Bridge the Gap – The Efficacy of β-Carotene in Immune Sensing of LPS and Intestinal Barrier Integrity in Colon Epithelial Cells

Abstract

\n \n \n Gastrointestinal (GI) disorders are causing significant global health care burden. Lipopolysaccharide (LPS), a structural component of Gram-negative bacteria, induces low-grade inflammation and disturbs GI homeostasis. While the anti-inflammatory function of β-Carotene (BC), a provitamin A carotenoid, has been explored in multiple systems, a limited information exists on the roles of BC in modulating LPS-induced inflammation in colonocytes. Therefore, we aimed to mechanistically investigate the role of BC in LPS-induced colonic inflammation and intestinal barrier dysfunction.\n \n \n \n Human colon epithelial cells (HT-29) were primed with interferon-γ at 50\xa0ng/mL for 12 hours, then treated with 1\xa0µg/mL LPS and BC at 1, 10, 100\xa0nM, and 1, 10 µM for 15 hours. Inflammatory cytokines were quantified with an ELISA assay. The fold change in mRNA levels was determined by qPCR. Changes in proteins of interest were evaluated using both Western blot and immunocytochemistry (ICC) assays.\n \n \n \n LPS-stimulated production of IL-6 and TNFα levels was inhibited by BC treatment at 10\xa0nM – 1\xa0μM; IL-1β in whole cell lysates and supernatant IL-6 levels were decreased by BC treatment at all dosages. LPS-induced elevation of C-reactive protein and cyclooxygenase-1 mRNA was reversed with 1\xa0μM and 10\xa0μM BC treatment. Claudin-1 and occludin are major tight junction proteins that contribute to maintaining colonic barrier integrity. Intriguingly, BC at 10\xa0nM – 1\xa0μM significantly enhanced claudin-1 and occludin mRNA and protein levels. The up-regulation of claudin-1 was also validated in the ICC assay. A further exploration of the underlying mechanisms showed that BC inhibited the LPS-activated TLR4-NF-κB p65 pathway to alleviate inflammation in the cells. CD14, a membrane protein expressed on the surfaces of epithelial cells, could restore the impaired intestinal barrier function. In this study, an increased CD14 protein level was found in BC-treated cells, which was correlated with the improved claudin-1 and occludin levels, indicating that BC may promote colonic barrier integrity via up-regulating CD14.\n \n \n \n BC plays a role in modulating LPS-induced TLR4 signaling pathway and enhancing gut barrier integrity. These novel findings will shed light on the role of BC in alleviating endotoxin-induced GI disorders.\n \n \n \n USDA.\n