Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages

Abstract

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150-450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1β, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/μL for IL1R1 and COX-2 (p < 0.01) and 300 ng/μL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

Keywords: gene expression; interleukin; permeability; pro-inflammatory cytokine; transepithelial electrical resistance.

Figure 1

The scheme of experiments performed on Caco-2 cells (A) and Caco-2/THP-1 or Caco-2 macrophages co-culture models (B,C).

Figure 2

The level of IL-1β (A), IL-6 (B), IL-8 (C) and TNF-α (D) after incubation of Caco-2 cells with LPS (1 µg/mL), osthole (150–450 ng/mL) and its mixtures. Horizontal line shows mean and bars depict standard error of mean. Statistically significant differences in comparison to control (###—p < 00.01, ####—p < 0.0001) and to cells treated with LPS (*—p < 0.05, ***—p < 0.001, ****—p < 0.0001) are marked.

Figure 3

The level of IL-1β (A), IL-6 (B), IL-8 (C) and TNF-α (D) in Caco-2/THP-1 co-culture after incubation with LPS (1 µg/mL), osthole (150–450 ng/mL) and its mixtures. Horizontal line shows mean and bars depict standard error of mean. Statistically significant differences in comparison to control (#—p < 0.05, ##—p < 0.01, ###—p < 0.001, ####—p < 0.0001) and to cells treated with LPS (**—p < 0.01, ***—p < 0.001, ****—p < 0.0001) are marked.

Figure 4

The level of IL-1β (A), IL-6 (B), IL-8 (C) and TNF-α (D) in Caco-2/macrophages co-culture after incubation with LPS (1 µg/mL), osthole (150–450 ng/mL) and its mixtures. Horizontal line shows mean and bars depict standard error of mean. Statistically significant differences in comparison to control (#—p < 0.05, ##—p < 0.01, ###—p < 0.001, ####—p < 0.0001) and to cells treated with LPS (*—p < 0.05, **—p < 0.01, ***—p < 0.001, ****—p < 0.0001) are marked.

Figure 5

Gene expression level after Caco-2 monoculture (A), Caco-2/THP-1 (B) and Caco-2/macrohpages (C) co-cultures incubation with LPS (1 µg/mL) and with LPS and different concentrations of osthole (150–450 ng/mL). Horizontal line shows mean and bars depict standard error of mean. Gene expression levels were scaled to the control sample (expression level = 1). Statistically significant differences in comparison to control (#—p < 0.05, ##—p < 0.01, ###—p < 0.001, ####—p < 0.0001) and to cells treated with LPS (*—p < 0.05, **—p < 0.01, ***—p < 0.001, ****—p < 0.0001) are marked.

Figure 6

Effects of LPS, osthole (A) and its mixtures (B) on transepithelial electrical resistance in Caco-2 monolayer). Horizontal line shows mean and bars depict standard error of mean. Statistically significant differences in comparison to control are marked (*—p < 0.05, **—p < 0.01, ****—p < 0.0001).

Figure 7

The scheme of LPS-induced inflammatory response.