Gastrointestinal epithelial barrier loss due to tight junction (TJ) dysfunction and bile acid‐induced diarrhea are common in patients with inflammatory diseases. Although excess colonic bile acids are known to alter mucosal permeability, few studies have compared the effects of specific bile acids on TJ function. We report that the primary bile acid, chenodeoxycholic acid (CDCA), and its 7α‐dehydroxylated derivative, lithocholic acid (LCA) have opposite effects on epithelial integrity in human colonic T84 cells. CDCA decreased transepithelial barrier resistance (pore) and increased paracellular 10 kDa dextran permeability (leak), effects that were enhanced by proinflammatory cytokines (PiC [ng/mL]: TNF α[10] + IL‐1ß[10] + IFN γ[30]). CDCA reversed the cation selectivity of the monolayer and decreased intercellular adhesion. In contrast, LCA alone did not alter any of these parameters, but attenuated the effects of CDCA ± PiC on paracellular permeability. CDCA, but not PiC, decreased occludin and not claudin‐2 protein expression; CDCA also decreased occludin localization. LCA ± CDCA had no effects on occludin or claudin expression/localization. While PiC and CDCA increased IL‐8 production, LCA reduced both basal and PiC ± CDCA‐induced IL‐8 production. TNF α + IL1ß increased IFN γ, which was enhanced by CDCA and attenuated by LCA. CDCA±PiC increased production of reactive oxygen species (ROS) that was attenuated by LCA. Finally, scavenging ROS attenuated CDCA's leak, but not pore actions, and LCA enhanced this effect. Thus, in T84 cells, CDCA plays a role in the inflammatory response causing barrier dysfunction, while LCA restores barrier integrity. Understanding the interplay of LCA, CDCA, and PiC could lead to innovative therapeutic strategies for inflammatory and diarrheal diseases.

Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl Ϫ secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 M, 15 min) drastically (Ͼ90%) inhibited FSK-stimulated short-circuit current (Isc), it did not alter carbachol-stimulated Isc. LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the I sc stimulated by the cAMP-dependent secretagogues 8-bromocAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP] i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i), chelation by BAPTA-AM did not reverse LCA's effect on I sc. LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on I sc. Finally, although LCA had no effect on basal I sc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl Ϫ current and inhibits a basolateral K ϩ current. In summary, 50 M LCA greatly inhibits cAMP-stimulated Cl Ϫ secretion, making low doses of LCA of potential therapeutic interest for diarrheal diseases.

Epithelial apoptosis and tight junction (TJ) changes contribute to intestinal barrier dysfunction in Crohn's disease and colitis. We have reported that in T84 monolayers, CDCA decreased transepithelial resistance (TER) and caused dose‐ and time‐related cytotoxicity. CDCA increased cascade blue 10kDa dextran (CB10D) flux from apical to basal surfaces and this was enhanced by pro‐inflammatory cytokines (PiC: TNFα+IL‐1ß+IFNγ; FASEB J:28, 1113.3, '14). In this study we examined if LCA, a dehydroxylated product of CDCA, had similar effects. Confluent T84 cells (TER >1000 Ω.cm2) were treated with 5‐500µM LCA for 1 and 24 hr ± PiCs. Cell viability and apoptosis (Accuri flow cytometry), toxicity (lactate dehydrogenase), and paracellular permeability (TER and CB10D fluxes) were assessed. One hour or overnight (O/N) LCA (5 – 500μM) exposure neither affected cell viability nor apoptosis. LCA (1 Hr or O/N) did not increase LDH release and therefore was not cytotoxic. Time‐dependent studies (0.5 – 18 Hr) show that LCA alone did not affect TER (18 Hr: Control: 925+15; 50µM LCA: 848+41 Ω.cm2 n=6) or CB10D fluxes. In contrast, 50µM LCA attenuated PiC‐induced decreases in TER (data not shown) and increases in CBD10 fluxes ± 500 μM CDCA (18 Hr, in μg: Control: 7±1; LCA: 5±1; PiC: 51±4; PiC+LCA: 10±1; CDCA: 110±2; PiC+CDCA: 180±2; CDCA+LCA: 63±1 PiC+CDCA+LCA: 37.8±0.3; n>3). Surprisingly, unlike CDCA, LCA neither causes cell death nor disrupts tight junction function. On the contrary, LCA restores epithelial barrier integrity that is disrupted by cytokines ± CDCA. This study suggests that LCA could be a target therapeutic drug for inflammatory and diarrheal diseases.

Polyphenols are naturally occurring compounds in plants which are typically involved in defense against ultraviolet radiation or pathogens. Previous studies have shown that various polyphenols have anti‐proliferative properties in a variety of human cancers. The purpose of this study is to determine the effect of Quercetin (QC), Epigallocatechin‐3‐gallate (EGCG), Catechin and Resveratrol (RVL) on cell death and apoptosis in human colon carcinoma, T84, cells.Cell death and apoptosis was measured by using Annexin V: FITC Apoptosis Detection Kit I and a flow cytometry. Short term (1 hour) and long term (overnight; O/N) effects were observed. Cells were grown in 6 well plates, serum starved O/N and treated with different concentrations of polyphenols for varying time. Linear Sweep Voltammetry (LSV) was used to electrochemically characterize the antioxidant capacity and to quantify active RVL and QC (the polyphenols acting as reducing agents) for the time frame of the study.While EGCG and Catechin did not have an effect, RVL and QC showed an interesting dose and time‐dependent change in cell death and apoptosis. O/N exposure to 200 µM QC caused an increase in cell death and apoptosis (control: 5.5+ 1.0%; QC: 15.5+ 4.3%). However, 30 µM RVL increased cell death and apoptosis at 1 hr (control: 4.3+ 0.8; RVL: 13.9+ 3.2) and returned back to control levels with time. Based on LSV, QC has a higher antioxidant capacity than RVL. However, the antioxidant effect of RVL decreased over time. In summary, QC and RVL both have anti‐proliferative properties with QC having prolonged effects in human colon carcinoma cells. Thus, a combination of polyphenols might have a better effect in preventing colon cancer.