TSLP-RhoA/ROCK通路调控纤维细胞转分化在哮喘气道重塑中的作用

An emerging concept suggests that the airway epithelium is central to asthma pathogenesis. The damage to the airway epithelium with a dysregulated repair process is considered to be the underlying cause of airway remodeling. Human fibrocytes, which exhibit mixed morphological and molecular characteristics of mesenchymal stem cells, are progenitors and can further transdifferentiate its phenotype into fibroblasts and myofibroblasts both in vitro and in vivo under permissive microenvironmental conditions. RhoA/ROCK signaling pathway is a pivotal regulator in the phenotype transdifferentiation of fibrocytes, which has a central role in pathogenesis of ischemic myocardial remodeling. However, the role of fibrocytes transdifferentiation in airway remodeling is unknown. An increase in the number of fibrocytes in both the airway smooth muscle bundle and the peripheral blood, as well as upregulation of TSLP level, were reported in subjects with severe refractory asthma, and were positively assiciated with airflow obstruction.Our group demonstrated that TSLP mediates Th2 responses, is a pivotal contributor to airway remodeling in mice with chronic allergen-induced asthma. In addition, an increase of fibrocyte numbers and TSLP receptor upregulation on fibrocytes were both observed in the BALF of our models. Interestingly, pretreatment with anti-TSLP mAb dramatically inhibited TSLP receptor expression on fibrocytes, and prevented airway structrual alterations (PLoS One 8(1),2013 and our previous studies). These data above indicated that Th2 responses and structrual cells alterations were both necessary factors for airway remodeling, which may serve as software and hardware in these processes, respectively. We attempt to explore in this project that whether RhoA/ROCK signaling pathway activated by TSLP is to be the underlying mechanism for the fibrocytes to transdifferentiate into fibroblasts and myofibroblasts, using biochemical techniques such as RNA interference and confocal imaging, through the estabished asthmatic model and fibrocytes in peripheral blood in subjects with asthma. The outcomes of this study will be a supplement for asthma pathogenesis and the major targets for therapeutic intervention.

哮喘气道重塑是上皮损伤后病态修复的结果，纤维细胞（FC）具有干细胞特征，可转分化为（肌）成纤维细胞，是组织病态修复的成因。RhoA/ROCK通路介导FC转分化参与缺血损伤后心肌重塑，但FC与气道重塑关系未明。重症哮喘者气道平滑肌束有FC浸润，外周血FC数及胸腺淋巴细胞生成素（TSLP）增高，与气道阻塞程度正相关。课题组研究发现：哮喘动物TSLP表达增加，介导Th2细胞极化；肺泡灌洗液FC数增多且高表达TSLP受体，拮抗TSLP能够抑制FC数及气道重塑（PLoS One 8(1),2013及研究基础），提示Th2优势极化和结构细胞改变是气道重塑的"软件"和"硬件"。本项目利用动物模型和哮喘患者外周血FC，应用RNA干扰、共聚焦成像等技术，研究RhoA/ROCK通路在FC转分化为（肌）成纤维细胞中的作用，阐明TSLP诱导FC转分化参与气道病态修复的机制，为完善哮喘发病机理和干预靶点开辟新思路。

哮喘是常见的慢性气道炎症性疾病之一，其发病机制尚不十分清楚。气道重塑是哮喘除气道炎症外的另一重要病理特征，也是导致哮喘难治的主要原因。因此，揭示气道重塑的发生发展机制是改善哮喘预后的关键。Th2细胞优势极化反应和结构细胞改变是气道重塑的“软件”和“硬件”。我们前期发现，过敏原可刺激支气管上皮细胞分泌胸腺基质淋巴细胞生成素（TSLP），用外源性抗体拮抗TSLP可抑制过敏原诱导的哮喘气道炎症和气道重塑。在此基础上，本课题进一步探讨了TSLP与Th2型炎症反应、结构细胞转分化的关系及其在哮喘气道重塑中的作用。通过细胞共培养发现，TSLP促进DC表达OX40L，进而从转录因子水平诱导Naïve T细胞向Th2方向定向分化。通过动物模型发现，经鼻给予TSLP抑制剂可同时改善过敏原诱导的上下气道炎症。肌成纤维细胞是导致哮喘气道重塑的关键结构细胞，纤维细胞作为其前体细胞，在上皮细胞损伤时，接收损伤信号后发生迁移和表型转分化的机制未明。因此，我们进一步阐明了TSLP诱导纤维细胞转分化参与气道病态修复的相关机制。我们在哮喘小鼠气道组织和肺泡灌洗液中检测到显著升高的TSLP，TSLP在肺组织中的表达量与浸润的循环纤维细胞数量成正相关，拮抗TSLP可减少循环纤维细胞向哮喘小鼠气道组织募集，抑制上皮源性促纤维化细胞因子TGF-β1、IL-25、IL-33表达；此外，纤维细胞浸润的部位气道平滑肌层明显增厚，肌成纤维细胞数量显著增加，气道高反应性显著增强，用外源性抗体拮抗TSLP可抑制纤维细胞转分化引起的哮喘气道结构改变及气道高反应性。本项目的科学意义在于阐明哮喘气道损伤后，如何引起体内免疫病理和结构细胞发生改变及其相应机制，从气道重塑的“软件”和“硬件”两个途径出发探索潜在的气道重塑干预靶点，研究成果进一步明确并完善了TSLP在哮喘气道重塑发生发展中的重要作用，有望为寻找有临床应用价值的新药和治疗方法提供理论基础和实验依据。

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