Notch信号通路影响气道平滑肌细胞合成基质金属蛋白酶12以促进COPD的机制探讨

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation and airway remodeling.Some studies have revealed that tumor necrosis factor alpha (TNF-alpha) and interleukin-18 (IL-18), which increase in COPD, induce the protein synthesis of matrix metalloproteinase-12 (MMP-12) originated from airway smooth muscle cells. Meanwhile, many studies also revealed that abnormal regulation of MMPs was induced by Notch signaling pathway in many cells, such as cancer cells, vascular endothelial cells,and vascular smooth muscle cells. In our previous study, airway smooth muscle cells from mice were cultured and IL-18 or TNF-alpha protein was added to cell supernatant. Significant increase of MMP-12 and Delta-like 4 (DLL4) which is one of Notch receptor was observed. A positive correlation have been found between the increased DLL4 and MMP-12. Thus, we speculate that DLL4/MMP-12 signal pathway may be a bridge that connects airway inflammation and airway remodeling. However, there are still many problems among the role of DLL4/MMP-12 signal pathway. In present proposal, first, we are going to study the role of Notch-MMPs pathway by siRNA technic to inhibit moleculars in the pathway one by one in vitro. Secondly, The effect of the Notch-MMPs is to be observed in vivo by using animal COPD models. The results of this study could help us to discover the partial role of Notch pathway by affecting MMP-12 regulation in airway smooth muscle cells in the disease process of COPD and provide some new knowledge of airway remodeling. Thus, we maybe have a new way to reduce the COPD and improve the disease status.

气道慢性炎症与气道重塑是慢性阻塞性肺疾病（COPD）重要病理特点。研究表明：COPD代表性炎症因子：肿瘤坏死因子alpha（TNF-alpha）和白介素-18（IL-18）可引起气道平滑肌细胞合成基质金属蛋白酶12（MMP-12）增多，从而推动气道重塑，但具体调节机制并不完全清楚。近年文献报道Notch信号通路影响肿瘤细胞等多种细胞的MMPs合成异常。我们预实验发现体外培养的气道平滑肌细胞中给予TNF-alpha或IL-18干预后，细胞表面Notch配体DLL4增多，MMP-12合成增加。本课题拟通过分别在体外细胞试验和动物模型在体实验两个层次干预气道平滑肌DLL4/MMP-12信号通路中各个因子，观察对下游信号分子的影响，以此阐明此信号通路的具体调控途径，观察该信号通路对COPD疾病进程的影响。本研究将有助于进一步揭示COPD的发病机制，从而为疾病的防治提供新思路。

气道慢性炎症与气道重塑是慢性阻塞性肺疾病（COPD）重要病理特点。研究表明：COPD代表性炎症因子：肿瘤坏死因子alpha（TNF-alpha）和白介素-18（IL-18）可引起小气道平滑肌细胞合成基质金属蛋白酶12（matrix metalloproteinases-12, MMP12）增多，从而推动气道重塑，但其中具体调节机制并不完全清楚。近年文献报道Notch信号通路影响肿瘤细胞，血管内皮细胞及血管平滑肌细胞等多种细胞的MMPs异常。在本项研究中，建立吸烟诱导的COPD小鼠模型，分离小鼠气道平滑肌细胞，通过q-PCR和Western-blot技术检测样品中Notch1、Notch2、Notch 3、Notch 4、DLL-1、DLL-3、Jagged-1、Jagged-2、NF-κB、AP-1的表达水平，证实以上因子均有不同程度升高。收集小鼠支气管肺泡灌洗液，通过ELISA技术检测证实TIMP-1下降，而MMP-9、MMP-12，和IL-6的蛋白水平升高。体外培养人气道平滑肌细胞（HASAM），给予香烟提取物刺激，notch信号通路抑制剂DAPT干预，以及Notch siRNA干预，通过检测caspase-3/7活性，CCK-8以及细胞流式技术检测，我们发现在低浓度香烟提取物刺激下，Notch干预组，细胞凋亡增加（P<0.05），但通过Western-blot对细胞自噬因子Beclin-1，Atg-3等蛋白检测，发现Notch信号通路激活使细胞内自噬作用增加，维持细胞稳态，进一步使AP-1蛋白磷酸化增加，细胞合成MMP-12增加。通过本研究，我们首次证实香烟通过Notch-1……Jagged-1……AP-1……p-AP-1……MMP-12这一信号通路促进COPD的进程。这将有助于进一步了解COPD的发病机制。