Influence of natural substances on mediators of intestinal inflammation "in vitro"

Abstract

Natural plant extracts and their isolated phenolic substances have been shown to exhibit several functions including the anti-inflammatory activity in various tissues. EGCG, curcumin and resveratrol have been identified as possible anti-inflammatory agents while little is known about extracts of Boswellia serrata (BS) and Rosmarinus officinalis (RO). In this thesis project, several methods were applied in order to identify the anti-inflammatory effects of aforementioned substances and extracts in “inflamed” intestinal epithelial cells. The colon cancer cell lines, HT29 and T84, were stimulated with TNF-α in the presence of various concentrations of EGCG, BS, RO, curcumin and resveratrol. Their effects were analyzed at different levels: (1) on the expression of multiple genes involved in inflammatory responses; (2) on the generation of metabolites such as PGE2, and production of chemokines (e.g., IL-8 and MIP-3α); (3) and on the protein expression. The expression of genes was determined by quantitative RT-PCR and microarray technology. The production of IL-8, MIP-3α and PGE2 were measured by ELISA. The expression of proteins belonging to the NF-κB pathway was analyzed by immunoblots. In addition, NF-κB DNA binding was carried out using EMSA. Treatment of activated cells with EGCG, BS, RO, curcumin and resveratrol reduced the expression of most genes related to the inflammatory response of HT29 and T84 cells. Furthermore, EGCG, curcumin and resveratrol diminished the generation of PGE2 in HT29 cells. In addition, the secretion of IL-8 and MIP-3α were dose-dependently inhibited by candidate compounds in HT29 cells. EGCG reduced the production of these chemokines also in T84 cells while curcumin and resveratrol only diminished MIP-3α synthesis. Moreover, the expression of IκBα and NF-κB p65 were analyzed in HT29 cells. BS was not able to prevent IκBα degradation and subsequent NF-κB translocation whereas RO interfered with IκBα degradation, however, it did not impede the activation of NF-κB. Additionally, RO reduced NF-κB DNA binding tested by EMSA. Collectively, the presented data show that phenolic substances or whole plant extracts have the capacity to attenuate inflammatory features by modulating genes involved in inflammatory responses; by reducing the chemoattraction of immunocytes through the inhibition of IL-8 and MIP-3α; and by affecting specific pathways involved in inflammation such as the NF-κB. Furthermore, phenolic compounds might have a preventive and/or therapeutic utility in chronic inflammation such as IBD, and therefore, they should be further analyzed in in vivo studies to consolidate these findings.