Proceedings of The Physiological Society

Physiology 2014 (London, UK) (2014) Proc Physiol Soc 31, C70

Oral Communications

Evaluation of small molecule inhibitors of nuclear factor kappa B (NF+

L. R. Gurney1, J. Taggart1, S. Robson1, M. Taggart1

1. Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom.

  • Fig 1. Representative western blotting displaying COX2 protein expression at 4 time points. Myometrial cells were exposed to either IL1β alone or following 1 hour preincubation with indicated inhibitor (n=3).

Preterm birth is a significant and underexposed clinical problem[1]. Current treatment strategies are inadequate and there is a dearth of therapeutic agents available to ameliorate this situation[2]. There is a substantial body of evidence that implicates the activation of the transcription factor NFκB as part of a pro-inflammatory cytokine cascade within the uterine environment following infectious insult [3, 4] making the potential therapeutic value of small molecule inhibitors of the NFκB pathway to be of interest [5]. Using an in vitro cytokine cell stimulation model with primary human uterine cells the effectiveness of 2 such inhibitors was compared. Following written informed consent (REC:10/H0906/71) lower uterine tissue was sampled at elective Caesarean section and myometrial cells prepared and cultured to passages ≤4. Cells were exposed to the cytokine stimulant IL1β (10ng/ml) alone or following 1 hour pre-incubation (and subsequent co-incubation) with two concentrations of putative NFκB inhibitors: Sc514 and Curcumin. Cells were then lysed at selected points over a 4 hour time course. Following protein assay, the expression of the inhibitor of κB protein IκBα, and the inducible prostaglandin synthase enzyme COX 2, were investigated using western blotting / densitometry. Responses between groups were compared using 1-way ANOVA with post hoc Bonferonni test (p<0.05; means ± SEM). Experiments were completed using biopsies of three separate patients. Following cell exposure to IL1β, IκBα expression was considerably reduced within 15 minutes. This was succeeded at 4 hours by marked COX2 upregulation. IL1β stimulated COX2 signal at 4 hours (15.01±1.32 Arbitrary Units (Log Optical Density)) was reduced in the presence of Sc514 at 30uM (7.953±2.08AU) and 50uM (3.86±2.04AU) and Curcumin at 30uM (8.436±2.973AU) and 50uM (1.09±0.89AU) (Fig 1). Diminution of IL1β stimulated IκBα degradation was observed on 2 of 3 occasions with both doses of curcumin and Sc514.In summary both inhibitors were effective in consistently reducing IL1β-stimulated COX2 expression in human myometrial cells in a dose dependent fashion. Inhibitor dependent prevention of IκBα degradation did not always occur indicating that mechanisms other than the canonical NFκB pathway may be affected. It will be of interest to ascertain the actions of other small molecule putative inhibitors of NFκB and, also, whether these effects are stimulus-dependent.

Where applicable, experiments conform with Society ethical requirements