Inhibition of ERK1/2 worsens intestinal ischemia/reperfusion injury.
Inhibition of ERK1/2 worsens intestinal ischemia/reperfusion injury.
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DOI：
10.1371/journal.pone.0076790
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Kozar RA
中科院分区：
文献类型：
--
作者：
Ban K;Peng Z;Kozar RA
细胞外信号调节蛋白激酶（ERK）在肠道缺血/再灌注（I/R）损伤中的作用尚未得到充分研究。本研究的目的是在肠道 I/R 损伤的体外和体内模型中检查 ERK 通路抑制的效果。使用特异性抑制剂 U0126 抑制缺氧/复氧条件下的肠上皮细胞以及经历 1 小时肠缺血随后 6 小时再灌注的小鼠中的 ERK1/2 活性。在体外，通过MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑）测定评估细胞增殖，通过DNA片段化评估细胞凋亡，并使用体外肠伤口愈合模型评估细胞迁移。还用 p70S6K 质粒转染细胞，并类似地分析过表达的影响。在体内，测量了U0126对肠细胞增殖和凋亡、肠通透性、肺和肠中性粒细胞浸润和损伤以及血浆细胞因子水平的影响。 U0126 后还评估了生存率。通过蛋白质印迹法测量 p70S6 激酶 (p70S6K) 的活性。在体外，U0126 对 ERK1/2 的抑制显着降低了细胞增殖和迁移，但增强了细胞凋亡。 p70S6K 的过表达促进细胞增殖并减少细胞凋亡。在体内，U0126显着增加肠道细胞凋亡并减少细胞增殖，增加肠道通透性、肠道和肺部中性粒细胞浸润和损伤，以及全身促炎细胞因子TNF-α、IL-6和IL-1β。 U0126 也显着增加了死亡率。 U0126 对 ERK1/2 的抑制也消除了体外和体内模型中 p70S6K 的活性。 U0126 对 ERK1/2 的药理学抑制会加重肠道 IR 损伤。有害作用至少部分是通过抑制 p70S6K 介导的，p70S6K 是哺乳动物雷帕霉素途径靶标的主要效应子。
The role of extracellular signal-regulated protein kinase (ERK) in intestinal ischemia/reperfusion (I/R) injury has not been well investigated. The aim of the current study was to examine the effect of inhibition of the ERK pathway in an in vitro and in vivo model of intestinal I/R injury. ERK1/2 activity was inhibited using the specific inhibitor, U0126, in intestinal epithelial cells under hypoxia/reoxygenation conditions and in mice subjected to 1 hour of intestinal ischemia followed by 6 hours reperfusion. In vitro, cell proliferation was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, apoptosis by DNA fragmentation, and migration using an in vitro model of intestinal wound healing. Cells were also transfected with a p70S6K plasmid and the effects of overexpression similarly analyzed. In vivo, the effects of U0126 on intestinal cell proliferation and apoptosis, intestinal permeability, lung and intestinal neutrophil infiltration and injury, and plasma cytokine levels were measured. Survival was also assessed after U0126. Activity of p70S6 kinase (p70S6K) was measured by Western blot. In vitro, inhibition of ERK1/2 by U0126 significantly decreased cell proliferation and migration but enhanced cell apoptosis. Overexpression of p70S6K promoted cell proliferation and decreased cell apoptosis. In vivo, U0126 significantly increased cell apoptosis and decreased cell proliferation in the intestine, increased intestinal permeability, intestinal and lung neutrophil infiltration, and injury, as well as systemic pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β. Mortality was also significantly increased by U0126. Inhibition of ERK1/2 by U0126 also abolished activity of p70S6K both in vitro and in vivo models. Pharmacologic inhibition of ERK1/2 by U0126 worsens intestinal IR injury. The detrimental effects are mediated, at least in part, by inhibition of p70S6K, the major effector of mammalian target of rapamycin pathway.
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