高迁移率族蛋白1上调平滑肌ETB受体参与CTEPH肺小动脉痉挛和重塑的作用及机制

The pulmonary arteriole vasospasm and remodeling in chronic thromboembolic pulmonary hypertension (CTEPH) causes irreversible elevation of pulmonary artery pressure. The endothelin system plays an important role in the pathophysiological process of pulmonary circulation. However, its pathological effects in CTEPH still need to be studied. High-mobility group box 1 protein (HMGB1) is an important regulator of lung diseases. It activates downstream signaling pathways by interacting with receptors, participates in maintaining inflammatory response, and regulates cell growth, differentiation, migration and proliferation. Our previous study found that the HMGB1 level and ETBR expression increased in CTEPH pulmonary arteriole smooth muscles. Therefore, the present study aims to confirm that HMGB1 upregulates ETBR expression in pulmonary arteriole smooth muscles through TLR4/ERK1/2/CREB pathway, and contributes to the pathogenesis of CTEPH pulmonary arteriole vasospastic and remodeling. Firstly, CTEPH model is prepared, myograph will be used to examine the vasoconstriction of pulmonary arterioles, the mRNA and protein expressions of ETBR are examined by qRT-PCR, Western blotting and immunofluorescence, respectively. The serum and pulmonary arteriole HMGB1 levels and pulmonary artery pressure are measured. Analyze the correlation among the smooth muscle ETBR upregualtion, HMGB1 level and pulmonary artery pressure. Second, HMGB1 and ETBR RNAi technique are used to confirm that HMGB1 upregulates the ETBR expression, and participates in pulmonary arteriole vasospasm and vascular remodeling in vivo and in vitro. Finally, TLR4 and CREB gene silence, TLR4, ERK1/2 and CREB inhibitors will be used to examine the relationship among HMGB1 level, ETBR upregulation and TLR4/ERK1/2/CREB pathway. And we will further certify the effect of TLR4/ERK1/2/CREB pathway on HMGB1-induced ETBR upregulation in the progress of CTEPH in rat modals. The findings will provide new theoretical basis and experimental evidence for the pathogenesis of CTEPH, and provide new targets for the medicine research and development.

慢性血栓栓塞性肺动脉高压（CTEPH）血管痉挛和重塑，造成肺动脉压升高。研究显示CTEPH时肺动脉平滑肌ETBR高表达，高迁移率族蛋白1（HMGB1）增加。我们前期发现平滑肌ETBR高表达与HMGB1有关，提出假设HMGB1经TLR4/ERK1/2/CREB上调平滑肌ETBR，参与CTEPH肺小动脉痉挛和重塑。本研究拟制备大鼠CTEPH模型，检测肺动脉压、肺小动脉HMGB1水平和平滑肌ETBR介导的收缩和受体表达，分析三者相关性；再通过RNAi在体干预HMGB1或体外干预ETBR，研究HMGB1上调ETBR参与血管痉挛和重塑作用；最后用基因沉默和通路抑制剂，细胞水平研究HMGB1与受体上调和TLR4/ERK1/2/CREB关系，整体验证HMGB1上调平滑肌ETBR参与CTEPH肺小动脉痉挛和重塑，影响疾病进程的作用。本研究将为CTEPH预防和治疗提供理论基础，为其靶点药物研发提供实验依据。

慢性血栓栓塞性肺动脉高压（CTEPH）是由于机化血栓堵塞肺动脉及肺血管痉挛和重塑，导致肺动脉压增高的进展性疾病。CTEPH大鼠肺小动脉平滑肌HMGB1水平增加，平滑肌ETBR高表达，但两者之间如何相互影响，通过何种途径参与CTEPH的形成过程，尚不清楚。本项目以研究肺小动脉病变为主线，以平滑肌HMGB1和ETBR上调为关键连接点，首先，建立大鼠CTEPH模型，研究不同时间节点肺小动脉平滑肌HMGB1水平和平滑肌ETBR表达与肺动脉压力的相关性，发现随造模时间增加，大鼠肺动脉压升高，肺小动脉平滑肌ETBR表达增加，HMGB1水平在2周即达到高峰，三者表达呈正相关。其次，细胞水平证实HMGB1对平滑肌ETBR的调控作用及机制，发现HMGB1能促进细胞增殖并致使ETBR高表达，抑制剂TAK-242和U0126能显著抑制HMGB1引起的ETBR高表达， 验证HMGB1通过TLR4/ERK1/2/CREB通路对平滑肌ETBR的调控。最后，整体水平使用TLR4和ERK1/2通路抑制剂，其能降低CTEPH大鼠大鼠的肺动脉压，减少ETBR高表达，并减少肺小动脉重塑。本项目的完成将揭示CTEPH肺小动脉痉挛和重塑的分子机制，为CTEPH的临床治疗和药物干预靶点的选择提供实验依据。