淫羊藿苷对肺动脉高压的作用机制研究

Pulmonary arterial hypertension(PAH) is a fatal pulmonary circulation system disease. The physiopathological progress of PAH is associated with endothelial dysfunction，reduction of nitric oxide(NO), increase of phosphodiesterase-5 (PDE-5) expression and activity, suggesting that the NO / cGMP pathway dysfunction is involved in pathogenesis of PAH. In addition, evidence has been accumulated supporting the role of inflammation in the progression of PAH. Fractalkine (FKN) ,a unique chemokine that functions not only as a chemoattractant but also as an adhesion molecule expressed on endothelial cells, is involved in the pathogenesis of PAH. FKN is a key factor in inflammatory cell recruitment to the pulmonary vascular wall. NO and FKN are related with pulmonary vascular remodeling. Most current treatment of PAH targets endothelial dysfunction and inflammation including the inhalation of NO, the use of PDE-5 inhibitors sildenafil and anti-inflammatory agents. Icariin (ICA) is one of the main component of Chinese herbal medicine Epimedium, which has been shown to inhibit PDE-5, to promote NO release and to have anti-inflammatory functions. However, effects of ICA on PAH have not been reported. Our preliminary study found that ICA attenuates mean pulmonary artery pressure(mPAP) and reverse the pulmonary artery remolding in monocrotaline(MCT)-induced PAH model rats. Unfortunately, it is not clear whether the protective mechanisms are involved in NO/cGMP pathway and FKN. This application will further confirm the effect of ICA on mPAP and pulmonary arterial histo-morphology in MCT-induced PAH model rats, and mechanisms will be analyzed further from two aspects. First, to examine the effect of ICA on NO/cGMP pathway in MCT-induced PAH model rats in vivo and cultured pulmonary arterial smooth muscle cells in vitro. Secondly, to study the relationship between ICA and FKN in Fkn knockout mice and the role of FKN in PAH. These studies will help our understanding of pharmacological basis of ICA in protecting against PAH, and search new drugs for PAH treatment.

肺动脉高压（PAH）是严重的肺循环疾病，文献报道PAH内皮细胞功能障碍，NO生成不足，PDE-5活性增强，提示NO/cGMP通路功能障碍是PAH 的发病机制之一。且PAH伴有炎症反应，趋化因子Fractalkine（FKN）是将炎症细胞募集到肺血管壁的关键因素。NO和FKN还参与了肺血管的重构。吸入NO、口服PDE-5抑制剂西地那非和抗炎药物是常用的治疗措施。淫羊藿苷（ICA）是我省道地中药材淫羊藿的主要成分之一，毒性低，具有抑制PDE-5、促进NO的生成释放和较强的抗炎作用，但ICA治疗PAH的研究未见报道。本课题组预实验发现ICA能够降低野百合碱（MCT）诱导的PAH模型大鼠的平均肺动脉压，改善肺血管重构。我们认为ICA抗PAH的机制可能与NO/cGMP通路和FKN有关。本项目拟采用MCT模型，进一步观察ICA抗PAH作用，并利用FKN基因敲除小鼠和离体细胞探讨其作用机制。

PAH病因复杂，发病机制未完全阐明。但研究发现PAH与血管活性物质失衡、血管炎症、氧化损伤等有关。ICA具有抗炎、抗氧化、促进NO合成，抑制PDE5等药理作用，提示ICA可能具有抗PAH作用。本研究旨在探明ICA的抗PAH作用和可能的作用机制。我们通过血流动力学评价、组织形态学分析、ELISA、Western blot、免疫荧光、real time RT-PCR等技术手段，采用MCT诱导的PAH大鼠模型、CX3CL1-/-和野生型C57BL/6J小鼠模型，多次重复观察了ICA对PAH动物模型的保护作用。采用H2O2诱导的HUVEC氧化损伤模型、ET-1诱导的肺动脉平滑肌细胞（pulmonary artery smooth muscle cell，PASMC）增殖模型、TNF-α或外源性CX3CL1诱导的人急性单核细胞白血病细胞系THP-1向HUVEC迁移、黏附模型，并着重从氧化损伤、NO/cGMP通路及其上游PI3K/AKT调控信号、CX3CL1/CX3CR1等三个方面分析了ICA抗PAH血管重构和血管炎症的作用机制。结果发现ICA能够显著降低MCT诱导的PAH SD大鼠、CX3CL1-/-和野生型C57BL/6J小鼠的平均肺动脉压、改善肺循环血流动力学异常、减轻肺动脉重构，提示ICA确有改善MCT诱导的PAH作用。其作用机制涉及以下方面：①激活PI3K/AKT/NO/cGMP信号通路，增强该通路的扩血管功能，抑制PASMC增殖，抑制内皮细胞氧化损伤；②抑制NF-κB信号通路活化，减少CX3CL1生成，并可直接抑制CX3CL1所致的单核细胞在血管壁的黏附聚集，改善血管炎症反应；③降低PAH大鼠血清中AngⅡ、ET、PGI2、PGF2α和TXA2的含量。研究结果与预期基本一致。本项目的系列发现为ICA在PAH或其他血管重构、血管炎症性疾病可能的应用提供了基础药理学依据，具有潜在的临床应用前景。