外泌体介导抗菌肽LL-37调控支气管上皮细胞-成纤维细胞对话促进COPD气道重塑的机制研究

Airway remodeling is the most important causing of the airflow limitation of Chronic Obstructive Pulmonary Disease. Exosome plays a pivotal role in modulation of crosstalk between bronchial epithelial cells and lung fibroblasts which further promotes the airway remodeling in Chronic Obstructive Pulmonary Disease, but the molecular mechanism still remains unclear. Our previous study confirmed that the expression of TLR4 and antimicrobial peptide LL-37 was significantly elevated in bronchial epithelial cells of COPD patients and the level of LL-37 was positively correlated with small airway wall thickness and collagen deposition. Cigarette smoke extract-induced epithelial secretion of LL-37 in exosome could up-regulate collagen expression of lung fibroblasts via activation of ERK signaling pathway. Based on the literature review and our previous study, we hypothesize that activation of TLR4 enhances exosomal shuttle of LL-37 in bronchial epithelial cells, LL-37 could be incorporated into exosome and transmitted to lung fibroblasts and then promote the proliferation and myofibroblast differentiation and the deposition of collagen in lung fibroblasts via activation of FPRL1-mediated ERK signaling pathway. To test this hypothesis, we will investigate the role and mechanism of exosome-transmitted LL-37 in regulation of crosstalk between bronchial epithelial cells and lung fibroblasts through the method of in vitro cell co-culture and knockout mice. This will further help us understand the pathogenesis of COPD, and will shed new lights on prevention and treatment of COPD.

气道重塑是COPD气流受限的主要原因，外泌体参与支气管上皮细胞-成纤维细胞对话促进COPD气道重塑发生，但机制不明。课题组前期研究发现：①COPD患者支气管上皮细胞TLR4及抗菌肽LL-37高表达，LL-37与小气道壁厚度及胶原沉积正相关；②CSE刺激支气管上皮细胞外泌体LL-37高表达，LL-37通过ERK通路促进成纤维细胞胶原合成。基于此，提出如下假说：TLR4调控支气管上皮细胞LL-37被包裹进入外泌体，LL-37经由外泌体途径被转运至成纤维细胞，与甲酰肽样受体结合激活ERK通路，促进成纤维细胞增殖、向肌成纤维细胞分化及胶原合成，促进COPD气道重塑。本课题拟采用细胞共培养、基因敲除小鼠等多种实验方法，从临床标本、细胞及动物等不同层面，探索LL-37经由外泌体途径调控支气管上皮细胞-成纤维细胞对话促进COPD气道重塑的分子机制，尝试以新的理论探讨COPD发病机理，为其防治提供科学依据

慢性阻塞性肺疾病（COPD）患病率高，气道重塑是COPD气流受限的主要原因之一。外泌体参与调控支气管上皮细胞与成纤维细胞间对话，促进COPD气道重塑，但机制不明。本课题收集COPD患者及健康吸烟者、健康非吸烟者诱导痰外泌体，分析外泌体中抗菌肽LL-37水平差异，并与患者肺功能FEV1%进行相关性分析；超速离心法提取支气管上皮细胞经CSE刺激后细胞培养上清液中的外泌体，进行鉴定；Western blot检测外泌体中LL-37水平，将外泌体加入成纤维细胞培养液中，观察其对成纤维细胞增殖及胶原合成的影响；最后进行动物实验在体内水平验证外泌体LL-37对于COPD气道重塑的作用。结果显示：COPD患者诱导痰外泌体中LL-37显著高于健康吸烟者及健康非吸烟者，健康吸烟者诱导痰外泌体中LL-37水平显著高于健康非吸烟者，COPD患者诱导痰外泌体中LL-37水平与患者肺功能FEV1%显著负相关；支气管上皮细胞经CSE刺激后LL-37表达升高，外泌体中LL-37水平显著升高，HLF-1能够摄取外泌体进入胞质中，支气管上皮细胞经CSE刺激后产生的外泌体可以促进成纤维细胞增殖及I型胶原、III型胶原的合成，MMP-9、MMP-12、Fibronectin mRNA和蛋白表达明显升高，将成纤维细胞中加入甲酰肽样受体FPRL-1的拮抗剂WRW4预处理，上述过程可被逆转。本研究系统探索支气管上皮来源的外泌体LL-37参与支气管上皮细胞-成纤维细胞对话，促进成纤维细胞增殖及胶原合成的过程，为研究COPD气道重塑机制提供了新的思路。