Arzanol, a natural phloroglucinol α-pyrone, protects HaCaT keratinocytes against H2O2-induced oxidative stress, counteracting cytotoxicity, reactive oxygen species generation, apoptosis, and mitochondrial depolarization

Abstract

Skin oxidative stress results in structural damage, leading to premature senescence, and pathological conditions such as inflammation and cancer. The plant-derived prenylated pyrone-phloroglucinol heterodimer arzanol, isolated from Helichrysum italicum ssp. microphyllum (Willd.) Nyman aerial parts, exhibits anti-inflammatory, anticancer, antimicrobial, and antioxidant activities. This study explored the arzanol protection against hydrogen peroxide (H₂O₂) induced oxidative damage in HaCaT human keratinocytes in terms of its ability to counteract cytotoxicity, reactive oxygen species (ROS) generation, apoptosis, and mitochondrial membrane depolarization. Arzanol safety on HaCaT cells was preliminarily examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic observation. The arzanol pre-incubation (5-100 μM, for 24 h) did not induce cytotoxicity and morphological alterations. The phloroglucinol, at 50 μM, significantly protected keratinocytes against cytotoxicity induced by 2 h-incubation with 2.5 and 5 mM H₂O₂, decreased cell ROS production induced by 1 h-exposure to all tested H₂O₂ concentrations (0.5-5 mM), as determined by the 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) assay, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS] method). The 2-h incubation of keratinocytes with H₂O₂ determined a significant increase of apoptotic cells versus control cells, evaluated by NucView® 488 assay, from the dose of 2.5 mM. Moreover, an evident mitochondrial membrane potential depolarization, monitored by fluorescent mitochondrial dye MitoView™ 633, was assessed at 5 mM H₂O₂. Arzanol pre-treatment (50 μM) exerted a strong significant protective effect against apoptosis, preserving the mitochondrial membrane potential of HaCaT cells at the highest H₂O₂ concentrations. Our results validate arzanol as an antioxidant agent for the prevention/treatment of skin oxidative-related disorders, qualifying its potential use for cosmeceutical and pharmaceutical applications.

Keywords: H2O2 oxidative stress; HaCaT cells; ROS; antioxidant activity; apoptosis; arzanol.