The Role of β-Carotene in Colonic Inflammation and Intestinal Barrier Integrity

Abstract

Background: Carotenoids are naturally occurring pigments accounting for the brilliant colors of fruits and vegetables. They may display antioxidant and anti-inflammatory properties in humans besides being precursors to vitamin A. There is a gap of knowledge in examining their role within colonic epithelial cells. We proposed to address this research gap by examining the effects of a major dietary carotenoid, β-carotene, in the in vitro epithelial cell model. Methods: We examined the function of β-carotene in the lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway. We conducted western blotting assays to evaluate expressions of TLR4 and its co-receptor, CD14. We also examined NF-κB p65 subunit protein levels in the model system. Furthermore, we studied the impact of β-carotene on the tight junction proteins, claudin-1, and occludin. We further carried out immunocytochemistry experiments to detect and visualize claudin-1 expression. Results: β-Carotene reduced LPS-induced intestinal inflammation in colonic epithelial cells. β-Carotene also promoted the levels of tight junction proteins, which might lead to enhanced barrier function. Conclusions: β-Carotene could play a role in modulating the LPS-induced TLR4 signaling pathway and in enhancing tight junction proteins. The findings will shed light on the role of β-carotene in colonic inflammation and also potentially in metabolic disorders since higher levels of LPS might induce features of metabolic diseases.

Keywords: colonic epithelial cells; colonic inflammation; tight junctions; vitamin A; β-carotene.

Figure 1

Inhibitory effect of β-carotene on LPS-induced inflammatory markers. (A–D) Graphical representations of changes in pro-inflammatory cytokines detected via ELISA: (A) Cytokine levels of IL-6 in HT-29 cell culture supernatants; (B) IL-6 levels in HT-29 whole cell lysates (WCL); (C) IL-1β levels in WCL; (D) TNF-α levels in WCL. (E) Western blot of CRP expression in WCL. Five treatment doses of β-carotene (1, 10, 100 nM, 1, 10 μM). Four replicates were used for statistical analysis in ELISA and western blot. Values are means ± SEMs. *Significance at p < 0.05 for comparison with the “No LPS” group, **significance at p < 0.01 for comparison with the “No LPS” group. ^#significance at p < 0.05 for comparison with the “LPS” group, ^##significance at p < 0.01 for comparison with the “LPS” group.

Figure 2

β-Carotene treatments induce tight junction proteins. (A,B) Western blot membranes with tight junction protein expression of (A) claudin-1 and (B) occludin. Positive control (+CTRL), obtained from A431 cells. (C,D) Fold changes in mRNA levels of (C) claudin-1 and (D) occludin generated from qPCR. Five treatment doses of β-carotene (1, 10, 100 nM, 1, 10 μM). Four replicates used for statistical analysis of western blot and qPCR. Values are means ± SEMs. *significance at p < 0.05 for comparison with the “No LPS” group. ^#significance at p < 0.05 for comparison with the “LPS” group, ^##significance at p < 0.01 for comparison with the “LPS” group.

Figure 3

β-Carotene promotes claudin-1 protein expression. Confocal microscope images of claudin-1 protein expression. (DAPI panel) HT-29 cell nuclei indicated via DAPI staining. (Claudin-1 panel) Protein expression of claudin-1 expression portrayed by fluorescent staining via Alexa Fluor 488. Panels are merged in the final panel for visualization of localization. Five treatment doses of β-carotene (1, 10, 100 nM, 1, 10 μM). Scale bar of 20 μm was included for size comparison.

Figure 4

ATRA minimally affects LPS-induced inflammatory markers. (A–C) Graphical representations of fold changes in pro-inflammatory cytokines detected via ELISA: (A) Cytokine levels of IL-1β in HT-29 cell culture supernatants; (B) IL-1β levels in HT-29 whole cell lysates (WCL); (C) TNF-α levels in WCL. (D) Western blot of CRP expression in WCL. Five treatment doses of ATRA (1, 10, 100 nM, 1, 10 μM). Four replicates were used for statistical analysis in ELISA and western blot. Values are means ± SEMs. *significance at p < 0.05 for comparison with the ‘No LPS’ group, **significance at p < 0.01 for comparison with the “No LPS” group. ^#significance at p < 0.05 for comparison with the “LPS” group.

Figure 5

ATRA is not as effective as in causing similar tight junction expression trends as β-carotene. (A,B) ATRA treatments on tight junction proteins or mRNA. (A) Claudin-1 WCL protein in western blot (B) and claudin-1 mRNA expressions in qPCR. Five treatment doses of ATRA (1, 10, 100 nM, 1, 10 μM). Four replicates were used for statistical analysis in western blot and PCR. Values are means ± SEMs. *Significance at p < 0.05 for comparison with the “No LPS” group.

Figure 6

β-Carotene inhibits the TLR4 and NF-κB pathway. (A–C) Western blots of β-carotene treated HT-29 WCL-graphical fold changes of (A) TLR4 expression, (B) CD-14 expression, and (C) NF-κB p65. Control images were re-used for illustrative purposes in (A,C). (D) Nuclear protein extractions from β-carotene treated samples-western blot and graphical fold changes of (D) NF-κB p65 expression. (E,F) Western blots of ATRA treated HT-29 WCL proteins-graphical fold changes of (E) TLR4 expression and (F) NF-κB p65. Four replicates were used for statistical analysis in western blot. Values are means ± SEMs. *Significance at p < 0.05 for comparison with the “No LPS” group, **significance at p < 0.01 for comparison with the “No LPS” group. ^#significance at p < 0.05 for comparison with the “LPS” group, ^##significance at p < 0.01 for comparison with the “LPS” group.

Figure 7

Graphical representation of proposed pathway in which β-carotene impacts colonic epithelial cells. β-Carotene effectively inhibits the LPS-induced inflammatory response within HT-29 colonic epithelial cells by downregulating TLR4, which may block the activation and nuclear translocation of the NF-κB p65 subunit and related downstream signaling. Thus, the release of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) is compromised. The β-carotene induced downregulation of TLR4 may also alleviate the inhibition of its co-receptor CD14. Enhanced CD14 levels by β-carotene may upregulate tight junction proteins (claudin-1 and occludin), leading to decreased intestinal permeability and enhanced barrier integrity.