Anti-inflammatory activity of plant sterols in a co-culture model of intestinal inflammation: focus on food-matrix effect

Abstract

This study investigates the gut anti-inflammatory activity of a plant sterol (PS) food supplement (PS-FS), alongside PS-enriched milk-based fruit beverage and PS-enriched rye bread. A co-culture model based on a dual-chamber system with differentiated intestinal-like Caco-2 cells (apical) and RAW264.7 macrophages (basolateral) was used. The bioaccessible fractions (BF) of the samples were obtained after INFOGEST 2.0 simulated gastrointestinal digestion. The BF were added to the apical part (diluted 1/20 v/v with culture medium to avoid cytotoxicity) for 90 min, followed by stimulation with lipopolysaccharide (LPS) (1 μg mL⁻¹, 24 h) on the basolateral side. The pharmacological interaction between samples and budesonide (1 μM, 90 min) was evaluated. Results indicate that PS-FS significantly attenuated LPS-induced secretion of IL-8 (28%) by Caco-2 cells, and TNF-α (9%) and IL-6 (54%) by RAW264.7 macrophages, whereas PS-enriched beverage and bread did not exhibit protective effects. Additionally, PS-FS demonstrated an improvement in oxidative status in Caco-2 cells, evidenced by reduced levels of reactive oxygen species (47%), iNOS protein expression (27%), and nitrite/nitrate secretion (27%). Mechanistically, PS-FS inhibited the NF-κB-COX-2-PGE₂ signaling pathway in macrophages, resulting in decreased NF-κB p65 nuclear translocation (39%), COX-2 protein expression (32%), and PGE₂ production (27%). Co-treatment with budesonide and PS-FS displayed an antagonistic effect (combination index 0.38–0.63). This study demonstrates the potent intestinal anti-inflammatory activity of a PS-FS, positioning it as a promising nutraceutical product for the management of inflammatory bowel diseases. However, the food matrix of the milk-based fruit beverage and rye bread appear to interfere with the anti-inflammatory activity of PS.