MRP4介导脓毒症时血管内皮屏障功能障碍的分子机制研究

Sepsis is a common complication with high mortality in patients with clinically severe diseases. Vascular endothelial barrier dysfunction is an important pathophysiological manifestation during sepsis. Previous studies demonstrated that vascular endothelial barrier dysfunction during sepsis is mainly attributed to the decrease in intracellular cAMP level and the sequential inactivation of Rac 1 and breakdown of cytoskeletal proteins. Multidrug resistance-associated protein 4(MRP4) is an important protein which can actively transport intracellular cAMP out of the cell. Our preliminary experiment in a rat model of sepsis showed that lung water content increased during sepsis,which was prevented by MK 571,a pharmacological inhibitor of MRP4. Furthermore,cell culture experiment revealed that lipopolysaccharide induced a significant increase in MRP4 protein expression in lung microvascular endothelial cells and thereby decreased intracellular cAMP level. Thus, MRP4 may play an important role in mediating vascular endothelial barrier during sepsis by regulating intracellular cAMP level. However,there is no report about the role and mechanism of MRP4 in mediating vascular endothelial barrier during sepsis so far. Therefore,through animal and cell culture experiments in our present program, we aim to investigate the role and mechanisms of MRP4 in mediating vascular endothelial barrier during sepsis and to look for new target for effectively preventing and treating vascular endothelial barrier dysfunction during sepsis.

脓毒症是临床危重患者的常见并发症，病情凶险，病死率高。血管内皮屏障功能障碍是脓毒症的重要病理生理表现，其发生机制为细胞内cAMP降低，进而诱导Rac 1失活，导致内皮细胞骨架破坏。多药耐药相关蛋白4（MRP4）是主动转运细胞内cAMP的重要蛋白分子。我们的前期实验研究发现，脓毒症大鼠肺组织含水量显著增加，应用MRP4抑制剂MK571明显减少肺含水量；细胞实验还显示，脂多糖刺激引起肺微血管内皮细胞MRP4表达增加，导致细胞内cAMP水平降低。因此，MRP4可能通过调节细胞内cAMP水平，对脓毒症时血管内皮屏障功能产生重要调控作用，但目前国内外尚无MRP4调控脓毒症时血管内皮屏障功能的研究报道。有鉴于此，本研究拟通过动物和细胞实验研究，探讨MRP4在脓毒症时血管内皮屏障功能障碍中的作用及机制，以期发现有效防治脓毒症时血管内皮屏障功能障碍的新靶点。

通过盲肠结扎穿孔（CLP）建立大鼠脓毒症模型分析MRP4对脓毒症时血管内皮屏障功能障碍的调控作用，对雄性SD清洁级大鼠分为假手术组、假手术MRP4抑制剂干预组、脓毒症组和脓毒症MRP4抑制剂组，术后24h麻醉处死后取材。培养大鼠肺微血管内皮细胞，用脂多糖建立细胞模型，并通过重组腺病毒载体Ad-MRP4、Ad-shMRP4转染细胞来探讨MRP4对肺微血管内皮细胞屏障功能的影响和具体作用机制。检测细胞通透性、细胞骨架形态、PKA活性、cAMP、Rac1、VE-cadherin、β-catenin水平来探讨MRP4对肺微血管内皮细胞屏障功能的影响和具体作用机制。动物在体实验证实脓毒症时MRP4表达显著上调，动脉血PH值和动脉血氧分压显著降低、血清TFN-α、肺组织湿/干比值增加，而MRP4抑制剂MK571可以使MRP4蛋白表达下调、动脉血PH值和动脉血氧分压升高、血清TFN-α、肺组织湿/干比值降低。细胞离体实验表明MRP4可以使细胞骨架形态改变应力纤维增多，细胞形态改变，降低细胞内cAMP 水平，促使Rac1失活，VE-cadherin、β-catenin水平降低，导致内皮细胞通透性增加。本实验从动物在体和细胞离体水平伤证实MRP4在脓毒症时血管内皮屏障功能的调控作用和机制，其抑制剂MK571通过下调MRP4表达可明显改善脓毒症大鼠肺功能，降低炎性因子水平，从而对脓毒症大鼠起到保护作用，为临床治疗MRP4作为脓毒症靶向治疗提供了实验依据。