Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA117

Poster Communications

Anti-apoptotic actions of lithocholic acid on colonic epithelial cells: implications for treatment of inflammatory bowel disease

N. K. Lajczak-McGinley1, A. O'Dwyer1, S. J. Keely1

1. Molecular Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.

Introduction: Although inflammatory bowel disease (IBD) represents a significant health and economic burden, current therapies are often ineffective. Dysregulated intestinal barrier function has been well-documented to contribute to IBD pathogenesis, with increased epithelial apoptosis being apparent. We have previously reported that ursodeoxycholic acid (UDCA) attenuates intestinal inflammation in the DSS model of colonic inflammation in mice, and that its efficacy is dependent on bacterial metabolism into lithocholic acid (LCA) (Ward et al, 2017). Here, we set out to examine the effects of LCA on apoptosis in colonic epithelial cells. Methods: C57BL/6 mice were administered dextran sodium sulphate (DSS, 2.5%) in their drinking water for 5 days, with or without treatment with lithocholic acid (30 mg/kg) via IP injection each day. T84 colonic epithelial cells were treated with LCA (30 µM) or the farnesoid X receptor (FXR) agonist, GW4064. Apoptosis was induced by treatment of the cells with a combination TNF-α (20 ηg/ml) and INF-γ (40 ηg/ml) for 24 hrs and was assessed by measuring levels of cleaved Poly (ADP-ribose) polymerase (PARP). miRNA profiling was performed on GW4064 (5 µM; 6 hrs)-treated T84 cells, with TargetScan being used to identify miRNA targets. Reverse transfection of T84 cells was performed using lipofectamine RNAimax and protein expression was assessed by western blotting or qPCR. Results: In the in vivo model of DSS-induced colonic inflammation, LCA treatment reduced levels of cleaved-PARP in the mucosa from 19.9 ± 10.9 to 3.9 ± 1.1 fold of that in non-DSS treated mice (n = 3-4; p ≤ 0.01). Similarly, in cultured monolayers of T84 cells, LCA prevented cytokine-induced apoptosis, an effect that was mimicked by the FXR agonist, GW4064. miRNA array profiling of GW4064-treated T84 cells revealed increased levels of miR-29a-3p. miR-29a3p is predicted to target PTEN, a pro-apoptotic protein, with a 92% context score as ascertained by TargetScan. Treatment of T84 cells with GW4064 resulted in decreased PTEN mRNA (n = 8; p ≤ 0.05) and PTEN protein levels (n = 3; p ≤ 0.05). Similarly, transfection of T84 cells with a miR-29a-3p mimic, but not a scrambled control, decreased PTEN levels (n=3; p ≤ 0.01). Discussion / Conclusion: Our data suggest that bacterial metabolism of UDCA to LCA in the colonic lumen may promote barrier function by prevention of cytokine-induced apoptosis. The mechanism by which LCA exerts anti-apoptotic actions may involve activation of FXR, induction of miR29a3p, and reduced expression of the pro-apoptotic protein, PTEN.

Where applicable, experiments conform with Society ethical requirements