NF-κB inhibition enhances peroxynitrite-induced enterocyte apoptosis
NF-κB inhibition enhances peroxynitrite-induced enterocyte apoptosis
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DOI：
10.1006/jsre.2002.6423
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发表时间：
2002-07-01
影响因子：
2.2
通讯作者：
Ford, HR
中科院分区：
文献类型：
--
作者：
Potoka, DA;Upperman, JS;Ford, HR
背景。在坏死性小肠结肠炎和炎症性肠病等疾病中，一氧化氮和过氧亚硝酸盐（ONOO-）的持续过量产生可能通过诱导肠上皮细胞凋亡而促进肠道屏障衰竭。炎症期间，NF-kappaB 在肠道中上调，除了其促炎作用外，还可能上调保护性或抗凋亡因子，例如凋亡蛋白抑制剂 (LAPS)。我们之前已经证明，NF-κB 抑制会增加细胞因子诱导的肠上皮细胞凋亡；然而，NF-κB对ONOO-诱导的肠上皮细胞凋亡的影响尚不清楚。材料和方法。用腺病毒载体 AdIkappaB 或 AdlacZ 转染大鼠肠上皮细胞 (IEC-6)。 AdIkappaB 包含 IkappaB 的突变形式，其功能是 NF-kappaB 的超阻遏蛋白。然后用50μM ONOO-或分解的ONOO-处理细胞。然后通过流式细胞术用膜联蛋白 V-FITC 和碘化丙啶染色测定细胞凋亡。使用蛋白质印迹分析检查 Caspase 激活以及 IAP、Bcl-2、Bad 和 Bax 表达，并通过电泳迁移率变动测定 (EMSA) 测定 NF-kappaB 激活。结果。用 AdIkappaB 抑制 NF-kappaB 显着增强 ONOO 诱导的 IEC-6 细胞凋亡。根据 EMSA 测定，ONOO-治疗并未激活 IEC-6 细胞中的 NF-kappaB。未转染细胞、AdlacZ 转染细胞和 AdIkappaB 转染细胞之间的 IAP、Bcl-2、Bad 和 Bax 表达没有差异。 AdIkappaB 转染细胞中的基线 procaspase 3 激活增加。结论。 NF-kappaB 抑制增强 ONOO 诱导的肠细胞凋亡，表明 NF-kappaB 上调保护因子。该保护因子似乎不是 IAP 或 Bcl-2 家族成员，并且可能是组成型表达，因为 ONOO- 不会激活超过基线激活水平的 NF-κB。 (C) 2002 年爱思唯尔科学（美国）。
Background. Sustained overproduction of nitric oxide and peroxynitrite (ONOO-) in conditions such as necrotizing enterocolitis and inflammatory bowel disease may promote gut barrier failure by inducing enterocyte apoptosis. NF-kappaB is upregulated in the gut during inflammation and, in addition to its proinflammatory effects, may upregulate protective or antiapoptotic factors such as inhibitor of apoptosis proteins (LAPS). We have previously demonstrated that NF-kappaB inhibition increases cytokine-induced enterocyte apoptosis; however, the effect of NF-kappaB on ONOO--induced enterocyte apoptosis is unknown.Materials and methods. Rat intestinal epithelial cells (IEC-6) were transfected with the adenoviral vector AdIkappaB or AdlacZ. AdIkappaB contains a mutated form of IkappaB which functions as a superrepressor of NF-kappaB. Cells were then treated with 50 muM ONOO- or decomposed ONOO-. Apoptosis was then determined by flow cytometry with annexin V-FITC and propidium iodide staining. Caspase activation and IAP, Bcl-2, Bad, and Bax expression were examined using Western blot analysis, and NF-kappaB activation was determined via electrophoretic mobility shift assay (EMSA).Results. Inhibition of NF-kappaB with AdIkappaB significantly enhanced ONOO--induced apoptosis in IEC-6 cells. ONOO- treatment did not activate NF-kappaB in IEC-6 cells as determined by EMSA. There was no difference in IAP, Bcl-2, Bad, and Bax expression between nontransfected, AdlacZ-transfected, and AdIkappaB-transfected cells. Baseline procaspase 3 activation was increased in AdIkappaB-transfected cells.Conclusions. NF-kappaB inhibition enhances ONOO--induced enterocyte apoptosis, suggesting that NF-kappaB upregulates a protective factor. This protective factor does not appear to be an IAP or Bcl-2 family member and may be expressed constitutively, since ONOO- did not activate NF-kappaB over baseline levels of activation. (C) 2002 Elsevier Science (USA).