Study Of EGCG Suppresses Gastroesophageal Reflux Contents-induced NF-κB Activation In Normal Human Esophageal Epithelial Cells

Objective:Gastroesophageal reflux (GER) contents are powerful inducer of inflammatory responses resulting in disruption of major cellular pathways with transcriptional and genomic alterations driving the cells towards carcinogenesis. Most of the studies indicate that green tea polyphenol EGCG possesses antiinflammatory, antioxidant and chemopreventive effects. This study mainly to investigate the effect of mixed refluxate (acid, bile acids and trypsin) on expression of NF-κB signaling pathway in normal human esophageal epithelial cells and the effect of EGCG pretreatment of cells on activation of NF-κB induced by mixed refluxate.Methods:Human esophageal epithelial cells (HEEC) were incubated in a media containing different concentrations of EGCG (0,5,10,20μmol/L) for4hours and they were divided into experimental and control groups. The experimental group were acidified media (pH6.5) treated with chenodeoxycholic acid (200μmol/L) for12h and trypsin (10U/mL) for the final hour. The control group were incubated without exposure to gastroesophageal refluxate. NF-κB DNA-binding activity was examined by EMSA and intracellular level of NF-κB was evaluated using ELISA, and the intracellular NF-κB reporter gene activity was measured with application of the luciferase reporter gene assay, and the level of expression of NFKB/p65, p-NFκB/p65, Iκa, p-IκBα, p-IKKα and proinflammatory cytokines such as IL-6, IL-8, COX-2and iNOS proteins were examined by Western blot analysis. Result:GER contents exposure to HEEC can result in a significant increase in NF-κB DNA-binding activity and intracellular level of NF-κB was elevated. Treatment with GER contents result in significant enhancement of luciferase reporter expression compared to control and also lead to an decrease in the total cytosolic NF-icB/p65levels with a concomitant increase in nuclear NF-κB/p65levels. The levels of NF-icB/p65, IκBα and IKKα phosphorylation and NF-κB-regulated proteins IL-6, IL-8, COX-2and iNOS in HEEC were increased. EGCG pretreatment of cells result in a significant inhibition of the GER contents-induced increase in the expression of these proteins and lead to an increase in the total cytosolic NF-κB/p65levels with a concomitant decrease in nuclear NF-KB/p65levels in experimental groups. The differences of the level of NF-κB between the experimental group and the control group were statistically significant (P<0.01).Conclusions:The activation of NF-κB signaling pathway is involved in the effect on the inflammatory response of HEEC induced by GER contents. Green tea polyphenol EGCG can inhibit GER contents-induced NF-κB activation in HEEC and downregulate NF-KB-regulated proteins expression, which may contribute to its ability to suppress inflammation and prevent the occurrence of an inflammatory environment induced by GER contents.