SIRT6在低氧性肺动脉高压肺血管重构中的作用及机制研究

There is increasing concern about the "Warburg effect" , characterized by the glucose metabolism shifted from aerobic to anaerobic pathways in pulmonary arterial hypertension (PAH), which indicate that energy metabolism is involved in the pathogenesis of PAH and may be a promising target for therapeutic intervention. Our previous study demonstrated that the key molecule in glucose metabolism, SIRT6, is significantly down-regulated in human pulmonary arterial smooth muscle cells (HPASMCs) under hypoxia, suggesting the potential role of SIRT6 in the pulmonary vascular remodeling in hypoxic PAH. In this project, through the methods of overexpression and RNA interference, we are going to study the role of SIRT6 in the maintenance of balance between proliferation and apoptosis in HPASMCs to elucidate the function of SIRT6 in hypoxic PAH pulmonary vascular remodeling. We will further perform co-immunoprecipitation assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and dual luciferase reporter assay to explore the possibility that SIRT6 might play its role via interaction with HIF-1α and repression of transcriptional activity of HIF-1α, followed by the inhibition of the HIF-1α-PDK4-PDH pathway. This project will clarify the pivotal role of SIRT6 in the molecular mechanism of hypoxic PAH, and may provide new treatment strategy and target for PAH from the aspect of energy metabolism.

以葡萄糖代谢类型从有氧途径向无氧途径转换为特征的"Warburg效应"在肺动脉高压（PAH）中的重要性越来越受到关注，从能量代谢角度研究PAH的发病机制有助于开发疗效更好的靶向治疗药物。我们前期研究发现能量代谢中的关键分子SIRT6在低氧诱导的人肺动脉平滑肌细胞（HPASMCs）中表达明显下调，提示其可能参与低氧性PAH肺血管重构过程。本项目拟进一步通过基因过表达及RNA干扰等手段研究SIRT6调控HPASMCs增殖与凋亡平衡的作用，明确SIRT6在低氧性PAH肺血管重构中的功能；采用免疫共沉淀、凝胶电泳迁移率变动分析、染色质免疫沉淀和双荧光素酶报告基因等技术，探讨SIRT6是否通过与HIF-1α相互作用并抑制其转录活性进而阻遏下游的HIF-1α-PDK4-PDH通路发挥作用。本项目对阐明低氧性PAH肺血管重构的分子机制具有重要意义，并有望从能量代谢角度为PAH的治疗提供新的靶点。

低氧诱导的肺血管重构是肺动脉高压持续发展且难以逆转的病理基础，低氧状态下肺动脉平滑肌细胞（HPASMCs）凋亡减少、肥大增生，最终导致肺动脉压力升高。SIRT6是一个在能量代谢过程中发挥关键作用的组蛋白去乙酰化酶，为了明确SIRT6及其下游通路在低氧诱导的肺动脉高压肺血管重塑中的作用，本课题围绕低氧诱导HPASMCs模型中SIRT6的表达变化、功能和机制开展了相关研究。我们发现，在低氧诱导的HPASMCs模型中，SIRT6的mRNA、蛋白表达及酶活性都呈现下降趋势，其下游HIF-1α和PDK4蛋白的表达增加。我们进一步利用基因干扰和过表达技术，研究了SIRT6对于低氧诱导HPASMCs细胞增殖和细胞凋亡的功能。结果表明，SIRT6抑制低氧诱导的HPASMCs细胞增殖，增加细胞凋亡，发挥对细胞分裂与增殖的负调控作用，这种作用依赖于其去乙酰化酶活性。最后，我们研究了SIRT6对其下游信号通路的影响，发现HIF-1α发挥促进HPASMCs细胞增殖的作用，而SIRT6能够负性调控下游HIF-1α-PDK4信号通路。.本课题研究表明SIRT6以酶活性依赖的方式调控了低氧诱导的HPASMCs细胞增殖和凋亡过程，SIRT6抑制低氧诱导的HPASMCs细胞增殖，促进细胞凋亡，SIRT6的这种作用可能是通过负性调控其下游HIF-1α-PDK4通路而实现的。通过本课题的实施，我们揭示了SIRT6参与低氧性肺动脉高压的功能和相关的分子机制，明确了SIRT6通过酶活性依赖方式参与低氧性肺动脉高压的负性调节，且与能量代谢关系密切，提示SIRT6可能成为低氧性肺动脉高压治疗的潜在靶点。