云药“七龙天”对HIF-1α介导AHH应激损伤的保护作用机制研究

In view of Acute high altitude disease(AHAD), as one regional major disease in Yunnan Plateau, which induced by Acute Hypobaric Hypoxia, take hypoxia-inducible factor-1α（HIF-1α）pathway as the research point,which is the core initiator of gene expression in anaerobic condition.Preparation of rat AHAD model through accelerated hypobaric hypoxia method, and choose Yunnan ethnic medicine QILONGTIAN as intervention drug. This compound medicine is composed of Diqing shangri-la alpine rhodiola, Wenshan pseudoginseng and other components. Early research has shown that QILONGTIAN reduced the happening of HPH( which caused by chronic normobaric hypoxia ) through inhibiting the angiogenesis induced by hypoxia significantly. Setting up an evaluation system for the damage and protective factors in AHH,expression of HIF-1α, vascular endothelial growth factor (VEGF) ,cytoglobin( CGB ) and erythropoietin (EPO) mRNA in rats lung and brain tissues were detected by RT-qPCR, the content of total antioxidant capacity(TAOC) and IL-1 β in serum is detected by ELISA,and the pathological changes of lung and brain tissues were observed to different extent. It is expected that HIF-1α pathway elucidates the protective mechanisms of QILONGTIAN in the prevention and treatment of acute hypobaric hypoxia(AHH) injury, providing experimental basis for dig out characteristic hypoxia-protect drug prescription from the rich ethnic medicine resources in Yunnan.

针对急性低压缺氧（AHH）损伤引发的云南高原区域性重大疾病问题—急性高原病（AHAD），把缺氧基因表达的核心启动因子----缺氧诱导因子-1α（HIF-1α）介导通路作为研究切入点，采用“急进低压低氧”法制备大鼠AHAD模型，选择基于云药特色、具有良好前期基础的复方“七龙天”（由迪庆香格里拉高山红景天、文山三七等组成，可通过抑制缺氧诱导血管新生显著降低“慢性常压低氧”法导致的肺动脉高压）进行干预性实验研究。建立针对AHH的损伤性及保护性因子的综合评价体系：运用RT-qPCR技术检测HIF-1α、VEGF、CGB、EPO在大鼠肺及脑组织中的mRNA表达；以ELISA法检测血清中TAOC、IL-1 β的含量；光镜下观测肺及脑组织病理学变化。以期观察七龙天对AHH应激损伤的保护作用，阐明其基于HIF-1α信号通路介导的多靶点作用机制，可望为从云药资源宝库中发掘缺氧保护特色药物组方提供实验依据。

主要研究内容：1.云药“七龙天”对AHAD模型大鼠的药效学研究；2.基于表达谱分析七龙天对AHH应激损伤的差异因子研究；3.“七龙天”对HIF-1α介导AHH应激损伤的保护作用机制研究。.重要结果：1.“急进低压低氧”方法可成功制备大鼠AHAD模型，七龙天对AHH 应激损伤具有保护性药效学作用，可提高总抗氧化能力（TAOC），降低血清IL-1β水平，减轻应激损伤及组织水肿；2.通过差异基因筛选，发现IL−17b、MMP8、MMP9、Timp1、Ccl2、Ccl7、Tnfsf9、Faim、S100a8、S100a9等因子呈显著变化，其表达在模型组中显著上升，而在七龙天组恢复正常。同时KEGG富集发现HIF-1ɑ、IL−17通路显著富集；3.基于HIF-1ɑ调控通路探讨七龙天对AHH应激损伤的保护机制。（1）七龙天对AHH 损伤相关基因的影响：七龙天可以显著下调HIF-1 ɑ，上调EPO、CGB基因表达，提高低氧大鼠氧结合能力、抗氧化能力等，从而发挥抗氧化应激等作用。（2）七龙天对AHH 损伤中炎症反应的作用：七龙天预先干预后，IL−17b、Ccl2、Ccl7、S100a8、S100a9各炎症因子的水平显著下降，炎症反应被一定程度抑制。（3）七龙天对血管通透性的影响：经七龙天预先干预后，VEGF、MMP9、TIMP1均被显著下调，从而发挥改善血管通透性减轻组织水肿的作用。（4）七龙天对细胞增殖与凋亡相关因子影响：被七龙天预先干预后，Tnfsf9、Faim水平显著下降，起到了稳定细胞增殖与凋亡平衡的作用。.科学意义：本研究聚焦区域性重大疾病AHAD，基于HIF-1ɑ调控通路初步阐示了“七龙天”对AHH应激损伤的保护作用机制，可望为发掘云药资源宝库中具有缺氧保护作用的特色组方提供依据，具有一定的科学意义和临床价值。