Inhibition of low-grade inflammation by anthocyanins from grape extract in an in vitro epithelial-endothelial co-culture model

Abstract

Background: Anthocyanins (ACNs) are the most prevalent flavonoids in berries and their health promoting effects on vascular functions are still discussed. The aim of the present study was to identify the anti-inflammatory effect of ACNs on activated human umbilical vein endothelial cells (HUVECs) after their transport across an epithelial monolayer. Study design: We established a transwell epithelial-endothelial co-culture system with Caco-2/HT29-B6 cells mimicking the intestinal layer and HUVECs as endothelial cells mimicking the vascular layer. Caco-2 were seeded alone (100%) or together with HT29-B6 cells (10 and 20%) on transwell inserts in order to simulate different metabolization sides of the gut. ACNs as well as malvidin-3-glucoside (M3G) were applied to the luminal compartment of the transwell-system. Transport and degradation rates were determined by high performance liquid chromatography with ultraviolet detection (HPLC-UV) or by ultra-PLC coupled to mass spectrometry (UPLC-MS). After 4 hours incubation time, co-cultured HUVECs were used immediately (short-term incubation) or after 20 hours (long-term incubation). Thereafter, HUVECs were stimulated for 3 hours with 1 ng mL⁻¹ TNF-α to mimic a low-grade or 10 ng mL⁻¹ to mimic a high-grade inflammation. Afterwards, (1.) leukocyte adhesion, (2.) expression of cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) and (3.) cytokine expression and secretion (IL-6 and IL-8) were determined using flow cytometry and real-time PCR. Results: Degradation and incubation studies revealed that ACNs were differently degraded depending on the ACN structure and the seeding densities. Incubation of ACNs and M3G to Caco-2 cells (100%) led to a fast decrease, which was not observed when HT29-B6 cells were co-cultured (10 and 20%). Concomitantly, anti-inflammatory effects were only observed using 100% Caco-2 cells, whereas mixtures of Caco-2 and HT29-B6 cells failed to induce an effect. ACN extract and M3G significantly attenuated TNF-α-stimulated low-grade leukocyte adhesion, expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 and cytokine expression and secretion (IL-8 and IL-6) as well as NF-κB mRNA expression. No effects were observed with high TNF-α (10 ng mL⁻¹) or after short-term incubation (4 hours). Conclusions: ACNs in physiological concentrations reached the serosal compartment and reduced inflammation-related parameters, which were related to the initial steps during the pathogenesis of atherosclerosis.