransferase 6 (PRMT6, Gene ID: 55170) is an evolutionarily conserved family of proteins responsible for catalyzing arginine methylation in proteins. Studies have found that PRMT6 is highly expressed in prostate cancer tissues and prostate cancer cell lines, and altered PRMT6 expression is positively correlated with the oncogenes PSA and KLK2, confirming that PRMT6 promotes prostate cancer development by regulating downstream target genes of arginine (AR). However, there are no reports on the application of PRMT6 or its encoding gene as a target in the preparation of drugs for treating psoriasis.

[0013] This invention, through experiments, is the first to demonstrate that PRMT6 is highly expressed in the skin lesions of psoriasis patients and its expression is elevated in a psoriatic keratinocyte model. The deficiency of protein arginine methyltransferase 6 leads to slowed proliferation of psoriatic keratinocytes and reduced production of inflammatory factors and chemokines. In animal experiments, the protein arginine methyltransferase 6 inhibitor EPZ020411 can alleviate imiquimod-induced skin inflammation in psoriatic mice, including improving scale and lesion thickness and reducing the production of inflammatory factors and chemokines. Therefore, this invention proposes a novel target for psoriasis treatment: protein arginine methyltransferase 6, whose therapeutic effect on psoriasis is clearly demonstrated.

[0014] In view of this, one embodiment of the present invention provides the use of the PRMT6 gene as a target in screening drugs for the treatment or inhibition of psoriasis.

[0015] In some embodiments of the present invention, the drug targets the PRMT6 gene to inhibit or silence the expression of the PRMT6 gene.

[0016] In some embodiments of the present invention, the application includes: Biological samples containing the PRMT6 gene were cultured in the presence of candidate reagents. Biological samples containing the PRMT6 gene were cultured in the absence of candidate reagents; Determining the thickness of scales and lesions, and the expression levels of inflammatory factors and chemokines in the biological samples with and without the candidate reagent, wherein the thickness of scales and lesions, and the expression levels of inflammatory factors and chemokines are lower in the presence of the candidate reagent than in the absence of the candidate reagent, is an indication that the candidate reagent is a drug for treating or inhibiting psoriasis.

[0017] In some embodiments of the present invention, the candidate reagent targets the PRMT6 gene to inhibit or silence its expression level.

[0018] In some embodiments of the present invention, the candidate reagent is siRNA targeting the PRMT6 gene; Alternatively, the candidate reagent is an antibody against the PRMT6 protein, which can inhibit the activity or quantity of the PRMT6 protein at the protein level.

[0019] In some embodiments of the present invention, the biological sample described above may be human keratinocytes (HEKa).

[0020] In another specific embodiment of the present invention, a pharmaceutical composition is provided, comprising an agent for inhibiting or silencing PRMT6 gene expression and pharmaceutically acceptable excipients.

[0021] In some embodiments of the present invention, the reagent for inhibiting or silencing PRMT6 gene expression is selected from any one of siRNA targeting the PRMT6 gene, small molecule inhibitors, or specific antibodies targeting the PRMT6 protein.

[0022] In some embodiments of the present invention, the sequence of the siRNA is as follows: sense 5′-GGGAACUGAAGAGGAAGAUTT-3′ (SEQ ID NO: 1); sense 5′-GGGAACUGAAGAGGAAGAUTT-3′ (SEQ ID NO: 2).

[0023] In some embodiments of the present invention, the sequence of the siRNA is as follows: sense 5′-GGCAUUCUGAGCAUCUUCUTT-3′ (SEQ ID NO: 3); Antisense 5′-AGAAGAUGCUCAGAAUGCCTT-3′ (SEQ ID NO: 4).

[0024] In some embodiments of the present invention, the sequence of the siRNA is as follows: Sense 5′-CGGAACAGGUGGAUGCCAUTT-3′ (SEQ ID NO: 5); Antisense 5′-AUGGCAUCCACCUGUUCCGTT-3′ (SEQ ID NO: 6).

[0025] In some embodiments of the present invention, the reagent for inhibiting or silencing PRMT6 gene expression is a small molecule inhibitor targeting the PRMT6 gene.

[0026] In some embodiments of the present invention, the small molecule inhibitor is EPZ020411.

[0027] A third aspect of the invention provides the use of the pharmaceutical composition described herein in the preparation of a product for treating psoriasis.

[0028] In some embodiments of the present invention, the product may be a test kit, a colloidal gold test strip, a gene chip, or a protein chip.

[0029] In this embodiment, medical-grade pure white petrolatum (brand: Lierkang) is used as the drug carrier in the matrix cream.

[0030] Imiquimod cream, brand name: Mingxinlidi, manufacturer: Sichuan Mingxin Pharmaceutical Co., Ltd.

[0031] In this embodiment, the expression levels of inflammatory markers, including the cytokines interleukin-1b (Il-1b) / interleukin-1β (IL-1β), interleukin-6 (Il-6 / IL-6), interleukin-8 (Il-8 / IL-8), interleukin-22 (Il-22 / IL-22), interleukin-23A (Il-23a / IL-23A), and interleukin-17A (Il-17a / IL-17A), were detected in mouse and human skin, respectively.

[0032] In this embodiment, all statistical analyses were performed using SPSS 23.0 and Graphpad Prism 9.5 software. Quantitative data are expressed as mean ± standard deviation. Differences between groups were determined using t-tests or one-way ANOVA. P < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicated no statistical difference.

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0034] Example 1 (1) Skin lesions in patients with psoriasis and normal skin tissue in healthy controls 1) Select 10 cases each of psoriasis lesion tissue and normal skin tissue from healthy controls, and homogenize them using a tissue homogenizer; 2) Add 1 mL of TRIzol reagent to the homogenized tissue or 6-well plate cells, and after tissue or cell lysis, transfer to a 1.5 mL enzyme-free EP tube and let stand for 5 min; 3) Add 200 μl of chloroform, shake vigorously, let stand for 10 min, and centrifuge at 12000 rpm at 4℃ for 15 min; 4) Transfer the supernatant to a new 1.5 mL enzyme-free EP tube, add 400 μl of isopropanol, mix by inverting, let stand for 10 min, and centrifuge at 12000 rpm at 4℃ for 15 min; 5) Discard the supernatant, add 1 mL of 75% DEPC-ethanol, and wash once by centrifugation; 6) After air drying, add 20-40 μL of DEPC. 7) RNA quantification: RNA concentration was detected using a spectrophotometer. When the OD260 / OD280 value was between 1.9 and 2.0, it indicated that the extracted RNA was of good quality. The RNA concentration of each sample was uniformly adjusted to 500 μg / mL. 8) Reverse transcription: Each component was added to a PCR tube according to the system in Table 1, with each tube containing 20 µL. After mixing, the system was centrifuged. The PCR tube was placed in a PCR instrument, and reverse transcription was performed according to the program in Table 2. After the reverse transcription was completed, the cDNA of the reverse transcription product was diluted with 80 µL of ddH2O and stored at -20℃ for later use.

[0035] Table 1 Reverse transcription system

[0036] Table 2 Reverse Transcription Procedure

[0037] (2) Real-time quantitative PCR 1) Primer sequences for the involved genes are shown in Table 3; 2) Add the reaction mixture from Table 4 to a 0.1 mL sterile PCR eight-tube set; 3) Mix the added samples thoroughly and centrifuge; 4) Place the PCR eight-tube set into a PCR instrument and perform real-time quantitative PCR according to Table 5; 5) After the reaction is complete, observe the amplification curve and melting curve, and record the Ct value of each gene, using β-actin as an internal control, and use 2... -ΔΔCt The method calculates the transcriptional level of the gene to be tested, and then performs statistical analysis.

[0038] Table 3 qPCR primer sequences

[0039] Table 4 RT-qPCR reaction system

[0040] Table 5 Real-time quantitative PCR reaction program

[0041] The results are as follows Figure 1 As shown, the expression of PRMT6 in the skin lesions of psoriasis patients was significantly higher than that in normal tissues.

[0042] Example 2 This embodiment investigates the expression level of the PRMT6 gene in a psoriatic keratinocyte model.

[0043] (1) Recovery and culture of HEKa keratinocytes 1) Cell thawing: Remove the frozen cells from the liquid nitrogen tank and quickly place them in a 37°C water bath. After the cells are completely thawed, transfer them to a 15 mL centrifuge tube using a pipette. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of culture medium to the centrifuge tube to resuspend the cells, and transfer them to a cell culture dish. Add another 5 mL of culture medium to the dish, shake well, and place it in a 37°C cell culture incubator containing 5% carbon dioxide. 2) Cell culture: Place the cells in an incubator and change the culture medium every 2 days. 3) Cell passage: When the cell density in the cell culture dish reaches 80%–90%, discard the culture medium, wash the cells 1–2 times, add trypsin to cover the cells, and place the dish in an incubator for 2–4 min. Remove the dish and observe under a microscope that the cells have become rounded and partially detached. Add an equal volume of culture medium to the trypsin to stop digestion. Gently pipette to remove all cells from the bottom of the dish and collect the cells in a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. After 1 minute, discard the supernatant, add fresh culture medium to resuspend the cells, and seed the cells into new culture dishes at a passage ratio of 1:4.

[0044] (2) Construction of a keratinocyte model of psoriasis 1) After digesting the cells into a single-cell suspension, cell counting was performed, and the cells were seeded in six-well plates at a cell density of 4 × 10⁶ cells / well. 5 1) After shaking the cells in each well, place them in a cell culture incubator; 2) Observe under a microscope. When the cell density reaches 60% after cell adhesion, replace with serum-free medium and starve for 12 h; 3) Discard the old medium and add complete medium containing a final concentration of 10 ng / mL M5 (a mixture of five cytokines: IL-17A, IL-22, IL-1α, TNF-α, and OSM) to the experimental group cells. Continue culturing in a cell culture incubator. After M5 stimulation for a specified time, extract total RNA and total protein from the cells.

[0045] (3) RNA extraction from cells Cellular RNA extraction does not require grinding; other steps are the same as step (1) in Example 1.

[0046] (4) Real-time quantitative PCR 1) Primer sequences for the involved genes are shown in Tables 6 and 7; 2) Add the reaction mixture from Table 4 to 0.1 mL sterile PCR eight-tube strips; 3) Mix the added samples thoroughly and centrifuge; 4) Place the PCR eight-tube strips into a PCR instrument and perform real-time quantitative PCR according to Table 5; 5) After the reaction is complete, observe the amplification curve and melting curve, and record the Ct value of each gene, using β-actin as an internal control, and use 2 -ΔΔCt The method calculates the transcriptional level of the gene to be tested, and then performs statistical analysis.

[0047] Table 6. PCR primer sequences (human origin)

[0048] Table 7 PCR primer sequences (mouse)

[0049] (5) Extraction of total cell protein Protein extraction: 1) Discard the cell culture medium and wash the cells twice with PBS; 2) Add RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors; 3) Scrape the cells with a cell scraper and transfer them to a 1.5 mL EP tube, shake vigorously, and let stand for 15 min; 4) Centrifuge at 12000 rpm at 4℃ for 15 min, and transfer the supernatant (protein sample) to a new 1.5 mL EP tube. b. Protein quantification: Take 5 μl of supernatant and quantify the protein using a BCA protein quantification kit. Balance all protein samples to the same concentration using RIPA at the lowest concentration. Add 5× Loading buffer to 1 / 4 of the total protein sample volume, mix well, and boil at 100℃ for 10 min.

[0050] (6) Immunoblotting a. SDS-PAGE gel electrophoresis: 1) Prepare SDS-PAGE gel: Prepare the separating gel and stacking gel systems separately, pour them sequentially into the gel glass plate's gel-specific holder, insert the comb, and wait for the gel to solidify before use for subsequent electrophoresis; 2) Electrophoresis: Install the gel plate into the electrophoresis tank, pour in 1× electrophoresis buffer, remove the comb, add different histone samples sequentially into the gel wells, add protein markers on both sides, connect the power supply, maintain a constant voltage of 80 V until the protein samples enter the separating gel, and then maintain a constant voltage of 120 V until the bromophenol blue reaches the bottom of the gel and stop electrophoresis. b. Transfer membrane: 1) Prepare 1× transfer buffer, cut out the SDS-PAGE gel, and place a sponge pad, two layers of filter paper, SDS-PAGE gel, nitrocellulose membrane, two layers of filter paper, and a sponge pad sequentially on the transfer clamp, place it in the transfer tank, add an ice pack and transfer buffer; 2) Connect the power supply, transfer membrane at a constant current of 300 mA for 90-120 min. c. Antibody incubation: 1) After transfer, place the nitrocellulose membrane in rapid blocking solution for 10 min; 2) After washing the membrane with 1×TBST, add diluted primary antibody and incubate overnight at 4℃; 3) The next day, wash the membrane three times with 1×TBST for 10 min each time, add diluted secondary antibody, and incubate at room temperature for 1 h; 4) Wash the membrane three times with 1×TBST for 10 min each time. d. Chemiluminescence: Prepare ECL chemiluminescence solution, evenly cover the membrane, place it in a chemiluminescence imaging system to luminesce and acquire images.

[0051] (7) Results Analysis In this embodiment, keratinocytes were stimulated with a mixture of cytokines M5 to simulate a psoriasis cell model, in order to evaluate the expression of PRMT6 in the psoriasis cell model. Real-time quantitative PCR was used to detect the mRNA level of PRMT6, and Western blotting was used to detect the protein expression of PRMT6. Results are as follows: Figure 2 As shown, compared with normal cells, the mRNA and protein expression levels of PRMT6 were increased in the psoriasis cell model.

[0052] Example 3 This embodiment investigates the effects of interfering with the PRMT6 gene on the proliferation activity of psoriatic keratinocytes and inflammatory factors in the cells.

[0053] (1) Cell transfection Before transfection, cells were arranged at a density of 4 × 10⁶. 5Seed cells per well in a six-well plate and place the plate in a cell culture incubator. When the cells have adhered and reached 60-70% confluence, perform cell transfection. 2) Add 5 μL of RNAimax to 100 μL of serum-reduced medium Opti-MEM, shake well by inversion, and incubate at room temperature for 5 min. 3) Take 5 μL of PRMT6 siRNA and negative control siRNA (the sequences of each siRNA are shown in Table 7) and add them to 100 μL of serum-reduced medium Opti-MEM. Mix well by inversion and incubate at room temperature for 5 min. 4) Mix the liquids from 2) and 3), invert well, and incubate at room temperature for 15 min. 5) Add the transfection mixture obtained in 4) to the wells of the six-well plate, add medium to make up to 2 mL / well, gently shake the plate to mix, label it, and place the plate in a cell culture incubator.

[0054] Table 8 siRNA Sequences

[0055] (2) Real-time quantitative PCR detection of cytokine expression levels Refer to steps (3) to (4) of Example 2.

[0056] (3) Protein detection Refer to steps (5) to (6) of Example 2.

[0057] (4) CCK-8 detection 1) Human primary keratinocytes in the logarithmic growth phase were collected, counted, and seeded into 96-well plates at a rate of 8000 cells / well; 2) After cell adhesion, PRMT6 was knocked down using siRNA and M5 was added to stimulate the cells to simulate the state of psoriasis cells; 3) 48 h after PRMT6 was knocked down using siRNA and M5 was added, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in a cell culture incubator in the dark for 2 h. The OD value of each well at 450 nm was detected and recorded using a microplate reader.

[0058] (5) Results Analysis In this embodiment, siRNA-mediated gene knockdown of PRMT6 was used, and the knockdown efficiency of PRMT6 was verified at both the mRNA and protein levels using real-time quantitative PCR and Western blotting methods. Figure 3 In the M5-treated psoriasis cell model, cell proliferation activity was significantly increased, consistent with the excessive proliferation of keratinocytes in psoriasis. In M5-treated cells, negative control siRNA and siPRMT6-1 were used respectively; the proliferation activity of psoriasis-like keratinocytes treated with siPRMT6-1 decreased. Figure 4This indicates that inhibiting the expression of the PRMT6 gene can suppress the proliferative activity of psoriatic keratinocytes.

[0059] The disease state of psoriasis includes not only keratinocyte proliferation but also the secretion of inflammatory factors. Therefore, the effect of PRMT6 deficiency on inflammatory factor secretion was further investigated. In the psoriasis cell model constructed using M5, the relative mRNA expression levels of IL1β, IL6, IL8, IL22, IL23A, and IL17A were significantly increased, while in cells mediated by siPRMT6-1, the production of these inflammatory factors was significantly decreased. Figure 5 This indicates that PRMT6 deficiency can comprehensively inhibit the production of multiple inflammatory factors in psoriatic keratinocytes.

[0060] Example 4 This embodiment investigates the therapeutic effect of PRMT6 inhibitors in a mouse model of psoriasis.

[0061] (1) Topical application of EPZ020411 to treat imiquimod psoriasis mice 1) Twenty wild-type C58BL / 6 mice aged 6-8 weeks were randomly divided into four groups: a matrix cream group, a matrix cream + 30 mg / kg EPZ020411 group, an imiquimod group, and an imiquimod + 30 mg / kg EPZ020411 group, with three mice in each group. Hair was removed from the backs of the mice. 2) Mice in the matrix cream group were applied 62.5 mg of matrix cream daily, followed by dimethyl sulfoxide (DMSO) 4 hours later. Mice in the matrix cream + 30 mg / kg EPZ020411 group were applied 62.5 mg of matrix cream daily, followed by 30 mg / kg EPZ020411 dissolved in DMSO 4 hours later. Mice in the imiquimod group were applied 62.5 mg of imiquimod cream daily, followed by DMSO 4 hours later. Mice in the imiquimod + 30 mg / kg EPZ020411 group were applied 62.5 mg of imiquimod cream daily, followed by DMSO 4 hours later. h later, apply EPZ020411 dissolved in DMSO at a concentration of 30 mg / kg; 3) Euthanize the mice on the 7th day and collect the skin tissue from the back of the mice for subsequent detection of cytokine expression levels. The detection method, primers and steps are the same as in Example 2.

[0062] (2) HE staining 1) Collect skin tissue from the back of mice, embed it in paraffin, and section the tissue. Dewax the sections. 2) Hematoxylin staining: Stain the sections with hematoxylin solution for 3 min, wash with tap water, differentiate with differentiation solution for 3-8 s, wash with tap water, and then wash with tap water after hematoxylin blueing solution. 3) Eosin staining: Dehydrate the sections in 85% and 95% graded alcohol solutions for 5 min each, and then stain with eosin solution for 5 min. 4) Dehydration and mounting: Soak the sections in 75%, 80%, 90%, and 100% ethanol for 5 min each, then place them in xylene for 5 min to clear them. After slightly drying, mount them with mounting glue. 5) Observation and analysis: Scan the sections with a tissue scanner after mounting to analyze the histopathological changes.

[0063] (3) Results Analysis This embodiment further constructed an imiquimod-induced psoriasis animal model and treated psoriasis mice with topical PRMT6 inhibitor EPZ020411. Clinical photographs of psoriasis mice under different treatments showed that the skin appearance of mice in the topical base cream group and the base cream + 30 mg / kg EPZ020411 group remained unchanged. Imiquimod successfully induced psoriatic skin inflammation, including scaling and epidermal thickening. Topical EPZ020411 treatment significantly improved symptoms such as scaling and epidermal thickening in both psoriasis mice. Figure 6 This embodiment further examined the histopathological changes of the skin on the back of mice. The skin appearance of mice treated with the topical matrix cream and the matrix cream + 30 mg / kg EPZ020411 group remained unchanged. Mice in the imiquimod group exhibited pathological changes similar to those in psoriasis patients, including hyperkeratosis, parakeratosis, acanthosis, and inflammatory cell infiltration. Topical treatment with 30 mg / kg EPZ020411 significantly improved these pathological changes in psoriasis mice. Figure 7 This embodiment further examined the levels of inflammatory factors in mouse skin lesions. Consistent with the appearance and pathological changes, the expression levels of skin inflammatory factors in mice treated with the topical base cream group and the base cream + 30 mg / kg EPZ020411 group were basically the same. The expression levels of Il1b, Il6, Il8, Il22, Il23a, and Il17a in mice treated with imiquimod were significantly increased, while topical treatment with 30 mg / kg EPZ020411 in psoriasis mice significantly reduced the production of the above-mentioned inflammatory factors. Figure 8 The above results indicate that there was no significant difference between the topical base cream group and the topical base cream + 30 mg / kg EPZ020411 group, suggesting that EPZ020411 has good safety. The topical imiquimod group successfully induced a psoriasis mouse model, and the topical EPZ020411 has a therapeutic effect on imiquimod-induced psoriasis mice.

[0064] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of PRMT6 gene as a target in screening drugs for the treatment or inhibition of psoriasis.

2. The application as described in claim 1, characterized in that, The drug targets the PRMT6 gene, inhibiting or silencing its expression.

3. The application as described in claim 1 or 2, characterized in that, The applications include: Biological samples containing the PRMT6 gene were cultured in the presence of candidate reagents. Biological samples containing the PRMT6 gene were cultured in the absence of candidate reagents; Determining the thickness of scales and lesions, and the expression levels of inflammatory factors and chemokines in the biological samples with and without the candidate reagent, wherein the thickness of scales and lesions, and the expression levels of inflammatory factors and chemokines are lower in the presence of the candidate reagent than in the absence of the candidate reagent, is an indication that the candidate reagent is a drug for treating or inhibiting psoriasis.

4. The application as described in claim 3, characterized in that, The candidate reagent targets the PRMT6 gene to inhibit or silence its expression level.

5. The application as described in claim 4, characterized in that, The candidate reagent is siRNA targeting the PRMT6 gene; Alternatively, the candidate reagent is an antibody against the PRMT6 protein, which can inhibit the activity or quantity of the PRMT6 protein at the protein level.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains an agent that inhibits or silences PRMT6 gene expression and pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 6, characterized in that, The reagent used to inhibit or silence PRMT6 gene expression is selected from any one of siRNA targeting the PRMT6 gene, small molecule inhibitors, or specific antibodies targeting the PRMT6 protein.

8. The pharmaceutical composition according to claim 7, characterized in that, The reagent used to inhibit or silence PRMT6 gene expression is a small molecule inhibitor targeting the PRMT6 gene.

9. The application as described in claim 8, characterized in that, The small molecule inhibitor is EPZ020411.

10. Use of the pharmaceutical composition according to any one of claims 6-9 in the preparation of a product for treating psoriasis.

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