Synaptotagmin1介导囊泡转运拮抗百草枯蓄积及其肺损伤的作用和机制研究

It is widely concerned that paraquat (PQ) induced lung injury is the main cause of death for the patients with PQ poisoning. The active accumulation of PQ in the lung tissue contributes to the persistent and exacerbated lung toxicity, and the underlying molecular mechanism of PQ accumulation remains largely unknown. We successfully constructed a PQ-resistant A549 cell line, in which surprisingly existed an antagonistic effect on PQ accumulation. Based on the results of DNA microarray, we found overexpression of synaptotagmin1 (SYT1) led to the reduction of PQ accumulation and the resistance to its toxicity, suggesting that STY1 plays an important role in regulating the intracellular accumulation of PQ. It is known that STY1 regulates the vesicle transport activity which governs transport of biochemical signal molecules in the cell and seems involved in the pathogenesis of PQ poisoning. Therefore, we hypothesize that STY1 promotes the excretion of PQ by regulating the vesicle transport activity, and reduce its intracellular accumulation and lung toxicity. Here, we will explore whether SYT1 mediated vesicle transport plays a role in preventing PQ accumulation and its toxicity in A549 cells, ICR mice and clinical patients, using various methods of UPLC-MS/MS, siRNA, flow cytometry, real-time PCR, western blot, and confocal microscopy. This study will provide new insights into the PQ-induced lung injury and its treatment.

百草枯（PQ）中毒引起肺损伤是造成患者死亡的主要原因，受到广泛关注。目前认为，PQ在肺组织的主动蓄积是持续和加剧肺损伤的关键因素，具体机制有待阐明。我们前期成功构建了对PQ抵抗的A549细胞系，其具有拮抗PQ在细胞内蓄积的特性。通过基因芯片筛查对比，发现高表达synaptotagmin1（SYT1）是减少PQ蓄积和抵抗其毒性的关键原因，提示STY1对PQ胞内蓄积有重要调控作用。已知STY1调节囊泡转运活性，而后者作为重要的生物分子转运途径被提示可能参与PQ致病的过程。因此，推测STY1可能通过调控囊泡转运活性，促进PQ跨膜排出，减轻其蓄积和肺毒性。本研究应用UPLC-MS/MS、siRNA、流式细胞术、定量PCR、western blot、共聚焦显微镜等技术，在A549细胞、ICR小鼠及临床患者多个层次阐述SYT1介导囊泡转运拮抗PQ蓄积和肺损伤的作用与机制，旨在为PQ中毒干预提供新思路。

百草枯（PQ）中毒救治是近年急诊医学领域突出难题，患者病死率高，缺乏有效的救治药物。本项目通过用 PQ 处理 A549 细胞获得对百草枯 (PQ) 具有中度抗性的 A549/PQ 细胞, 其生长速度减慢, PQ 的积累浓度和生长抑制水平, 损伤和早期凋亡诱导。 PQ 显着低于亲代 A549 细胞。芯片筛选和RT-qPCR检测发现耐药细胞中Synaptotagmin-1（SYT1）表达显着增加，PQ进一步增强其表达。抑制A549/PQ细胞SYT1表达后，细胞活力、胞内PQ浓度和Bcl-2、SNAP25、RAB26的表达显着降低，而死亡率、早期凋亡率和Bax表达显着升高。体内实验还进一步表明，百草枯促进百草枯中毒小鼠体内SYT1、SNAP25和RAB26的表达；当抑制SYT1表达时，肺组织中PQ浓度显着升高，肺损伤和细胞凋亡水平也显着增强，而SNAP25和RAB26的表达显着降低。这就说明 PQ 中毒导致体内 SYT1 等囊泡转运相关蛋白的代偿性上调,从而促进 PQ 跨膜转运,进而减少 PQ 的肺蓄积和 PQ 引起的肺损伤。在此基础上，我们发现细胞代谢物草酰乙酸 (OAA)与α-酮戊二酸二甲酯（DMK）能够改善线粒体功能来抑制 PQ 诱导的细胞焦亡，减轻PQ中毒小鼠的肺损伤。此外，我们还构建了两种PQ中毒患者预后评估的方法，可为 PQ 中毒患者预后判断提供参考。本项目研究成果从理论和实践上丰富了PQ的中毒机制和治疗策略。