Lu Jing, Caplan Michael S, Saraf Anita P, Li Dan, Adler Luba, Liu Xuesong, Jilling Tamas
Department of Pediatrics, Northwestern University, Feinberg School of Medicine, 2650 Ridge Ave., Evanston, IL 60201, USA.
Am J Physiol Gastrointest Liver Physiol. 2004 Feb;286(2):G340-50. doi: 10.1152/ajpgi.00182.2003. Epub 2003 Sep 25.
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.
血小板活化因子（PAF）是炎症性肠病（IBD）发病机制中的关键介质，但PAF诱导黏膜损伤的机制尚不清楚。为了确定凋亡及凋亡调节基因产物的Bcl-2家族是否在PAF诱导的黏膜损伤中起作用，我们在乳糖诱导型启动子的控制下，在大鼠小肠上皮细胞-6中稳定且条件性地过表达bcl-2。采用蛋白质免疫印迹分析和免疫组织化学来验证可诱导的Bcl-2，并分析Bcl-2和Bcl-2家族的促凋亡成员Bax的亚细胞分布。通过酶联免疫吸附测定法定量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'-四乙基苯并咪唑基羰花青碘化物（JC-1）和荧光数字成像测定线粒体膜电位。Bcl-2表达可被乳糖类似物异丙基-β-(d)-硫代半乳糖苷（IPTG）高度诱导，且主要定位于线粒体。在未过表达bcl-2且用PAF处理后，Bax转位至线粒体，线粒体膜电位在1小时内崩溃，随后半胱天冬酶-3激活，在6小时达到峰值，随后DNA片段化在18小时达到最大值。在IPTG诱导bcl-2表达后，PAF未能诱导DNA片段化、半胱天冬酶-3激活、Bax转位或线粒体膜电位崩溃。这些数据首次表明，PAF可通过涉及Bax转位和线粒体膜电位崩溃的机制激活肠上皮细胞中的凋亡机制，且这两个事件均受bcl-2表达水平的控制。更好地理解PAF和凋亡调节因子的Bcl-2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。
