解偶联蛋白2介导代谢重编程调控巨噬细胞极化在ARDS中的作用及机制研究

Macrophage polarization is important in acute respiratory distress syndrome (ARDS). Recent studies have found that metabolic reprogramming plays a key role in macrophage polarization, but its mechanism is unclear. We first found that uncoupling protein 2 (UCP2) can not only increased ARDS inflammatory injury, but also promoted glycolysis via inhibiting GAPDH which is the key enzyme of glycolysis nuclear translocation. Thus we speculated that UCP2 could translocate oxidative phosphorylation to glycolysis，and promote the macrophages to M1 macrophage polarization by inhibition of GAPDH nuclear。M1 macrophage increases inflammatory factors to aggravate ARDS. In this project, we intend to construct adenovirus vector to overexpress or lowered the expression of UCP2, and mutation GAPDH phosphorylation sites, to discussed the mechanism of UCP2 mediated nuclear translocation of GAPDH.ARDS animal model will used to reseach UCP2 mediated metabolic reprogramming in macrophage polarization and ARDS, which may help to find new potential therapeutic target for ARDS and other critically illness.

摘要: 巨噬细胞极化是急性呼吸窘迫综合征（ARDS）炎症损伤的重要因素。新近发现代谢重编程在巨噬细胞极化中起关键作用，但其机制不明。本课题组前期首次证实解偶联蛋白2（UCP2）不仅能加重ARDS炎症损伤，还可抑制糖酵解关键酶GAPDH核易位，促进糖酵解，可能成为治疗ARDS的关键。据此我们推测UCP2可通过调控GAPDH核易位使氧化磷酸化向糖酵解转化，促进巨噬细胞向经典活化型巨噬细胞(M1)极化，导致促炎因子增多参与ARDS。本研究拟构建腺病毒载体上调和下调UCP2的表达，在细胞水平探讨代谢重编程对巨噬细胞极化和功能的影响；通过气管内注射UCP2腺病毒转染小鼠肺组织，研究ALI动物模型中UCP2对巨噬细胞极性、肺炎症损伤的效应；突变GAPDH磷酸化位点，探讨UCP2介导GAPDH核易位的作用机制。阐明UCP2诱导的巨噬细胞极化在ARDS中的作用和机制，为ARDS等炎症相关危重病奠定基础。

解偶联蛋白2 (UCP2)对炎症和线粒体能量代谢至关重要，在全身炎症和感染患者中表达上调。然而，UCP2在急性呼吸窘迫综合征中的潜在作用仍不清楚。本研究通过腹腔注射脂多糖建立急性呼吸窘迫综合征小鼠模型。构建ShRNA-UCP2腺病毒用于抑制UCP2在肺中的表达。研究评估了评估肺病理变化、肺水肿、肺部炎症、肺泡巨噬细胞极化标记物和能量代谢分子。结果显示ShRNA-UCP2减轻LPS诱导的小鼠肺损伤、肺水肿和炎症介质表达。同时，M1型巨噬细胞标记物(iNOS)的表达减少，M2型巨噬细胞标记物(CD206)的表达增加。此外，转染shRNA-UCP2后，LPS诱导的巨噬细胞糖酵解、葡萄糖转运蛋白1 (GLUT1)、丙酮酸激酶亚型M2 (PKM2)表达、乳酸脱氢酶(LDH)和乳酸被抑制。此外，在小鼠巨噬细胞中转染shRNA-UCP2后，线粒体氧化磷酸化(mt-OXPHOS)复合物ⅰ、ⅱ、ⅲ和ⅴ以及线粒体耗氧量都得到了改善。综上所述，我们的结果表明，在ARDS发生后，抑制UCP2的表达，可以调控巨噬细胞极化，从而抑制肺损伤和肺部炎症，这与急性呼吸窘迫综合征中糖酵解向mtOXPHOS途径的代谢转移有关。因此，我们的发现表明UCP2可能是ARDS的潜在治疗靶点。我们的研究结果证实了基金申请中的主要关键问题，在ARDS 救治中有潜在应用价值。