HIF-1α上调LOXs在百草枯致肺纤维化中的机制研究

Paraquat(PQ) poisoning remains a major cause of death among patients with acute poisoning in developing countries.The key to successful treatment of victims suffering from high mortality rates in PQ poisoning is how to early prevent the development of extensive fibrosis in the lung. Recent findings by our group have indicated that the expression HIF-1α in the alveolar epithelia was significantly upregulated during the early phase of PQ poisoning, accompanying by obvious collagen deposition, which implies a correlation between HIF-1α activation and pulmonary fibrosis. Recent studies suggest HIF-1α enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes (LOXs). We presume whether HIF-1α-LOXs promote fibrogenesis in PQ-induced pulmonary fibrosis. One major interest of our laboratory is to understand the role and mechanisms of HIF-1α upregulated lysyl oxidase in PQ-induced pulmonary fibrosis based on an in vivo rat PQ poisoning model and an in vitro PQ-induced epithelia EMT model with immunohistochemical, Western blot, RNAi and Transwell techniques, investigating the HIF signaling in the development of PQ induced early pulmonary fibrosis.The goal of this study is to search for measures aimed at preventing pulmonary fibrosis and provide theoretical and experimental evidence for finding new targets used in the management of PQ poisoned patients.

百草枯中毒仍然是发展中国家急性中毒患者的主要死亡原因。如何阻止早期肺间质纤维化的发生，是降低百草枯中毒患者死亡率的关键。前期工作发现，HIF-1α肺部表达在百草枯中毒早期即出现显著变化，同时伴随着胶原沉积增加，提示HIF-1α的活性增强可能与肺纤维化存在相关性。有研究发现，HIF-1α活化可上调赖氨酰氧化酶(LOXs)活性，LOXs既影响细胞外基质蛋白交联，还可诱导上皮－间质转化。因而推测百草枯导致的肺纤维化是否与HIF-1α上调LOXs有关。本研究拟采用免疫组织化学、Western Blot、RNAi及Tranwell细胞迁移检测等技术探讨HIF-1α是否通过上调LOXs诱导上皮－间质转化及影响细胞外基质代谢，从而在百草枯导致肺纤维化中发挥作用，并探讨上述作用的信号调控通路，为百草枯中毒寻找新的治疗靶点提供理论和实验依据。

百草枯(PQ)中毒仍然是发展中国家急性中毒患者的主要死亡原因。如何阻止早期肺纤维化的发生，是降低PQ中毒患者死亡率的关键。文献报道提示：HIF-1α活化可上调赖氨酰氧化酶(LOXs)活性，LOXs既影响细胞外基质蛋白交联，还可诱导上皮－间质转化。本项目主要探讨HIF-1α是否通过上调LOXs诱导上皮－间质转化及影响细胞外基质代谢，从而在PQ导致肺纤维化中发挥作用，并探讨上述作用的信号调控通路，为PQ中毒寻找新的治疗靶点提供理论和实验依据。本项目通过体内及体外研究证实了PQ中毒后HIF-1α表达明显增高，体外应用siRNA抑制HIF-1α表达后，肺泡上皮细胞EMT变化程度明显减轻，调控EMT的关键分子Snail和β-catenin表达明显下降，说明HIF-1α可能通过Snail和β-catenin途径参与调控PQ诱导的EMT。同时，我们也证实了PQ中毒后LOX表达也明显增高，且应用LOX抑制剂BAPN及siRNA抑制LOX后，肺泡上皮细胞EMT变化程度明显减轻，细胞迁移能力显著降低，Snail表达下降及GSK-3β表达升高，提示LOX可能通过GSK-3β和Snail参与调控PQ诱导的EMT。然而我们发现在PQ中毒，沉默HIF-1α后LOX表达明显下降，而沉默LOX后HIF-1α表达则无明显变化，说明在PQ致肺纤维化中HIF-1α可调控LOX的表达变化。因此，我们认为在PQ中毒高表达的HIF-1α不仅可以增强LOXs 酶活性，使分泌至细胞外的LOXs引起细胞外基质重构，直接影响肺的组织结构；细胞内的LOXs还可以通过激活EMT相关信号通路诱导肺泡上皮细胞发生上皮－间质转化，转化的成纤维细胞可迁移至间质，产生更多的细胞外基质，促进肺纤维化的发生。最后，我们还回顾分析了2011-2013年PQ中毒患者的临床资料，应用高效液相检测PQ血药浓度，同时检测血清中HIF-1α的水平，并做相关性分析，结果提示PQ血药浓度与HIF-1α的水平呈正相关。本研究发现PQ中毒引起的肺纤维化在急性肺损伤的早期，打破传统认为急性肺损伤分为渗出期、增生期、纤维期的观点，为临床早期揭示纤维化形成提供了理论依据，同时也为其他原因引起的肺纤维化的救治提供了一定的理论指导意义。