Pharmacological SIRT2 Inhibition by AGK2 Attenuates Hapten-Induced Keratinocyte Inflammation via NF-κB/NLRP3 Suppression and Nrf2 Reactivation.

Background: Allergic contact dermatitis (ACD) is initiated by hapten-induced keratinocyte oxidative stress and inflammatory signaling, commonly involving NF-κB activation, NLRP3 inflammasome priming-associated signaling, and inadequate Nrf2-dependent antioxidant responses. Identification of druggable upstream regulators that integrate these pathways could support host-directed pharmacological strategies for dermatitis-like inflammation. Objective: To investigate whether pharmacological inhibition of SIRT2 using AGK2 mitigates hapten-triggered inflammatory-oxidative stress signaling in human keratinocytes and to define the associated molecular mechanisms. Methods: Human Ker-CT keratinocytes were cultured alone or in transwell co-culture with CCD-1064Sk dermal fibroblasts and stimulated with 2,4-dinitrochlorobenzene (DNCB); nickel sulfate served as a comparator sensitizer. Cells were treated with the selective SIRT2 inhibitor AGK2, and target engagement was verified by acetyl-α-tubulin accumulation. Outcomes included viability (MTT), ROS generation (DCFDA), cytokine expression/secretion (qPCR/ELISA), and pathway signaling (Western blotting, immunofluorescence, nuclear-cytoplasmic fractionation), focusing on NF-κB (p65/IκBα), NLRP3 priming-associated readouts (NLRP3 expression and IL-1 family cytokine output), and Nrf2 localization with downstream antioxidant enzymes (HO-1, SOD). To strengthen specificity beyond pharmacological inhibition, SIRT2 involvement was genetically validated using siRNA-mediated knockdown (siSIRT2) in Ker-CT cells. In addition, AGK2-alone control groups (without DNCB) were included across major readouts to assess baseline effects on NF-κB, ROS, and Nrf2 signaling, and Keap1 expression was examined as mechanistic support for altered Nrf2 regulation. Results: DNCB produced a reproducible sub-toxic keratinocyte stress phenotype characterized by increased ROS, activation of NF-κB signaling with IκBα degradation and p65 nuclear translocation, upregulation of NLRP3 expression with elevated IL-1β/IL-18 secretion consistent with enhanced inflammasome-associated inflammatory responses, and impaired Nrf2 nuclear accumulation with reduced antioxidant enzyme expression. AGK2-mediated SIRT2 inhibition significantly attenuated NF-κB activation and reduced pro-inflammatory cytokine outputs, while reducing NLRP3 priming-associated responses and lowering IL-1β/IL-18 secretion. In parallel, AGK2 restored Nrf2 nuclear translocation and enhanced expression of HO-1 and SOD, resulting in partial normalization of intracellular redox balance. Importantly, siSIRT2 phenocopied AGK2 effects by reducing DNCB-induced NF-κB activation, ROS accumulation, and restoring nuclear Nrf2 enrichment. AGK2 alone did not significantly alter basal NF-κB signaling, ROS levels, or Nrf2 nuclear localization, supporting that AGK2 effects were stimulus-dependent rather than intrinsic pathway suppression. Consistent with impaired antioxidant competence under DNCB challenge, Keap1 levels increased with DNCB and were partially normalized by SIRT2 inhibition/knockdown, providing mechanistic context for the ROS-Nrf2 paradox. Conclusion: Pharmacological inhibition of SIRT2 with AGK2 is associated with anti-inflammatory and antioxidant effects in hapten-stimulated human keratinocytes, including attenuation of NF-κB signaling and reduction of NLRP3 priming-associated IL-1 family cytokine responses, together with restoration of Nrf2-dependent cytoprotective signaling. Collectively, these findings suggest that SIRT2 may have pharmacological relevance in ACD-like inflammatory skin stress and warrant further evaluation in preclinical dermatitis models and therapeutic development studies.