不同免疫抑制剂经核因子-κB信号途径调节肠上皮细胞防御素的表达

目的探讨4种不同免疫抑制剂（他克莫司、环孢素A、雷帕霉素、雷公藤多甙）对Caco-2和HT-29细胞人β防御素1和2（hBD-1／2）的表达影响及机制。方法通过实时定量聚合酶链反应（Real-timePCR）和Western blot检测终质量浓度为10μmol／L的4种不同免疫抑制剂对白细胞介素-1p（IL-1p，20μg／L）诱导的人肠上皮细胞hBD-1／2表达的影响，应用凝胶电泳迁移率实验法（EMSA）和酶联免疫吸附试验（ELISA）检测Caeo-2细胞核因子-κB（NF—κB）的表达。结果肠上皮细胞组成性表达hBD-1；他克莫司、环孢素A和雷帕霉素组在Caco-2细胞中hBD-2mRNA促进率分别为（20．58±1．02）％、（139．49±6．97）％和（425．97±21．29）％，雷公藤多甙组hBD-2mRNA抑制率为（48．59±2．42）％（P〈0．05）。HT-29细胞中上述指标分别为（62．52±3．25）％、（20．56±1．10）％、（46．79±2．28）％和（82．73±7．88）％（P〈0．05）。Caco-2细胞中hBD-2蛋白的表达与mRNA的表达趋势一致。EMSA和ELISA检测活化的NF-κB显示他克莫司、环孢素A和雷帕霉素组促进NF—κB的活化，雷公藤多甙组抑制NF—κB的活化。并与Ser276磷酸化有关。结论他克莫司、环孢素A和雷帕霉素促进人肠系细胞在IL-1β诱导下表达hBD-2，雷公藤多甙抑制hBD-2的表达。其机制与NF—κB信号途径有关。

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.