HMGB1诱导PASMC表型转化在肺血管重构中的作用及分子机制研究

The high mobility group protein box 1 (HMGB1) plays key role in many acute and chronic pulmonary diseases. Our previous study demonstrated that HMGB1 could induce the proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) and which are involved in the pulmonary vascular remodeling (PVR) of endotoxin acute lung injury rat model. Phenotypic switching is the key step of SMC to gain the capability of proliferation, migration and secretion of extracellular matrix. But till now the effect of HMGB1 on the phenotypic switching of PASMC is unknown. Our preliminary test shown that HMGB1 could induce the phenotypic switching of PASMC, however, the molecular mechanism of it is still to be explored. So, we proposed a hypothesis that HMGB1 is involved in the process of PVR by inducing the phenotypic switching of PASMC to regain the ability of proliferation, migration and secretion of extracellular matrix. To test this hypothesis, we will utilize the primary cultured PASMC and PVR model induced with endotoxin from wild or gene knock-out Wistar rats, the methods of real-time PCR, immunohistochemistry, western blot, co-immunoprecipitation, siRNA, ChIP, Oligonucleotide pull down, Luciferase assay to explore the role and its mechanism of HMGB1 on the phenotypic switching of PASMC and PVR in level of molecule, cell, tissue and whole animal. A new view of PVR will be disclosed from the effect of HMGB1 on PASMC phenotypic switching, and this will provide us a new clue to deal with the pulmonary arterial hypertension and its complications.

HMGB1在多种急慢性肺病中起作用。我们前期研究发现HMGB1可诱导PASMC增殖/迁移参与PVR。表型转化是SMC重获增殖/迁移能力的前提，迄今HMGB1对PASMC表型转化的影响尚不清楚。我们的预实验结果提示HMGB1可诱导PASMC表型转化。但其作用机制还有待进一步探讨。为此，我们提出假说：HMGB1可能通过诱导PASMC表型转化，增强其增殖/迁移/基质分泌能力，参与PVR。为验证这一假说，我们将用野生和基因敲除大鼠原代PASMC、PVR模型，采用实时PCR、免疫组化/印迹/共沉淀、siRNA、ChIP、pull down、Luciferase等手段，从分子、细胞、组织、动物整体多层面，探讨HMGB1在PASMC表型转化和PVR形成中的作用，明确HMGB1诱导PASMC表型转化的调控机制。本研究将从HMGB1这个新视点揭示PVR发生机制，为肺动脉高压及其并发症的防治提供新的思路。

目的：PVR是多种因素引发的肺血管增厚性结构改变。PVR与血管收缩和血栓形成相互作用导致PAH和右心室衰竭。在PVR形成中，肺小动脉肌化是引起肺血管增厚病理基础。前期研究显示，HMGB1可诱导PASMC增殖和迁移参与PVR。但是HMGB1如何调控PASMC及其参与PVR的分子机制尚不清楚。我们设想HMGB1可能通过诱导PASMC表型转化，增强其增殖、迁移和基质分泌能力，参与PVR。.方法：本研究以野生、基因敲除大鼠及原代培养大鼠PASMC和ALI-PVR模型为研究对象，利用基因敲除、特异抑制剂、MTT、transwell小室、免疫组化/印迹/共沉淀、实时PCR、ChIP等实验方法，在分子、细胞和动物整体水平观察细胞形态学、SMC特异表型基因表达、细胞功能改变、以及参与调控的信号因子和转录因子表达和肺小血管肌化程度观察。.结果：HMGB1可刺激PASMC细胞形态由长棱形变得偏圆、OPN基因表达增强、α-SMA和SM22α表达下调；刺激后的PASMC 增殖、迁移和胶原I,III合成增加，且存在浓度效应和时间效应；同时，膜受体RAGE、转录因子KLF4表达增加；CoIP和ChIP显示，HMGB1刺激下KLF4与SM22α结合位点1发生脱磷酸化和脱乙酰化，与SM22α启动子区结合弱化，抑制其转录和表达。siRAGE、siKLF4或者PI3K、Akt抑制剂处理后，HMGB1诱导细胞形态、表型基因表达、细胞增殖迁移和基质分泌的能力均有不同程度的减弱。动物实验显示，HMGB1抑制剂，或RAGE、KLF4基因敲除可部分逆转LPS诱导的肺小动脉肌化，抑制PVR形成。.结论：本研究表明HMGB1可诱导PASMC向分泌型转化，参与PVR形成；HMGB1通过RAGE-MAPKs通路调控转录因子KLF4蛋白修饰，抑制表型基因SM22α转录和表达，调控PASMC表型转化；HMGB1和RAGE-MAPKs-KLF4可作为PVR防止的关键点。