VitaminD3介导线粒体自噬调控巨噬细胞极化在脓毒症肺损伤中的保护作用及机制

Acute lung injury(ALI) is a common critical illness,especially sepsis-induced lung injury,and the treatment of them is very difficult. The key mechanism of sepsis-induced lung injury is inflammation and immune dysfunction caused by mitochondrial autophagy dysfunction and M1 /M2 polarization imbalance in macrophages. Previous studies showed that Vitamin D3 can regulate macrophage polarization and promote autophagy, but the exact mechanism is still not clear. Our previous studies have found that Vitamin D3 can regulate macrophage polarization and autophagy and have remedial effects on sepsis. Because Vitamin D3 can activate the BMP/Smads/HO-1 pathway, we speculated that Vitamin D3 can regulate macrophage polarization and play a protective role by mitochondrial autophagy which is mediated through BMP/Smads/HO-1 pathway. This project intends to clarify the effects of Vitamin D3 on sepsis-induced acute lung injury in vivo, in cells and in molecular level by establishing models of acute lung injury via LPS peritoneal injection, cecal ligation and puncture (CLP) in animals and LPS interference in cells and investigate the specific mechanism of Vitamin D3-BMP / Smads / HO-1 pathway to regulate mitochondrial autophagy and promote macrophage polarization, then provides a new way for the prevention and treatment of acute lung injury induced by sepsis.

急性肺损伤是临床常见的危重症,特别脓毒症肺损伤最常见,救治非常困难。巨噬细胞线粒体自噬功能的紊乱、M1/M2极化失衡导致的炎症反应和免疫功能障碍是其关键机制。近年研究显示维生素D3能调节巨噬细胞极化、促进自噬，但具体机制尚不清楚。我们前期研究发现维生素D3可调控脓毒症巨噬细胞极化平衡、促进自噬，对脓毒症有治疗作用，结合Vitamin D3可以激活BMP/Smads/HO-1通路，推测VitaminD3可能通过BMP/Smads/HO-1介导线粒体自噬，调控巨噬细胞极化，发挥保护作用。本项目拟通过LPS 腹腔注射、盲肠结扎穿刺术（CLP）及LPS处理巨噬细胞的在体和离体模型，从整体、细胞、分子三个层次阐明Vitamin D3对脓毒症急性肺损伤的有效性，探讨VitaminD3-BMP/Smads/HO-1通路介导线粒体自噬、调节巨噬细胞极化平衡的具体机制，为脓毒症肺损伤的防治提供新思路。

脓毒症导致的急性肺损伤/急性呼吸窘迫综合征 (Acute lung injury/acute respiratory distress syndrome, ALI/ARDS)是一种伴有肺部广泛炎症的危重临床综合征。巨噬细胞M1/M2极化失衡导致炎症因子风暴是脓毒症急性肺损伤发病的重要原因，然而如何调控巨噬细胞M1/M2极化平衡缓解脓毒症急性肺损伤尚未阐明。维生素D3 (Vitamin D3, VD3) 是一种脂溶性维生素，能够减轻炎症反应，调控机体免疫平衡，进而发挥保护作用。本课题使用脂多糖（Lipopolysaccharide, LPS）分别构建体内、外脓毒症急性肺损伤模型，通过生信分析预测和分子生物学技术进一步探究VD3在脓毒症急性肺损伤中的保护作用及机制。结果发现VD3可以抑制肺泡巨噬细胞的M1极化，减少炎症因子生成，减轻脓毒症小鼠肺部炎症症状，降低肺水肿程度和死亡率，缓解脓毒症急性肺损伤。线粒体自噬与巨噬细胞极化失衡密切相关。我们进一步探究发现VD3可以通过增强肺泡巨噬细胞线粒体自噬，减少ROS的产生，降低线粒体DNA到胞质的释放，进而抑制cGAS-STING促炎通路的激活，最终减轻炎症反应。糖代谢重编程在巨噬细胞极化失衡中发挥关键作用, 以糖酵解为靶点调控巨噬细胞极化平衡，可以减轻肺损伤。利用体外脓毒症急性肺损伤模型研究发现，VD3可以降低肺泡巨噬细胞糖酵解水平，减少炎症因子产生。此外，铁死亡作为一种调节性细胞死亡方式，其特征是细胞内铁蓄积、活性氧 （ROS） 产生和谷胱甘肽过氧化物酶4（GPX4）表达量降低。我们研究发现VD3可以通过降低脂钙蛋白-2（lipocalin-2, LCN2）的表达，减少LPS导致的肺泡巨噬细胞内ROS的产生和亚铁离子的蓄积，抑制铁死亡。综上所述，本课题研究了VD3通过促进线粒体自噬，抑制铁死亡，抑制糖酵解进而调控巨噬细胞极化平衡缓解脓毒症急性肺损伤的作用机制，为脓毒症急性肺损伤的临床治疗提供了新的思路和方向。