Shikonin suppresses fibrotic phenotype and oxidative stress in keloid fibroblasts via EGFR-targeted AKT/STAT3/PTGS2 modulation.

Abstract

Keloids are fibroproliferative disorders with excessive extracellular matrix (ECM) deposition, fibroblast hyperproliferation, oxidative stress, and a lack of effective therapies. Shikonin, a natural naphthoquinone, exhibits anti-tumor and antioxidant activities, yet its role in keloid fibroblasts (KFs) remains unclear. In this study, putative Shikonin targets were predicted from public databases and intersected with keloid-associated genes, followed by PPI network construction and GO enrichment analysis. Primary KFs and normal skin fibroblasts (NFs) were treated with Shikonin and assessed for proliferation by CCK-8 assay, apoptosis by flow cytometry, ECM-related protein and HIF-1α expression by Western blotting/immunofluorescence, and intracellular ROS by DCFH-DA staining. Target engagement and functional dependence were validated through molecular docking, molecular dynamics simulations, MM-PBSA analysis, biotin pull-down, CETSA, proteasome inhibition, and EGFR overexpression rescue experiments. Network analysis identified AKT1, EGFR, CASP3, STAT3, BCL2, PTGS2, and HIF-1α as core targets, with enrichment in oxidative stress and PI3K-centered pathways. Shikonin inhibited KF proliferation, induced apoptosis, downregulated Bcl-2 while upregulating Cleaved caspase-3, and suppressed ECM proteins (α-SMA, Fibronectin, Collagen I) and HIF-1α expression. Mechanistically, Shikonin directly bound to EGFR and promoted its proteasomal degradation, consequently suppressing p-AKT, p-STAT3, and PTGS2 expression. EGFR overexpression significantly reversed Shikonin's inhibitory effects on proliferation, ECM production, and oxidative stress, as well as its pro-apoptotic activity. To conclude, Shikonin exerts anti-KF effects by directly targeting EGFR, inducing its proteasomal degradation, and thereby inhibiting downstream AKT/STAT3 signaling and PTGS2 expression.