CRF通过CRF2受体介导肠上皮细胞中TLR4的表达

目的:研究促肾上腺皮质释放因子(CRF)介导肠上皮细胞中Toll样受体4(toll-like receptor4,TLR4)的表达,并探讨其可能通过的受体途径.方法:常规培养人结肠上皮细胞株HT-29细胞,将HT-29细胞分为正常对照组(不加刺激剂),脂多糖(lipopolysaccharide,LPS)刺激组(LPS20g/L刺激24h),促肾上腺皮质释放因子(corticotrophin-releasing factor,CRF)刺激组(CRF20g/L刺激24h),CRF+LPS刺激组(预先CRF20g/L刺激12h,更换细胞液后再与LPS20g/L刺激12h),CRF+Antalarmin 组(CRF与Antalarmin20g/L共刺激24h),CRF+LPS+Antalarmin组(CRF与Antalarmin20g/L共刺激12h后再以LPS刺激12h),CRF+Astressin2B组(CRF与Astressin2B20g/L共刺激24h),CRF+LPS+Astressin2B组(CRF与Astressin2B20g/L共刺激12h后再以LPS刺激12h).刺激结束后,收取各组HT-29细胞,RT-PCR法和免疫印迹法检测各组上皮细胞中TLR4mRNA和蛋白的表达.ELISA法检测各组细胞上清液中IL-8的表达.结果:CRF可以诱导人结肠上皮细胞株HT-29细胞中TLR4表达导致IL-8分泌增多(P<0.05),C R F1受体拮抗剂不能有效地阻滞C R F对TLR4的诱导(P>0.05,CRF+LPS组vs CRF组),CRF2受体拮抗剂可阻滞CRF对TLR4的诱导(P<0.05,CRF+LPS组vs CRF组).结论:CRF通过CRF2受体通路介导肠上皮细胞中TLR4的表达.

Activation of TLR4 by CRF in human intestinal epithelial cells is mediated by the CRF2 receptor

AIM:To investigate the effect of corticotrophinreleasing factor(CRF) on the expression of tolllike receptor 4(TLR4) in human intestinal epithelial cell line HT-29.METHODS:HT-29 cells were divided into eight groups:non-treated group,LPS group(treated with 20 g/L LPS for 24 h),CRF group(treated with 20 g/L CRF for 24 h),LPS plus CRF group(pretreated with 20 g/L CRF for 12 h and then treated with 20 g/L LPS for 12 h),Astressin 2B plus CRF group(pretreated with 20 g/L Astressin 2B for 12 h and then treated with 20 g/L CRF),Antalarmin plus CRF group(pretreated with 20 g/L Antalarmin for 12 h and then treated with 20 g/L CRF),Astressin 2B plus LPS group(pretreated with 20 g/L Astressin 2B for 12 h and then treated with 20 g/L LPS),and Antalarmin plus LPS group(pretreated with 20 g/L Antalarmin for 12 h and then treated with 20 g/L LPS).The expression of TLR4 mRNA and protein was detected by reverse transcriptionpolymerase chain reaction(RT-PCR) and Western blotting,respectively.The secretion of interleukin-8 in the culture supernatants was determined by enzyme-linked immunosorbent assay(ELISA).RESULTS:CRF could induce the expression of TLR4 in HT-29 cells and result in increased interleukin-8 secretion(P < 0.05).CRFR2 antagonist Astressin 2B inhibited the expression of LR4(P < 0.05,CRF+LPS group vs CRF group),while CRF1 antagonist Antalarmin had no significant effect on the expression of TLR4(P > 0.05,CRF+LPS group vs CRF group).CONCLUSION:The induction of TLR4 expression by CRF in human intestinal epithelial cells is mediated by the CRF2 receptor.