Assessing the Intestinal Permeability and Anti-Inflammatory Potential of Sesquiterpene Lactones from Chicory

Abstract

Cichorium intybus L. has recently gained major attention due to large quantities of health-promoting compounds in its roots, such as inulin and sesquiterpene lactones (SLs). Chicory is the main dietary source of SLs, which have underexplored bioactive potential. In this study, we assessed the capacity of SLs to permeate the intestinal barrier to become physiologically available, using in silico predictions and in vitro studies with the well-established cell model of the human intestinal mucosa (differentiated Caco-2 cells). The potential of SLs to modulate inflammatory responses through modulation of the nuclear factor of activated T-cells (NFAT) pathway was also evaluated, using a yeast reporter system. Lactucopicrin was revealed as the most permeable chicory SL in the intestinal barrier model, but it had low anti-inflammatory potential. The SL with the highest anti-inflammatory potential was 11β,13-dihydrolactucin, which inhibited up to 54% of Calcineurin-responsive zinc finger (Crz1) activation, concomitantly with the impairment of the nuclear accumulation of Crz1, the yeast orthologue of human NFAT.

Keywords: 11β,13-dihydrolactucin; 11β,13-dihydrolactucopicrin; NFAT; calcineurin; lactucin; lactucopicrin.

Figure 1

Chemical structure of tested sesquiterpene lactones (SLs). (1) Costunolide; (2) parthenolide; (3) lactucin; (4) lactucopicrin; (5) 11β,13-dihydrolactucin; (6) 11β,13-dihydrolactucopicrin; (7) 15-oxalyl-lactucin; (8) 15-oxalyl-lactucopicrin; (9) 15-oxalyl-11β,13-dihydrolactucin; (10) 15-oxalyl-11β,13-dihydrolactucopicrin.

Figure 2

Cytotoxic profile of parthenolide, costunolide, lactucopicrin, lactucin, 11β,13-dihydrolactucin and 11β,13-dihydrolactucopicrin in Caco-2 cells. Cytotoxicity was evaluated using the PrestoBlue^® cell viability assay, by testing the compounds between 12.5 and 500 μM. Data are presented as means ± SD, n = 3.

Figure 3

Transepithelial electrical resistance (TEER) of Caco-2 cells. TEER measurements were made before and after the 4 h permeability experiment and at the 24 h recovery period after the end of the assay. SLs were tested at a concentration of 10 μM and fluorescein at 2.7 μM. Data are presented as means ± SD, n = 3.

Figure 4

Cytotoxicity of chicory SLs in yeasts. Yeast cells were incubated with different concentrations (6.25–100 µM) of SLs to determine the highest nontoxic concentration. Cell viability for each compound was determined using the Cell Titer Blue assay. Data are presented as means ± SD, n = 3.

Figure 5

Anti-inflammatory potential of 11β,13-dihydrolactucin as demonstrated by the inhibition of the calcineurin-Crz1 pathway in S. cerevisiae. Statistical differences are noted as ** p < 0.01, *** p < 0.001 relative to the activated control (cells treated with MnCl₂).

Figure 6

11β,13-dihydrolactucin inhibits Crz1-GFP nuclear accumulation. YAA3 cells were first treated or not with 3.6 µM of 11β,13-dihydrolactucin, challenged with 3 mM MnCl₂ and Crz1-GFP subcellular distribution was monitored using fluorescence microscopy. The immunosuppressant FK506 was used as a positive control. Approximately 1800 cells for each condition were counted. Representative imagens are shown, and the values represent the mean of percentage of nuclear Crz1-GFP ± SD of three biological replicates. Statistical differences are denoted as *** p < 0.001 relative to activated cells.

Figure 7

11β,13-dihydrolactucin modulates the downregulation of endogenous Crz1 target genes. BY4741 cells were subjected to 3.6 µM of 11β,13-dihydrolactucin, induced with 3 mM MnCl₂ and the mRNA levels of Crz1 target genes PMR1 and GSC2 were assessed by qPCR. The immunosuppressant FK506, which inhibits calcineurin and prevents Crz1 activation, was used as a positive control. The values represent the mean ± SEM of at least three biological replicates, ** p < 0.01, *** p < 0.001.