Use of siglec-15 in the preparation of a medicament for treating atopic dermatitis and psoriasis

Abstract

The application discloses application of Siglec-15 in preparation of a drug for treating atopic dermatitis and psoriasis, belongs to the technical field of biological medicine, and the dosing amount of the Siglec-15 protein is 0.025-0.1 mg / kg. The administration mode is subcutaneous injection administration at a lesion or external application administration. The administration frequency is once a day, and the continuous administration lasts for 4-7 days. The Siglec-15 can be immunologically controlled from a T cell activation source, has more fundamental and persistent treatment effect, and effectively solves the problems of poor treatment effect and high price in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Siglec-15 in the preparation of drugs for treating atopic dermatitis and psoriasis. Background Technology

[0002] Atopic dermatitis (AD) and psoriasis (PSO) are two common chronic inflammatory skin diseases that seriously affect patients' quality of life, and current treatments still have many shortcomings.

[0003] (I) Pathogenesis and current treatment status of atopic dermatitis Atopic dermatitis (AD) is a chronic, relapsing, inflammatory skin disease characterized by dry skin, intense itching, and eczematous rashes. The pathogenesis of AD is complex, involving the interaction of multiple factors, including impaired skin barrier function, immune dysregulation, genetic susceptibility, and environmental triggers. Immune imbalance is a core element in AD pathogenesis: excessive activation of the Th2 immune response leads to the infiltration of CD4+ T cells and mast cells into the skin lesions, elevated IgE levels, and abnormal secretion of Th2 cytokines such as IL-4, IL-5, and IL-13, further exacerbating skin inflammation and barrier damage.

[0004] Currently, the main treatments for Alzheimer's disease (AD) include topical therapy (corticosteroids, calcineurin inhibitors), phototherapy (UVB / UVA / PUVA), and systemic therapy (methotrexate, cyclosporine, biologics). Corticosteroids, as first-line therapy, can cause serious side effects with long-term use, such as skin atrophy, telangiectasia, folliculitis, and adrenal suppression. While biologics that have been marketed in recent years (such as dupilumab and anti-IL-4 / IL-13 monoclonal antibodies) have shown significant efficacy, they are expensive, require long-term injections, and have poor responses in some patients. Therefore, the development of novel therapeutic drugs with high safety, proven efficacy, and clearly defined mechanisms of action remains an urgent need in the field of AD treatment.

[0005] (II) Pathogenesis and Current Treatment Status of Psoriasis Psoriasis is a common chronic inflammatory autoimmune skin disease. Its core pathological features are abnormal proliferation of keratinocytes and infiltration of inflammatory cells, resulting in well-defined erythematous and squamous skin lesions. Immunological studies have shown that the IL-23 / Th17 axis plays a central driving role in the pathogenesis of psoriasis: IL-23 induces Th17 cell differentiation and activation, promoting the production of large amounts of pro-inflammatory factors such as IL-17A, IL-17F, IL-22, and TNF-α. These cytokines further stimulate keratinocyte proliferation and chemokine release, forming a vicious cycle of positive feedback between inflammation and proliferation.

[0006] Current treatments for psoriasis include topical medications (vitamin D3 analogs, corticosteroids), phototherapy, traditional systemic therapy (methotrexate, cyclosporine, acitretin), and biologics (anti-TNF-α monoclonal antibodies, anti-IL-17 monoclonal antibodies, anti-IL-23 monoclonal antibodies). While biologics offer strong targeting, they suffer from drawbacks such as inconvenient injection administration, increased risk of infection, high cost, and primary / secondary failure.

[0007] In summary, discovering new therapeutic targets and developing novel therapeutic drugs for atopic dermatitis and psoriasis has significant clinical value and market potential. Summary of the Invention

[0008] In view of the above-mentioned shortcomings in the prior art, the present invention provides the application of Siglec-15 in the preparation of drugs for treating atopic dermatitis and psoriasis. Siglec-15 can regulate immunity from the source of T cell activation, and has a more fundamental and lasting therapeutic effect, effectively solving the problems of poor treatment effect and high price in the prior art.

[0009] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: Application of Siglec-15 protein in the treatment of atopic dermatitis and / or psoriasis.

[0010] Furthermore, the dosage of Siglec-15 protein is 0.025-0.1 mg / kg.

[0011] Furthermore, the dosage of Siglec-15 protein was 0.05 mg / kg.

[0012] Furthermore, the administration method is subcutaneous injection into the lesion or topical application.

[0013] Furthermore, the administration frequency is once daily for 4-7 consecutive days.

[0014] A medication for treating atopic dermatitis and / or psoriasis, comprising the Siglec-15 protein.

[0015] Furthermore, when the drug dosage form is a topical preparation, the drug also includes at least one of a transdermal penetration enhancer, a stabilizer, an excipient, a preservative, and a pH adjuster.

[0016] Furthermore, transdermal penetration enhancers include azone, oleic acid, propylene glycol, and laurocapram; stabilizers include trehalose, mannitol, or human serum albumin.

[0017] The beneficial effects of this invention are as follows: 1. Siglec-15 protein preparation can significantly improve skin symptoms in DNFB-induced BALB / c mouse AD model, and has a significant therapeutic effect on symptoms such as erythema, edema, scaling, and lichenification of the model mouse skin; it can significantly reduce the number of mast cells and the degree of CD4+ T cell infiltration in the skin lesions of model mice; at the same time, it can inhibit the number of inflammatory factors such as IgE, IL-4, IL-5 and IL-13 in serum, and block the pathological cascade reaction of atopic dermatitis from the source of inflammation.

[0018] 2. Siglec-15 protein preparation can significantly improve skin symptoms of imiquimod (IMQ)-induced PSO model in BALB / c mice, and has obvious therapeutic effects on symptoms such as erythema, scaling, thickness and infiltration in model mice; it can significantly inhibit excessive epidermal proliferation in model mice; at the same time, it inhibits the production of inflammatory factors such as IL-17, IL-23, TNF-α and IL-6 in serum, thus blocking the vicious cycle of PSO pathology from upstream.

[0019] 3. The Siglec-15 protein preparation has good safety. It has no effect on the weight, food intake, water intake, mental state and activity level of experimental mice. It has no significant effect on the heart, liver, lungs and kidneys of mice. The spleen index of mice is significantly lower than that of the model control group, which can alleviate the problem of disease-related splenomegaly.

[0020] 4. This dosage form is administered topically, with the drug acting directly on the site of inflammation. It has low systemic exposure, avoiding the risk of infection caused by systemic immunosuppression, and has higher safety.

[0021] 5. Unlike existing downstream cytokine antagonists, this formulation can regulate immunity from the source of T cell activation, potentially leading to more fundamental and lasting therapeutic effects. This formulation has broad-spectrum regulatory capabilities on different T cell subsets and is suitable for a variety of T cell-mediated inflammatory skin diseases. Attached Figure Description

[0022] Figure 1 A diagram showing the DNFB induction modeling method and drug administration time schedule; Figure 2 Photographs showing dermatitis symptoms on the backs of AD mice treated with Siglec-15. Figure 3 Statistical graph of SCORAD scores for AD mice in each group; Figure 4 Figures showing the trends in body weight and dietary and water intake of AD mice in each group; Figure 5 Comparison of spleen size among AD mice in each group; Figure 6 Statistical graph of spleen index in AD mice of each group; Figure 7Representative photographs of HE staining and TB staining of the dorsal skin of AD mice in each group, and statistical graphs of epidermal / spinous layer thickness; Figure 8 Representative photographs and cell count statistics of the dorsal skin of AD mice in each group are shown. Figure 9 The image shows the ELISA results of serum IgE, IL-4, IL-5, and IL-13 levels in each group of AD mice. Figure 10 Immunohistochemical tissue section of Ki67 on the back of a mouse and statistical diagram of positive cells; Figure 11 Fluorescent staining image and statistical graph of CD86 / CD206 in mouse dorsal skin tissue; Figure 12 Photographs of skin lesions on the backs of PSO mice treated with Siglec-15; Figure 13 A statistical comparison chart of PASI scores for PSO mice in each group; Figure 14 Figures showing the trends in body weight and diet of PSO mice in each group; Figure 15 Photographs of the heart, liver, lungs, kidneys, and spleen of each group of PSO mice, and a statistical chart of spleen index. Figure 16 Representative photographs and epidermal thickness statistics of HE-stained dorsal skin of PSO mice in each group; Figure 17 Representative photographs of immunofluorescence staining of the dorsal skin of PSO mice in each group and statistical graphs of IL-17A+ Th17 cell counts; Figure 18 The image shows the ELISA results of serum IL-17, IL-23, TNF-α, and IL-6 levels in PSO mice from each group. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0024] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0025] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0026] The features and performance of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0027] Example 1 A drug for treating atopic dermatitis and / or psoriasis, comprising Siglec-15 protein, is prepared as a lyophilized powder for injection. The specific preparation method is as follows: 1.0 mg of Siglec-15 protein is dissolved in PBS, 50 mg of trehalose and 5 mg of human serum albumin are added, and the mixture is gently mixed and filtered through a 0.22 μm sterile filter. The filtrate is aliquoted into sterile vials, 1.0 mL per vial (containing 1.0 mg of Siglec-15), partially stoppered, and lyophilized. Lyophilization conditions: pre-freezing temperature -45℃ for 4 hours; primary drying temperature -20℃, vacuum 10 Pa for 20 hours; secondary drying temperature 25℃, vacuum 5 Pa for 8 hours. After lyophilization, the vials are stoppered, capped with aluminum, labeled, and stored at 2–8℃ protected from light. The resulting lyophilized powder for injection appears as a white, loose, lumpy substance. After reconstitution, it becomes clear and transparent, with a pH of 7.2–7.6, protein purity >95%, and endotoxin <0.1 EU / mg.

[0028] Example 2 A medicament for treating atopic dermatitis and / or psoriasis, comprising Siglec-15 protein. When formulated into an external cream, the preparation method is as follows: mixing 15 g of stearic acid, 5 g of glyceryl monostearate, 10 g of white vaseline, 10 g of liquid paraffin, and 2 g of lanolin, heating to 70-75 ℃ for melting, and using the mixture as the oil phase. Dissolving 5 g of glycerin, 1 g of triethanolamine, and 0.1 g of ethyl nipagin ester in distilled water, heating to 70-75 ℃, and using the solution as the aqueous phase. Slowly adding the aqueous phase to the oil phase while stirring, emulsifying for 10 minutes, adding 10 mg of Siglec-15 protein (equivalent to 0.1%, w / w, pre-dissolved in a small amount of PBS) and 2 g of azone (transdermal penetration enhancer) when the temperature is lowered to 40 ℃, continuing stirring until the mixture reaches room temperature, and the cream is obtained. The cream is a white fine paste with a pH of 6.5-7.0 and good stability, and the protein activity remains >90% after storage at 4 ℃ for 6 months.

[0029] Example 3 A medicament for treating atopic dermatitis and / or psoriasis, comprising Siglec-15 protein. When formulated into an external gel, the preparation method is as follows: uniformly sprinkling 1.0 g of Carbomer 940 into distilled water, allowing it to swell at room temperature for 4 hours to obtain a transparent gel matrix. Adding 10 g of propylene glycol and 0.1 g of methyl nipagin, and stirring uniformly. Slowly adding 1.2 g of triethanolamine dropwise while stirring, adjusting the pH to 6.5-7.0 to form the gel. Finally adding 10 mg of Siglec-15 protein solution (pre-dissolved in a small amount of PBS), stirring gently and uniformly, and the gel is obtained. The gel is colorless and transparent, easy to spread, and the protein activity remains >85% after storage at 4 ℃ for 6 months.

[0030] Test Example 1. Evaluation of the therapeutic effect of Siglec-15 on atopic dermatitis 1.1 Experimental animals: SPF-grade female BALB / c mice, 6-8 weeks old, weighing 18-22 g, purchased from a certain experimental animal center (Laboratory Animal Production License No.: SCXK (Chuan) 2025-0034). The animals were housed in an SPF-grade animal room with a temperature of 22±2℃, a humidity of 50%-60%, 12-hour light-dark alternation, and free access to food and water. All animal experiments were approved by the institutional animal ethics committee.

[0031] 1.2 Main Reagents and Instruments: Siglec-15 recombinant protein (purity ≥95%, endotoxin <0.1 EU / μg, lyophilized powder); 2,4-dinitrofluorobenzene (DNFB, AbMole, catalog number M40775); dexamethasone (Dex, positive control, Shanghai Yuanye Biotechnology Co., Ltd., catalog number A10077); acetone (analytical grade); olive oil (pharmaceutical grade, aladdin catalog number 0108686); mouse IgE ELISA kit (Ruixin Biotechnology Co., Ltd., catalog number: RX202737M), mouse IL-4 ELISA kit (Ruixin Biotechnology Co., Ltd., catalog number: RX203051M), mouse IL-5 ELISA kit (Ruixin Biotechnology Co., Ltd., catalog number: RX203050M), mouse IL-13... ELISA kit (Ruixin Biotechnology Co., Ltd., catalog number: RX203070M); anti-mouse CD4 antibody (primary antibody), anti-mouse CD8 antibody (primary antibody), FITC-labeled goat anti-mouse IgG (secondary antibody), DAPI staining solution; toluidine blue staining solution; Saiviewer image analysis software; GraphPad Prism 10.1.2 statistical analysis software.

[0032] 1.3 Preparation of Siglec-15 solution: Take 50 μg of Siglec-15 lyophilized powder and centrifuge at 12000 rpm for 30 seconds at 4 °C to allow the powder to gather at the bottom of the tube. Add 500 μL of PBS buffer (pH 7.4) containing 5% (w / v) trehalose, gently shake the lyophilized powder tube to dissolve the powder, and place it at 4 °C for 2 min to allow it to fully dissolve, thus preparing a 100 μg / mL Siglec-15 stock solution. Aliquot the stock solution into at least 20 μL tubes and store at -80 °C for later use, avoiding repeated freeze-thaw cycles. Before use, thaw at 4 °C and use directly.

[0033] Preparation of 0.3% DNFB solution: Using the subtraction method, weigh 0.0300 g of DNFB powder into a 15 mL centrifuge tube containing 7.5 mL using an electronic balance. Vortex for 1 min, add 2.5 mL of olive oil solution, and vortex again for 1 min to fully dissolve it into a clear and transparent solution. Store the prepared solution in a refrigerator at 4°C with aluminum foil.

[0034] Preparation of 0.4% DNFB solution: Using the subtraction method, weigh 0.0400 g of DNFB powder into a 15 mL centrifuge tube containing 7.5 mL using an electronic balance. Vortex for 1 min, add 2.5 mL of olive oil solution, and vortex again for 1 min to fully dissolve it into a clear and transparent solution. Store the prepared solution in a refrigerator at 4°C with aluminum foil.

[0035] 1.4 Dosage Design and Administration Regimen: Based on preliminary experimental results, the half-maximal effective concentration (EC50) of Siglec-15 in inhibiting anti-CD3 antibody-induced peripheral blood mononuclear cell (PBMC) proliferation is 0.48–0.57 μg / mL, with the median value of 0.50 μg / mL taken. Mice have a blood volume of approximately 6% of their body weight; for a standard 20 g mouse, the blood volume is approximately 1.2 mL. To achieve the EC50, 0.5 × 1.2 = 0.6 μg of Siglec-15 is required. Considering a subcutaneous bioavailability of approximately 60%, the actual dose required is 0.6 ÷ 60% = 1.0 μg, equivalent to 0.05 mg / kg. A dose gradient was established at a ratio of 4:2:1, determining the experimental doses as follows: a medium dose of 0.05 mg / kg and a high dose of 0.1 mg / kg. The administration route was subcutaneous injection at the center of the inflammatory foci, once daily for 7 consecutive days (days 8 to 14 after modeling). The positive control group was given dexamethasone (Shanghai Yuanye Biotechnology Co., Ltd., catalog number A10077) at a dose of 2 mg / kg, administered subcutaneously once daily for 7 consecutive days (from day 8 to day 14 of modeling), with the same route and frequency of administration as the Siglec-15 treatment group.

[0036] 1.5 Construction and Grouping of AD Mouse Model: A classic AD mouse model induced by DNFB was used. The modeling period lasted for 14 days: the first 7 days were the sensitization phase, on days 1, 4, and 7, 50 μL of 0.3% DNFB solution (solvent: acetone:olite = 3:1, v / v) was applied to the shaved area on the back of the mice; the next 7 days were the challenge phase, on days 10 and 12, 50 μL of 0.4% DNFB solution was applied to the same area. The mice that successfully developed the model were randomly divided into 5 groups (n=5 per group): (1) Normal control group (Ctrl): normal mice were given an equal volume of solvent (PBS containing 5% trehalose and Dex solvent); (2) Model control group (Model): AD model mice were given an equal volume of solvent; (3) Positive control group (Dex): AD model mice were given dexamethasone (2 mg / kg); (4) Siglec-15 medium dose group (SM): AD model mice were given Siglec-15 at a dose of 0.05 mg / kg; (5) Siglec-15 high dose group (SH): AD model mice were given Siglec-15 at a dose of 0.1 mg / kg.

[0037] 1.6 General Indicator Monitoring: Digital photographs of the mouse dorsal skin were taken every two days during treatment. Two independent observers performed a blinded scoring process using the SCORAD scoring criteria. The SCORAD score assessed three dimensions: erythema / edema, scaling, and lichenification. Each dimension was scored from 0 to 3 points based on severity, for a total score of 0 to 9 points. Higher scores indicated more severe skin lesions. Mouse weight, food intake, and water intake were recorded daily, and the mice's mental state and activity level were observed.

[0038] 1.7 Sample Collection: 24 hours after the last administration, mice were fasted but allowed free access to water for 12 hours. After anesthesia with an intraperitoneal injection of 0.5% sodium pentobarbital at 40 mg / kg, approximately 0.5–0.8 mL of blood was collected from the eyeballs. The blood was allowed to stand at room temperature for 2 hours, then centrifuged at 4000 rpm for 20 minutes at 4°C. The serum was separated and centrifuged again at 8000 rpm for 5 minutes at 4°C, for a total of two centrifugations. The serum was then aliquoted and stored at -80°C for later use. Mice were euthanized by cervical dislocation, and a small amount of lesioned skin tissue (approximately 0.5 cm × 0.5 cm) was quickly collected from the back. One portion was fixed in 4% paraformaldehyde fixative (for H&E staining, toluidine blue staining, immunofluorescence, and immunohistochemical staining), and the other portion was stored at -80°C. Meanwhile, the heart, liver, spleen, lungs, and kidneys were harvested, weighed, and their organ indices (organ mass / body weight, g / g) were calculated. A portion of the visceral tissue was cryopreserved at -80℃, while another portion was fixed in 4% paraformaldehyde for HE staining biocompatibility evaluation.

[0039] 1.8 Histological examination: (1) H&E staining: HE staining procedure for cell smears: (1.1) Fixation: Add tissue fixative to the well plate and fix for 15 min. Then, aspirate the fixative from the wells and wash with ultrapure water 4 times. (1.2) Membrane perforation: Add membrane perforation solution to the well plate and perforate for 15 min. Aspirate the membrane perforation solution from the wells and wash with ultrapure water 4 times. (1.3) Pretreatment: Add an appropriate amount of high-resolution pretreatment solution to the well plate and treat for 1 min. (1.4) Hematoxylin staining: Add an appropriate amount of hematoxylin staining solution to the well plate and stain for 5 min. Aspirate the staining solution from the wells and wash with tap water 4 times. Use differentiation solution for differentiation for 2 s and wash with tap water 4 times. Use blue solution for blueing for 2 s and wash with tap water 4 times. (1.5) Eosin staining: Add anhydrous ethanol for 1 min pretreatment, remove the anhydrous ethanol, add eosin staining solution for 3 min staining, remove the staining solution, wash 4 times with anhydrous ethanol, (1.6) Mounting: After drying, take a clean glass slide, write the number on it, add ultra-clean quick-drying mounting medium in the middle of the glass slide, take out the slide, place the side with the cells facing down, and mount with neutral resin. HE staining steps for sections: (2.1) Dewaxing paraffin sections to water: Put the sections into environmentally friendly dewaxing solution I for 15 min - environmentally friendly dewaxing solution II for 15 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water. Frozen sections are thawed and fixed: Take the frozen sections out of the -20℃ freezer and restore them to room temperature, fix with acetone for 1 min, and then wash with water. (2.2) Pretreatment: Put the sections into high-definition constant staining pretreatment solution for 1 min. (2.3) Hematoxylin staining: Immerse sections in hematoxylin staining solution for 3 min, rinse with tap water, differentiate with differentiation solution for 3-5 s, rinse with tap water, apply blue solution for 3-6 s, and rinse with running water. (2.4) Eosin staining: Immerse sections in 95% alcohol for 1 min to dehydrate, then stain with eosin staining solution for 15 s. (2.5) Dehydration and mounting: Immerse sections sequentially in anhydrous ethanol I for 1 min, anhydrous ethanol II for 1 min, anhydrous ethanol III for 1 min, n-butanol I for 1 min, n-butanol II for 1 min, xylene I for 1 min, and xylene II for 1 min for clearing, then mount with neutral resin. (2.6) Microscopic examination and image acquisition and analysis: Use Saiviewer image analysis software to measure epidermal thickness and spinous layer thickness. Five mice were counted in each group, and five discontinuous fields of view were selected for each mouse. The mean ± standard deviation was calculated.

[0040] (2) Toluidine Blue (TB) Staining: (1.1) After tissue fixation and embedding, the embedded tissue was cut into sections using a paraffin microtome. The sections were then flattened and attached to poly-L-lysine-treated glass slides and placed in a 37°C oven overnight to ensure firm adhesion. (1.2) Dewaxing to Water: The baked sections were placed sequentially into two staining jars containing xylene, each for 15 minutes, to completely remove the paraffin from the sections. The sections were then transferred sequentially to two staining jars containing anhydrous ethanol, each for 5 minutes, to wash away the xylene. The sections were then immersed in 95% ethanol for 3 minutes and 75% ethanol for 3 minutes to complete the gradient dehydration. The sections were then rinsed slowly with tap water for 2 minutes, and then rinsed twice with distilled water for 1 minute each time to ensure full hydration. (1.3) Then proceed with the staining step. Immerse the hydrated sections in a pre-prepared 0.5% toluidine blue staining solution and stain at room temperature for 30 minutes. After staining, remove the sections and rinse them quickly twice with distilled water for 30 seconds each time to remove excess staining solution from the surface of the sections. Immerse the rinsed sections in 0.5% glacial acetic acid differentiation solution for about 30 seconds. During differentiation, the staining effect of the sections needs to be monitored in real time under a microscope. When the mast cell granules are clearly purplish-red and the background tissue is pale blue, remove the sections immediately to terminate differentiation. (1.4) After differentiation is terminated, rinse the sections twice with distilled water for 1 minute each time to thoroughly remove the differentiation solution and prevent the differentiation reaction from continuing. Then perform rapid dehydration and clearing operations. Place the sections into two staining jars containing anhydrous ethanol in sequence. The immersion time in each staining jar is strictly controlled within 1 minute. If the dehydration time is too long, the purplish-red metachromaticity of the mast cells will disappear and turn blue. After dehydration, the sections were sequentially placed into two staining jars containing xylene, each for 5 minutes, until the sections were completely transparent. Finally, mounting and microscopic examination were performed. A suitable amount of neutral resin was applied to the tissue section using a dropper, and a coverslip was gently placed on top, avoiding air bubbles. The mounted sections were then placed in a well-ventilated area to air dry, and subsequently observed and photographed under an optical microscope. The mast cell cytoplasmic granules appeared purplish-red; the number of mast cells per field of view was counted under an optical microscope.

[0041] (3) CD4 Immunohistochemical (IHC) staining: (1.1) Cut the embedded skin tissue paraffin blocks using a microtome, attach the sections to poly-L-lysine-coated glass slides, and bake the sections at 37°C overnight to ensure firm adhesion. Then proceed with the following steps: (1.2) Dewaxing to water: Xylene I: 15 minutes - Xylene II: 15 minutes - Anhydrous ethanol I: 5 minutes - Anhydrous ethanol II: 5 minutes - 95% ethanol: 3 minutes - 85% ethanol: 3 minutes - 75% ethanol: 3 minutes - Rinse with tap water: 2 minutes - Rinse with PBS: 3 times, 3 minutes each time. (1.3) Antigen retrieval: Place the sections in an antigen retrieval box containing preheated citrate buffer (pH 6.0) to boiling, ensuring the buffer completely submerges the sections. Cover the retrieval box and place it in an autoclave. Start timing after the pressure valve releases steam and continue heating for 2 minutes. Turn off the power and allow it to cool naturally to room temperature (about 30 minutes). Do not cool rapidly. Remove the slides and rinse with PBS 3 times, 3 minutes each time. (1.4) Endogenous peroxidase blocking: Gently blot away excess liquid around the slides with absorbent paper (do not wipe the tissue dry). Add 3% hydrogen peroxide blocking solution to completely cover the tissue and incubate at room temperature in the dark for 10 minutes. Rinse with PBS 3 times, 3 minutes each time. (1.5) Endogenous peroxidase blocking: Gently blot away excess liquid around the slides with absorbent paper (do not wipe the tissue dry). Add 3% hydrogen peroxide blocking solution to completely cover the tissue and incubate at room temperature in the dark for 10 minutes. Rinse with PBS 3 times, 3 minutes each time. (1.6) Primary antibody incubation: Dilute the anti-CD4 primary antibody with PBS according to the ratio determined in the preliminary experiment (dilution ratio 1:500). Add the diluted primary antibody to completely cover the tissue, place the slides in a humidified chamber (with a small amount of distilled water in the chamber for humidification), and incubate overnight (16 hours) at 4°C. The next day, remove the humidified chamber, warm to room temperature for 30 minutes, and rinse with PBS 3 times, 5 minutes each time. (1.7) Secondary antibody incubation: Add EnVision HRP-labeled secondary antibody complex to completely cover the tissue and incubate at room temperature for 30 minutes. Rinse three times with PBS for 5 minutes each time. (1.8) DAB staining: Prepare DAB working solution according to the kit instructions before use (e.g., 20 μl DAB substrate in 1 ml buffer), mix well, and store in the dark. Add DAB working solution to completely cover the tissue and incubate at room temperature in the dark for 3-10 minutes. Monitor under a microscope in real time: When positive cells show clear brownish-yellow staining and the background shows no obvious staining, immediately immerse the section in tap water to stop the staining. (1.9) Hematoxylin counterstaining: Immerse the section in hematoxylin staining solution and stain at room temperature for 3-5 minutes. Rinse with tap water for 1 minute to remove excess staining solution. (1.10) Differentiation and blueing: Immerse in 0.2% ammonia water for 30 seconds until the cell nuclei turn blue.(1.11) Dehydration, clearing, and sealing: Perform the following steps in sequence: 75% ethanol for 1 minute - 85% ethanol for 1 minute - 95% ethanol for 1 minute - anhydrous ethanol I for 2 minutes - anhydrous ethanol II for 2 minutes - xylene I for 5 minutes - xylene II for 5 minutes - seal with neutral resin, air dry in a ventilated place, and then examine under a microscope. Observe the infiltration of CD4+ T cells under an optical microscope and count them.

[0042] (4) Ki67 immunohistochemical staining: (1.1) Dewaxing paraffin sections or chips to water: Place paraffin sections in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and wash with distilled water. (1.2) Antigen retrieval: Heat retrieval with EDTA (pH 8.0) at 70℃ for 4 h. During this process, excessive evaporation of buffer should be prevented, and the slides should not be dried. After natural cooling, place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker, 5 min each time. Block endogenous peroxidase: Place the slides in 3% methanol-hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker, 5 min each time. (1.3) Serum blocking: Add 3% BSA to the histochemistry zone and evenly cover the tissue. Block at room temperature for 30 min. (Use rabbit serum to block primary antibodies from goat sources, and BSA to block primary antibodies from other sources.) (1.4) Add primary antibody (Ki67:1:2000 dilution): Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. (1.5) Add secondary antibody (S-vision immunohistochemical polymeric secondary antibody (goat anti-rabbit): Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add the secondary antibody (HRP-labeled) of the corresponding species to the primary antibody to the zone and cover the tissue. Incubate at room temperature for 50 min. (1.6) DAB staining: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time. After slightly drying the sections, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. The positive result is brownish-yellow. Rinse the sections with tap water to stop the staining. (1.7) Counterstain cell nuclei: Counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, use hematoxylin blue solution to return to blue, and rinse with running water. (1.8) Dehydration and mounting: Sections and cell smear samples: Place the sections in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol I for 2, 8, 1.5 minutes, anhydrous ethanol II for 5 minutes, n-butanol for 5 minutes, and xylene I for 5 minutes to dehydrate and clear them. Take the sections out of xylene and let them dry slightly. Mount them with mounting gel.

[0043] (5) CD4 / CD8 immunofluorescence detection: (1.1) Antigen retrieval: Heat retrieval at 70℃ for 4 hours. During the retrieval process, prevent excessive evaporation of the buffer solution and do not dry the slide. After retrieval, allow it to cool naturally. Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. (1.2) Circle drawing: Use a hydrogen peroxide blocking pen to draw circles around the tissue. Immerse the slide in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes to block endogenous peroxidase. Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. (1.3) Serum blocking: Shake off the PBS, add BSA (the primary antibody is of goat origin and is blocked with 10% rabbit serum), and block for 30 minutes. (1.4) Add the first primary antibody (CD4:1:3000 dilution): Remove the blocking solution, add the prepared primary antibody, and incubate it overnight at 4°C in a humidified chamber. (1.5) Add the corresponding HRP-labeled secondary antibody (S-vision immunohistochemical polymeric secondary antibody (goat anti-rabbit): Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add the corresponding HRP-labeled secondary antibody to the circle and incubate at room temperature for 50 min. (1.6) Add the corresponding 488TSA dye: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add TSA dye to the circle. A. Incubate at room temperature in the dark for 10 min. After incubation, place the slide in TBST and wash three times on a destaining shaker for 5 min each time. (1.7) Antibody elution: After slightly drying the slide, add immunostaining antibody elution buffer to cover the entire tissue. Incubate at room temperature for 5 min, then remove the antibody elution buffer. Add sufficient immunostaining antibody elution buffer again to completely cover the tissue. Incubate at 37℃ for 30 min. After incubation, place the slide in TBST and wash three times on a pendulum shaker for 5 min each time. (1.8) (1.9) Add the corresponding HRP-labeled secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add the corresponding HRP-labeled secondary antibody to the slide and incubate at room temperature for 50 min. (1.10) Add the corresponding 555TSA dye: Place the slide in PBS (pH 7.4) and wash it on a decolorizing shaker for 5 min each time. Wash 3 times, 5 min each time. After slightly drying the slide, add TSA to the circle and incubate at room temperature in the dark for 10 min. After incubation, place the slide in TBST and wash 3 times on a decolorizing shaker, 5 min each time. (1.12) Counterstain cell nuclei with DAPI: After slightly drying the slide, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 min. (1.13) Quenching autofluorescence: After slightly drying the slide, place the slide flat in a humidified box in the dark, add autofluorescence quencher to the circle for 5 min, and rinse with running water for 10 min.(1.14) Mounting: Mount with anti-fluorescence quenching mounting medium.

[0044] (6) CD206 / CD86 immunofluorescence staining: (1.1) Dewaxing paraffin sections or chips to water: Place paraffin sections in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and wash with distilled water. (1.2) Antigen retrieval: Heat retrieval with EDTA (pH 8.0) at 70℃ for 4 h. During the retrieval process, prevent excessive evaporation of the buffer solution and do not dry the slides. After retrieval, allow the slides to cool naturally. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker, 5 min each time. (1.3) Circle drawing: Use a hydrogen peroxide blocking histochemical pen to draw circles around the tissue. Immerse the slides in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min to block endogenous peroxidase. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker, 5 min each time. (1.4) Serum blocking: Shake off the PBS, add BSA and block for 30 min. (1.5) Add the first primary antibody: Remove the blocking solution, add the prepared primary antibody, and incubate overnight at 4°C in a humidified chamber. (1.6) Add the corresponding HRP-labeled secondary antibody (S-vision immunohistochemical polymeric secondary antibody (goat anti-rabbit)): Place the slide in PBS (pH 7.4) and wash 3 times on a decolorizing shaker for 5 min each time. Shake off the slide slightly, add the corresponding HRP-labeled secondary antibody in the circle, and incubate at room temperature for 50 min. (1.7) Add the corresponding TSA dye: Place the slide in PBS (pH 7.4) and wash 3 times on a decolorizing shaker for 5 min each time. Shake off the slide slightly, add TSA in the circle, and incubate at room temperature in the dark for 10 min. After incubation, place the slide in TBST and wash 3 times on a decolorizing shaker for 5 min each time. (1.8) Antibody elution: After slightly drying the slides, add immunostaining antibody elution buffer to cover the entire tissue. Incubate at room temperature for 5 min, then remove the antibody elution buffer. Add sufficient immunostaining antibody elution buffer again to completely cover the tissue. Incubate at 37°C for 30 min. After incubation, place the slides in TBST and wash three times on a pendulum shaker for 5 min each time. (1.9) Add the second primary antibody (CD206: dilution ratio: 1:2000): Add the prepared primary antibody and incubate the slides flat in a humidified chamber at 4°C overnight. (1.10) Add the corresponding HRP-labeled secondary antibody: Place the slides in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 min each time. After slightly drying the slides, add the corresponding HRP-labeled secondary antibody to the circle and incubate at room temperature for 50 min. (1.11) Add the corresponding TSA dye: Place the slides in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add TSA to the circle and incubate at room temperature in the dark for 10 minutes. After incubation, place the slide in TBST and wash it three times on a decolorizing shaker for 5 minutes each time.(1.12) Counterstaining cell nuclei with DAPI: After slightly drying the sections, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 min. (1.13) Quenching autofluorescence: After slightly drying the sections, place them flat in a humidified box in the dark and add autofluorescence quencher to the circle for 5 min, then rinse with running water for 10 min. (1.14) Mounting: Mount the slide with antifluorescence quenching mounting medium.

[0045] (7) Serological detection: The levels of IgE, IL-4, IL-5 (10-fold dilution), and IL-13 in serum were detected using an ELISA kit, strictly following the kit instructions. The procedure is as follows: All reagents and components were brought to room temperature. The required strips were removed from the aluminum foil bag, and the remaining strips were sealed in a resealable bag and returned to the refrigerator. Standard wells, zero-value wells, blank wells, and sample wells were set up. 50 μL of different concentrations of standard solution were added to each standard well, 50 μL of sample diluent was added to the zero-value wells, no diluent was added to the blank wells, and 50 μL of the sample to be tested was added to the sample wells. Except for the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to the standard wells, zero-value wells, and sample wells. The reaction plate was covered with a sealing film and incubated in a 37°C water bath or incubator in the dark for 60 min. Remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing buffer, let stand for 20 seconds, shake off the washing buffer, pat dry on absorbent paper, and repeat this process 5 times. If using an automatic plate washer, follow the washer's operating procedure and add a 30-second soaking time to improve detection accuracy. After washing, before adding the substrate, thoroughly pat the reaction plate dry on clean, lint-free paper. Mix substrates A and B thoroughly in a 1:1 volume ratio, and add 100 μL of the substrate mixture to all wells. Cover the reaction plate with the sealing film and incubate at 37°C in a water bath or incubator in the dark for 15 minutes. Add 50 μL of stop solution to all wells and read the absorbance (OD value) of each well at 450 nm using a microplate reader.

[0046] 1.9 Statistical Analysis: Data analysis was performed using GraphPad Prism 10.1.2 software. Quantitative data were expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons among multiple groups, and P < 0.05 was considered statistically significant.

[0047] 1.10 Experimental Results (1) Improvement of skin symptoms: After DNFB provocation, the dorsal skin of mice in the model control group showed obvious erythema, edema, scaling, and lichenification, and the SCORAD score was significantly increased. Skin symptoms began to improve in the medium-dose and high-dose Siglec-15 groups from day 4 of administration. By the end of treatment (day 14), the SCORAD scores in both groups were significantly lower than those in the model control group, and this improvement was dose-dependent. The SCORAD score of the high-dose group was close to that of the positive control dexamethasone group. The skin of mice in the normal control group was smooth and intact, without obvious lesions (see...). Figure 2 and Figure 3 ).

[0048] (2) General condition: During the treatment period, the body weight, food intake, and water intake of mice in each Siglec-15 dosage group remained stable or showed a slow upward trend, with no significant difference from the normal control group. Mice in the model control group had decreased appetite and slow weight gain due to the disease itself. Mice in the Siglec-15 treatment group had good mental state, normal activity level, and no obvious abnormal behavior was observed (see [link to treatment group]). Figure 4 A is a graph showing weight changes, B is a graph showing daily food intake, and C is a graph showing daily water intake.

[0049] (3) The spleen index (spleen mass / body weight) of mice in the medium-dose Siglec-15 group (**p<0.01), high-dose Siglec-15 group (***p<0.001), and positive control dexamethasone group (***p<0.001) was significantly lower than that in the model control group, suggesting that Siglec-15 can alleviate AD-related splenomegaly. There were no statistically significant differences in the heart index, liver index, lung index, and kidney index among the groups (P>0.05), indicating that Siglec-15 had no significant toxic effects on major organs at the experimental dose (see [link to relevant documentation]). Figure 5-6 ).

[0050] (4) Histopathological improvement: HE staining showed that the epidermis in the model control group was significantly thickened, with acanthosis, hyperkeratosis, and parakeratosis, and a large number of inflammatory cells infiltrated the superficial dermis. The epidermal and acanthosis thicknesses in the Siglec-15 treatment group were significantly lower than those in the model control group (****p<0.0001), the epidermal structure was closer to normal, and inflammatory cell infiltration was reduced. Toluidine blue staining showed that the number of mast cells in the model control group was significantly increased, mainly distributed in the superficial dermis. The number of mast cells in the Siglec-15 treatment group was significantly lower than that in the model control group (****p<0.0001) (see...) Figure 7 A shows HE staining images of the back skin of mice in each group; B shows TB staining images of the back skin of mice in each group; and C shows a statistical chart of epidermal and spinous layer thickness and mast cell positivity rate.

[0051] (5) Immune cell infiltration: Immunofluorescence assay showed that CD4+ T cells infiltrated extensively in the skin lesions of the model control group, mainly accumulating in the dermis. The degree of CD4+ T cell infiltration was significantly lower in the medium-dose Siglec-15 treatment group (***p<0.001) and the high-dose Siglec-15 treatment group (****p<0.0001) compared to the model control group (***p<0.001), and this was dose-dependent. There was no significant difference in CD8+ T cell count among the groups (P>0.05), suggesting that Siglec-15 has a selective inhibitory effect on CD4+ T cells (see [link to relevant documentation]). Figure 8 The top image shows immunofluorescence staining of CD4+ T cells and CD8+ T cells, while the bottom image shows statistical graphs of CD4+ T cells and CD8+ T cells.

[0052] (6) Inflammatory factor levels: Serum ELISA showed that serum IgE, IL-4, IL-5, and IL-13 levels in the model control group were significantly higher than those in the normal control group. In the medium- and high-dose Siglec-15 treatment groups, IL-4 (**p<0.01, ***p<0.001), IL-5 (*p<0.05, ***p<0.001), IL-13 (**p<0.01, ***p<0.001), and IgE (*p<0.05) levels were significantly lower than those in the model control group, suggesting that Siglec-15 may alleviate AD symptoms by affecting the release of inflammatory factors (see...). Figure 9 ).

[0053] (7) Immunohistochemistry of keratinocytes Ki67: Immunohistochemistry of Ki67 showed that the proliferation of Ki67 in the model control group was relatively lower than that in the medium-dose (*p<0.05) and high-dose (*p<0.05) Siglec-15 treatment groups. Figure 10 A is a qualitative morphological image of Ki67 immunohistochemical staining on mouse dorsal skin, and B is a quantitative statistical image of Ki67 immunohistochemical staining on mouse dorsal skin.

[0054] (8) Immunofluorescence of M1 / M2 macrophages: Immunofluorescence of M1 macrophages showed that the proportion of M1 macrophages in the model control group was lower than that in the medium-dose Siglec-15 group (*p<0.05) and the high-dose Siglec-15 group (***p<0.001). Immunofluorescence of M2 macrophages showed that the proportion of M2 macrophages in the model control group was lower than that in the medium-dose Siglec-15 group (***p<0.001) and the high-dose Siglec-15 group (***p<0.001). Figure 11 ).

[0055] II. Evaluation of the therapeutic effect of Siglec-15 on psoriasis 2.1 Experimental Animals: SPF-grade female BALB / c mice, 6–8 weeks old, weighing 18–22 g, were housed in the SPF-grade barrier system at the Animal Experiment Center of Southwest Medical University. The room temperature was 21–25℃, the relative humidity was 55–60%, and the mice were allowed free access to food and water under 12-hour alternating light and dark conditions. All mice were allowed one week of acclimatization before subsequent experiments. The animal experiment ethics involved were approved by the Animal Use and Protection Committee of Southwest Medical University.

[0056] 2.2 Main reagents: Siglec-15 recombinant protein (HY-P70744, MCE); imiquimod cream (IMQ, H20030129, Sichuan Mingxin Pharmaceutical Co., Ltd.); mouse IL-17 ELISA kit (RXW203067M, Ruixin Biotechnology), mouse IL-23 ELISA kit (RXW203058M, Ruixin Biotechnology), mouse TNF-α ELISA kit (RXW202412M, Ruixin Biotechnology), mouse IL-6 ELISA kit (RXW203049M, Ruixin Biotechnology).

[0057] 2.3 Preparation of Siglec-15 solution: Take 50 μg of Siglec-15 lyophilized powder and centrifuge at 12000 rpm for 30 seconds at 4 °C to allow the powder to gather at the bottom of the tube. Add 500 μL of PBS buffer (pH 7.4) containing 5% (w / v) trehalose, gently shake the lyophilized powder tube to dissolve the powder, and place it at 4 °C for 2 min to allow it to fully dissolve, thus preparing a 100 μg / mL Siglec-15 stock solution. Aliquot the stock solution into at least 20 μL tubes and store at -80 °C for later use, avoiding repeated freeze-thaw cycles. Before use, thaw at 4 °C and use directly.

[0058] 2.4 Dosage Design and Administration Regimen: The PSO experiment was conducted with three dosage groups: low dose (0.025 mg / kg), medium dose (0.05 mg / kg), and high dose (0.1 mg / kg). The administration route was subcutaneous injection at the center of the inflammatory focus, once daily. Day 0 was recorded on the day of model establishment, and administration continued for 5 days (Day 5 to Day 9). The positive control group received dexamethasone (MCE, catalog number HY-14648) at a dose of 1 mg / kg, subcutaneously, once daily for 5 days (Day 5 to Day 9).

[0059] 2.5 PSO Mouse Model Construction and Grouping: A classic psoriasis mouse model induced by imiquimod (IMQ) was used. The model was established for 10 days: 3.125 mg / kg IMQ cream (approximately 62.5 mg of cream, calculated based on a mouse weight of 20 g) was applied to the shaved area on the back of the mice daily. The mice that successfully developed the model were randomly divided into 6 groups (6 mice in each group): (1) Normal control group (Ctrl): normal mice, which were given an equal volume of solvent during the intervention; (2) Model control group (Model): PSO model mice, which were given an equal volume of solvent during the intervention; (3) Positive control group (Dex): PSO model mice, which were given dexamethasone 1 mg / kg during the intervention; (4) Low-dose Siglec-15 group (SL): PSO model mice, which were given Siglec-15 0.025 mg / kg; (5) Medium-dose Siglec-15 group (SM): PSO model mice, which were given Siglec-15 0.05 mg / kg; (6) High-dose Siglec-15 group (SH): PSO model mice, which were given Siglec-15 0.1 mg / kg.

[0060] 2.6 General Indicator Monitoring: Digital photographs of the mouse dorsal skin were taken daily during the treatment period, and a blinded scoring method was used according to the PASI scoring criteria. The PASI score was assessed on three dimensions: erythema, scaling, and thickness. Each dimension was scored from 0 to 4 points according to severity, with a total score of 0 to 12 points. Mouse weight, food intake, and water intake were also recorded daily.

[0061] 2.7 Sample Collection: Blood was collected from the eyeballs the day after the last administration, and the serum was separated and frozen at -80 ℃. After euthanizing the mice, the diseased skin tissue from the back and the major organs (heart, liver, spleen, lung, and kidney) were collected.

[0062] 2.8 Histological examination: After the mice were sacrificed, skin tissue from their backs was collected, fixed in 4% paraformaldehyde, and sent to Chengdu Aochuang Biotechnology Co., Ltd. for HE staining. The steps are as follows: (1) Preparation of paraffin sections: After trimming the fixed tissue blocks, rinse them under running water for 4 h. Then, perform graded ethanol dehydration in sequence: 70% ethanol (2 h), 80% ethanol (overnight), 90% ethanol (2 h), anhydrous ethanol I (1 h), and anhydrous ethanol II (1 h); then clear them with xylene for about 30 min, and finally immerse them in a mixture of xylene and paraffin at 60℃ for 2 h. Place the paraffin-impregnated tissue in a metal mold containing molten paraffin, cool and solidify it, and then embed it into blocks. Observe for any air bubbles. Use a microtome to continuously section the tissue, using 5 μm thick sections. Flatten the sections in a warm water bath and attach them to glass slides, then bake them at 60℃ for 2 h. Immerse the sections in xylene I and II for 5 min each, anhydrous ethanol I and II for 5 min each, then graded ethanol in 95%, 85%, and 75% for 2 min each, and finally soak them in distilled water for 2 min, ready for staining.

[0063] (2) Immerse the sections in hematoxylin staining solution for 5 min, then soak in distilled water for 5 min to remove excess stain. Next, differentiate in 1% hydrochloric acid alcohol for 3 s, immediately rinse with running water for 20 min to restore blue color, and then soak in distilled water for 2 min. After completing the above steps, immerse the sections in eosin staining solution for 3 min. After staining, dehydrate the sections by passing them through ethanol of progressively increasing concentrations, and then immerse them in xylene I and II for 10 min each to clear them. Finally, mount the sections with neutral resin and allow them to air dry at room temperature. Observe the morphology of the skin tissue under an optical microscope and measure the epidermal thickness using Saiviewer image analysis software. Six mice were counted in each group, and three discontinuous fields of view were selected for each mouse.

[0064] 2.9 Immunofluorescence detection: The coated paraffin-embedded skin blocks were sent to Chengdu Aochuang Biotechnology for sectioning. They were placed at room temperature for 15 min, soaked in PBS, air-dried, and the OCT embedding gel was removed. The sections were then incubated with blocking buffer at room temperature for 1 h. The corresponding primary antibody was added, and the sections were incubated at room temperature for 2 h or overnight at 4°C, ensuring the antibody remained submerged in the tissue. The next day, the humidified chamber was removed, and the sections were allowed to warm to room temperature for 30 min before recovering the primary antibody. Secondary antibody was added, and the sections were incubated in an opaque humidified chamber for 1 h. A small amount of glycerol mounting medium (containing DAPI) was added to completely submerge the tissue. The sections were then covered, and finally examined, photographed, and stored using a fluorescence microscope. Three fields of view were taken for quantitative analysis using Saiviewer software. The prepared sections can be stored in an opaque humidified chamber at 4°C.

[0065] 2.10 Serological testing: After collecting mouse blood samples, allow them to coagulate at room temperature for 2 hours. Centrifuge at 4000 rpm for 20 minutes at 4°C, collect the supernatant, centrifuge at 12000 g for 5 minutes at 4°C, aliquot the supernatant, and store at -80°C for later use. Dilute the serum 5-10 times and follow the instructions for the mouse IL-6, IL-17A, IL-23, and TNF-α ELISA kit. Measure the absorbance at 450 nm using a microplate reader, plot a standard curve, and calculate the concentration of the diluted serum sample based on the standard curve equation. Calculate the initial concentration of the serum sample.

[0066] 2.11 Statistical Analysis: Statistical analysis was performed using GraphPad 8 software. The t-test was used for comparisons between two groups, and the one-way ANOVA was used for multiple comparisons of three or more groups. Results are expressed as mean ± standard error. p < 0.05 was considered statistically significant.

[0067] 2.12 Experimental Results (1) Improvement of skin symptoms: After IMQ application, the skin on the backs of mice in the model control group showed obvious erythema, thick scales and thickening, and the PASI score was significantly increased. The PASI scores of the low, medium and high dose groups of Siglec-15 were significantly lower than those of the model control group (p<0.001), and the degree of PASI improvement was comparable to that of the positive control dexamethasone group (see Figure 12 and Figure 13 ).

[0068] (2) General condition: The body weight of mice in each Siglec-15 dose group showed an increasing trend during the treatment period. Their diet and water intake were normal, and no obvious abnormalities were observed (see Figure 14 A is a chart of food intake, B is a chart of water intake, and C is a chart of weight change.

[0069] (3) Organ safety: The spleen index in the low, medium, and high dose Siglec-15 groups and the dexamethasone group was significantly lower than that in the model control group (p<0.001), while there were no statistically significant differences in other organ indices (heart, liver, lung, kidney) among the groups (P>0.05) (see Figure 15 A shows the statistical chart of heart weight in different groups; B shows the statistical chart of liver weight in the same group; C shows the statistical chart of spleen weight in different groups; D shows the statistical chart of lung weight in different groups; E shows the statistical chart of kidney weight in different groups; F shows the actual image of spleen in different groups; G shows the actual image of heart, liver, lung, kidney and spleen in different groups.

[0070] (4) Histopathological improvement: HE staining showed that the epidermis in the model control group was significantly thickened, hyperkeratotic, and showed obvious inflammatory cell infiltration in the superficial dermis. The epidermal thickness in the three Siglec-15 dose groups was significantly lower than that in the model control group (p<0.001) (see Figure 16 A is an HE staining image of mouse dorsal skin, and B is a statistical graph of epidermal thickness of mouse dorsal skin in each group.

[0071] (5) Th17 cell infiltration: Immunofluorescence assay showed that IL-17A+ Th17 cells infiltrated extensively in the skin lesions of the model control group, mainly distributed in the superficial dermis and around the epidermal ridges. The number of Th17 cells in the Siglec-15 treatment group was significantly reduced compared with that in the model control group, and this reduction was dose-dependent (see [link to study]). Figure 17 Image A shows an immunofluorescence double staining image of CD4+ IL-17A+ cells (Th17 cells) on the back of a mouse, and image B shows a statistical graph of the positive cell rate of CD4+ IL-17A+ cells on the back of a mouse.

[0072] (6) Inflammatory factor levels: Serum ELISA detection showed that the serum levels of IL-17, IL-23, TNF-α, and IL-6 in the model control group were significantly higher than those in the normal control group. The levels of these inflammatory factors in all three Siglec-15 dose groups were significantly lower than those in the model control group, and this was dose-dependent. The high-dose group showed the most significant decrease in IL-17 and IL-23 (see...). Figure 18 , A is IL-6, B is IL-17, C is IL-23, D is TNF-α).

Claims

1. Application of Siglec-15 protein in the treatment of atopic dermatitis and / or psoriasis.

2. The application as described in claim 1, characterized in that, The dosage of the Siglec-15 protein is 0.025-0.1 mg / kg.

3. The application as described in claim 2, characterized in that, The dosage of the Siglec-15 protein is 0.05 mg / kg.

4. The application as described in any one of claims 1-3, characterized in that, The administration method is subcutaneous injection at the lesion site or topical application.

5. The application as described in any one of claims 1-3, characterized in that, The administration frequency is once daily for 4-7 consecutive days.

6. A medicine for treating atopic dermatitis and / or psoriasis, characterized in that, Including the Siglec-15 protein.

7. The medicament for treating atopic dermatitis and / or psoriasis as described in claim 6, characterized in that, When the drug dosage form is a topical preparation, the drug also includes at least one of the following: a transdermal penetration enhancer, a stabilizer, an excipient, a preservative, and a pH adjuster.

8. The medicament for treating atopic dermatitis and / or psoriasis as described in claim 7, characterized in that, The transdermal penetration enhancer includes azone, oleic acid, propylene glycol, and laurocapram; the stabilizer includes trehalose, mannitol, or human serum albumin.

Images