基于Ca2+-Calcineurin-NFAT信号通路的益气化痰祛瘀方对COPD肺血管重构的机制研究

Pulmonary vascular remodeling is the important pathophysiology foundation in the occurrence and development of pulmonary arterial hypertension (PAH). Ca2+-Calcineurin-NFAT signaling pathway is closely related to pulmonary arterial smooth muscle cell hyperplasia of pulmonary vascular remodeling in COPD. Regulation of the key molecule expression of Ca2+-Calcineurin-NFAT signaling pathway is one of the important strategies to intervene in pulmonary vascular remodeling of COPD. Traditional Chinese medicine believes that COPD belongs to the syndrome of root deficiency and branch excess，and “Qi deficiency，phlegm and blood stasis” is the key pathogenesis of PAH formation. The therapy for nourishing Qi，eliminating phlegm and resolving stasis (Yiqi Huatan and Quyu treatment) is the basic therapeutic method of COPD. The previous studies showed that the Yiqi Huatan Quyu recipe — Qibaipingfei capsule could effectively inhibit the over-expression of endothelin-1 and fibrinogen，meanwhile increased the expression levels of NO and relieve pulmonary arterial hypertension. However，the molecular mechanism was left to be further proved. Based on the previous studies，this project will use research methods of combining in vivo and in vitro. The modern molecular biology techniques will be used to observe the expression of key molecules related Ca2+-Calcineurin-NFAT signaling pathway，and the correlation between the effect of Yiqi Huatan Quyu recipe on pulmonary vascular remodeling and Ca2+-Calcineurin-NFAT signaling pathway will be in-depth studied. This study will further reveal its pathways to work and targets about intervention pulmonary vascular remodeling in COPD，and provide a new breakthrough point on prevention and treatment of COPD.

肺血管重构是PAH发生发展的重要病理生理基础。Ca2+-Calcineurin-NFAT 信号通路与COPD肺血管重构过程中肺动脉平滑肌细胞异常增殖密切相关。调控Ca2+-Calcineurin-NFAT信号通路关键分子表达，是干预COPD肺血管重构的重要策略之一。中医认为COPD属本虚标实之证，气虚痰凝血瘀为PAH形成的关键病机，益气化痰祛瘀为其基本治法。既往研究显示，益气化痰祛瘀方—芪白平肺胶囊能有效下调ET-1、FIB的高表达，上调NO，缓解PAH，但其分子机制有待进一步探明。本课题在前期研究基础上，拟采用体内外相结合的研究方法，运用现代分子生物学技术，观察Ca2+-Calcineurin-NFAT 信号通路相关分子表达，深入研究益气化痰祛瘀方干预肺血管重构与该信号通路的相关性，进一步明确其干预COPD肺血管重构的起效途径和作用靶点，为COPD防治提供新的突破口。

本课题探讨了益气化痰祛瘀方对慢性阻塞性肺疾病（COPD）肺血管重构的作用及机制。主要研究内容与结果如下：1）通过观察COPD模型大鼠肺功能、血气分析、肺血管病理结构等，发现益气化痰祛瘀方能显著改善肺功能，改善COPD肺血管的结构及功能，从而干预肺血管重构。2）RT--qPCR及Western blot技术检测Ca2+-Calcineurin-NFATc3信号通路相关蛋白在COPD大鼠体内的表达，证实了益气化痰祛瘀方能抑制Ca2+-Calcineurin-NFATc3信号通路的表达，从而明确了益气化痰祛瘀方干预COPD肺血管重构的作用靶点。3）益气化痰祛瘀方含药血清作用于肺动脉平滑肌细胞，可通过降低细胞内Ca2+浓度，抑制calcineurin和NFATc3蛋白表达，抑制肺动脉平滑肌细胞增殖。4）采用SiRNA NFATc3基因沉默技术，能有效抑制低氧状态下肺动脉平滑肌细胞增殖，进一步验证了益气化痰祛瘀方对COPD肺血管重构的干预作用与Ca2+-Calcineurin-NFATc3信号通路的相关性。课题组通过体内外相结合的研究方法，逐步验证课题假说，完成了预期计划。