Itch-related signaling pathway analysis method

Abstract

This invention relates to the field of biomedical technology and discloses a method for analyzing pruritus-related signaling pathways, comprising the following steps: S1, pruritus phenotype anchoring and standardized sample preparation stage; S2, multidimensional omics detection and data preprocessing stage; S3, pruritus-related differential molecular screening and functional screening stage; S4, core signaling pathway anchoring stage; S5, pathway regulatory network and crosstalk analysis stage; S6, core pathway multidimensional experimental verification stage; S7, pathway specificity and universality final verification stage. This invention effectively solves the core pain points of traditional pruritus-related signaling pathway analysis, such as inaccurate phenotypic quantification, strong one-sidedness of single-omics analysis, high false positive rate, and disconnect between phenotypic and molecular analysis. Through multidimensional omics joint analysis, it achieves a panoramic capture of molecular changes during the occurrence of pruritus, and combined with a pruritus-specific analysis system, it significantly improves the targeting and accuracy of pathway screening.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for analyzing pruritus-related signaling pathways. Background Technology

[0002] Itching is one of the most common somatic sensory abnormalities in the body. It is a core clinical manifestation of inflammatory skin diseases such as atopic dermatitis, psoriasis, and chronic urticaria, and is also commonly secondary to various systemic diseases such as diabetic peripheral neuropathy, cholestatic liver disease, chronic kidney disease, and tumors. Among them, chronic pruritus with a course of more than 6 weeks is characterized by recurrent attacks and difficulty in curing. Current clinical treatments are mostly non-specific symptomatic treatments such as antihistamines and glucocorticoids, which have limited efficacy, significant side effects, and easy drug resistance with long-term use. There is still no specific radical cure for chronic pruritus, which seriously damages the physical and mental health and quality of life of patients and has become a clinical problem that urgently needs to be solved worldwide.

[0003] The occurrence and development of chronic pruritus is a complex process involving multiple links and multiple cells working together, including peripheral skin effector cells, dorsal root ganglion sensory neurons, spinal cord conduction pathways and central regulatory networks. Its pathological process is accompanied by molecular abnormalities at multiple levels, such as gene transcription, protein expression, post-translational modification and metabolic regulation. The precise analysis of pruritus-related signaling pathways is the core foundation and necessary prerequisite for elucidating the pathogenesis of chronic pruritus, identifying specific therapeutic targets and developing new anti-pruritus drugs.

[0004] Currently, research and analysis of pruritus-related signaling pathways mainly involve two mainstream technical approaches: one is the targeted validation model, which, based on existing literature reports and research foundations, pre-determines candidate signaling pathways and verifies their regulatory role in pruritus occurrence through cell or animal experiments. This model is limited by the cognitive boundaries of existing research, making it impossible to achieve unbiased screening and discovery of novel pruritus regulatory pathways, and it is also difficult to clarify the location and crosstalk relationships of target pathways in the overall pruritus regulatory network. The other is the bioinformatics screening model, which screens differentially expressed genes through single transcriptome sequencing and combines it with general functional databases to complete pathway enrichment analysis. This model can only capture changes at the molecular transcriptional level and cannot reflect the core links of signaling pathway activation. Furthermore, it lacks a standardized phenotypic anchoring and full-chain validation system, resulting in defects such as high false positive rates, weak correlation with pruritus phenotypes, and poor reproducibility of research results, failing to meet the practical application needs of chronic pruritus mechanism research and targeted drug development. Summary of the Invention

[0005] The purpose of this invention is to provide a method for analyzing pruritus-related signaling pathways to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The method for analyzing pruritus-related signaling pathways includes the following steps:

[0008] S1. In the stage of anchoring pruritus phenotype and preparing standardized samples, a multi-group pruritus research system was constructed, the quantitative collection and verification of pruritus phenotypes were completed, and biological samples for multi-dimensional analysis were prepared and quality-controlled.

[0009] S2, Multi-dimensional omics detection and data preprocessing stage: complete multi-omics high-throughput detection of homologous samples, perform quality control filtering and standardization correction on the raw offline data, and output expression matrix in a unified format;

[0010] S3, the screening and initial functional screening of differentially expressed molecules related to pruritus: the screening of differentially expressed molecules between groups is completed based on the standardized expression matrix, and functional annotation and preliminary pathway enrichment are performed on the differentially expressed molecules to narrow the scope of target analysis.

[0011] S4, Core signaling pathway anchoring stage: Based on the pruritus-specific reference gene set, target pathway enrichment is completed, and combined with phenotypic association analysis and kinase substrate cascade verification, the core signaling pathways related to pruritus are identified.

[0012] S5, Pathway Regulation Network and Crosstalk Analysis Stage: Analyze the upstream and downstream regulatory relationships of the core pathway, identify the interaction nodes between the core pathway and the classic pruritus pathway, and construct a complete regulatory network map.

[0013] S6. Multi-dimensional experimental verification stage of core pathways: complete the functional verification and data collection of core pathways from four dimensions: molecular expression, cell function, in vivo phenotype, and clinical relevance.

[0014] S7. Final stage of pathway specificity and universality verification: Pathway function verification is completed in a multi-subtype pruritus model, the sensory regulation specificity of the pathway is detected, and the applicability and evaluation results of the pathway are output.

[0015] As a further improvement to this technical solution: In the pruritus phenotype anchoring and standardized sample preparation stage, a normal control group and multiple pruritus model groups are set up to complete the model construction of each group of experimental animals. Pruritus behavioral data of each group of experimental animals are collected, including the number of effective scratches per unit time, the average duration of a single scratch, the frequency of scratching attacks, and the percentage of scratches on the target area. The standardized pruritus phenotype score (SIS) of each group of samples is calculated using the standardized pruritus phenotype scoring formula to complete the quantitative verification of the pruritus phenotype. Target tissue samples are collected from each group of experimental animals, and total RNA, total protein, phosphorylated protein, and metabolites are extracted from the samples. The concentration, purity, and integrity of the extracted biomolecules are tested. Unqualified samples are discarded, and qualified samples are retained for subsequent testing. The standardized pruritus phenotype scoring formula is: Explanation of formula symbols: The number of times the target animal effectively scratches per unit of time; The maximum number of effective scratches by all animals in the same batch of experiments; : Average duration of a single scratch by the target animal; : The maximum average duration of scratching for all animals in the same batch of experiments; : The frequency of scratching attacks in the target animal per unit of time; : The maximum frequency of scratching attacks among all animals in the same batch of experiments; The percentage of scratches on the target area of ​​the target animal out of the total number of scratches; The percentage of animals scratching the largest target area in the same batch of experiments.

[0016] As a further improvement to this technical solution: in the multi-dimensional omics detection and data preprocessing stage, transcriptome sequencing, quantitative proteome detection, phosphorylated proteome detection, and targeted metabolome detection are performed on qualified homologous samples to obtain raw data from various omics. Low-quality data filtering, low-abundance molecule removal, and outlier sample screening are performed on the raw data from various omics to complete basic quality control. Median centering correction is performed on the various omics data that have completed basic quality control to complete data standardization and output the multi-omics standardized expression matrix of homologous samples.

[0017] As a further improvement to this technical solution: In the screening and initial functional screening stage of pruritus-related differentially expressed molecules, the difference analysis between the pruritus model group and the control group is completed based on the multi-omics standardized expression matrix to obtain the fold change and corrected p-value for each molecule. The reported datasets of pruritus-related molecules are integrated, and a corresponding pruritus functional weight coefficient is matched for each molecule. The pruritus association score (SDS) for each molecule is calculated using the differential molecular pruritus association scoring formula. High-confidence differentially expressed molecules are screened according to a preset threshold. GO functional annotation and KEGG pathway enrichment analysis are performed on the screened high-confidence differentially expressed molecules, and pathways unrelated to pruritus biological function are removed, retaining pathways and differentially expressed molecules related to pruritus biological function to narrow the scope of the target analysis. The differential molecular pruritus association scoring formula is as follows: Explanation of formula symbols: The fold difference in molecular weight between the pruritus model group and the control group; : Corrected P-value for differences between molecular groups; W: Weighting coefficient of pruritus function of molecules, ranging from 1.0 to 3.0.

[0018] As a further improvement to this technical solution: In the screening and initial functional screening stage of pruritus-related differentially expressed molecules, the difference analysis between the pruritus model group and the control group is completed based on the multi-omics standardized expression matrix to obtain the fold change and corrected p-value for each molecule. The reported datasets of pruritus-related molecules are integrated, and a corresponding pruritus functional weight coefficient is matched for each molecule. The pruritus association score (SDS) for each molecule is calculated using the differential molecular pruritus association scoring formula. High-confidence differentially expressed molecules are screened according to a preset threshold. GO functional annotation and KEGG pathway enrichment analysis are performed on the screened high-confidence differentially expressed molecules, and pathways unrelated to pruritus biological function are removed, retaining pathways and differentially expressed molecules related to pruritus biological function to narrow the scope of the target analysis. The differential molecular pruritus association scoring formula is as follows: Explanation of formula symbols: The fold difference in molecular weight between the pruritus model group and the control group; : Corrected P-value for differences between molecular groups; W: Weighting coefficient of pruritus function of molecules, ranging from 1.0 to 3.0.

[0019] As a further improvement to this technical solution: In the pathway regulation network and crosstalk analysis stage, the transcription factor binding relationship of the core molecules of the locked core signaling pathway is predicted and verified, the non-coding RNA targeted regulation relationship of the core molecules of the pathway is predicted and verified, the upstream and downstream regulatory network of the core pathway is constructed, the core regulatory nodes of the pathway are identified, a protein interaction network is constructed, the interaction nodes between the core pathway and the classic pruritus pathway are identified, the crosstalk relationship analysis between the core pathway and the classic pruritus pathway is completed, and a complete regulatory map of the core pathway is drawn.

[0020] As a further improvement to this technical solution: in the multi-dimensional experimental verification stage of the core pathway, the expression level of key molecules in the core pathway is verified by nucleic acid quantitative detection and protein immunoassay; the cellular functional verification of the core pathway is verified by primary cell culture and gene or drug intervention; the in vivo phenotypic verification of the core pathway is verified by gene intervention or drug administration in animal models; the correlation verification between the expression of core pathway molecules and the clinical pruritus phenotype is verified by clinical sample detection; and the full dataset for core pathway functional verification is collected and archived.

[0021] As a further improvement to this technical solution: in the final stage of the pathway specificity and universality verification, histamine-dependent and non-histamine-dependent acute pruritus models and various chronic pruritus models are constructed. Intervention experiments on the core pathway are performed in various pruritus models to complete the functional verification of the pathway in different pruritus subtypes. The mechanical pain threshold, thermal pain threshold, and cold pain threshold of experimental animals after core pathway intervention are detected to complete the sensory regulation specificity detection of the pathway. The universality and specificity of the pruritus regulation of the core pathway are quantitatively evaluated, and the applicable scope and final evaluation results of the pathway are output.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. This invention effectively addresses the core pain points of traditional pruritus-related signaling pathway analysis, such as inaccurate phenotypic quantification, strong bias in single-omics analysis, high false positive rate, and disconnect between phenotypic and molecular analysis. Through multi-dimensional omics joint analysis, it achieves a panoramic capture of molecular changes during the occurrence of pruritus. Combined with a pruritus-specific analysis system, it significantly improves the targeting and accuracy of pathway screening, effectively eliminates interference information unrelated to the biological function of pruritus, and fully covers the entire chain of signaling pathway analysis needs from unbiased screening and mechanism analysis to functional verification. It provides a standardized and systematic execution framework for the study of pruritus-related signaling pathways.

[0024] 2. This invention addresses the dual application needs of discovering novel pathways and deeply analyzing known pathways. It can achieve unbiased screening of novel regulatory pathways for various chronic pruritus diseases, as well as refined functional verification and mechanism analysis of specific pathways. Furthermore, through systematic verification of multi-subtype pruritus models and detection of sensory modulation specificity, it can clarify the applicability and clinical translation potential of target pathways, providing reliable technical support for the screening of therapeutic targets and the development of targeted drugs for pruritus diseases. The standardized process design also effectively reduces the result bias between different experimental systems and different research batches, improving the reproducibility and universality of research results. It can be widely adapted to basic research and preclinical translational research scenarios for various pruritus-related diseases.

[0025] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0026] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0027] Figure 1 This is a schematic diagram of the method structure for analyzing pruritus-related signaling pathways. Detailed Implementation

[0028] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. The invention is described more specifically in the following paragraphs by way of example with reference to the accompanying drawings. It should be noted that the drawings are in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of the present invention.

[0029] Please see Figure 1 In this embodiment of the invention, the method for analyzing pruritus-related signaling pathways includes the following steps: S1, the pruritus phenotype anchoring and standardized sample preparation stage, constructing a multi-group pruritus research system, completing the quantitative collection and verification of pruritus phenotypes, and preparing and quality-controlling biological samples for multi-dimensional analysis.

[0030] S2, Multi-dimensional omics detection and data preprocessing stage: complete multi-omics high-throughput detection of homologous samples, perform quality control filtering and standardization correction on the raw offline data, and output expression matrix in a unified format;

[0031] S3, the screening and initial functional screening of differentially expressed molecules related to pruritus: the screening of differentially expressed molecules between groups is completed based on the standardized expression matrix, and functional annotation and preliminary pathway enrichment are performed on the differentially expressed molecules to narrow the scope of target analysis.

[0032] S4, Core signaling pathway anchoring stage: Based on the pruritus-specific reference gene set, target pathway enrichment is completed, and combined with phenotypic association analysis and kinase substrate cascade verification, the core signaling pathways related to pruritus are identified.

[0033] S5, Pathway Regulation Network and Crosstalk Analysis Stage: Analyze the upstream and downstream regulatory relationships of the core pathway, identify the interaction nodes between the core pathway and the classic pruritus pathway, and construct a complete regulatory network map.

[0034] S6. Multi-dimensional experimental verification stage of core pathways: complete the functional verification and data collection of core pathways from four dimensions: molecular expression, cell function, in vivo phenotype, and clinical relevance.

[0035] S7. Final stage of pathway specificity and universality verification: Validate pathway function in a multi-subtype pruritus model, detect the sensory modulation specificity of the pathway, and output the scope of application and evaluation results of the pathway.

[0036] Specifically, the pruritus phenotype anchoring and standardized sample preparation module provides a standardized research system and qualified analytical samples for this method. It is the foundation of the entire analytical process and ensures that the samples and phenotypic data in subsequent analyses are consistent and reliable.

[0037] Multidimensional omics detection and data preprocessing module: realizes panoramic acquisition of multidimensional molecular information of pruritus-related samples, eliminates systematic errors and batch effects in the data, and provides a high-quality standardized data foundation for subsequent analysis;

[0038] The pruritus-related differential molecular screening and functional screening module: screens out high-confidence differential molecules related to pruritus from massive molecular data, eliminates irrelevant data, narrows the target range of subsequent pathway analysis, and improves analysis efficiency and targeting.

[0039] Core signaling pathway anchoring module: This is the core central module of this method. It accurately identifies core signaling pathways that are strongly associated with the pruritus phenotype and have complete activation cascades from the enriched pathways, realizing the transformation from massive data to core research objectives;

[0040] Pathway regulation network and crosstalk analysis module: Clarify the upstream and downstream regulatory relationships of core pathways and their location in the pruritus regulation network, providing a complete regulatory logic for subsequent mechanism research and target development;

[0041] Multi-dimensional experimental verification module for core pathways: Verify the reliability of bioinformatics analysis results through multi-dimensional experiments, confirm the regulatory role of core pathways in the occurrence of pruritus, and eliminate false positive results of bioinformatics analysis;

[0042] The pathway-specificity and universality final verification module clarifies the applicable pruritus subtypes and regulatory specificity of the core pathway, completes the closed loop of the entire analysis process, and provides a clear scope of application and safety basis for subsequent clinical translation.

[0043] In the pruritus phenotype anchoring and standardized sample preparation stage, a normal control group and multiple pruritus model groups were set up to complete the model construction of each group of experimental animals. Pruritus behavioral data of each group of experimental animals were collected, including the number of effective scratches per unit time, the average duration of a single scratch, the frequency of scratching attacks, and the percentage of scratches on the target site. The standardized pruritus phenotype score (SIS) for each group of samples was calculated using the standardized pruritus phenotype scoring formula to complete the quantitative verification of the pruritus phenotype. Target tissue samples were collected from each group of experimental animals, and total RNA, total protein, phosphorylated protein, and metabolites were extracted from the samples. The concentration, purity, and integrity of the extracted biomolecules were tested. Unqualified samples were discarded, and qualified samples were retained for subsequent testing. The standardized pruritus phenotype scoring formula is: Explanation of formula symbols: The number of times the target animal effectively scratches per unit of time; The maximum number of effective scratches by all animals in the same batch of experiments; : Average duration of a single scratch by the target animal; : The maximum average duration of scratching for all animals in the same batch of experiments; : The frequency of scratching attacks in the target animal per unit of time; : The maximum frequency of scratching attacks among all animals in the same batch of experiments; The percentage of scratches on the target area of ​​the target animal out of the total number of scratches; The percentage of animals scratching at the largest target site in the same batch of experiments;

[0044] Specifically, the small animal behavior recording and analysis device, equipped with a high-definition infrared camera unit and a dark box-type behavior observation box, is used to collect video data of pruritus behavior in experimental animals and complete the basic identification and data statistics of scratching actions; the pathological tissue processing device, including a paraffin sectioner, a cryostat, and a fully automated staining instrument, is used to verify the pathological characteristics of the pruritus model and ensure the successful construction of the model; the biological sample extraction and quality control device, including a high-speed refrigerated centrifuge, a fully automated nucleic acid extractor, an ultra-micro spectrophotometer, a biological analyzer, and a protein quantification instrument, is used for the extraction of biomolecules from target tissues and the quality control detection of concentration, purity, and integrity.

[0045] The formula normalizes multidimensional pruritus behavioral indicators into standardized scores in the 0-1 range, eliminates systematic errors between different batches of experiments and different pruritus models, and provides unbiased and comparable phenotypic input data for subsequent phenotypic-molecular association analysis. It is the core foundation of phenotypic quantification in this method.

[0046] The detailed annotations of the formulas are as follows: Explanation of formula symbols: : The number of effective scratches by the target animal per unit time, referring to the count of compliant scratching actions after excluding non-itchy related actions; The maximum number of effective scratches by all animals in the same batch of experiments, used to normalize the scratch count index; The average duration of a single scratch by the target animal reflects the intensity of the itching behavior. The maximum average scratching duration of all animals in the same batch of experiments was used to normalize the duration index. The frequency of scratching episodes in the target animal per unit of time, with continuous scratching actions counted as one episode, reflects the frequency of itching episodes. The maximum scratching frequency of all animals in the same batch of experiments was used to normalize the frequency index. The percentage of scratches on the target area of ​​the target animal out of the total number of scratches reflects the targeting of the scratching action and excludes interference from non-specific whole-body movements. The percentage of animals scratching the maximum target area in the same batch of experiments is used to normalize the targeting indicators. The weight coefficients 0.4, 0.3, 0.2, and 0.1 in the formula are determined based on the correlation analysis between the clinical pruritus VAS score and animal behavioral indicators. The number of scratches is the core weight item, matching the core evaluation criteria for the severity of clinical pruritus.

[0047] A normal control group and multiple pruritus model groups were set up to construct a control analysis system and ensure the reliability of subsequent differential analysis. Total RNA, total protein, phosphorylated protein, and metabolites were extracted to provide homologous analytical substrates for subsequent multi-omics detection. Sample quality control steps were used to remove unqualified samples to avoid low-quality samples affecting the accuracy of subsequent detection and analysis results.

[0048] In the multi-dimensional omics detection and data preprocessing stage, transcriptome sequencing, quantitative proteome detection, phosphorylated proteome detection, and targeted metabolome detection are performed on qualified homologous samples to obtain raw data from various omics. Low-quality data filtering, low-abundance molecule removal, and outlier sample screening are performed on the raw data from various omics to complete basic quality control. Median centering correction is performed on the various omics data that have completed basic quality control to complete data standardization and output the multi-omics standardized expression matrix of homologous samples.

[0049] Specifically, the high-throughput sequencing device includes a second-generation high-throughput sequencer and a fully automated library preparation system, used to construct sequencing libraries and perform high-throughput sequencing of the transcriptome of samples, obtaining whole transcriptome expression profile data; the quantitative proteomics detection device includes a high-resolution liquid chromatography-tandem mass spectrometer and a nano-liquid chromatography system, used to perform TMT-labeled quantitative proteomics and label-free phosphorylated proteomics detection, obtaining whole protein expression profiles and phosphorylation modification profile data; the targeted metabolomics detection device includes a triple quadrupole liquid chromatography-mass spectrometry system, used to perform targeted quantitative detection of core mediators of pruritus such as histamine, serotonin, and neuropeptides, obtaining pruritus-related metabolite expression profile data; and the bioinformatics high-performance computing and analysis platform, equipped with a Linux operating system and an R language analysis environment, is used to perform quality control filtering and standardization correction of omics data.

[0050] The study employs four omics testing methods—transcriptome sequencing, quantitative proteomics, phosphorylated proteomics, and targeted metabolomics—performing tests on the same homologous sample to ensure the relevance and accuracy of subsequent multi-omics joint analyses. Basic quality control steps, including low-quality data filtering, low-abundance molecule removal, and outlier sample screening, are used to eliminate noise and interference data in the original data, improving data reliability. Median centering correction is used to eliminate systematic errors and batch effects in the omics data, achieving dimensional uniformity across different samples and omics data, and providing a standardized expression matrix for subsequent differential analysis.

[0051] In the screening and initial functional screening stage of pruritus-related differentially expressed molecules, the difference between the pruritus model group and the control group was analyzed based on the multi-omics standardized expression matrix to obtain the fold change and corrected p-value for each molecule. Reported pruritus-related molecule datasets were integrated, and a corresponding pruritus function weight coefficient was matched for each molecule. The pruritus association score (SDS) of each molecule was calculated using the differential molecular pruritus association scoring formula. High-confidence differentially expressed molecules were screened according to a preset threshold. GO functional annotation and KEGG pathway enrichment analysis were performed on the screened high-confidence differentially expressed molecules, and pathways irrelevant to pruritus biological function were removed, retaining pathways and differentially expressed molecules related to pruritus biological function to narrow the scope of the target analysis. The differential molecular pruritus association scoring formula is as follows: Explanation of formula symbols: The fold difference in molecular weight between the pruritus model group and the control group; : Corrected p-value for differences between molecular groups; W: Weighting coefficient of pruritus function of molecules, ranging from 1.0 to 3.0;

[0052] Specifically, the high-performance computing and analysis platform for bioinformatics, along with analysis packages such as DESeq2, limma, and clusterProfiler, is used to complete intergroup difference analysis, functional annotation, and pathway enrichment analysis.

[0053] The formula integrates three dimensions: the magnitude of molecular expression changes, statistical significance, and pruritus function weight. It quantifies the degree of pruritus association for each molecule, effectively eliminating false positive molecules that only have statistical differences but are not related to pruritus function. This improves the association between the differential molecule set and the pruritus phenotype, providing a highly reliable molecule set for subsequent pathway enrichment analysis.

[0054] The detailed annotations of the formulas are as follows: Explanation of formula symbols: The fold difference in molecular expression between the pruritus model group and the control group reflects the magnitude of changes in molecular expression under pruritus conditions. : Corrected P-value for differences between molecular groups, i.e., false discovery rate, reflects the statistical significance of differences in molecular expression. The smaller the FDR value, the higher the statistical reliability of the difference; W: The pruritus function weighting coefficient of the molecule, ranging from 1.0 to 3.0, where 3.0 corresponds to core molecules that have been clearly reported to participate in pruritus regulation, 2.0 corresponds to molecules that have been reported to participate in pruritus-related biological processes such as pain, inflammation, and sensory transmission, and 1.0 corresponds to molecules that have no clear reports of pruritus-related functions;

[0055] Intergroup differential analysis was used to obtain the magnitude and statistical significance of expression changes for each molecule; GO functional annotation and KEGG pathway enrichment analysis were used to classify differentially expressed molecules by function, preliminarily screen pathways related to pruritus biological functions, eliminate irrelevant pathways, and narrow down the target scope for subsequent analysis.

[0056] In the core signaling pathway anchoring stage, a reference gene set for pruritus was constructed by integrating previously reported pruritus-related functional genes. GSEA gene enrichment analysis was performed based on the standardized expression matrix and the reference gene set to screen for significantly enriched pathways related to pruritus. A weighted gene co-expression network was constructed based on the standardized expression matrix, and the core gene modules related to the pruritus phenotype were screened. Core genes within the core gene modules were extracted, and intersection screening was performed with molecules within the significantly enriched pathways to narrow down the target pathway range. Kinase enrichment analysis was performed based on phosphorylated proteome data to verify the activation status of the kinase substrate cascade of the target pathway, thus completing the identification of the core signaling pathways related to pruritus.

[0057] Specifically, the high-performance computing and analysis platform for bioinformatics, equipped with analysis software such as GSEA, WGCNA, and Cytoscape, and databases such as STRING and PhosphoSitePlus, is used to complete pathway enrichment, co-expression network construction, and kinase enrichment analysis.

[0058] This study constructs a pruritus-specific reference gene set, integrating reported pruritus-related functional genes to cover the entire biological process of pruritus development. This replaces the universal KEGG reference gene set, enabling targeted enrichment of pathways and avoiding interference from numerous irrelevant pathways, thus improving the targeting and accuracy of enrichment analysis. GSEA gene set enrichment analysis, based on the whole-genome expression matrix, performs unbiased enrichment analysis without pre-setting differential molecule thresholds. It can identify pathways with changing overall expression trends but insignificant single-molecule differences, overcoming the shortcomings of traditional differential enrichment analysis and comprehensively screening for significantly enriched pruritus-related pathways. Weighted gene co-expression network construction and core module screening cluster genes with similar expression patterns into modules, analyzing the relationship between modules and pruritus... The correlation between pruritus phenotype and core gene modules strongly associated with pruritus phenotype was screened to identify core genes directly related to pruritus phenotype, thus achieving a strong correlation between molecules and phenotype. Intersection screening involved intersecting the phenotype-associated core genes with molecules from enriched pathways to further narrow down the target pathway range, ensuring that the identified pathways were directly related to the pruritus phenotype. Kinase enrichment analysis and kinase substrate cascade validation were performed based on phosphorylated proteome data to analyze core kinases with significant activity changes in the pruritus model, verifying the activation status of the phosphorylation cascade of the target pathway. This confirmed the actual activation status of the pathway at the protein modification level, eliminating false positive results of pathway enrichment without actual functional activation, ultimately achieving precise identification of the core signaling pathway.

[0059] In the pathway regulation network and crosstalk analysis stage, the transcription factor binding relationship of the core molecules of the identified core signaling pathway was predicted and verified, the non-coding RNA targeted regulation relationship of the core molecules of the pathway was predicted and verified, the upstream and downstream regulatory network of the core pathway was constructed, the core regulatory nodes of the pathway were identified, a protein interaction network was constructed, the interaction nodes between the core pathway and the classical pruritus pathway were identified, the crosstalk relationship between the core pathway and the classical pruritus pathway was analyzed, and a complete regulatory map of the core pathway was drawn.

[0060] Specifically, the bioinformatics analysis platform, equipped with JASPAR, TRRUST, TargetScan, miRanda, STRING databases and Cytoscape software, is used to complete the analysis and visualization of transcriptional regulation, non-coding RNA regulation, and protein interaction networks;

[0061] This study focuses on: predicting and validating transcription factor binding relationships to identify upstream transcriptional regulators of key molecules in the core pathway and elucidating the regulatory mechanisms at the transcriptional level; predicting and validating non-coding RNA (mRNA) targeted regulation relationships to clarify the targeted regulatory effects of non-coding RNAs such as miRNAs on core pathway molecules and supplementing the post-transcriptional regulatory mechanisms of the pathway; constructing upstream and downstream regulatory networks to integrate transcriptional regulation, non-coding RNA regulation, and protein cascade relationships, mapping the complete upstream and downstream regulatory networks of the core pathway, identifying the core regulatory nodes of the pathway, and providing clear targets for subsequent interventions; constructing protein interaction networks and identifying interaction nodes to identify common interacting proteins between the core pathway and reported classical pruritus pathways, i.e., crosstalk nodes, and clarifying the cross-regulatory relationships between pathways; and analyzing crosstalk relationships and mapping regulatory maps to clarify the location of the core pathway in the entire pruritus regulatory network, analyze its synergistic or antagonistic effects with classical pruritus pathways, and map a complete regulatory map, providing a complete logical framework for subsequent mechanistic studies.

[0062] In the multi-dimensional experimental verification phase of the core pathway, the expression level of key molecules in the core pathway is verified by nucleic acid quantification and protein immunoassay; the cellular functional verification of the core pathway is verified by primary cell culture and gene or drug intervention; the in vivo phenotypic verification of the core pathway is verified by gene intervention or drug administration in animal models; the correlation verification between the expression of core pathway molecules and the clinical pruritus phenotype is verified by clinical sample testing; and the full dataset for core pathway functional verification is collected and archived.

[0063] Specifically, the nucleic acid expression detection device includes a real-time quantitative PCR instrument and a digital PCR instrument, used to quantitatively detect the expression level of core pathway molecules' mRNA and verify transcriptome analysis results; the protein expression and localization detection device includes a Western blot system, a fully automated immunohistochemical staining instrument, and a laser confocal scanning microscope, used to detect the expression level of core pathway proteins and verify cell localization; the cell function detection device includes a primary cell culture system, a calcium imaging system, a whole-cell patch-clamp system, a flow cytometer, and an enzyme-linked immunosorbent assay (ELISA) reader, used to culture pruritus-related cells and detect cell functional phenotypes after pathway intervention; and the in vivo animal function verification device includes a small animal stereotaxic instrument, a microinjection pump, and a small animal in vivo imaging system, used to perform targeted drug delivery, viral intervention, and in vivo pathway activation level detection in animals.

[0064] Expression level validation involves verifying the mRNA and protein expression levels of core pathway molecules in independent animal models and clinical samples, confirming their consistency with bioinformatics analysis results, which is the foundation of pathway function validation. Cellular level function validation involves regulating pathway activity through gene or drug intervention in pruritus-related primary cell models, detecting pruritus-related functional phenotypes in cells, and clarifying the pathway's regulatory role and direction at the cellular level, which is the core step in pathway function validation. In vivo animal phenotype validation involves regulating pathway activity through gene or drug administration in animal models, detecting changes in animal pruritus behavior, and confirming the pathway's direct regulatory role on pruritus phenotypes in vivo, which is a key step in pathway function validation. Clinical relevance validation involves detecting the correlation between the expression of core pathway molecules and patients' pruritus phenotypes in clinical samples, confirming the pathway's clinical significance in human pruritus diseases, and providing a basis for subsequent clinical translation. Dataset collection and archiving involves systematically archiving all validation data, providing complete experimental evidence for subsequent research and patent examination.

[0065] In the final stage of pathway specificity and universality verification, histamine-dependent and non-histamine-dependent acute pruritus models and various chronic pruritus models were constructed. Intervention experiments on the core pathway were performed in various pruritus models to verify the function of the pathway in different pruritus subtypes. The mechanical pain threshold, thermal pain threshold and cold pain threshold of experimental animals after core pathway intervention were detected to complete the sensory regulation specificity of the pathway. The universality and specificity of the core pathway in pruritus regulation were quantitatively evaluated, and the applicable scope and final evaluation results of the pathway were output.

[0066] Specifically, the small animal husbandry and modeling device is used to construct different subtypes of pruritus animal models; the sensory function testing device includes a dynamic plantar tactile sensor, a hot plate, a cold plate, and a tail-flicking device, used to detect the mechanical pain threshold, thermal pain threshold, and cold pain threshold of experimental animals and to assess the sensory modulation specificity of the pathway; and the behavioral recording and analysis device is used to detect behavioral changes in different pruritus model animals after pathway intervention.

[0067] This study constructs multiple subtype pruritus models, including histamine-dependent and non-histamine-dependent acute pruritus models, as well as various chronic pruritus models such as atopic dermatitis, psoriasis, and diabetic peripheral neuropathy, covering common clinical pruritus subtypes and providing a model basis for the universality validation of the pathway. Multi-model functional validation involves intervening in the core pathway in different pruritus subtype models, detecting changes in pruritus behavior, clarifying whether the pathway specifically regulates a particular type of pruritus or has a broad-spectrum pruritus regulatory effect, and determining the pathway's applicability. Sensory modulation specificity detection involves detecting changes in the animal's baseline pain threshold, touch, and temperature sensation after pathway intervention, assessing whether the pathway specifically regulates pruritus sensation without affecting normal physiological sensory transmission, and clarifying the safety and clinical translation potential of pathway-targeted intervention. Quantitative evaluation and result output involve quantitatively evaluating the universality, specificity, and safety of the pathway's pruritus regulation, outputting the final applicability and evaluation results, completing the entire closed-loop process of the pruritus-related signaling pathway analysis method.

[0068] The method of use and working principle of this invention are as follows:

[0069] Methods of Use: First, a standardized pruritus research system with multiple subgroups is constructed to complete the quantitative collection and validation of pruritus phenotypes. Simultaneously, the biological samples required for analysis are prepared and quality control is performed. Then, high-throughput multi-dimensional omics detection is conducted on qualified homologous samples. The acquired raw data is subjected to quality control filtering and standardization correction, and a uniformly formatted expression matrix is ​​output. Subsequently, based on the standardized expression matrix, pruritus-related differentially expressed molecules are screened between groups. Functional annotation and preliminary pathway enrichment are performed on the screened differentially expressed molecules to narrow the scope of target analysis. Then, based on a pruritus-specific reference gene set, targeted pathway enrichment is completed. Combined with phenotypic association analysis and kinase substrate cascade validation, the core signaling pathways related to pruritus are identified. Next, the upstream and downstream regulatory relationships of the core pathways are analyzed, the interaction nodes between the core pathways and classic pruritus pathways are identified, and a complete regulatory network map is constructed. Then, the functional validation and data collection of the core pathways are completed from four dimensions: molecular expression, cellular function, in vivo phenotype, and clinical association. Finally, pathway functional validation is completed in a multi-subtype pruritus model, the sensory regulatory specificity of the pathways is detected, and the applicability and evaluation results of the pathways are output.

[0070] Working Principle: Based on the technical logic of combining phenotypic standardization and anchoring with multi-omics joint analysis, this method first eliminates the systematic errors of traditional behavioral analysis through a multi-dimensional pruritus phenotypic quantification system, providing a stable and reliable phenotypic anchor for subsequent molecular analysis. Then, through multi-dimensional omics detection of homologous samples, it captures molecular changes at multiple levels, including transcription, protein, post-translational modification, and metabolism, during the occurrence of pruritus. Combined with a molecular screening system specific to pruritus, it accurately eliminates interfering molecules unrelated to the biological function of pruritus, identifying a set of differentially expressed molecules highly correlated with the pruritus phenotype. Next, through multi-dimensional cross-validation enrichment analysis, it eliminates false positive results from traditional pathway analysis, accurately anchoring core signaling pathways with complete activation cascades. Simultaneously, it analyzes the complete regulatory network of core pathways and their location in the overall pruritus regulatory system. Then, through progressive multi-dimensional experimental verification, it confirms the biological function of the pathways. Finally, through validation using a multi-subtype pruritus model, it clarifies the regulatory specificity and applicability of the pathways, forming a closed-loop analysis logic from unbiased screening to functional confirmation, achieving accurate and comprehensive analysis of pruritus-related signaling pathways.

[0071] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the description and drawings above. However, any modifications, alterations, and variations made by those skilled in the art without departing from the scope of the present invention using the disclosed technical content are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A method for analyzing pruritus-related signaling pathways, characterized in that, Includes the following steps: S1. In the stage of anchoring pruritus phenotype and preparing standardized samples, a multi-group pruritus research system was constructed, the quantitative collection and verification of pruritus phenotypes were completed, and biological samples for multi-dimensional analysis were prepared and quality-controlled. S2, Multi-dimensional omics detection and data preprocessing stage: complete multi-omics high-throughput detection of homologous samples, perform quality control filtering and standardization correction on the raw offline data, and output expression matrix in a unified format; S3, the screening and initial functional screening of differentially expressed molecules related to pruritus: the screening of differentially expressed molecules between groups is completed based on the standardized expression matrix, and functional annotation and preliminary pathway enrichment are performed on the differentially expressed molecules to narrow the scope of target analysis. S4, Core signaling pathway anchoring stage: Based on the pruritus-specific reference gene set, target pathway enrichment is completed, and combined with phenotypic association analysis and kinase substrate cascade verification, the core signaling pathways related to pruritus are identified. S5, Pathway Regulation Network and Crosstalk Analysis Stage: Analyze the upstream and downstream regulatory relationships of the core pathway, identify the interaction nodes between the core pathway and the classic pruritus pathway, and construct a complete regulatory network map. S6. Multi-dimensional experimental verification stage of core pathways: complete the functional verification and data collection of core pathways from four dimensions: molecular expression, cell function, in vivo phenotype, and clinical relevance. S7. Final stage of pathway specificity and universality verification: Pathway function verification is completed in a multi-subtype pruritus model, the sensory regulation specificity of the pathway is detected, and the applicability and evaluation results of the pathway are output.

2. The method for analyzing pruritus-related signaling pathways according to claim 1, characterized in that, In the pruritus phenotype anchoring and standardized sample preparation stage, a normal control group and multiple pruritus model groups were set up to complete the model construction of each group of experimental animals. Pruritus behavioral data of each group of experimental animals were collected, including the number of effective scratches per unit time, the average duration of a single scratch, the frequency of scratching attacks, and the percentage of scratches on the target area. The standardized pruritus phenotype score (SIS) for each group of samples was calculated using the standardized pruritus phenotype scoring formula to complete the quantitative verification of the pruritus phenotype. Target tissue samples were collected from each group of experimental animals, and total RNA, total protein, phosphorylated protein, and metabolites were extracted from the samples. The concentration, purity, and integrity of the extracted biomolecules were tested. Unqualified samples were discarded, and qualified samples were retained for subsequent testing. The standardized pruritus phenotype scoring formula is: Explanation of formula symbols: The number of times the target animal effectively scratches per unit of time; The maximum number of effective scratches by all animals in the same batch of experiments; : Average duration of a single scratch by the target animal; : The maximum average duration of scratching for all animals in the same batch of experiments; : The frequency of scratching attacks in the target animal per unit of time; : The maximum frequency of scratching attacks among all animals in the same batch of experiments; The percentage of scratches on the target area of ​​the target animal out of the total number of scratches; The percentage of animals scratching the largest target area in the same batch of experiments.

3. The method for analyzing pruritus-related signaling pathways according to claim 1, characterized in that, In the multi-dimensional omics detection and data preprocessing stage, transcriptome sequencing, quantitative proteome detection, phosphorylated proteome detection, and targeted metabolome detection are performed on qualified homologous samples to obtain raw data from various omics. Low-quality data filtering, low-abundance molecule removal, and outlier sample screening are performed on the raw data from various omics to complete basic quality control. Median centering correction is performed on the various omics data that have completed basic quality control to complete data standardization and output the multi-omics standardized expression matrix of homologous samples.

4. The method for analyzing pruritus-related signaling pathways according to claim 1, characterized in that, In the screening and initial functional screening stage of pruritus-related differentially expressed molecules, the difference between the pruritus model group and the control group was analyzed based on the multi-omics standardized expression matrix to obtain the fold change and corrected p-value for each molecule. Reported pruritus-related molecule datasets were integrated, and a corresponding pruritus function weight coefficient was matched for each molecule. The pruritus association score (SDS) for each molecule was calculated using the differential molecular pruritus association scoring formula. High-confidence differentially expressed molecules were screened according to a preset threshold. GO functional annotation and KEGG pathway enrichment analysis were performed on the screened high-confidence differentially expressed molecules, and pathways irrelevant to pruritus biological function were removed, retaining pathways and differentially expressed molecules related to pruritus biological function to narrow the scope of the target analysis. The differential molecular pruritus association scoring formula is as follows: Explanation of formula symbols: The fold difference in molecular weight between the pruritus model group and the control group; : Corrected P-value for differences between molecular groups; W: Weighting coefficient of pruritus function of molecules, ranging from 1.0 to 3.

0.

5. The method for analyzing pruritus-related signaling pathways according to claim 1, characterized in that, In the core signaling pathway anchoring stage, a pruritus-specific reference gene set was constructed by integrating previously reported pruritus-related functional genes. GSEA gene enrichment analysis was performed based on the standardized expression matrix and the pruritus-specific reference gene set to screen for significantly enriched pruritus-related pathways. A weighted gene co-expression network was constructed based on the standardized expression matrix, and the core gene modules related to the pruritus phenotype were screened. Core genes within the core gene modules were extracted, and intersection screening was performed with molecules within the significantly enriched pathways to narrow down the target pathway range. Kinase enrichment analysis was performed based on phosphorylated proteome data to verify the activation status of the kinase substrate cascade of the target pathway, thus completing the identification of the pruritus-related core signaling pathways.

6. The method for analyzing pruritus-related signaling pathways according to claim 1, characterized in that, The pathway regulation network and crosstalk analysis stage involves predicting and verifying the transcription factor binding relationships of core molecules in the identified core signaling pathway, predicting and verifying the non-coding RNA targeted regulation relationships of core molecules in the pathway, constructing the upstream and downstream regulatory networks of the core pathway, identifying the core regulatory nodes of the pathway, constructing a protein interaction network, identifying the interaction nodes between the core pathway and the classic pruritus pathway, performing crosstalk analysis between the core pathway and the classic pruritus pathway, and drawing a complete regulatory map of the core pathway.

7. The method for analyzing pruritus-related signaling pathways according to claim 1, characterized in that, The multi-dimensional experimental verification phase of the core pathway involves verifying the expression levels of key molecules in the core pathway through nucleic acid quantification and protein immunoassay, verifying the cellular function of the core pathway through primary cell culture and gene or drug intervention, verifying the in vivo phenotype of the core pathway through gene intervention or drug administration in animal models, verifying the correlation between the expression of core pathway molecules and the clinical pruritus phenotype through clinical sample testing, and collecting and archiving the full dataset for the functional verification of the core pathway.

8. The method for analyzing pruritus-related signaling pathways according to claim 1, characterized in that, In the final stage of the pathway specificity and universality verification, histamine-dependent and non-histamine-dependent acute pruritus models and various chronic pruritus models were constructed. Intervention experiments on the core pathway were performed in various pruritus models to verify the function of the pathway in different pruritus subtypes. The mechanical pain threshold, thermal pain threshold, and cold pain threshold of experimental animals after core pathway intervention were detected to detect the sensory regulation specificity of the pathway. The universality and specificity of the core pathway in pruritus regulation were quantitatively evaluated, and the applicable scope and final evaluation results of the pathway were output.

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