Evaluating the Antioxidant, Cytoprotective and Wound-Healing-Associated Effects of Haberlea rhodopensis Ethanolic Extract in Human Skin Keratinocytes

The resurrection plant Haberlea rhodopensis is a rare species endemic to Greece and Bulgaria, renowned for its exceptional desiccation tolerance and rich phytochemical composition. This study investigated the antioxidant, cytoprotective, and wound-healing-associated effects of H. rhodopensis ethanolic extract (HEE) in human keratinocytes (HaCaT cells) under oxidative and cytotoxic stress conditions. Antioxidant capacity was initially evaluated using a plasmid DNA protection assay, in which HEE attenuated oxidative DNA damage induced by a Fenton reaction system and preserved the native supercoiled structure of pUC19 plasmid DNA. Cytotoxicity screening using the sulforhodamine B (SRB) assay and real-time proliferation monitoring (HoloMonitor® M4) identified 20 μg/mL as a non-toxic pre-treatment concentration (EC10). Under hydrogen peroxide (H2O2)-induced oxidative stress, HEE pre-treatment maintained cell viability and significantly reduced intracellular reactive oxygen species (ROS) levels, indicating a protective effect. In vitro wound-healing assays demonstrated enhanced scratch closure in keratinocyte monolayers. RT-qPCR analysis revealed modulation of antioxidant-related genes (CAT, SOD1, HMOX1, NQO1, GPX, GSR), while mRNA sequencing suggested selective stress-adaptive responses, involving extracellular matrix (ECM)-, metabolic-, and tissue-repair/aging-associated pathways. Overall, HEE exhibits antioxidant and cytoprotective effects in keratinocytes and is associated with transcriptional changes linked to cellular stress responses and wound closure. These findings support its potential relevance for dermatological, pharmaceutical, and cosmeceutical applications, while further studies are required to establish the underlying molecular mechanisms.