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首页> 外文期刊>American Journal of Physiology >Platelet-activating factor-induced apoptosis is blocked by Bcl-2 in rat intestinal epithelial cells.
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Platelet-activating factor-induced apoptosis is blocked by Bcl-2 in rat intestinal epithelial cells.

机译：血小板活化因子诱导的凋亡被大鼠肠上皮细胞中的Bcl-2阻断。

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Plateletactivating factor (PAF) is a key mediator in pathogenesis of inflammatory bowel diseases (IBDs) but mechanisms of PAF-induced mucosal injury are poorly understood. To determine whether apoptosis and the Bcl-2-family of apoptosis regulatory gene products play a role in PAF-induced mucosal injury, we stably and conditionally overexpressed bcl-2 in rat small intestinal epithelial cells-6 under the control of a lactose-inducible promoter. Western blot analysis and immuno-histochemistry were used to verify inducible Bcl-2 and to analyze Bcl-2 and a proapoptotic member of the Bcl-2 family, Bax, subcellular distribution. DNA fragmentation was quantified by ELISA, caspase activity was measured by using fluorogenic peptide substrates, and mitochondrial membrane potential was assayed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and fluorescence digital imaging. Bcl-2 expression was highly inducible by lactose analog isopropyl-beta-(d)-thiogalactoside (IPTG) and was localized predominantly to mitochondria. In the absence of bcl-2 overexpression and after treatment with PAF, Bax translocated to mitochondria, and mitochondrial membrane potential collapsed within 1 h, followed by caspase-3 activation, which peaked at 6 h with an ensuing DNA fragmentation maximizing at 18 h. After IPTG-induction of bcl-2 expression, PAF failed to induce DNA fragmentation, caspase-3 activation, Bax translocation, or a collapse of mitochondrial membrane potential. These data are the first to show that PAF can activate apoptotic machinery in enterocytes via a mechanism involving Bax translocation and collapse of mitochondrial membrane potential and that both of these events are under control by bcl-2 expression levels. A better understanding of the role of PAF and Bcl-2 family of apoptosis regulators in epithelial cell death might aid design of better therapeutic or preventive strategies for IBDs.
机译：血小板激活因子（PAF）是炎症性肠病（IBDs）发病机理中的关键介体，但对PAF诱导的粘膜损伤的机制了解甚少。为了确定凋亡和凋亡调控基因产物的Bcl-2家族在PAF诱导的粘膜损伤中是否起作用，我们在乳糖诱导型调控下稳定和条件性地在大鼠小肠上皮细胞-6中过表达bcl-2启动子。 Western印迹分析和免疫组化被用于验证诱导型Bcl-2，并分析Bcl-2和Bcl-2家族的凋亡前成员，Bax，亚细胞分布。通过ELISA定量DNA片段化，使用荧光肽底物测量半胱天冬酶活性，并通过5,5'，6,6'-四氯-1,1'，3,3'-四乙基苯并咪唑基碳菁碘（JC）测定线粒体膜电位-1）和荧光数字成像。乳糖类似物异丙基-β-（d）-硫代半乳糖苷（IPTG）可高度诱导Bcl-2表达，并且主要定位于线粒体。在不存在bcl-2过表达的情况下，并且在用PAF处理后，Bax易位到线粒体，线粒体膜电位在1小时内崩溃，随后caspase-3激活，在6小时达到峰值，随后的DNA断裂在18小时达到最大。 IPTG诱导bcl-2表达后，PAF无法诱导DNA片段化，caspase-3激活，Bax易位或线粒体膜电位降低。这些数据首次表明，PAF可以通过涉及Bax易位和线粒体膜电位塌陷的机制激活肠上皮细胞的凋亡机制，并且这两个事件均受bcl-2表达水平的控制。更好地了解PAF和Bcl-2凋亡调节因子家族在上皮细胞死亡中的作用可能有助于设计更好的IBD治疗或预防策略。

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