LOX促进胶原交联调控ILK信号通路在百草枯诱导EMT的机制研究

Epithelial-mesenchymal transition(EMT) plays an important role in Paraquat (PQ)-induced pulmonary fibrosis. Our group have demonstrated that the expression of GSK-3β (the key molecule of EMT) phosphorylation and the degree of EMT were reduced after silencing lysyloxidase (LOX) with siRNA, the mechanism is unclear. Recent studies suggest that LOX could initiate the process of covalent crosslinking of extracellular collagen, and crosslinked collagen could activate integrin-linked kinase (ILK), which inactivated GSK-3β (GSK-3β phosphorylation), and promote EMT. Our studies have found that collagen deposition occurs prior to the progression of EMT. We inferred that collagen corsslinked by LOX binds with integrin, which activats ILK signaling pathway and ILK promotes GSK-3β phosphorylation, leading to EMT and aggravates pulmonary fibrosis. We will use overexpression and inhibition of LOX, collagen, ILK and GSK-3β to confirm their roles in PQ-induced EMT. Through Western Blot and reverse blocking technology, we will clarify the molecular mechanism of LOX crosslinked collagen regulating EMT through ILK signaling pathway. The goal is to provide theory basis and therapeutic target to ameliorate PQ-induce pulmonary fibrosis.

上皮-间质转化（EMT）在百草枯（PQ）中毒导致的肺纤维化中发挥重要作用。课题组发现siRNA沉默赖氨酰氧化酶（LOX）可以抑制EMT的关键分子GSK-3β磷酸化的表达，降低EMT的程度，具体机制有待阐明。文献报道LOX可以催化细胞外的胶原共价交联；胶原交联后能激活整合素相关激酶（ILK），磷酸化GSK-3β使其失活，介导EMT的发生。我们前期研究发现胶原表达升高早于EMT。因此推测PQ中毒后，LOX促进胶原交联，胶原交联沉积后与整合素结合，激活细胞内ILK信号通路，从而磷酸化GSK-3β，促进EMT基因表达，加重肺纤维化。本项目拟在整体和细胞水平上，通过诱导和抑制LOX、胶原、ILK和GSK-3β，研究其在PQ诱导肺泡上皮细胞EMT中的作用；通过蛋白质印迹法及反向阻断技术等，阐明LOX促进胶原交联通过ILK信号通路调控EMT的分子机制，为减轻PQ中毒导致的肺纤维化提供理论依据和干预靶点。

上皮-间质转化（EMT）在百草枯（PQ）中毒导致的肺纤维化中发挥重要作用。课题组发现siRNA沉默赖氨酰氧化酶（LOX）可以抑制EMT的关键分子GSK-3β磷酸化的表达，降低EMT的程度。文献报道LOX可以催化细胞外的胶原共价交联；胶原交联后能激活整合素相关激酶（ILK），磷酸化GSK-3β使其失活，介导EMT的发生。本项目主要探讨LOX是否通过交联胶原调控ILK信号通路诱导肺泡上皮细胞发生EMT，从而在PQ导致肺纤维化中发挥作用。通过体外研究发现了P Q中毒后LOX、胶原、ILK表达增加，GSK-3β表达下降，上皮标志物E-cadherin、ZO-1表达降低；间质标志物α-SMA、Vimentin表达增加，细胞发生EMT。应用siRNA抑制ILK、GSK-3β表达后，肺泡上皮细胞EMT变化程度减轻，提示ILK可能通过调控GSK-3β参与PQ诱导的EMT。同时发现，应用siRNA抑制LOX表达后，胶原、ILK表达降低，GSK-3β表达增加；再进行PQ处理后，对比单纯PQ组，上皮标志物表达增加；间质标志物α-SMA表达减少，提示LOX可能通过调控ILK信号通路参与诱导EMT相关基因的表达。我们进一步研究发现，胶原刺激A549细胞后，ILK表达增加，GSK-3β表达下降，上皮标志物E-cadherin、ZO-1表达降低；间质标志物α-SMA、Vimentin表达增加，细胞发生EMT。因此我们认为在百草枯中毒中，LOX促进胶原交联可能通过调控ILK信号通路参与诱导 EMT 相关基因的表达，加重肺纤维化的发生。最后，课题组在前期工作证实LOX在百草枯致肺纤维化的EMT中发挥重要的基础上，进一步探讨LOX上游分子HIF1α与LOX相互调控关系，证实 PQ导致肺纤维化EMT中，LOX表达变化可能单向受调于HIF1α的表达，为临床上救治PQ导致的肺纤维化提供一定得理论指导意义。