Confounder-Adjusted Transcriptomic Signature Reversal and Multi-Database Mechanistic Triangulation of Phytochemical Perturbagens in Human Osteoarthritic Synovial Fibroblasts: An Integrative Study of GSE29746, LINCS L1000, HERB 2.0, and the Comparative Toxicogenomics Database

Background: Synovial fibroblast activation drives synovitis, matrix remodeling, and pain in osteoarthritis (OA), yet connectivity screens rarely audit demographic confounding, resampling stability, permutation controls, or curated human directional gene reversal. Objective: To computationally prioritize phytochemical perturbagens reversing the human OA synovial fibroblast transcriptomic signature by integrating GSE29746, LINCS L1000, HERB 2.0, and the Comparative Toxicogenomics Database (CTD). Methods: Passage-4 synovial fibroblast microarrays from GSE29746 (N = 22: 11 OA vs. 11 healthy controls; 19,261 genes) were analyzed via limma with and without age/sex adjustment, audited by jackknife/bootstrap resampling and external synovial validation (GSE55235/GSE55457), and queried in L1000CDS2 against 10,000-permutation controls, curated HERB 2.0/CTD links, and ADME/docking benchmarks. Results: Sex was balanced (6F/5M; p = 1.000), whereas OA donors were older (73.0 ± 7.1 vs. 59.0 ± 19.0 years; Δ = +14.0 years, Welch p = 0.040). Age/sex adjustment attenuated FDR < 0.05 hits by 93.5% (from 46 to 3 downregulated genes: MMP27 [log2FC = −1.733, FDR = 0.033], IFIT3 [−1.238, FDR = 0.033], and MX2 [−2.057, FDR = 0.048]; Spearman ρ = 0.914; jackknife concordance = 98.4%; external p ≤ 0.0041). Among 52 unique LINCS compounds, 5 (9.6%) matched curated HERB 2.0 phytochemicals. Celastrol ranked first (Tier B; score = 0.0350, p = 0.0038; HME1, 10 μM, 3 h) with curated CTD reversal on MDH2 and TNFSF10 (p = 0.0019) and GRP94/IKKβ/MDH2 docking (RMSD ≤ 0.91 Å). Reserpine (0.0435), emetine (0.0435), and homoharringtonine (0.0400) replicated across models (Tier C) without CTD reversal; isoeugenitol (0.0300) ranked Tier D. Reactome rRNA processing was enriched (p = 0.028). Zero candidates reached Tier A. Conclusions: Triangulation prioritizes celastrol for in vitro (IL-1β/TNF-α-stimulated OASF measuring IL-6, MMP-13, and COX-2) and in vivo testing before clinical interpretation.

celastrol; Comparative Toxicogenomics Database; connectivity mapping; HERB 2.0; LINCS L1000; osteoarthritis; synovial fibroblasts

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