FGF23/FGFR-1通过激活血管内皮细胞NOX2-ROS参与重症中暑急性肺损伤的机制研究

Our previous study revealed that severe heat stroke (sHS) could lead to acute lung injury (ALI), and it was associated with ROS accumulation in vascular endothelial cells (VECs). However, the initiation factors and intermediate mechanisms of ROS accumulation in VECs caused by sHS are not clear. We conducted a preliminary experiment to screen the target genes of sHS-induced ALI through in vivo and in vitro cross comparison and firstly found out that fibroblast growth factor 23 (FGF23) might be an upstream initiating factor in sHS-induced ALI. Besides, sHS-induced FGF23/FGFR-1 could lead to NOX2 activation and ROS accumulation in VECs and subsequent ALI through Klotho independent pathway. Based on that, we speculated that sHS could lead to an increasing of FGF23 expression and FGFR-1 activation through Y766 phosphorylation in pulmonary vascular endothelial cells and phospho-FGFR-1 could bind to PLC-γ 2 and promote PLC-γ 2 phosphorylation, then activate p47phox phosphorylation and translocation to cell membrane to assemble with NOX2、p22phox, resulting in NOX2 activation and ROS accumulation in VECs and subsequent ALI. We propose a study in normal, FGF23-/-, FGFR-1-/- and FGFR-1 Y766 site mutation in VECs，combined with FGF23 and FGFR-1 knockout mice：1. To validate that FGF23 is a key upstream initiator of sHS-induced ALI; 2. To clarify that FGF23/FGFR-1 are involved in sHS-induced ALI by inducing ROS accumulation in VECs through Klotho independent pathway; 3. To explore the intermediate molecular mechanism of FGFR-1-mediated ROS accumulation in VECs. This study might shed a light on profound understanding of the mechanism of sHS-induced ALI and provide a new idea for the treatment of clinical sHS-induced ALI.

我们前期研究发现重症中暑可以导致急性肺损伤，与高热刺激诱导的VECs中ROS累积有关，但这一过程中高热刺激导致VECs中ROS累积的启动因素以及具体中间机制，目前并不明确。预实验通过体内外交叉对比，筛选介导重症中暑过程中急性肺损伤的靶基因，首次发现FGF23可能是其上游启动的一个关键因素，并且，FGF23/FGFR-1通过Klotho非依赖途径，促进NOX2活化，诱导VECs中ROS爆发而参与急性肺损伤。基于上述发现，结合既往文献报道，我们假设重症中暑后，肺组织中FGF23表达增加，激活肺血管内皮细胞FGFR-1，使FGFR-1 Y766磷酸化，磷酸化的FGFR-1 Y766与PLC-γ2结合后促使PLC-γ2磷酸化，进一步诱导p47phox磷酸化、膜转位，并完成与NOX2、p22phox的组装，通过引起NOX2活化，最终导致VECs中ROS累积。本研究拟在正常、FGF23和FGFR-1敲除、以及FGFR-1 Y766位点突变的VECs模型，并利用FGF23和FGFR-1基因敲除小鼠，1.验证FGF23是重症中暑急性肺损伤的一个关键上游启动因素；2.明确FGF23/FGFR-1通过Klotho非依赖途径，介导高热刺激后VECs中ROS累积，进而参与重症中暑急性肺损伤；3.探索FGFR-1介导VECs中ROS累积的分子机制。研究结果不仅有助于全面了解重症中暑过程中急性肺损伤的分子机制，更为临床早期有针对性的预防和治疗重症中暑，甚至MODS方面提供新的策略和药物靶点。

重症中暑的高死亡率主要与多器官功能障碍综合征（multiple organ dysfunction syndrome，MODS）有关，急性肺损伤导致的呼吸功能衰竭是重症中暑过程中并发MODS的重要促发因素。既往研究显示，重症中暑急性肺损伤主要与高热刺激诱导的血管内皮细胞（vascular endothelial cells，VECs）中活性氧（reactive oxygen species，ROS）累积有关，但具体启动因素以及中间机制并不明确。前期实验，通过体内外交叉对比筛选介导重症中暑过程中急性肺损伤的靶基因，发现成纤维细胞生长因子23（fibroblast growth factor 23，FGF23）可能是其上游启动的一个关键因素，但其作用机制仍有待阐明。结合文献报道，FGF23具有促进ROS累积的作用，我们假设重症中暑后FGF23/FGFR-1通过Klotho依赖途径诱导FGFR-1Y766磷酸化，导致VECs中NOX2-ROS累积，参与重症中暑急性肺损伤的形成。研究通过在正常、FGF23和 FGFR-1敲除、以及FGFR-1Y766位点突变的VECs模型，并利用各抑制剂处理小鼠，从动物和细胞水平、正反两方面阐明了：FGF23是重症中暑急性肺损伤的关键上游启动因素；并且，FGF23/FGFR-1通过诱导PLC-γ2磷酸化，促进NOX2活化以及ROS累积，进而参与重症中暑急性肺损伤；更为重要的是，FGFR-1Y766位点磷酸化是FGF23/FGFR-1诱导PLC-γ2磷酸化，并进一步启动高热刺激后VECs中NOX2-ROS累积，参与重症中暑急性肺损伤的关键。研究结果将为临床重症中暑急性肺损伤的救治提供新思路。