Abstract / Summary
Abstract Background Feng et al. (2022, J Neuroinflammation) demonstrated that Wnt/beta-catenin signaling promotes neuropathic pain (NP) through microglial NLRP3 inflammasome activation. The present study extends this finding computationally by investigating astrocyte-specific Wnt-pyroptosis co-activation at single-cell resolution, providing human genetic association evidence, and systematically prioritizing drug candidates targeting these pathway nodes. Methods Seven parallel computational analyses were performed using publicly available data (GEO, UK Biobank, STRING, DrugBank, PDB), with acknowledged limitations for each: (1) cross-dataset differential expression of two rat spared nerve injury (SNI) transcriptomic datasets (n = 8 each) integrated by Robust Rank Aggregation; (2) single-cell RNA-seq analysis (GSE134003, mouse SNI spinal cord) with CellChat ligand-receptor detection and pseudotime trajectory inference; (3) high-confidence STRING protein-protein interaction network analysis; (4) descriptive three-tier drug-target classification based on DrugBank annotations (no statistical testing performed due to incomplete target coverage of 21 targets across 20 drugs); (5) weighted gene co-expression network analysis (WGCNA) with cross-dataset module preservation testing (Z-summary); (6) conservative instrumental variable analysis using published multi-instrument Mendelian randomization (MR) estimates (OR = 1.12–1.31) rather than single-instrument Wald ratios (which yield inflated OR = 2.47–2.78); and (7) structural analysis of four PDB structures (MD simulations not completed; binding energies sourced from published literature). Negative and null results are explicitly reported throughout. Results Of 32 Wnt pathway genes tested, 8 were differentially expressed (DEGs) and 20 were NOT, indicating pathway-level rather than universal gene-level activation. For pyroptosis, 15 of 16 pathway genes were detected and 6 were DEGs. WGCNA across 5,000 variable genes identified 9 co-expression modules; Wnt/pyroptosis genes distributed across 4 modules, with the primary module (M8, 4 genes) showing no cross-dataset preservation (Z-summary = 1.00, threshold Z < 2). Single-cell analysis confirmed astrocyte-enriched expression of Wnt (Ctnnb1, Tcf4, Lef1) and pyroptosis (Nlrp3, Gsdmd, Casp1) genes; CellChat detected Wnt-Fzd ligand-receptor pairs between astrocytes and neurons/microglia. PPI analysis (216 nodes, 189 edges) identified Il6, Ccl2, Wnt2, and Nfkb1 as top hubs; JASPAR predicted TCF4 binding sites in Nlrp3 and Gsdmd promoters. Descriptive three-tier drug classification identified nine Tier 1 candidates directly targeting Wnt/pyroptosis nodes: MCC950 (Kd = 5 nM, NLRP3), disulfiram (FDA-approved GSDMD inhibitor), XAV939 (Kd = 46 nM, TNKS2), and six others. Conservative published MR estimates supported associations between NLP3 (OR = 1.31, 95% CI 1.14–1.50), IL1B (OR = 1.28, 1.12–1.46), and CTNNB1 (OR = 1.22, 1.08–1.38) expression and chronic pain risk; 4 of 13 genes had CIs crossing 1.0. Conclusions This computational study extends the Wnt-NLRP3-NP axis from microglia (Feng et al., 2022) to astrocytes, with human genetic support and systematic drug prioritization. All findings are hypothesis-generating computational predictions requiring independent experimental validation. Key limitations include: extension of prior work rather than de novo discovery; single-instrument genetic analysis; small sample sizes (n = 8); incomplete drug target data (descriptive classification only); MD simulations not completed; and WGCNA modules showing no cross-dataset preservation (Z = 1.00). No simulated data were used.