A novel 2-oxopyrrolidine derivative (LN-53) efficiently induces Nrf-2 signaling pathway activation in human epidermal keratinocytes.

Background: The skin is a pivotal organ that serves as a physical barrier, protecting the body from harmful substances such as pathogens, allergens, and other environmental irritants. Chronic inflammation in the skin, along with the anthropogenic effects, can cause reactive oxygen species (ROS) overproduction. Prolonged exposure to elevated ROS levels and inadequate antioxidant defenses in the skin can contribute to the onset of various skin disorders. The nuclear factor erythroid 2-related factor-2 (Nrf-2) signaling pathway plays a key role in enhancing antioxidant capacity by promoting the production of antioxidant and detoxifying molecules. Consequently, pharmacological activation of the Nrf-2 pathway may help restore the oxidant-antioxidant balance, thereby improving therapeutic outcomes for chronic skin disorders. This study aimed to investigate the potential effect of novel agent: (5-((4-(4-(methoxycarbonyl)-2-oxopyrrolidin-1-yl)phenyl)carbamoyl)benzene-1,2,3-triyl triacetate (LN-53), synthesized based on the structure of previously developed by our team lead compound SK-119, on Nrf-2 signaling pathway in human epidermal keratinocytes (HEKs) at mRNA and protein level.

Methods: The cytotoxicity of LN-53 was evaluated by MTT, LDH, live/dead cell staining, and caspase-3,-8,-9 multiplex activity assays. Intracellular ROS production was assessed by DCFH-DA staining. The Nrf-2 gene was silenced by transient transfection using human Nrf-2 siRNA. Nrf-2 and related factors (heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase: quinone-1 (NQO1)) were evaluated at the mRNA level by qPCR and protein level in nuclear and cytosolic fractions by Nrf-2 activation assay and Western blot. The levels of inflammatory cytokines (IL-6 and IL-8) in supernatants were determined by ELISA.

Results: Our results indicate that LN-53 effectively reduces intracellular ROS production triggered by tert-butyl hydroperoxide (TBHP), without leading to any noticeable cell damage. It promoted the nuclear translocation of Nrf-2 and induced the production of Nrf-2, HO-1, and NQO1 at both the mRNA and protein levels. LN-53-mediated alterations in antioxidant gene expressions were blocked by Nrf-2 knockdown. LN-53 treatment also suppressed the release of IL-6 and IL-8 cytokines mediated by TBHP exposure. Additionally, novel compound LN-53 was found to be more stable than the parent compound SK-119.

Conclusion: LN-53 can effectively induce antioxidant mechanisms by promoting Nrf-2 nuclear translocation and suppressing ROS production in human epidermal keratinocytes. These data may suggest that LN-53 can contribute to maintaining redox balance and homeostasis in the skin.