Fig. 2. Conjugated BAs protect epithelial monolayers from unconjugated BA-induced permeability.

(A) Cecal extracts from CDAHFD-fed rats at early time points caused increased epithelial permeability. Caco-2 monolayer permeability measured by FITC-dextran passage into the basolateral chamber after exposure to indicated purified cecal extracts over 12 hours. DMSO, dimethyl sulfoxide. (B) Forty-eight–hour and 1-week CDAHFD BA pool induced significantly higher permeability than the HFD BA pool. (C) Conjugated BAs alone did not increase Caco-2 monolayer permeability at concentrations found in the cecum of CDAHFD rats, and when combined with unconjugated BAs, conjugated BAs are protected against unconjugated BA-induced permeability. (D) Schematic of in vitro permeability experiment. (E) Conjugated BAs protected against unconjugated BA-induced Caco-2 monolayer permeability at physiologic concentrations (n = 6 per group). (F) Conjugated BAs protected epithelial monolayers from unconjugated BA-induced cell death. Cell viability of Caco-2 cells measured by MTT assay (n = 6 per group). (G) Conjugated BAs protected the physical integrity of epithelial monolayers from unconjugated BA-induced damage. Representative light microscopy images of hematoxylin and eosin (H&E)–stained Caco-2 monolayers after exposure to BA pools. Scale bars, 20 μm. (H) Conjugated BAs prevented the development of unconjugated BA-induced tight junction dilatation. Representative transmission electron microscopy (TEM) images of Caco-2 cells from transwells after exposure to BA pools at indicated concentrations. The white arrow points to tight junction dilatation. Scale bars, 500 nm. See the Supplementary Materials for additional representative TEM images. Unless otherwise specified, all experiments were performed in biological triplicate. See Materials and Methods for statistical analyses. *P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001. Bars represent means ± SEM.