Two sesquiterpene aminoquinones protect against oxidative injury in HaCaT keratinocytes via activation of AMPKα/ERK-Nrf2/ARE/HO-1 signaling

Abstract

AimsTo investigate the cytoprotective effects of two sesquiterpene aminoquinones isolated from the marine sponge Dysidea fragilis, Dysidaminone H (DA8) and 3′-methylamino-avarone (DA14), we examined their effects against hydrogen peroxide (H2O2)-induced oxidative injury in human keratinocyte cell line and elucidated the underlying mechanisms. Main methodsCell viability was detected using a CCK-8 assay kit. Intracellular reactive oxygen species (ROS) production was measured by fluorescence of 2, 7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA). Messenger RNA and protein expression were measured by real-time quantitative PCR and western blotting analysis. Immunocytochemistry was performed to determine the intracellular location of nuclear factorerythroid 2 p45 related factor 2 (Nrf2). The antioxidant response element (ARE)-luciferase reporter gene assay and RNA interference were used to establish the role of ARE and Nrf2. Key findingsDA8 and DA14 (DAs) resisted H2O2induced decline of cell viability by inhibiting the accumulation of ROS. Meanwhile, DAs increased HO-1 expression and ARE activity and induced Nrf2 expression, as well as the accumulation of Nrf2 in the cell nucleus. However, silencing of Nrf2 abolished DAs-induced HO-1 expression and ARE luciferase activation. In addition, DAs induced the phosphorylation of both cyclic AMP-activated protein kinase-α (AMPKα) and extracellular signal-regulated kinase (ERK), while specific inhibitors of AMPKα and ERK abrogated HO1 upregulation and Nrf2 activation. SignificanceDAs provided cytoprotective effects against H2O2-induced cytotoxicity by activation of the Nrf2/ARE/HO-1 pathway via phosphorylation of AMPKα and ERK. The findings suggested that DA8 and DA14 might be the candidate therapeutic agents for skin diseases caused by oxidative injury.