基于RAGE介导的IRE1-XBP1-Smad通路探讨HMGB1在肺泡上皮细胞间质转化中的作用

Pulmonary fibrosis (PF) is a chronic interstitial lung disease and its exact mechanisms have not yet been elucidated. Epithelial-mesenchymal transition (EMT) of alveolar epithelial cells plays an important role in the process of PF. Endoplasmic reticulum stress (ERS) and its signaling inositol requiring enzyme 1 (IRE1) takes part in EMT and Smad2/3 phosphorylation. High mobility group box 1 (HMGB1) can induce ERS, and its role may be involved in X-box binding protein 1 (XBP1) and the receptor for advanced glycation end products (RAGE), but the regulatory mechanism is unclear. Our preliminary experiments found that EMT was existed while the protein expression of XBP1 and HMGB1 was upregulated in rats with PF. In vitro data showed that HMGB1 could induce EMT of alveolar epithelial cells, however, XBP1 siRNA (siXBP1) inhibited tunicamycin-induced EMT. Therefore, we have reasons to hypothesize that HMGB1 may accelerate EMT by activating RAGE-mediated IRE1-XBP1-Smad pathway. This project was conducted to investigate the role of RAGE on the activation of IRE1-XBP1-Smad pathway and the process of EMT by the intervention of soluble RAGE (sRAGE) in rats with PF, while the role of RAGE-mediated IRE1-XBP1-Smad pathway on HMGB1-modulated EMT was evaluated in vitro. It will clarify the molecular mechanisms of PF from novel perspective and provide new thoughts for the clinical treatment of PF.

肺纤维化（PF）是一种慢性间质性肺病，其确切发病机制尚未阐明。肺泡上皮细胞间质转化（EMT）在PF进程中起重要作用。内质网应激(ERS)及其信号IRE1参与EMT和Smad2/3磷酸化。高迁移率族蛋白1（HMGB1）可诱导ERS，且可能与XBP1、RAGE有关,但其调控机制不明。前期预实验发现PF大鼠发生EMT时XBP1和HMGB1表达上调。体外HMGB1可诱导肺泡上皮细胞发生EMT，而siXBP1能抑制衣霉素诱导的EMT。因此，有理由推测HMGB1可能通过RAGE介导IRE1-XBP1-Smad通路的活化，加速EMT过程。本项目拟在可溶性RAGE干预的PF大鼠模型上探索RAGE如何调节IRE1-XBP1-Smad通路的活化诱导EMT的发生；体外探讨RAGE介导的IRE1-XBP1-Smad通路在HMGB1调控EMT过程中的作用。从新的视角阐释PF发生的分子机制，为临床防治PF提供新思路。

本课题首先在建立的大鼠肺纤维化模型上，采用HE染色、Masson染色观察肺组织的病理变化及胶原表达情况，并运用免疫组化、Western blot检测上皮-间质转化（EMT）标志蛋白、HMGB1、IRE1/XBP1、TGF-β1/Smad2/3 等信号分子的表达变化情况。其次，在肺泡上皮细胞系A549及RLE-6TN上，运用Western blot、免疫荧光、基因沉默等方法研究HMGB1、IRE1/XBP1、TGF-β1/Smad2/3、Nrf2对肺纤维化中EMT的调控机制。研究结果表明：HMGB1参与肺纤维化进程中肺泡上皮细胞的EMT发生，且此过程与激活TGF-β1/Smad2/3通路有关；内质网应激IRE1/XBP1通路能通过诱导snail转录因子表达促进肺泡上皮细胞发生EMT；并证实Nrf2能通过下调HMGB1的表达抑制肺泡上皮细胞发生EMT。在对肺纤维化的干预研究中发现，黄芪甲苷可通过抑制HMGB1的活性，改善大鼠肺纤维化。本项目进一步阐明了肺纤维化发生的分子机制，且HMGB1、IRE1、XBP1等蛋白作为肺纤维化过程中调控EMT发生的关键因子，可能是防治肺纤维化的潜在靶点。这为开发脏器纤维化的靶向治疗药物提供了新的理论证据，具有重要临床意义。