Reactive oxygen species (ROS) Play a Key Role in Bile Acid (BA)‐Induced Barrier Dysfunction And Alters the Leak Function of Tight Junctions (TJ) in Human Colon Carcinoma T84 Cells
Authors: Sirajuddin Hafsa , Nair Tanushree , Ao Mei , Haery Ladan , Alendry Sharyl , Rao Meena , Sarathy Jayashree 
Published: November 2023

Dysfunction of the pore and leak pathways of TJ plays a vital role in the pathogenesis of inflammatory and diarrheal diseases. Excess colonic BAs are known to alter mucosal permeability. We have shown that chenodeoxycholic acid (CDCA; 500μM) and its derivative, lithocholic acid (LCA; 50μM) have opposing effects on the barrier integrity of T84 cells (FASEB J 2016 30:1223.29). While CDCA‐induced changes in pore and leak function of TJs are further enhanced by pro‐inflammatory cytokines (PiC [ng/ml]: TNFα[10]+IL‐1β[10]+IFNγ[30]), LCA reversed CDCA± PiC's effects only on TJ leak function. As ROS is known to increase paracellular permeability, we examined its role in BA regulation of TJ dysfunction.Confluent T84 cells (TransEpithelial Resistance; TER >1000Ωcm2) were treated apically with DMSO, 500μM CDCA, 50μM LCA alone, in combination, or ± PiCs for 0.5–18 hours (H). Mitochondrial ROS (mROS, CellRox, flow cytometry), H2O2 release (colorimetry) and apoptosis (Annexin V, flow cytometry) were measured. ROS contribution to BA regulation of TJ was assessed by examining the effect of ROS scavenger, N‐acetyl cysteine (NAC; 1mM)±BA±PiC on i. Oxidative stress; ii. Pore function measured as TER or dilution potential (Dψ, upon 50% reduction in apical [NaCl]); iii. Leak function measured as fluorescein isothiocyanate‐10 kDa dextran flux (F10D).CDCA increased mROS and this was unaltered by PiC (CellRox+ cells, in %, DMSO: 8±1; CDCA: 24±3; PiC: 11±1; PiC+CDCA: 25±3, n=3). Similarly, LCA did not alter CDCA‐induced mROS changes. CDCA ± PiC increased H2O2 release over time (4 H: fold–change, DMSO: 1; PiC: 2, CDCA: 1.5, CDCA+PiC: 2; 18 H: PiC: 4.5, CDCA: 17, CDCA+PiC: 14; n=6). LCA reversed H2O2 release by CDCA or PiC at 4 and 18H or CDCA+PiC at 4H (p<0.05). However, at 18H LCA only inhibited ~50% of CDCA+PiC‐induced H2O2 release. NAC inhibited ROS production in all of the above conditions. In contrast, NAC had no significant effect on BA‐induced apoptosis (18 H: DMSO 8±0.3%, CDCA: 18±1%; LCA: 8±0.2%; CDCA+LCA: 12±0.3%; NAC: 9±0.4%; CDCA+NAC: 15±1%; LCA+NAC: 7±0.1%; CDCA+LCA+NAC: 14±0.2%, p>0.05, n=6).In terms of pore function, NAC neither altered CDCA±LCA‐induced decreases in TER (% decrease compared to DMSO; 1H CDCA: 83±7; CDCA+NAC: 84±5; CDCA+LCA: 80±3; CDCA+LCA+NAC: 81±7, n=3) nor ion selectivity (DΨ, 25 min, in mV; DMSO: 8 ± 1, CDCA: − 7 ± 2, LCA: 8 ± 1; CDCA+LCA: −5 ± 2, NAC: 7±1, CDCA+NAC: −6±1, LCA+NAC: 9±2, CDCA+LCA+NAC: −5±0.4, n≥3). In contrast, NAC altered the leak function over time causing ~65% reduction in CDCA‐induced F10D flux (18 H, μg of F10D: Control: 8±1; CDCA: 120±2; NAC: 7±0.1; CDCA+NAC: 41±4; p<0.05, n=3). LCA decreased CDCA's effect by 60%, which was further attenuated by NAC (18 H (μg): LCA: 13±2; NAC+LCA: 6±0.2; CDCA+LCA: 50±3; NAC+CDCA+LCA: 17±1; p<0.05, n≥3). We demonstrate that ROS plays a role in CDCA‐induced TJ dysfunction by altering leak, but not pore function in T84 cells. Equally important, LCA may be acting as an antioxidant as it reverses CDCA action on H2O2 release and the leak function which restores barrier integrity. Understanding the role of ROS in BA action can lead to novel therapeutic strategies for inflammation‐associated diarrheas.Support or Funding InformationThis was supported by APS‐UGSRF to HS, NSF‐MRI: DBI‐1427937 and Institutional Funds: BU to JS, UIC to MR.