Standardized Thunbergia laurifolia Extract Inhibits PM2.5-Induced Oxidative Stress by Regulating p62-KEAP1-NRF2 Signaling Pathway.

Abstract

Fine particulate matter (PM2.5) in air pollution causes skin damage through the induction of oxidative stress in the epidermis. Antioxidants help counteract cellular oxidant species and maintain cell homeostasis. This study aimed to examine the protective effect of standardized ethanolic extract of Thunbergia laurifolia leaves on PM2.5-mediated oxidative stress in epidermal keratinocytes. The extract was standardized with rosmarinic acid. Effects of standardized T. laurifolia extract (STLE) (0-400 μg/ml) and PM2.5 (0-32 μg/ml) on cell viability after 24 h of treatment were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. PM2.5 (0-32 μg/ml) induction of intracellular reactive oxygen species (ROS) at 6 h was monitored using 2',7'-dichlorodihydrofluorescein diacetate. Cells were co-treated for 6 h with PM2.5 (32 μg/ml) and STLE (25-100 μg/ml) and monitored for oxidative stress inhibition. Proteins related to cellular antioxidant defense system were examined by western blot analysis, after co-treatment and STLE treatment for 6 h and 24 h, respectively. Nuclear expression of nuclear factor erythroid 2-related factor (NRF2) and p62 were determined by immunofluorescence after co-treatment of 6 h. PM2.5 (32 μg/ml) remarkably induced ROS production within 6 h. The co-treatment dramatically inhibited PM2.5-induced oxidative stress at 6 h. In addition, STLE enhanced cellular defense system by increasing the levels of p62, NRF2 and superoxide dismutase 1 proteins. STLE stimulated nuclear localization and function of NRF2 and p62 proteins, while suppressing Kelch-like ECH-associated protein 1. STLE exhibits promising natural antioxidant activity against oxidative stress induced by PM2.5 in keratinocytes.