Application of regulating Notch / Wnt signal channel to block formation of hidradenitis suppurativa active sinus tract

Abstract

The invention discloses an application of regulating and controlling a Notch / Wnt signal channel in blocking formation of hidradenitis suppurativa active sinus tracts, and further discloses an application of regulating and controlling the Notch / Wnt signal channel in screening a medicine for blocking formation of the hidradenitis suppurativa active sinus tracts. The screening is realized by detecting the influence of a candidate substance on the activity of the Notch / Wnt signaling pathway. The influence on the activity of the Notch / Wnt signal path comprises activation of the Notch signal path and / or inhibition of the Wnt signal path. According to the invention, the S0 epithelial cell subgroup and a molecular marker combination (such as COL4A1, LAMC2, ITGA6, MMP1 and the like) specifically expressed by the S0 epithelial cell subgroup are identified, and the key regulation function of a Notch / Wnt signal channel in the subgroup is defined, so that a clear direction and a target spot are provided for deeply researching a molecular mechanism formed by a sinus tract; and a key theoretical basis and a feasible technical path are provided for subsequent establishment of a targeted drug screening system and discovery of effective candidate substances.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically, it relates to the application of regulating the Notch / Wnt signaling pathway to block the formation of active sinus tracts in hidradenitis suppurativa. Background Technology

[0002] Hidradenitis suppurativa (HS), also known as acne inversa (AI), is a chronic inflammatory skin disease that commonly occurs in areas where apocrine glands are distributed, such as the armpits, groin, perineum, and perianal region. It manifests as painful nodules, abscesses, sinus tracts, and scars. The reported prevalence in European and American populations ranges from 0.05% to 4.1%, while the prevalence in my country is significantly lower, at approximately 0.033%. The presence of sinus tracts in HS patients increases the likelihood of progression to Hurley stage 3, and sinus tracts are a hallmark of the aggressive development of HS. Sinus tracts are an important source of inflammatory mediators in HS, releasing foul-smelling secretions onto the skin surface and causing severe pain and discomfort in patients.

[0003] Currently, the exact mechanism of sinus tract formation remains unclear, and treatment for HS sinus tracts is ineffective. Surgical resection of HS is an important treatment for moderate to severe or recurrent cases, but it has a high recurrence rate. Antibiotic treatment (such as clindamycin and rifampin) can relieve acute inflammation, but its effect on existing sinus tracts is limited. Biologics (such as adalimumab) can reduce inflammation and sinus tract formation, but they are ineffective for late-stage fibrotic sinus tracts. Active HS sinus tracts are characterized by active inflammation, purulent discharge, and secretions, while inactive sinus tracts are characterized by relatively quiescent inflammation, no significant secretions, and have less impact on the patient's quality of life. One of the treatment goals for HS is to prevent the formation of active sinus tracts or to keep existing sinus tracts in a quiescent stage. Therefore, how to intervene and block the formation of active sinus tracts has important clinical value for the treatment of HS patients. Summary of the Invention

[0004] This invention addresses the problem of unclear formation mechanism and lack of effective intervention methods in the prior art for active sinus tract formation in hidradenitis suppurativa, and provides an application of regulating the Notch / Wnt signaling pathway to block the formation of active sinus tracts in hidradenitis suppurativa.

[0005] Therefore, in a first aspect, the present invention provides an application of regulating the Notch / Wnt signaling pathway in blocking the formation of active sinus tracts in hidradenitis suppurativa.

[0006] A second aspect of the present invention provides the use of regulating the Notch / Wnt signaling pathway in screening drugs for blocking active sinus tract formation in hidradenitis suppurativa.

[0007] According to a preferred embodiment of the present invention, the screening is achieved by detecting the effect of candidate substances on the activity of the Notch / Wnt signaling pathway.

[0008] Furthermore, the effects on the Notch / Wnt signaling pathway activity include: activating the Notch signaling pathway and / or inhibiting the Wnt signaling pathway.

[0009] According to a preferred embodiment of the present invention, the screening is achieved by detecting the expression level of S0 subgroup-specific molecular markers; the S0 subgroup-specific molecular markers are selected from at least one of the following: COL4A1, COL17A1, LAMA3, LAMB3, LAMC2, INHBA, PLAU, ITGA6, ITGB4, ITGA3, ITGB1, MMP1, MMP10.

[0010] Furthermore, the screening is achieved by detecting the effect of candidate substances on the phenotype of the S0 subpopulation of sinus tract epithelium; wherein the S0 subpopulation phenotype is characterized by high expression of neonatal basement membrane components and / or high cell migration ability.

[0011] The present invention has the following beneficial effects:

[0012] 1. This invention identifies the S0 epithelial cell subset and its specific expression of molecular markers (such as COL4A1, LAMC2, ITGA6, MMP1, etc.), and clarifies the key regulatory role of the Notch / Wnt signaling pathway in this subset, providing a clear direction and target for in-depth research on the molecular mechanism of sinus tract formation.

[0013] 2. This invention clarifies that regulating Notch / Wnt pathway activity and the expression level of S0 subset-specific biomarkers are the core detection indicators, providing a key theoretical basis and feasible technical path for the subsequent establishment of targeted drug screening systems and the discovery of effective candidate substances. Attached Figure Description

[0014] Figure 1 The morphological differences between active and inactive sinus tract epithelium, among which... Figure 1 Image a is a hematoxylin-eosin (HE) staining image showing dermal sinus tracts in DT and NDT. Blue dashed lines mark the sinus tract-dermal junction, and black dashed lines mark the dermal-epidermal junction. Sinus tract epithelium (TE) is divided into three morphological types: Type I (keratinized), Type II (non-keratinized), and Type III (neo-epithelial). Abbreviations: L for sinus tract cavity, D for dermis. Scale bar: 200 μm (low power field), 20 μm (high power field). Figure 1 b shows the composition ratio of the three sinus tract epithelial types in DT and NDT.

[0015] Figure 2 This is a map showing the major cell types in tissues for spatial transcriptomics analysis. Among them, Figure 2 a is the Uniform Manifold Approximation and Projection (UMAP) diagram, which is visualized based on the spatial transcriptomics data of two tissue sections (bin size set to 8 μm). It annotates 11 major cell types, including fibroblasts, endothelial cells (ECs), melanocytes, parietal cells, neutrophils (NE), epithelial cells, macrophages (MPs), mast cells, T cells, B cells, and plasma cells. Figure 2 b、 Figure 2 c is a spatial location map, showing the distribution of the 11 cell types identified by spatial transcriptomics on tissue sections. Scale bar: 200 μm.

[0016] Figure 3 This is a clustering and localization map of sinus tract epithelial cell subsets. Figure 3 a is the UMAP diagram, which shows the five subgroups S0, S1, S2, S3, and S4 formed by performing second-order unsupervised clustering on all sinus tract epithelial cells. Figure 3 b、 Figure 3 c is a spatial location map showing the distribution of the five sinus tract epithelial cell subsets on the tissue section. Abbreviations: E for epidermis, L for sinus tract lumen, D for dermis. Scale bar: 200 μm. Figure 3 d is a high-power field spatial transcriptome diagram, showing the sinus tract epithelial cell subpopulations corresponding to the three types of sinus tract epithelium, scale bar: 10 μm.

[0017] Figure 4 This is a validation diagram of the functional characteristics and biomarker expression of the S0 subgroup. Among them, Figure 4 a is a Gene Ontology (GO) pathway enrichment analysis diagram, showing the upregulated pathways of the S0 subset compared with other sinus tract epithelial subsets. The X-axis represents the number of differentially expressed genes, and the color represents the P-value. Figure 4 b shows the expression of genes representing the components of the newly formed basement membrane in five sinus tract epithelial cell subsets. The color represents the average expression level, and the size represents the percentage of cells expressing the gene. Figure 4 c shows the expression of cell migration-related genes in five sinus tract epithelial cell subsets. The color represents the average expression level, and the size represents the percentage of cells expressing the gene. Figure 4 d shows the expression of inflammation-related genes in five sinus tract epithelial cell subsets; Figure 4 e shows the expression of cell differentiation-related genes in five sinus tract epithelial cell subsets; Figure 4 f shows the expression of genes related to the Notch and WNT signaling pathways that regulate cell differentiation in five sinus tract epithelial cell subsets; Figure 4g shows the expression differences of LAMC2 and LAMB3 in DT and NDT detected by immunohistochemistry, and the expression levels of LAMC2 and LAMB3 in DT and NDT are shown by box plot.

[0018] Figure 5 Experimental validation diagram for constructing the S0 phenotype by combining CLDN1 knockdown with IL-36γ regulation of the Notch / Wnt pathway, in which... Figure 5 Figure a shows a heatmap of differential gene clustering in HaCaT cells treated with CLDN1 knockdown or IL-36γ. Figure 5 b is a graph showing the verification of CLDN1 knockdown and IL-36γ treatment of HaCat cells by qPCR detection, demonstrating the mRNA expression levels of LAMC2, INHBA, PLAU, IVL, KRT16 and NOTCH1 in different treatment groups; Figure 5 c shows the Transwell experimental diagram and quantitative analysis diagram, demonstrating the effects of IL-36γ and IL-36RA on HaCaT cell migration; Figure 5 d shows the scratch assay and quantitative analysis diagrams, illustrating the effect of sh-CLDN1 on HaCaT cell migration; Figure 5 e shows the immunostaining and quantitative analysis diagrams, illustrating the effect of blocking the IL-36γ receptor on LAMC2 expression in the 3D-SeboSkin model.

[0019] Figure 6 This diagram illustrates the experimental validation of regulating the Notch / Wnt pathway to block the S0 phenotype in epithelial cells. Figure 6 Figure a shows the experimental verification of the inhibition of LAMC2 expression in HaCaT cells by IL36 receptor antagonist (HB0034), Notch agonist (JAG1), and Wnt antagonist (IWP2). Figure 6 Figure b shows the experimental verification of the effect of IL36 receptor antagonists, Notch agonists, and Wnt antagonists on inhibiting LAMC2 expression in a 3D skin model constructed from fresh skin lesions of HS patients. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0021] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] Example 1: Screening and determination of specific molecular marker combinations for neonatal sinus tract epithelium in HS

[0024] 1.1 Sample Collection and Quality Control

[0025] Sample Source: Six patients with hepatic sclerosis (HS) were selected. Formalin-fixed paraffin-embedded (FFPE) tissue samples were surgically obtained from three draining sinus tracts (DT, active sinus tract) and three non-draining sinus tracts (NDT, inactive sinus tract). The sampling sites were confirmed by the clinicians, avoiding necrotic areas and normal skin tissue. Sample collection was approved by the Ethics Committee of Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine (Approval No.: KY2021-113-B). Each participant was fully informed of the experimental research content, process, and related risks, and signed a written informed consent form.

[0026] Sample quality control: Pre-assessment of FFPE tissue using hematoxylin-eosin (HE) staining is required. The complete sinus tract lumen and surrounding epithelial tissue must be clearly observed, and the type of sinus tract epithelium must be distinguishable as follows: Type I (keratotic type, composed of basal layer, spinous layer, granular layer and stratum corneum, similar to the epidermal structure, with a single layer of columnar basal cells in the basal layer and covered by a cellular layered stratum corneum), Type II (non-keratotic type, containing a basal layer and a thinner spinous layer, but lacking granular layer and stratum corneum, with a large number of immune cells infiltrating the dermis below), and Type III (neonatal type, composed only of basal layer and 3-4 layers of flattened suprabasal cells, with darker staining of suprabasal cell nuclei and fewer keratin granules in the cytoplasm). Ensure that the sample does not have severe autolysis, necrosis or tissue deformation caused by improper fixation. Figure 1 A morphological phenotypic diagram of the sinus tract epithelium, such as... Figure 1 As shown in Figure a, HE staining clearly reveals the dermal sinus tract structure and three different types of sinus tract epithelium in DT and NDT. Furthermore, stacked bar charts can be used to understand the compositional ratio of the three sinus tract epithelial types in DT and NDT. Figure 1 As shown in b, the proportion of type II (non-keratinized epithelium) and type III (neo-embryonic epithelium) epithelium in active sinus tracts is higher than that in inactive sinus tracts, while immune cells such as macrophages and neutrophils mainly accumulate around type II (non-keratinized epithelium) and type III (neo-embryonic epithelium). Therefore, active sinus tracts are the lesion sites that trigger strong inflammatory responses in HS and are also the main target areas for treating inflammatory damage in HS.

[0027] 1.2 Spatial Transcriptome Sequencing and Data Processing

[0028] S1. Tissue Sectioning and RNA Quality Control: FFPE tissue blocks were cut into 5μm thick continuous sections. Two structurally intact sections were selected (one for sequencing and one for HE staining and localization). Sequencing sections were dewaxed (10 min each with xylene I / II), hydrated (5 min each with 75% / 80% / 100% ethanol), and protected with RNA. RNA purity (A260 / A280 = 1.8~2.0) and integrity (RIN ≥ 6.0) were then tested to ensure they met sequencing requirements.

[0029] S2. Spatial Transcriptome Sequencing: A 10x Visium HD spatial transcriptome sequencing chip was used. The tissue slices were attached to the chip containing spatial barcodes for tissue permeation, allowing intracellular RNA to bind to the probe. Subsequently, cDNA synthesis, amplification, and library construction were completed. Sequencing was performed using the Illumina NovaSeq 6000 sequencing platform in PE150 mode, with a sequencing depth of ≥5 million reads per sample (bin size set to 8μm).

[0030] S3. Bioinformatics Analysis: The raw data was filtered (low-quality reads, adapter contamination, and rRNA sequences were removed) to obtain clean reads, which were then aligned to the human reference genome (GRCh38.p13) to calculate the gene expression matrix. PCA dimensionality reduction and UMAP visualization were performed using Seurat 4.0 software, and 11 major cell types, including fibroblasts, endothelial cells (ECs), and epithelial cells, were annotated based on cell markers. Epithelial cell data were extracted and subjected to secondary unsupervised clustering to divide the data into five subgroups: S0, S1, S2, S3, and S4. Differential expression analysis was performed on the S0 subgroup and other subgroups, and GO pathway enrichment analysis was combined to explore the core functional pathways related to the S0 subgroup. Figure 2 This is a map showing the major cell types in tissues for spatial transcriptomics analysis, where... Figure 2 a is the Uniform Manifold Approximation and Projection (UMAP) diagram, which is visualized based on spatial transcriptomics data from two tissue sections (bin size set to 8 μm). It annotates 11 major cell types, including fibroblasts, endothelial cells (ECs), melanocytes, parietal cells, neutrophils (NE), epithelial cells, macrophages (MPs), mast cells, T lymphocytes, B lymphocytes, and plasma cells. Figure 2 b and 2c are spatial location maps, showing the distribution of 11 cell types identified by spatial transcriptomics on tissue sections. According to... Figure 2 It is known that spatial transcriptome data can annotate 11 major cell types, including fibroblasts, endothelial cells, and epithelial cells, and clarify the localization of each cell type on tissue sections. Spatial transcriptome sequencing can obtain the gene expression profiles and spatial localization of various cells in HS sinus tract tissue specimens, providing data support for identifying key cell subpopulations that trigger invasive sinus tract growth.

[0031] Figure 3 This is a clustering and localization map of sinus tract epithelial cell subsets, in which... Figure 3 a is the UMAP diagram, which shows the five subgroups S0, S1, S2, S3, and S4 formed by performing second-order unsupervised clustering on all sinus tract epithelial cells. Figure 3 b and 3c are spatial location maps, showing the distribution of the five sinus tract epithelial cell subpopulations on the tissue section; Figure 3 Image d is a high-power field spatial transcriptome map, showing the sinus tract epithelial cell subsets corresponding to the three types of sinus tract epithelium. Based on... Figure 3 It can be seen that two-dimensional unsupervised clustering can divide sinus tract epithelial cells into 5 subpopulations, and the distribution of each subpopulation on tissue sections and their correspondence with different types of sinus tract epithelium can be determined. Based on the subpopulation classification of sinus tract epithelium using spatial transcriptome sequencing data, a unique subpopulation S0 in active HS sinus tracts was screened out, while this subpopulation is almost non-existent in inactive sinus tracts.

[0032] 1.3 Screening and Validation of Molecular Marker Combinations

[0033] 1.3.1 Biomarker Screening:

[0034] Based on the "high expression of newly formed basement membrane (BM) components (COL4A1, laminin α3β3γ2) and cell migration functional molecules (integrin α6β4 / α3β1, metalloproteinase 1 / 10) in S0 cells," and considering both expression specificity (significantly higher expression levels in the S0 subpopulation than in other subpopulations) and detection feasibility (detectable by conventional techniques), a combination of core biomarkers was screened.

[0035] 1) Components of the newly formed basement membrane (BM): COL4A1, COL17A1, LAMA3, LAMB3, LAMC2, INHBA, PLAU;

[0036] 2) Cell migration functional molecules: ITGA6, ITGB4, ITGA3, ITGB1, MMP1, MMP10, CALD1, FSCN1.

[0037] 1.3.2 Verification Experiment:

[0038] S1. Immunohistochemical staining: FFPE sections were dewaxed, hydrated, subjected to 3% hydrogen peroxide to block endogenous peroxidase, antigen retrieval by water bath heating, incubated with primary antibody, incubated with HRP secondary antibody, DAB staining, and counterstained with hematoxylin.

[0039] S2. Immunofluorescence co-localization verification: Double-label immunofluorescence staining showed that the S0 marker co-localized with the epithelial basal cell marker, confirming that the S0 subset is an activated basal cell subset in the sinus tract epithelium.

[0040] S3, qPCR validation: Total RNA was extracted from DT and NDT tissues and reverse transcribed into cDNA. The mRNA expression level of S0 marker in DT and NDT was detected by qPCR, and the consistency with the spatial transcriptome sequencing results was validated. Figure 4 This is a validation diagram of the functional characteristics and biomarker expression of the S0 subgroup, in which... Figure 4 a is a gene ontology (GO) pathway enrichment analysis diagram, showing the upregulated pathways of the S0 subset compared to other sinus tract epithelial subsets; Figure 4 b shows the expression of genes representing the components of the newly formed basement membrane in five sinus tract epithelial cell subsets; Figure 4 c shows the expression of cell migration-related genes in five sinus tract epithelial cell subsets; Figure 4 d shows the expression of inflammation-related genes in five sinus tract epithelial cell subsets; Figure 4 e shows the expression of cell differentiation-related genes in five sinus tract epithelial cell subsets; Figure 4 f shows the expression of genes related to the Notch and WNT signaling pathways that regulate cell differentiation in five sinus tract epithelial cell subsets; Figure 4 g demonstrated the expression levels of LAMC2 and LAMB3 in DT and NDT using immunostaining and box plots. Figure 4 It was found that genes related to newly formed basement membrane components and cell migration were specifically expressed in the S0 subset, and immunostaining showed that the expression levels of LAMC2 and LAMB3 in DT were higher than those in NDT. Compared with other epithelial subsets, S0 was in a state of low differentiation and was jointly regulated by the Notch and WNT pathways. Analysis of the molecular characteristics of S0 cells revealed that this cell subset has strong migration and inflammatory mediator secretion capabilities. Combined with its low differentiation characteristics, this indicates that S0 is not involved in normal tissue repair, but is closely related to pathological epithelial repair and is a key cell in the invasive growth of HS sinus tracts.

[0041] Example 2: Construction of a S0 phenotype regulatory model for newly formed sinus tract epithelial cells in HS and verification of the Notch / Wnt signaling pathway

[0042] 2.1 Construction of in vitro cell model (induction of HaCaT cell S0 phenotype)

[0043] Basic cell culture conditions: The immortalized human keratinocyte cell line HaCaT was used and cultured in DMEM high glucose medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a constant temperature incubator at 37°C and 5% CO2. The medium was changed every 2-3 days, and the cells were passaged when the confluence reached 80%-90%.

[0044] S0 phenotype induction methods:

[0045] IL36G stimulation induction: HaCaT cells were seeded in 6-well plates and cultured until the confluence reached 70%-80%. The experimental group was given medium containing recombinant human IL-36G, while the control group was given medium without IL-36G. The cells were cultured for another 24 hours. The mRNA expression levels of S0 markers (LAMC2, INHBA, PLAU) and the Notch signaling gene (NOTCH1) were detected by qPCR. Cell migration ability was detected by Transwell assay to verify the S0 phenotype induction effect.

[0046] CLDN1 gene knockdown induction: A CLDN1-specific shRNA lentiviral vector was constructed, transfected into 293T cells, and the viral fluid was collected to infect HaCaT cells. After selection with puromycin, a stable CLDN1 knockdown cell line was obtained. The expression level of CLDN1 protein was detected by Western blot, and cell migration ability was detected by scratch assay to verify the S0 phenotype induction effect.

[0047] 2.2 Verification of Notch / Wnt signaling pathway regulation

[0048] Experimental Design: Notch signaling activator JAG1 and Wnt signaling inhibitor IWP2 were selected as candidate compounds. Preliminary cytotoxicity experiments were conducted to determine the experimental concentrations (JAG1 10 μmol / L, IWP2 10 μmol / L) with no significant toxicity. S0 phenotype HaCaT cells were divided into a model control group, a JAG1 treatment group, and an IWP2 treatment group, and cultured for 48 h for multidimensional validation.

[0049] 1) Molecular level verification:

[0050] mRNA levels: The mRNA expression levels of S0 markers (LAMC2, ITGA6, MMP1), Notch pathway genes (NOTCH1, HES1), and Wnt pathway genes (CTNNB1, MYC) in each group were detected by qPCR.

[0051] Protein levels: The expression levels of S0 markers (LAMC2, ITGA6), Notch pathway-related proteins (NOTCH1, HES1), and Wnt pathway-related proteins (CTNNB1) in each group were detected by Western blot.

[0052] Pathway activity: The expression levels of Notch downstream protein (HES1) and Wnt downstream protein (nuclear β-catenin) in each group were detected by Western blot to verify the regulatory effect of the compound on the Notch / Wnt pathway.

[0053] 2) Cell function verification:

[0054] Transwell assay: The migration ability of cells in each group was detected, and the difference in the number of migrating cells between the model control group and the treatment group was compared.

[0055] Scratch test: The healing rate of cells in each group was detected, and the differences in cell migration function between the model control group and the treatment group were compared.

[0056] 3) 3D skin model verification:

[0057] The 3D-SeboSkin model was used to simulate the in vivo skin microenvironment. After the complete model was constructed, it was divided into a control group, a model group (containing IL-36G), a JAG1 treatment group (containing IL-36G+JAG1), and an IWP2 treatment group (containing IL-36G+IWP2). The samples were tested after 7 days of culture.

[0058] Histological analysis: HE staining was used to observe the differences in epidermal structure and sinus tract epithelial-like structure among the groups; S0 marker detection: Immunostaining was used to detect the expression level of LAMC2 in each group, and the differences between the treatment group and the model group were quantitatively analyzed to verify the pathway intervention effect. Figure 5 Experimental validation diagram for constructing the S0 phenotype by combining CLDN1 knockdown with IL-36γ regulation of the Notch / Wnt pathway, in which... Figure 5 a is a heatmap of differential gene clustering in HaCaT cells with CLDN1 knockdown or IL-36γ treatment; Figure 5 b shows CLDN1 knockdown and IL-36γ treatment; 5a shows the qPCR detection verification diagram, displaying the mRNA expression levels of LAMC2, INHBA, PLAU, and NOTCH1 in different treatment groups. Figure 5 c shows the Transwell experimental diagram and quantitative analysis diagram, demonstrating the effects of IL-36γ and IL-36RA on HaCaT cell migration; Figure 5 d shows the scratch assay and quantitative analysis diagrams, illustrating the effect of sh-CLDN1 on HaCaT cell migration; Figure 5 e shows the immunostaining and quantitative analysis diagrams, illustrating the effect of blocking the IL-36γ receptor on LAMC2 expression in the 3D-SeboSkin model. According to... Figure 5 It is known that CLDN1 knockdown or IL36G stimulation can affect the activity of the Notch / Wnt pathway, promote the expression of S0 markers and cell migration ability in HaCaT cells.

[0059] Figure 6 This diagram illustrates the experimental validation of regulating the Notch / Wnt pathway to block the S0 phenotype in epithelial cells. Figure 6Figure a shows the experimental verification of the inhibition of LAMC2 expression in HaCaT cells by IL36 receptor antagonist (HB0034), Notch agonist (JAG1), and Wnt antagonist (IWP2). Figure 6 Figure b shows the experimental validation of the inhibition of LAMC2 expression in a 3D skin model constructed from fresh skin lesions of HS patients by IL36 receptor antagonists, Notch agonists, and Wnt antagonists. According to... Figure 6 It is known that regulating the activity of the Notch / Wnt pathway can block the expression of S0 markers and inhibit the formation of S0-like cells.

[0060] In summary, this invention, through spatial transcriptomics analysis, clarified the core functional characteristics of the S0 epithelial cell subset in active sinus tracts of hidradenitis suppurativa (HS), confirming that the Notch / Wnt signaling pathway can block the formation of active sinus tracts in HS by regulating the phenotypic characteristics and cell migration ability of the S0 subset. This provides a reliable target and scientific basis for the development of targeted drugs for active sinus tracts in HS. The aforementioned Notch / Wnt signaling pathway and its associated S0 subset-specific markers (such as COL4A1, COL17A1, LAMC2, ITGA6, MMP1, etc.) can be further used for screening drugs for the treatment of active sinus tracts in HS using methods known in the art. For example, a drug screening model can be constructed with Notch / Wnt pathway activity regulation and S0 subset phenotypic inhibition as core indicators. By detecting the effects of candidate drugs on the expression of key genes in the pathway (such as NOTCH1 and CTNNB1), S0 marker levels, and cell migration function, drugs with the potential to block the formation of active sinus tracts can be screened. This is obvious to those skilled in the art.

[0061] The above description is only a partial embodiment of the present invention and is not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made based on the description of the present invention fall within the protection scope of the present invention. All aspects not described in detail in this invention are conventional technical content.

Claims

1. An application of regulating the Notch / Wnt signaling pathway in blocking active sinus tract formation in hidradenitis suppurativa.

2. The application of a method for regulating the Notch / Wnt signaling pathway in screening drugs for blocking active sinus tract formation in hidradenitis suppurativa.

3. The application according to claim 2, characterized in that, The screening is achieved by detecting the effect of candidate substances on the activity of the Notch / Wnt signaling pathway.

4. The application according to claim 3, characterized in that, The effects on the Notch / Wnt signaling pathway activity include: activating the Notch signaling pathway and / or inhibiting the Wnt signaling pathway.

5. The application according to claim 2, characterized in that, The screening is achieved by detecting the expression level of S0 subgroup-specific molecular markers; the S0 subgroup-specific molecular markers are selected from at least one of the following: COL4A1, COL17A1, LAMA3, LAMB3, LAMC2, INHBA, PLAU, ITGA6, ITGB4, ITGA3, ITGB1, MMP1, MMP10.

6. The application according to claim 2, characterized in that, The screening is achieved by detecting the effect of candidate substances on the phenotype of the S0 subpopulation of sinus tract epithelium; wherein the S0 subpopulation phenotype is characterized by high expression of neonatal basement membrane components and / or high cell migration ability.

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