Multi-Target Systems Pharmacology and Multi-Cohort Human Transcriptomic Triangulation of Withania somnifera Withanolides in Chondrocyte Senescence and Osteoarthritic Cartilage Degradation: A Computational Study Anchored in Curated ChEMBL Bioassays and Public GEO Datasets (GSE114007, GSE117999, and GSE246425)

Background: Withania somnifera (Ashwagandha) withanolides are proposed as multi-target candidates for osteoarthritis (OA) and chondrocyte senescence, yet network pharmacology studies frequently rely on unverified target predictions. This computational study triangulated curated ChEMBL bioactivities against multi-cohort human OA and senescence transcriptomes. Methods: Six withanolides (Withanolide A, D, E, F, Withaferin A, Withanone) were audited across PubChem, SwissADME, ChEMBL v34 (confidence score = 9), and STRING (score ≥ 0.70). Differential expression (Benjamini–Hochberg FDR < 0.05, |log2FC| ≥ 1.0), SenMayo/CellAge senescence benchmarking, 10,000-permutation random-target controls, and g:Profiler (g:SCS p < 0.05) enrichment were executed across N = 70 human samples: GSE114007 (n = 38), GSE117999 (n = 20), and GSE246425 (n = 12). Results: All six withanolides satisfied Lipinski's Rule-of-Five (100.0%, bioavailability score = 0.55, P-gp substrates) and mapped to 11 ChEMBL records across seven human proteins (BRD4, BRD3, AKT1, NFKBIA, CASP3, HSPA6, CYP1A1), led by non-covalent Withanolide E–BRD4/BRD3 (IC50 = 556.2/720.3 nM) and Withaferin A–AKT1 (2.5 μM). Covariate-adjusted models identified 1,897 (GSE114007), 23 (GSE117999), and 984 (GSE246425) DEGs, with significant SenMayo (2.44×, p = 2.84 × 10-4) and CellAge (1.85×, p = 7.42 × 10-4) enrichment. While 0/7 direct targets reached primary DEG significance (permutation p = 1.000) and KDM6B was the sole strictly replicated OA gene (log2FC = −1.49 and −1.18), pathway convergence across extracellular matrix organization (57 shared terms, Jaccard = 0.238, p < 1.0 × 10-4), collagen turnover, PI3K–AKT (p = 0.005), apoptosis, and MAPK exceeded random-target expectations (9.76×, permutation p = 0.0018). Conclusions: Withanolide pharmacology in OA operates via post-translational signaling and chromatin modulation rather than target mRNA shifts, requiring prospective wet-lab validation in IL-1β/senescent chondrocytes and DMM animal models. Keywords: Withania somnifera; withanolides; osteoarthritis; chondrocyte senescence; BRD4; KDM6B; SenMayo; systems pharmacology.

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