The Study On Expression And Regulation Of TLR4 And β-defensin 2 In Colonic Epithelium Of Colitis

Effects of TNF-α, IL-1β, IFN-γ and LPS on the Expression and Regulation of TLR4 and HBD2 in HT-29 CellObjectivesThe normal intestinal epithelium serves as a physical barrier to limit access of enteric microbes. Intestinal epithelial cells (IEC) express low levels of TLR4 and MD-2 and are lipopolysaccharide (LPS)-unresponsive. At the same time, IEC contribute to innate host defense by producing effector molecules against luminal microbes. Antimicrobial peptides, such as the human β-defensin 2(HBD2) is one kind of effector molecules, the expression of which is regulated by bacterial infection or proinflammatory agonists. In this study, HT-29 intestinal epithelial cell line was stimulated by cytokines of TNF-α, IL-1β, IFN-γ and LPS in order to investigate expressions of TLR4, MD-2 and HBD2 by reverse transcription-PCR and western blot and to characterize their correlation with the NF-κB transcriptional activation.Materials and Methods1 HT-29 cells were divided into 8 groups. Each were treated with RPMI1640, TNF-α (20ng/ml), IL-1β(20ng/ml), IFN-γ(20ng/ml), LPS (50ng/ml), TNF-α(20ng/ml)+ LPS (50ng/ml) ,IL-1β(20ng/ml) +LPS (50ng/ml) and IFN-γ (20ng/ml) + LPS (50ng/ml) separately.2 Supernatants of each group were harvested to measure IL-8 by ELISA.3 The expressions of TLR4, MD2 and HBD2mRNA were assayed by RT-PCR.4 The expressions of TLR4 and NF-κB protein of each group were determined by western blot.