Nootkatone Attenuates Oxidative Stress, Focal Adhesion Abundance, and Profibrotic Gene Expression in Human Keloid...

Keloids are driven by persistent fibroblast activation characterized by excessive matrix deposition, abnormal motility, and oxidative/inflammatory signaling. Nootkatone, a natural sesquiterpenoid present in grapefruit, has reported antioxidant and anti-inflammatory activities, but its effects on human keloid fibroblasts (HKFs) remain unclear. SV40-immortalized HKFs and non-keloid HSFs were exposed to nootkatone. Dose-response analysis showed that HKFs were more sensitive to nootkatone than HSFs; therefore, 20 and 40 microg/ml nootkatone (hereafter termed NKT20 and NKT40, respectively) were selected for subsequent HKF-focused assays, including wound closure, Transwell invasion, immunofluorescence analyses of F-actin organization, alpha-SMA, vinculin-positive focal adhesions, and intracellular reactive oxygen species (ROS), as well as RT-qPCR assays for extracellular matrix- and inflammation-related genes. HKFs were more sensitive to nootkatone than HSFs in the viability assay. Nootkatone reduced scratch-wound closure and Transwell invasion, decreased phalloidin signal, cell aspect ratio, alpha-SMA intensity, vinculin-positive focal adhesion counts, and intracellular ROS. NKT20 reduced the mRNA expression of COL1A1, COL3A1, and FN1, whereas NKT20 and NKT40 reduced IL8 and HMGB1. Nootkatone attenuated multiple profibrotic features in an SV40-immortalized HKF model, including motility-associated readouts, cytoskeletal organization, focal adhesion abundance, oxidative stress, and extracellular matrix/ inflammatory gene expression. These findings support further mechanistic evaluation of nootkatone in primary, non-immortalized, multi-donor keloid fibroblast models. Key words Nootkatone " Keloid fibroblast " Oxidative stress " Focal adhesion " alpha-SMA " Extracellular matrix.