基于JAK-STAT信号通路研究益气活血通络法干预COPD肺血管重构的分子机制

Pulmonary vascular remodeling is one of the main pathological feature and pathological basis of chronic obstructive pulmonary diease(COPD) .Reversing pulmonary vascular remodeling is one of the method for preventing and treating COPD.Based on the previous study,which we found that tonifying Qi, promoting blood flow and dredging collaterals treatment on COPD can regulate the expression of VEGF in smooth muscle cells of pulmonary vascular.This study focuses on the JAK/STAT signaling pathway which is close related to cell growth and migration of pulmonary arterial smooth muscle cells(PASMC). We plan to determine the mechanism of tonifying Qi, promoting blood flow and dredging collaterals treatment to inhibit the proliferation of PASMC by directly regulating the JAK/STAT signaling pathway.Firstly, the rat model of COPD will be established by intratracheal instillation of LPS and daily exposure to cigarette-smoking.Then, PASMC is taking for target cells.Using the methods such as immunohistochemistry, immunocytochemistry signaling pathway phosphorylation level of detection and signal blocking technology.The major signaling pathways and cytokine balance control of BaoFei DingChuan decoction will be determined according to the quantitative alteration of factors which related with pulmonary vascular remodeling such as JAK、STAT、IL-6、VEGF and PCNA,as well as pulmonary vascular histopathological changes in the cell of animal models.The results are expected to elucidate gene target mechanism of tonifying Qi, promoting blood flow and dredging collaterals treatment on pulmonary vascular remodeling of COPD,and provide the theoretical and experimental evidences for this treatment to cure COPD in clinic.

慢性阻塞性肺疾病（COPD）主要病理改变之一是肺血管重构，减轻肺血管重构是防治COPD及其并发症的方法之一。在我们前期研究发现益气活血通络法（保肺定喘汤）对肺动脉平滑肌细胞（PASMC)表达VEGF有抑制作用的基础上，本项目紧扣与血管平滑肌细胞增殖和迁移密切相关的JAK-STAT信号传导通路，从益气活血通络法对JAK-STAT信号传导通路的调控来抑制PASMC增殖的研究思路出发，首先采用气道滴注LPS联合香烟烟雾暴露的方法制备大鼠COPD模型，然后以PASMC为研究靶细胞，采用免疫组织化学、免疫细胞化学、信号通路磷酸化水平检测及信号阻断等技术，观察保肺定喘汤治疗后JAK、STAT及IL-6、VEGF、PCNA等肺血管重构相关因子的表达水平及肺动脉的病理变化，阐明益气活血通络法干预COPD肺血管重构及调控肺动脉平滑肌细胞增殖相关信号传导通路的分子机制，为其临床推广提供理论和实验依据。

研究背景：肺血管重构是COPD主要病理改变之一，寻找能有效干预肺血管重构药物是治疗COPD、减少其并发症的关键所在。本项目紧扣与血管平滑肌细胞增殖和迁移密切相关的JAK/STAT信号传导通路，从中医益气活血通络法对JAK/STAT信号传导通路的调控来抑制PASMCs增殖的研究思路出发，研究中医治法对于COPD肺血管重构的作用机制。.研究内容：研究分为在体实验以及离体实验两部分。首先在体实验观察益气活血通络法（保肺定喘汤）对大鼠肺血管重构病理形态学变化，血清IL-6含量、JAK/STAT信号通路磷酸化水平和VEGF、PCNA在肺动脉上表达的影响。其次在细胞实验中以PASMCs为研究靶细胞，用CSE刺激PASMCs，观察PASMCs增殖以及凋亡的变化。观察保肺定喘汤含药血清对CSE刺激后PASMCs增殖、凋亡水平变化的影响,以及PASMCs中PCNA蛋白、JAK-STAT信号通路磷酸化水平的影响。.重要结果：.1.动物实验：.1.1、保肺定喘汤可以明显减轻COPD大鼠肺血管重构病理形态学变化。.1.2、COPD大鼠血清IL-6含量、肺动脉PCNA及VFGF表达量增加，保肺定喘汤可显著减少血清IL-6含量，抑制JAK/STAT信号通路的磷酸化水平，减少VEGF、PCNA在肺血管组织的表达。.2.细胞实验：.2.1、CSE刺激下的PASMCs增殖程度逐渐增加，在10%CSE作用下达到峰值；随着CSE浓度的增高PASMC的凋亡程度逐渐增加，在20%CSE作用下已经产生了明显的凋亡。.2.2、保肺定喘汤含药血清能够抑制10%CSE 刺激下的PASMCs的增殖，可能与保肺定喘汤含药血清抑制JAK-STAT信号通路从而导致PCNA表达的下降有关；同时研究还发现保肺定喘汤含药血清剂量依赖性的抑制了PASMCs的凋亡，其作用机制之后需要进一步的实验研究。.2.3、CSE刺激可以导致PASMCs中JAK/STAT信号通路的磷酸化水平升高，同时出现PCNA蛋白表达的增强；而保肺定喘汤含药血清可以抑制由于CSE刺激而激活的JAK/STAT信号通路的磷酸化水平并以此来降低PCNA的表达。.总之：保肺定喘汤能抑制肺血管平滑肌细胞增殖，减少PCNA及VEGF表达，从而减轻肺血管重构，其分子机制可能通过抑制JAK-STAT信号传导通路在肺血管平滑肌细胞中的表达有关。