基于NF-κB-BMP信号通路探讨黄芩苷对肺动脉高压血管重构的作用及机制研究

Pulmonary arterial hypertension (PAH) is characterized by functional and structural changes in the pulmonary vasculature, leading to increased pulmonary vascular resistance, right ventricular dysfunction, lung vascular remodeling and loss of the distal pulmonary vasculature. There are large numbe of herapies have been proven useful in decreasing pulmonary arterial pressure, improving exercise tolerance and quality of life, but an effective therapy of the long-term outcome in this disorder is lacking. In recent years, studies have shown that endothelial dysfunction is an indispensable feature and an early event in the pathogenesis of PAH. Vascular remodeling is the common response in various classes of PAH and proliferation of pulmonary arterial smooth muscle cells (PASMCs) contributes to vascular remodeling mostly. Presistent high pulmonary arterial pressure leads to increased vascular resistence, right ventricle hypertrophy, heart failure and even death.There is growing interest in identifying the signal mechanism underlying the development of vascular remodeling and the transition to heart failure. Nuclear factor-κB (NF-κB) is a key transcription regulator in various cardiac disorders but the role of NF-κB remains limited in PAH-induced vascular remodeling and RVH. Bone morphogenetic protein (BMP) signaling plays a critical role in PAH. Dysfunction of BMP signaling has been thought as an important reason of PAH. Studies have shown that in animal models of PAH, BMP signaling pathway and NF-κB signaling pathways has important connection and involved in vascular remodeling process. The regulating of NF-κB-BMP signaling pathway in the lungs providing new mechanistic information about MCT-induced PAH and RVH. In our previous studies showed that baicalin can decrease the pulmonary artery pressure, reduce right ventricular hypertrophy, and attenuate pulmonary vascular remodeling. The mechanism may be through inhibiting the inflammatory reaction and regulation NF-κB-BMP signaling pathway, but the underlying mechanism remains elusive. Based on our previous work, this research intends to through blocking signal pathways and siRNA technology to study the regulating mechanism of NF-κB-BMP signaling pathway in the development of PAH induced vascular remodeling. On the other hand, the aim was to explore the protective effects of baicalin on PAH through regulating NF-κB-BMP signal pathway, these would provide beneficial exploration for Chinese medicine in the treatment of PAH.

肺动脉高压(PAH)是一种进行性恶化疾病，WHO功能分级为Ⅳ的PAH患者，预后较差。肺小动脉内膜增厚，血管阻力增加导致的右心室衰竭是主要病理改变。近几年血管重构越来越引起重视，但其确切机制尚不完全清楚。我们前期研究证实抑制NF-κB信号通路能逆转PAH造成的肺血管重构，黄芩苷通过下调NF-κB信号通路，能抑制血管重构对PAH产生保护作用。最新研究发现BMP与NF-κB信号通路共同参与了PAH血管重构的发生过程，可能存在互作位点，但具体机制研究较少。结合我们前期工作基础与国内外研究现状，本研究拟应用蛋白酶体抑制剂阻断NF-κB信号通路，利用siRNA技术沉默NF-κB p65和BMPR2基因，从不同角度对NF-κB-BMP信号通路中起调控作用的细胞因子、蛋白、基因进行评价，筛选作用位点。并基于NF-κB-BMP信号通路探讨黄芩苷抑制PAH血管重构的作用机制，为中药治疗PAH提供有益的探索。

肺动脉高压(PH)是由不同病因导致的以肺动脉压力和肺血管阻力渐进性增高为特点的一组疾病或临床综合征，最终导致患者出现右心衰竭而死亡。PH发病机制复杂，肺动脉平滑肌细胞和肺血管内皮细胞增殖与凋亡平衡失调导致的肺血管重构是PAH不可逆发展的基本病理改变。因此，如何逆转肺血管细胞的增殖与凋亡平衡失调，从而抑制肺血管重构是有效防治PAH的关键环节，已经成为目前治疗PH的一个研究关注点。目前，临床上对PH的治疗效果不佳，进一步寻找有效抑制肺血管重构，改善心肺功能，提高患者生存率的药物十分必要。我们前期研究显示中药黄芩苷能明显改善野百合碱诱导的大鼠PH的右心室收缩压和右心室肥厚指数，减少炎症反应，但是作用机制不十分明确。结合研究现状与前期工作基础，本研究将应用组织学、免疫组织化学、实时定量PCR、Western blotting等技术检测黄芩苷对野百合碱诱导大鼠PH的防治作用。结果显示PH组与正常组相比大鼠活动减少，毛发晦涩无光泽，呼吸急促，消瘦，体重下降或不增；而黄芩苷治疗组特征有所改善，一般情况良好；PH模型组双侧肺脏明显肿大，颜色紫红，表面凹凸不平，弹性差，局部可见瘀血，黄芩苷治疗组上述损伤改善明显。不同剂量黄芩苷能使PH大鼠右心室测量右心室收缩压（RVSP）明显降低，右心室重量比（RV/LV+S）明显增加（P<0.05）；肺小动脉管壁增厚，管腔狭窄程度减轻（P<0.05）。免疫组织化学检测显示黄芩苷能明显降低肺血管增殖现象，表现为增殖细胞核抗原（PCNA）阳性细胞数量减少（P<0.05）；RT-PCR与western blot结果显示黄芩苷上调凋亡抑制基因Bcl2，下调促凋亡基因Bax和凋亡执行蛋白酶caspase-3表达；同时上调NF-κB-BMP信号通路中IκB-α、BMP2、BMP4、BMP9、BMPR2、Id1和Id3表达（P < 0.05），而下调NF-κB p65表达（P < 0.05）。Western blot与免疫荧光显示黄芩苷明显提高内皮细胞标记物CD31和E-cadherin 的表达，降低α-SMA表达（P < 0.05）。结论：黄芩苷对肺动脉高压的治疗作用与NF-κB-BMP信号通路相关，通过抑制NF-κB信号通路进一步干预BMP信号通路，抑制EMT发生相关。