Effect of different immunosuppressive agents on defensin expression of intestinal epithelial cells via nuclear factor-κB signaling pathways

Abstract

Objective To investigate the effect of tacrolimus (FK506),cyclosporin A (CsA),rapamycin (Rapa) and Glucosida Tripterygii TOTA (GTT) on human β-defensin (hBD-1 and hBD-2) expression in intestinal epithelial cells (Caco-2 and HT-29 cells).Methods Quantitative real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were used to detect the effects of the final concentration of 10 μmol/L of FK506,CsA,Rapa and GTT on hBD-1 and hBD-2 expression in human intestinal epithelial cells stimulated by interleukin (IL)-1 β (20 μg/L).Nuclear factor-κB (NF-κB)binding activity in the Caco-2 cells was examined by using electrophoretic mobility shift assay (EMSA) and an enzyme linked immunosorbent assay (ELISA)-based assay with immobilized oligonucleotide.Results As compared with the IL-1β alone,the addition of FK506,CsA or Rapa in the culture medium in Caco-2 cells significantly increased the mRNA level of hBD-2 by (20.58 ± 1.02)％,(139.49 ± 6.97)％ and (425.97 ± 21.29)％ respectively (P ＜0.05).Meanwhile,the mRNA level of hBD-2 was decreased by (48.59 ± 2.42)％ in Caco-2 cells cultured with GTT (P＜ 0.05).In HT-29 cells,the value was (62.52±3.25)％,(20.56±1.10)％,(46.79 ±2.28)％ and (82.73 ±7.88)％ respectively (P＜0.05).Western blotting analysis revealed that the same results.However,GTT attenuated the expression of hBD-2.As compared with the control group,either FK506,CsA and rapamycin promnoted the activation of NF-κB,but GTr decreased the activation of NF-κB (P ＜ 0.05),which was related to the phosphorylation of Ser276.Conclusion The results presented in this paper show that FK506,CsA and Rapa activate NF-κB in intestinal epithelial cells,resulting in efficient induction of hBD-2 production in vitro. Key words: Intestinal epithelial cells; Defensin; Immunosuppressant; Nuclear factor-κB