(006) Inhibiting NFκB to Target Inflammation May Have Unintended Consequences on Calcium Channels in Vulvodynia

Abstract Introduction Localized provoked vulvodynia (LPV) is a poorly understood condition of the vulva that results in chronic and severe pain at the vulvar vestibule (ring of tissue surrounding the vaginal opening) and significantly compromises quality of life. Inflammation, which is regulated through the nuclear factor-kappa B (NF-кB) pathway in vulvodynia is both necessary and sufficient for the activation of pain signaling. Pain is signaled through calcium channels (eg, transient receptor potential vanilloid 4; TRPV4) downstream of NF-кB. The relationship between NF-кB activity and activation of TRPV channels remains unclear. Objective The objective of our investigation was to better understand the molecular basis of vulvodynia and identify potential molecular targets in the disease mechanism to explore therapeutic options. Methods To investigate the role of NF-кB in regulating inflammation in this disease mechanism, we used an in vitro primary vulvar fibroblast model of LPV primed with endogenous pro-inflammatory cytokine, IL-1β. We treated fibroblasts with NF-кB inhibitors (Bay-11-7082, andrographolide) and assessed inflammation and TRPV channel activity via enzyme-linked immunosorbent assays (ELISAs) and calcium imaging. Bay-11-7082 is a synthetic anti-inflammatory compound that prevents IкB-α from releasing NF-кB in the cytoplasm, which inhibits NF-кB translocation into the nucleus. To ensure TRPV activity from inhibiting NF-кB was not compound-specific, we used andrographolide, which exerts anti-inflammatory effects by inhibiting several intracellular signaling pathways such as blocking the signal transduction pathway that leads to NF-кB activation or inhibiting NF-кB binding activity to target genes. Results Inhibiting inflammation ablates downstream calcium influx when fibroblasts cells are pretreated with an NF-кB inhibitor (Bay-11-7082) 24 hours or 1 hour before imaging. Contrastingly, calcium influx increases significantly when NF-кB is inhibited acutely. When we pretreated fibroblast cells (1 hour before imaging) with Bay-11-7082 but did not remove the supernatant from overnight IL-1β pretreatment containing inflammatory mediators, we observed no significant difference in calcium levels. Using real-time calcium imaging, when we pulsed Bay-11-7082 and TRPV4 agonists over cells, we observed a non-transient increase in calcium signals that fall back down to near baseline only when calcium is removed from the buffer. The calcium signal elevation following acute treatment with an NF-кB inhibitor persists even when TRPV1 and TRPV4 channels are inhibited. We observed a similar pattern of calcium response when we repeated experiments with andrographolide, an NF-кB inhibitor that acts via different mechanisms from Bay-11-7082. Conclusions Based on experimental data, the timing-dependent calcium response to NF-кB inhibition is not specific to a particular inhibitory compound. Rather, our investigation suggests a unique mechanism of NF-кB activating downstream calcium channels. Following acute NF-кB inhibition, calcium channels other than TRPV1 and 4 are likely involved in the resulting elevated calcium influx. This study provides evidence that NF-кB inhibits calcium signaling by preventing inflammation, indicating the effects of NF-κB inhibition are not the result of direct effects on the channel. Overall, this study contributes to our understanding of the molecular relationship between inflammation and pain signaling in vulvodynia. Disclosure No.