P-糖蛋白下调引发早期内毒素易位在全肠外营养诱发肠屏障损伤进程中的作用与机制

Intestinal P-glycoprotein (P-gp) plays a profound role in antagonizing lipopolysaccharide (LPS) translocation via transcellular route, but its changes and significance under the context of total parenteral nutrition (TPN) remain unclear. By using a rat model of TPN, a significant down-regulation of intestinal P-gp function was observed prior to the disruption of epithelial barrier function (EBF), which suggested the possibility that LPS translocation may occur when P-gp function is impaired. Thus, we propose a hypothesis that TPN leads to down-regulation of P-gp first, which faciliates LPS translocation via transcellular route. LPS-induced inflammatory response is then activated, leading to severe mucosal injury and further LPS translocation via papracellular route as a result. Since TPN is often accompanied by deprivation of enteral nutrition, we also speculate that regulation of P-gp may involve the inactivation of AP-1 due to polyamine depletion. In this study, we will clarify the role of P-gp in TPN-induced LPS translocation; explore the mechanisms of P-gp regulation by TPN; and define the interaction between P-gp function and barrier function. This study will be helpful in revealing the mechanism of TPN-associated complications and providing new ideas for the prevention and treatment from the new perspective of P-gp.

P-糖蛋白（P-gp）是肠道抵御内毒素（LPS）经跨细胞途径易位的重要蛋白，但P-gp在全肠外营养（TPN）中的变化及意义尚不清楚。我们在前期研究中发现，TPN造成动物肠道内P-gp功能损伤，且先发于肠屏障损伤，提示TPN导致的LPS易位很可能在肠屏障损伤前便已发生，而由此引发的炎症反应对肠屏障的损伤进程具有推动作用。据此我们提出假说：TPN导致P-gp功能损伤，致使LPS早期经跨细胞途径少量易位，随即促发炎症反应，损伤肠屏障再经细胞旁途径大量易位。肠内营养的缺失可能是P-gp下调的重要原因，其机制可能与肠细胞内多胺耗竭引发的转录因子AP-1失活相关。本课题拟从分子、细胞及动物等多层次明确P-gp在TPN诱发LPS易位中的作用；探讨TPN调控P-gp表达的分子机制；揭示P-gp下调与LPS易位及肠屏障损伤间的内在联系，从P-gp这个新视点为完善TPN相关并发症的致病机制及防治提供新思路。

背景.肠屏障损伤是长期全肠外营养（TPN）诱发的严重并发症，前期研究表明上皮细胞间紧密连接崩坏是造成肠屏障损伤的重要诱因，但具体机制尚未明确。P-糖蛋白（P-gp）是哺乳动物细胞膜表面的重要外排转运蛋白，我们假设TPN状态下肠道细胞P-gp外排功能损伤，诱发肠道内环境紊乱是引发肠屏障损伤的可能途径。.方法.动物水平构建TPN大鼠模型，考察P-gp表达与肠导通透性变化；细胞水平采用P-gp抑制剂考察肠上皮Caco-2细胞对内毒素（LPS）通透性与大肠杆菌（E.coli）粘附性的影响；分子水平探究TPN处方脂肪乳剂（SOLE）对P-gp表达调控的信号途径及分子机制。.结果.动物水平我们发现TPN处理3天后即可见P-gp表达显著下调，7天后可见肠屏障损伤，紧密连接蛋白Occludin表达显著下调，提示早期P-gp功能下调可能是肠屏障损伤的先行诱因。细胞水平我们通过Transwell模型发现，Caco-2细胞内P-gp功能抑制后LPS的通透性显著增加，提示P-gp功能损伤可诱发内毒素移位，是后者激活肠道炎症反应的潜在入口；同时我们还发现E.coli对Caco-2细胞黏附力的影响也受P-gp功能调控，P-gp功能抑制后E.coli在细胞表面的粘附力显著增加，细胞毒性反应增强；分子水平我们发现SOLE引起P-gp表达下调的途径可能受ERK依赖的FOXO3a转位影响，即SOLE激活ERK信号途径后，后者磷酸化FOXO3蛋白诱导其转位出核继而抑制P-gp转录。.结论.TPN引发的肠屏障损伤与早期P-gp表达下调相关，针对SOLE激活的ERK/FOXO3信号通路可能是抑制P-gp下调从而补救肠屏障损伤的有效途径