参附益心颗粒调控PI3K/Akt通路改善肺动脉高压机制研究

Pulmonary arterial hypertension is a sort of malignant pulmonary vascular disease which present right heart failure as major clinical manifestations and caused by a variety of reasons. Preliminary studies have shown that shenfuyixin granule which got the effects of warming yang and supplementing Qi, promoting blood circulation and promoting urination have a significant effect in the treatment of right heart failure due to pulmonary arterial hypertension. PI3K/Akt pathway plays a major part in pulmonary angiogenesis which can reduce pulmonary artery pressure.It is still indistinct that Shenfuyixin granule can promote pulmonary angiogenesis by regulating the PI3K/Akt pathway and reduce pulmonary artery pressure or not. In this study, we established the animal model of pulmonary arterial hypertension by injecting monocrotaline subcutaneously,and to observe the influence of hemodynamics and pulmonary angiogenesis in the model by interventions of Shenfuyixin granule and EPCs transplantation ;obtained EPCs in separation of pulmonary hypertensive rats' bone marrow and gave different doses of serum containing Shenfuyixin granule in vitro intervention. Then we investigated the paracrine mechanism of shenfuyixin granule induced EPCs differentiation and PI3K/Akt signal pathways by angiogenesis-related protein overexpression and Western blot techniques, So as to explore its mechanism to provide new ideas and methods therapy of pulmonary arterial hypertension.

肺动脉高压是由多种原因引起的一种恶性肺血管疾病，其临床表现以右心衰竭为主。前期研究显示温阳益气、活血利水方药参附益心颗粒对肺动脉高压及其所致右心衰竭有明显的治疗作用。肺动脉血管新生能够降低肺动脉压力，PI3K/Akt通路在血管新生中起重要作用。参附益心颗粒能否通过调控PI3K/Akt通路促进肺动脉血管新生、降低肺动脉压力尚不明确。本研究采用野百合碱皮下注射建立大鼠肺动脉高压模型，给予参附益心颗粒和EPCs移植干预，观察其对模型大鼠肺血流动力学和肺动脉血管新生的影响；分离肺动脉高压模型大鼠骨髓，获取EPCs，给予不同剂量的参附益心颗粒含药血清体外干预，利用血管新生相关蛋白过表达和Western blot技术，研究参附益心颗粒诱导EPCs分化的旁分泌机制和PI3K/Akt信号传导通路，从而探讨其作用机制，为肺动脉高压的治疗提供新的思路和方法。

目的：通过研究参附益心颗粒对内皮祖细胞血管新生的影响，揭示其降低肺动脉高压大鼠肺血流动力学的作用机制。方法：皮下注射野百合碱制作肺动脉高压模型，给予参附益心颗粒灌胃，经尾静脉移植内皮祖细胞。测定肺血流动力学及毛细血管密度，ELISA法及qPCR法检测内皮祖细胞分化与旁分泌功能。体外培养内皮祖细胞，制备参附益心颗粒含药血清，不同剂量刺激，观察其对内皮祖细胞功能的影响，Western blot 法检测参附益心颗粒通过PI3K/Akt通路调节内皮祖细胞功能的作用机制。结果：内皮祖细胞移植组及参附益心颗粒组大鼠肺血流动力学明显减轻，右心室肥厚指数下降；内皮祖细胞移植组肺血管密度明显增高；移植组血浆内血管新生相关生长因子明显升高。参附益心颗粒中、高剂量组内皮祖细胞增殖与迁移能力增强，血管新生相关生长因子的含量增高。参附益心颗粒可以上调PI3K/Akt信号通路的p-Akt和p-eNOS蛋白表达水平。结论：参附益心颗粒能够明显减轻肺动脉高压大鼠肺血流动力学，降低右心室肥厚指数，并有促血管新生的作用。参附益心颗粒加强内皮祖细胞的增殖、迁移，并促进其分泌相关生长因子。参附益心颗粒促进EPCs增殖、迁移的作用可能与其激活 PI3K/Akt 信号通路，上调 p-Akt和 p-eNOS 蛋白表达水平有关。完成研究任务。