Probiotics Play a Role in Alleviating Bile Acid (BA)‐induced Apoptosis and Oxidative Stress in Human Colonic T84 Cells
Authors: Mehmood Daniyal , Maestas Jade , Ala Fatima , Poch Mark , Sarathy Jayashree 
Published: May 2022

BA‐malabsorption diarrhea (BAD) has been identified in one‐third of patients with inflammatory bowel disease (IBD) and affects >200 million people worldwide. Probiotic supplements are used to promote gut health, restoring microbiome and bile acid composition in these patients. We have previously reported the yin and yang in bile acid (BA) action, wherein the primary BA, chenodeoxycholic acid (CDCA; 500μM), altered tight junctions (TJ) and increased paracellular permeability in T84 cells, while its secondary BA, lithocholic acid (LCA; 50μM), did not. High colonic concentrations (>500μM) of CDCA have been reported in patients with IBD which can modify the gut microflora. Here, we studied the effect of a probiotic supplement on BA action in T84 cells and hypothesized that probiotics could ameliorate BA‐induced apoptosis, oxidative stress and barrier disruption.To study the crosstalk between BA and microflora, probiotic strains in Up & Up extra strength supplement (30 billion CFUs) were grown in T84 epithelial cell culture media ± BA under anaerobic conditions at 37°C. Bacteria grew well in control media (CFU x 108: 6 ± 1) after 48h with Lactobacillus paracasei identified as the predominant species by 16S rRNA sequencing. Further, microbial growth was modified by BAs. 500μM CDCA caused a significant reduction in the growth rate (CFU x 108: 0.07± 0.3; p<0.001, n=4) with L. paracasei remaining predominant. 50μM LCA alone did not alter growth but reversed the effect of CDCA on bacterial growth (CFU x 108: LCA: 6 ± 0.3; CDCA+LCA: 6 ± 1, n=4). Interestingly, LCA ± CDCA changed the bacterial composition with L. rhamnosus becoming the prevalent species. Culture media ± BA was sterile filtered to remove bacteria and the conditioned media (CM) obtained was used in subsequent experiments.T84 cells were grown to confluency in 6‐well plates and treated overnight with CM± BA. Apoptosis (Annexin V, flow cytometry, fluorescence microscopy), cell death (lactate dehydrogenase (LDH) assay, colorimetry) and mitochondrial ROS (mROS, CellRox, flow cytometry) were measured.Flow cytometry and LDH assay revealed that CM caused a dose‐dependent increase in T84 cell apoptosis with 100% CM causing significant (35‐fold) T84 cell death (n=3). The optimal CM dilution with minimal cell death was identified to be 1:8. As seen previously, CDCA (500μM) caused a small increase in apoptosis, while LCA (50μM) did not; CM± LCA reversed CDCA’s effect on cell death (% cell death, CTRL: 10±3 vs CM: 9±4; LCA: 9±3 vs CM+LCA: 10±2; CDCA: 19±4 vs CM+CDCA: 6±3; CDCA+LCA: 16±6 vs CM+CDCA+LCA: 8±3, p<0.05, n=5). Similarly, CDCA increased mROS and this was attenuated by CM (CellRox+ cells, in %, DMSO: 8±1; CDCA: 18±1; CM: 9±1; CM+CDCA: 9±2, n≥3). Though LCA alone did not have an effect, CM+LCA attenuated CDCA‐induced mROS release (LCA: 9±1; CDCA+LCA: 15±1 CM+LCA: 11±1, CM+CDCA+LCA: 9±2; n=4). We demonstrate that BA altered bacterial growth and probiotic CM±LCA reverses CDCA‐induced apoptosis and oxidative stress in T84 cells. We are currently studying the effect of CM on BA‐induced increases in paracellular permeability and TJ dysfunction. Delineating the role played by probiotics in alleviating BAD and understanding the mechanism of action could lead to novel therapeutic strategies for inflammation‐associated diarrheas.