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Program Number: B284
Session Name: Poster Session

N-acetylcysteine attenuates tobacco-induced NFkB expression in human keratinocytes

William Kamm, BS; Chelsea A Gelboin-Burkhart, MD; Beverly Wuertz, BS; Frank Ondrey, MDPhD; University of Minnesota

It is well established that tobacco exposure is associated with increased postoperative complications including impaired wound healing. Cigarette smoke impedes both the inflammatory phase and the proliferative phases of wound healing via inflammatory dysregulation, oxidative stress and diminished vascular supply. Keratinocytes play a key role in wound healing, inducing reepithelialization through migration and proliferation and mediate the inflammatory response through toll-like receptor activation which induce the NFkB signaling pathway. Tobacco smoke induces NFkB expression in a variety of skin types including keratinocytes leading to immune dysregulation. In our prior work, we have shown that N-acetylcysteine, an antioxidant, improves wound healing in the setting of inhaled tobacco smoke. In this study, we aim to investigate the effect of NAC treatment on tobacco-induced NFkB expression in vitro in human keratinocytes.

We measured NFκB expression in human epithelial keratinocytes, treated with tobacco smoke extract, by a reporter plasmid assay.  Keratinocytes were cultured and plated in triplicate then co-transfected with pCMV Lac-Z reporter and IgκB plasmids. Following transfection, cells were then treated with empty vector, PMA (phorbol 12-myristate 13-acetate, an inducer of  NFkB expression), cigarette smoke concentrate (CSC), n-acetylcysteine, or cigarette smoke concentrate with n-acetylcysteine. After incubation, the cells in each well were lysed and activity of the reporter plasmid was measured by a luciferase activity with a luminometer.

Statistical analysis by a one-way ANOVA yielded a p-value < 0.0001. Tukey post hoc analysis showed each treatment group is significantly different from all other groups (p < 0.01) excluding the non-treated control vs NAC+CSC treatment. Relative expression of IgκB in each group is as follows: Non-treated: 1.000 (SE+0.019), PMA 2.380 (SE+0.093), CSC 1.370 (SE+0.019), NAC 0.541 (SE+0.048), and CSC+NAC 0.843 (SE+0.064). Our results suggest CSC upregulates and NAC downregulates NFκB expression in keratinocytes. NFκB expression is also shown to be significantly lower in CSC+NAC compared to CSC alone, suggesting NAC counters the increase in NFκB expression due to CSC exposure. Moreover, NAC appears to have a profound effect on NFκB expression induced by CSC as NFκB levels in this group are statistically indistinguishable from the non-treated group.

 

 

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