Effects of corticotrophin-releasing factor on the Toll-like receptor 4/nuclear factor-rB signaling pathway in human intestinal epithelial cell line HT-29

Abstract

Objective To investigate the effect of corticotrophin-releasing factor (CRF) on the regulation of Toll-like receptor 4/NF-κB signaling pathway expression in human intestinal epithelial cell line HT-29. Methods HT-29 cells were divided into four groups, normal control group, LPS group (LPS 20 μg/ml stimulated for 24 h), CRF group (CRF 20 ng/ml stimulated 24 h) and CRF+ LPS group (CRF incubated for 12 h then changed to LPS for another 12 h). After stimulation, the expression of TLR4 mRNA of each group was examined by reverse transcriptase polymerase chain reaction (RT-PCR). Total cell protein were extracted and the expression of TLR4 and NFκB p65 at protein level were detected by western blotting.Cell culture supernatant was collected and the secretion of interleukin-8 was detected by enzyme-linked immunoasorbent assay (ELISA). Results The expression of TLR4 in LPS group at mRNA and protein level were 0.31±0.04 and 0.48±0.17,there was no significant difference compared with normal control group (0.28±0.02 and 0.45±0.12,t＝0.216 and 0.712 , P＞0.05 ). In CRF group which were 1.05±0.06 and1. 08±0.21, significantly higher than normal control group (t＝3.721 and 3.802, P＜0.05). In CRF+LPS group which were 1.68±0.05 and 1.81±0. 18,significantly higher than CRF group (t＝4. 816 and 3. 918, P＜0.05).The results of NF-κB p65 expression at protein level and interleukin-8 expression of cell culture supernatant were consistent with the results of TLR4 expression at mRNA and protein level.Conclusion CRF not only activate TLR4/NF-κB signaling pathway in human intestinal epithelial cell,but enhance the reaction of intestinal epithelial cell to LPS as well, which resulting in increased interleukin-8 secretion. Key words: Corticotrophin-releasing hormone; lipopolysaccharides; Toll-like receptor 4; NFkappa B; Colon; Epithelial cells; lnterleukin-8