Heparin-binding EGF-like growth factor down regulates proinflammatory cytokine-induced nitric oxide and inducible nitric oxide synthase production in intestinal epithelial cells
Heparin-binding EGF-like growth factor down regulates proinflammatory cytokine-induced nitric oxide and inducible nitric oxide synthase production in intestinal epithelial cells
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DOI：
10.1006/niox.2001.0393
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发表时间：
2002-03-01
影响因子：
3.9
通讯作者：
Besner, GE
中科院分区：
文献类型：
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作者：
Lara-Marquez, ML;Mehta, V;Besner, GE
我们以前已经表明，肝素结合EGF样生长因子（HB-EGF）保护肠上皮细胞（IEC）的坏死和凋亡在体外和肠缺血/再灌注损伤在体内，然而，HB-EGF细胞保护的机制尚不清楚。诱导型一氧化氮合酶和一氧化氮的过度产生与几种形式的缺血/再灌注损伤的发病机制有关。因此，我们研究了HB-EGF是否可以下调促炎性精氨酸诱导的肠上皮细胞在体外的iNOS和NO的生产。将DLD-1人肠上皮细胞暴露于促炎细胞因子白细胞介素-1 β（IL-1 β）（20 ng/ml）和干扰素-γ（IFN-γ）（10 ng/ml）以刺激iNOS诱导和NO产生。在暴露于细胞因子之前或暴露于细胞因子的情况下，用HB-EGF（0-100 ng/ml）处理细胞，并在24和48小时后收获细胞和上清液。通过化学发光法测定上清液中累积的NO。从细胞裂解液中提取总RNA，采用实时逆转录-聚合酶链反应（RT-PCR）进行iNOS mRNA定量，并从细胞裂解液中提取总蛋白，用于检测iNOS蛋白。HB-EGF以剂量依赖性方式显著降低精氨酸诱导的NO产生，并且NO减少与mRNA和蛋白水平上的iNOS抑制相关。虽然细胞因子暴露导致这些细胞中iNOS mRNA表达显著增加（109 +/-9倍），但HB-EGF使iNOS表达减少5.7倍（P < 0.05）。这些结果表明，HB-EGF可能发挥其细胞保护作用，部分，通过下调iNOS和NO的生产，并提供了进一步的理由HB-EGF在体内肠缺血/再灌注损伤的治疗中的作用的额外测试。(C)2001 Elsevier Science（美国）。
We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) protects intestinal epithelial cells (IEC) from necrosis and apoptosis in vitro and from intestinal ischemia/reperfusion injury in vivo; however, the mechanisms of HB-EGF cytoprotection are unclear. Overproduction of iNOS and NO have been implicated in the pathogenesis of several forms of ischemia/reperfusion injury. We therefore studied whether HB-EGF could down-regulate proinflammatory cytokine-induced iNOS and NO production in intestinal epithelial cells in vitro. DLD-1 human intestinal epithelial cells were exposed to the proinflammatory cytokines interleukin-1beta (IL-1beta) (20 ng/ml) and interferon-gamma (IFN-gamma) (10 ng/ml) to stimulate iNOS induction and NO production. Cells were treated with HB-EGF (0-100 ng/ml) either before or with cytokine exposure, and cells and supernatants were harvested 24 and 48 h later. Accumulated NO was measured in supernatants by chemiluminescence. Total RNA was extracted from cell lysates for iNOS mRNA quantification using real-time reverse transcription-polymerase chain reaction (RT-PCR), and total protein was extracted from cell lysates for detection of iNOS protein. HB-EGF significantly decreased cytokine-induced NO production in a dose dependent manner, and NO reduction was associated with iNOS suppression at both the mRNA and protein levels. While cytokine exposure resulted in a significant increase in iNOS mRNA expression in these cells (109 +/- 9 fold), HB-EGF reduced iNOS expression by 5.7-fold (P < 0.05). These results suggest that HB-EGF may exert its cytoprotective effects, in part, by down-regulating iNOS and NO production, and provides further rationale for additional testing of the effects of HB-EGF in the treatment of intestinal ischemia/reperfusion injury in vivo. (C) 2001 Elsevier Science (USA).