ROS-Mediated Cytotoxic Effects of Hypericin in Gingival Fibroblasts

Objective: This study investigated the cytotoxic and pro-apoptotic effects of hypericin on human gingival fibroblasts (HGF) under non-photoactivated conditions. Methods: HGF cells (ATCC® PCS-201-012TM) were exposed to increasing concentrations of hypericin (1–100 µM), and cell viability was assessed using the MTT assay at 24, 48, and 72 h. Long-term proliferative capacity was evaluated by clonogenic assays, while DNA synthesis was determined using BrdU/EdU incorporation. Intracellular reactive oxygen species (ROS) production was measured using the DCFH-DA fluorescent probe. Apoptotic responses were further analyzed by caspase-3/7 activity assays and Annexin V-FITC/PI flow cytometry. Results: Hypericin induced a significant, concentration- and time-dependent reduction in HGF cell viability, colony formation, and DNA synthesis. Exposure to higher concentrations (≥10 µM) resulted in a marked increase in intracellular ROS levels. In parallel, caspase-3/7 activation and apoptotic cell populations were significantly elevated in treated cells compared with controls. Importantly, these effects were observed in the absence of photodynamic activation, suggesting that hypericin may exert intrinsic cytotoxic and oxidative effects independent of light-induced ROS generation. Conclusion: Overall, hypericin demonstrates dose-dependent pro-oxidant and pro-apoptotic activity in gingival fibroblasts. These findings provide new insight into the biological responses of normal oral fibroblasts to hypericin and highlight the importance of defining safe exposure levels for potential oral and dental applications.