Biomarker for prurigo nodularis and application of biomarker

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a biomarker of prurigo nodularis and application of the biomarker. The invention discloses that protease activated receptor 2 (PAR2) regulates expression of Th2 type cytokines through a G alpha12-RhoA-ERK signal channel for the first time, and further participates in morbidity of prurigo nodularis (PN). According to the embodiment of the invention, various experimental means such as qRT-PCR, Western Blot, IHC, IF and the like are utilized to verify expression up-regulation of PAR2 and related molecules in skin tissues of a PN patient and the correlation of the PAR2 and related molecules with clinical symptoms. Molecules such as PAR2, RhoA, ERK, IL-4, IL-31 and the like are proved to be highly related to pathological characteristics of PN. The molecules can be used as diagnostic markers or target spots for treating prurigo nodularis, and can be used for early diagnosis of PN, disease assessment or drug development.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomarker for nodular prurigo and its application. Background Technology

[0002] Nodular prurigo (PN) is a debilitating, difficult-to-treat, and persistent skin condition characterized by multiple papules and nodules accompanied by parakeratosis and hyperkeratosis, along with intense itching. The itching and subsequent scratching create a vicious cycle, making the condition difficult to treat, impacting patients' sleep quality and overall quality of life, and increasing their financial burden. Treatment options for PN are limited, and traditional therapies are often ineffective, highlighting the need to develop novel treatment methods. The pathogenesis of PN is complex and not yet fully understood.

[0003] Recent studies have shown that neurofibrillary pruritus (PN) involves complex interactions between neural, immune, and epidermal components. The Th2 immune response, centered on interleukin (IL)-4 and IL-31, is considered a key pathological driver of chronic inflammation, neurofibrillation, and intractable pruritus. However, the upstream signaling mechanisms that trigger and maintain this Th2 response remain a focus of research and potential targets for clinical intervention. Increasing research indicates that protease-activated receptor 2 (PAR2) plays a crucial role in the Th2 immune response.

[0004] Protease-activated receptor 2 (PAR2) is a transmembrane protein in the G protein-coupled receptor (GPCR) family, activated by irreversible cleavage of its N-terminus by various endogenous and exogenous proteases. This activation releases an anchored ligand (TL), which subsequently binds to and activates the receptor. PAR2 is widely expressed in various tissues, including the skin, and is a promising drug target for the treatment of arthritis, asthma, and neuropathic pain. PAR2 is involved in skin inflammatory responses, promoting Th2 inflammation, delaying skin barrier repair, and affecting keratinocyte differentiation. It is also involved in the transmission of sensations of itching and pain. Emerging evidence suggests that PAR2 plays a crucial role in the pathogenesis of inflammatory skin diseases such as acne vulgaris, rosacea, psoriasis, and atopic dermatitis; however, its role in pruritus (PN) has been rarely reported. PAR2 functions through multiple signaling pathways. A cascade associated with Gα12 / 13 or β-repressor proteins can activate Rho family member A (RhoA), nuclear factor κB (NF-κB), or mitogen-activated protein kinase (MAPK / ERK). These pathways regulate skin homeostasis, immune and inflammatory responses, and tumor surveillance. Multiple protease allergens, including those from Aspergillus fumigatus, house dust mites, and Staphylococcus aureus V8 protease, mediate PAR2 sensing, triggering a strong inflammatory response and releasing pro-Th2 cytokines (IL-4, IL-6, IL-25, IL-31, IL-1β, TSLP). In mouse models, PAR2-deficient mice failed to develop airway inflammation after house dust mite (HDM) sensitization and challenge, lacked HDM-specific IgG1 production, and had lower lung IL-4 mRNA levels than wild-type mice. Blocking PAR2 activation during sensitization in wild-type mice reduced lung Th2 mediators, including IL-4, IL-5, and IL-13. Serine protease allergens promote Th2 responses in mice by activating PAR2 and STAT3.

[0005] The regulation of Th2-type inflammatory response in PN by PAR2 has not been reported. This study aims to explore the pathogenesis of PN using clinical samples in order to discover potential targets for PN treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker for nodular prurigo and its application. The biomarker PAR2 provided by this invention can be used as a diagnostic marker or a therapeutic target for nodular prurigo, for early diagnosis, disease assessment or drug development of PN.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a biomarker for nodular prurigo, wherein the biomarker is PAR2.

[0008] Preferably, PAR2 regulates the Th2-dominated response pathogenesis of nodular prurigo by mediating the RhoA-ERK signaling axis.

[0009] More preferably, the expression level of PAR2 is strongly positively correlated with the RhoA-ERK signal axis.

[0010] The present invention also provides a kit for detecting nodular prurigo, the kit comprising reagents for detecting the above-mentioned biomarkers.

[0011] The present invention also provides a pharmaceutical composition for treating and / or preventing nodular prurigo, said pharmaceutical composition comprising an effective amount of an inhibitor of PAR2.

[0012] The present invention also provides a method for diagnosing nodular prurigo, specifically comprising the following steps: S1. Extract RNA or protein from the skin tissue of the test subject; S1. Detect the expression level of PAR2 molecules; If the PAR2 expression level of the subjects is significantly higher than that of healthy controls, it suggests that the subjects have PN or are at risk of PN.

[0013] This invention also provides the application of the above-mentioned biomarkers in the prognostic assessment of nodular prurigo.

[0014] The present invention also provides the application of a biomarker in the preparation of a reagent for detecting nodular prurigo, wherein the biomarker is PAR2.

[0015] Preferably, the nodular prurigo detection reagent is selected from probes that specifically recognize PAR2; Or primers that specifically amplify PAR2; Or antibodies or ligands that specifically bind to the protein encoded by PAR2.

[0016] Beneficial effects: This invention identifies eight significantly differentially expressed genes associated with the pathogenesis of nodular prurigo (NP): PAR2, STAT3, Gα12, RhoA, AKT1, ERK1, IL-4, and IL-31. Correlation analysis revealed a strong positive correlation between PAR2 and STAT3, Gα12, RhoA, ERK1, IL-4, and IL-31, and these genes were closely related to skin erosion, nodule number and extent, epidermal keratinization and hyperplasia, inflammatory cell type, and degree of inflammation in NP. These results indicate that PAR2 mediates the Th2-dominated pathogenesis of NP through the RhoA-ERK signaling axis. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A: Differentially expressed genes identified by qRT-PCR in nodular prurigo; A: Significantly differentially expressed genes in the initial screening (7 control cases and 11 nodular prurigo cases), values ​​are mean ± standard deviation, * P <0.05,** P <0.01; B: Validation of differentially expressed genes (11 control cases and 27 cases of nodular prurigo), values ​​are mean ± standard deviation, ns indicates no statistical difference, * P <0.05,** P <0.01; C: Correlation analysis between differentially expressed genes. Numerical values ​​represent correlation coefficients (r), * P <0.05,** P <0.01, *** P <0.001; D: Correlation analysis with disease manifestations. Values ​​represent correlation coefficients (r), * P <0.05,** P <0.01, *** P <0.001; E: Correlation analysis between differentially expressed genes and disease symptoms, values ​​are correlation coefficients (r), * P <0.05,** P <0.01, *** P <0.001; Figure 2 Differential protein expression between the PN and Ctrl groups; A: Western blot detection of differentially expressed protein levels; B: Protein correlation analysis using Western blot grayscale values. Numerical values ​​show the correlation coefficient (r), * P <0.05,** P <0.01, *** P <0.001; C: Western blot band intensity is presented in graphical form after quantification. Values ​​are mean ± standard deviation. P <0.01, *** P <0.001; D: Differentially expressed proteins after immunohistochemical staining are quantified and presented as a heatmap. Values ​​are mean ± standard deviation. *** P <0.001; E: Immunohistochemical staining for differentially expressed proteins, scale bar: 50 μm; Figure 3 For the colocalization and interaction of differentially expressed proteins; A: Immunofluorescence staining (IF) detection of the expression and colocalization of PAR2, RhoA, and IL-31, scale bar: 50 μm; B: Quantification of fluorescence intensity by immunofluorescence staining, presented in graphical form, with values ​​expressed as mean ± standard deviation. P <0.01, *** P <0.001; C: Protein-protein interactions (PPIs) of eight differentially expressed proteins analyzed using the STRING database; D: Interactions (PPIs) between differentially expressed proteins and other proteins; E: Figure 3 C and Figure 3 Legend for D. Detailed Implementation

[0019] This invention reveals for the first time the regulatory role of the PAR2-RhoA-ERK signaling axis in the Th2 response of neural tube defects (PN). The protease-activated receptor 2 (PAR2) regulates the Th2 response through the RhoA-ERK signaling axis, thereby influencing the pathogenesis of PN. The results suggest that PAR2 is a novel target for the diagnosis and treatment of PN, providing a theoretical basis for the development of drugs targeting PAR2 or its downstream signaling molecules, enriching the neuro-immune interaction network of PN pathogenesis, and laying the foundation for subsequent functional validation and drug screening.

[0020] This invention demonstrates that Th2-type cytokines IL-4 and IL-31 are significantly upregulated in PN. IL-4 and IL-31 levels are negatively correlated with age and sex, but positively correlated with skin erosion, keratosis, epidermal and dermal hyperplasia, nodule number, inflammatory cell infiltration, and disease course. Furthermore, the upregulated IL-4 and IL-31 are strongly positively correlated with differentially expressed genes PAR2, STAT3, Gα12, RhoA, and ERK.

[0021] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0022] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0023] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0024] Example 1 Research on PN patients in routine clinical practice.

[0025] The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical University (KT2025-216-01), and all participants obtained written informed consent before participating in the study. Patients diagnosed with PN between October 2023 and December 2025 were included. Eligible patients were ≥18 years of age, and their PN diagnosis was confirmed by an experienced dermatopathologist as either newly diagnosed or previously diagnosed. The diagnosis was based on clinical examination and histological confirmation, excluding patients with systemic diseases such as anemia, chronic kidney disease, liver disease, thyroid disease, malignant tumors, and neuropsychiatric disorders.

[0026] Other exclusion criteria included: use of any systemic glucocorticoids, immunosuppressants, biologics, or phototherapy within the three months prior to the study participation. The healthy control group consisted of healthy adults who underwent reconstructive or excisional plastic surgery at the Affiliated Hospital of Guangdong Medical University, with no history of pruritus or comorbidities such as malignant tumors. Factors considered in the clinical data assessment included age, sex, biopsy site, erosion status, estimated number of lesions, nodule status, epidermal keratinization, epidermal hyperplasia, inflammatory cell infiltration, collagen hyperplasia, and disease duration. The clinical and pathological characteristics of the study participants are shown in Table 1.

[0027] Table 1 Clinical and pathological characteristics of study participants

[0028] 1.1 Skin biopsy Four 4 mm biopsy samples were obtained from 27 patients with nodular prurigo (PN) and 11 healthy volunteers (HV). Biopsies were performed on the most representative lesion sites. The samples were used for immunohistochemical analysis, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence assay.

[0029] 1.2 Immunohistochemistry Paraffin-embedded skin biopsy sections were dewaxed, hydrated, and then heat-retarded with sodium citrate for 5 minutes. The samples were blocked with an inhibitor for 30 minutes, and then incubated overnight at 4°C with IL-4 antibody (Santa Cruz, sc-53084, 1:50), IL-31 antibody (ThermoFisher, PA5-47405, 1:100), PAR2 antibody (Abcam, ab180953, 1:100), and RhoA antibody (Santa Cruz, sc-418, 1:50). The following day, the sections were incubated with biotinylated secondary antibody and developed with DAB solution (Beyotime, P0202). Subsequently, the sections were counterstained with hematoxylin and images were taken under a microscope. Semi-quantitative analysis of IL-4, IL-31, PAR2, and RhoA expression was performed by calculating the average optical density of the positive stain.

[0030] 1.3 Real-time quantitative PCR (qRT-PCR) Transcriptional profiles of PN skin were determined using lesion biopsy tissue from PN patients and control samples from healthy volunteers (CTRL). Total RNA was extracted from biopsy tissues using TRIzol reagent (Invitrogen), and then reverse transcribed into cDNA using a reverse transcription kit containing M-MLV reverse transcriptase (Aikerui Biotechnology, AG11728). qRT-PCR was performed using the SYBR Green Pro Taq HS premixed qPCR kit (Aikerui Biotechnology, AG11718).

[0031] Initial screening used 7 CTRL patients and 11 NP patients to detect the expression profiles of 32 genes associated with inflammatory response and pruritus. These 32 genes were selected for their pathogenic role in atopic dermatitis (AD), an inflammatory skin disease with Th2 dominance characteristics similar to PN, and which has been more thoroughly studied. Validation was performed using 11 CTRL patients and 27 NP patients to detect 10 differentially expressed genes identified in the initial screening. GAPDH was used as an endogenous control gene, and a 2... -ΔΔCT Methods were used to analyze gene mRNA differences, and GraphPadPrism 9.0 was used for statistical analysis and differential heatmaps. Pearson correlation coefficient analysis was used to analyze correlations and plot the results.

[0032] 1.4 Immunoblotting Tissue protein components were extracted on ice using a protein extraction kit from Shanghai Beyotime Biotechnology Co., Ltd., and the supernatant was collected after centrifugation at 12000 g for 10 minutes.

[0033] Protein concentration was determined using the ThermoFisher 23225 BCA protein quantification kit. Protein lysis buffer was separated by 10% SDS-PAGE electrophoresis and then transferred to a ThermoFisher 88520 PVDF membrane. The membrane was blocked with 5% skim milk powder and incubated with primary antibody overnight at 4°C.

[0034] The primary antibody dilution ratios were as follows: PAR2 antibody (Abcam, ab180953) 1:1000; p-STAT3 antibody (CST, 9145S) 1:2000; STAT3 antibody (CST, 30835S) 1:1000; RhoA antibody (Santa Cruz, sc-418) 1:500; p-AKT antibody (CST, 4060S) 1:2000; AKT antibody (CST, 4691S) 1:1000; p-ERK antibody (Santa Cruz, sc-7383) 1:1000; ERK1 / 2 antibody (CST, 4695S) 1:1000; IL-4 antibody (Santa Cruz, sc-7383) 1:1000; p-AKT ...060S) 1:2000; AKT antibody (CST, 4695S) 1:1000; IL-4 antibody (Santa Cruz, sc-7383) 1:1000; p-AKT antibody (CST, sc-7383) 1:1000; IL-4 antibody (CST, sc-7383) 1:1000; p-AKT antibody (CST, sc-7383) 1:1000; p-AKT antibody (CST, sc-7383) 1:1000; p-AKT antibody (CST, sc-7383) 1:1000; p-AKT antibody (CST, sc-7383) 1:1000; Cruz (sc-53084) 1:500; IL-31 antibody (ThermoFisher, PA5-47405) 1:1000; GAPDH internal control antibody (Proteintech, 10494) 1:2000.

[0035] After incubation with Proteintech's HRP-labeled secondary antibodies (SA00001-1, SA00001-2, SA00001-4), the membrane bands were detected using a Millipore ECL chemiluminescence assay kit. Protein expression levels were quantified using ImageJ software, and all experiments were independently repeated three times.

[0036] 1.5 Statistical Analysis GraphPad Prism 9 software was used to analyze experimental data. Values ​​are expressed as mean ± standard deviation. For comparisons of differences between groups of continuous data, when the data conform to normality and homogeneity of variance, the independent samples t-test was used for comparisons between two groups; analysis of variance (ANOVA) was used for comparisons between multiple groups; when the data do not conform to normality and homogeneity of variance, statistical analysis methods such as the rank-sum test can be used. The Pearson correlation coefficient was used to assess the correlation between clinical symptoms and pathological diagnoses and mRNA levels, and Pearson's mean clustering was used for visualization in a heatmap. The criteria for statistical significance were... P <0.05.

[0037] 1.6 Results 1.6.1 PAR2-related pathway genes are associated with the Th2 response in nodular prurigo. Thirty-two PAR2 and Th2-related genes were screened in 7 CTRL and 11 NP samples using qRT-PCR. Statistical analysis identified 10 significantly differentially expressed genes, such as... Figure 1 As shown in (A). Further validation of the 10 selected genes was performed using 11 CTRL cases and 27 NP cases, identifying 8 significantly differentially expressed genes associated with NP pathogenesis: PAR2, STAT3, Gα12, RhoA, AKT1, ERK1, IL-4, and IL-31. Figure 1 (B) Some of the qRT-PCR primer sequences used are shown in Table 2.

[0038] Table 2. Partial primer sequences for qRT-PCR

[0039] Correlation analysis revealed a strong positive correlation between PAR2 and STAT3, Gα12, RhoA, ERK1, IL-4, and IL-31. Figure 1 (CE) and was closely related to skin erosion, number and extent of nodules, epidermal keratinization and hyperplasia, inflammatory cell type and degree of inflammation in PN. Furthermore, the inflammatory cell type and degree of inflammation in PN were positively correlated with skin erosion, number and extent of nodules, epidermal keratinization and hyperplasia, and the onset time of PN, and were also positively correlated with the eight differentially expressed genes screened. These results suggest that PAR2-related pathway genes are associated with driving the Th2-type response in nodular prurigo.

[0040] 1.6.2 Expression of PAR2-related pathway proteins and Th2 inflammatory cytokines in nodular prurigo Western blot and intracranial immunoglobulin (IHC) were used to detect PAR2-related pathway proteins and Th2-type inflammatory factors in 6 CTRL and 6 PN specimens. Results showed that PAR2, RhoA, p-ERK, IL-4, and IL-31 were significantly upregulated in PN. Figure 2 (A and C), correlation analysis showed that PAR2-RhoA-ERK1 / 2 was strongly positively correlated with IL-4 and IL-31, such as... Figure 2 (B). IHC confirmed that PAR2, RhoA, IL-4, and IL-31 were significantly upregulated in PN and their expression locations were altered, such as... Figure 2 (DE).

[0041] 1.6.3 PAR2 / RhoA / IL-31 interaction regulates the pathogenesis of nodular prurigo Further investigation using immunofluorescence techniques to examine the expression and co-localization of PAR2, RhoA, and IL-31 revealed that all three were upregulated and their co-localization was enhanced in PN. Figure 3(AB). A differentially expressed protein-protein interaction network was constructed using the STING (Version: 12.0) database. The results showed that eight differentially expressed proteins may have direct or indirect interactions, such as... Figure 3 (CE).

[0042] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A biomarker for nodular prurigo, characterized in that, The biomarker is PAR2.

2. The biomarker according to claim 1, characterized in that, The PAR2 modulates the Th2-dominated response pathogenesis of nodular prurigo by mediating the RhoA-ERK signaling axis.

3. The biomarker according to claim 2, characterized in that, The expression level of PAR2 is strongly positively correlated with the RhoA-ERK signal axis.

4. A kit for detecting nodular prurigo, characterized in that, The kit includes reagents for detecting the biomarker of claim 1.

5. A pharmaceutical composition for treating and / or preventing nodular prurigo, characterized in that, The pharmaceutical composition includes an effective amount of PAR2 inhibitor.

6. A method for diagnosing nodular prurigo, characterized in that, Includes the following steps: S1. Extract RNA or protein from the skin tissue of the test subject; S1. Detect the expression level of PAR2 molecules; If the PAR2 expression level of the subjects is significantly higher than that of healthy controls, it suggests that the subjects have PN or are at risk of PN.

7. The application of the biomarker of claim 1 in the prognostic assessment of nodular prurigo.

8. The application of biomarkers in the preparation of reagents for detecting nodular prurigo, characterized in that, The biomarker is PAR2.

9. The application according to claim 8, characterized in that, The nodular prurigo detection reagent is selected from probes that specifically recognize PAR2; Or primers that specifically amplify PAR2; Or antibodies or ligands that specifically bind to the protein encoded by PAR2.

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