Proteogenomics Research Institute for Systems Medicine (PRISM), 505 Coast Blvd. South, La Jolla, CA 92037, USA.
Cells. 2024 Mar 15;13(6):515. doi: 10.3390/cells13060515.
Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease characterized by the relentless deposition of extracellular matrix (ECM), causing lung distortions and dysfunction. Animal models of human IPF can provide great insight into the mechanistic pathways underlying disease progression and a means for evaluating novel therapeutic approaches. In this study, we describe the effect of bleomycin concentration on disease progression in the classical rat bleomycin model. In a dose-response study (1.5, 2, 2.5 U/kg i.t), we characterized lung fibrosis at day 14 after bleomycin challenge using endpoints including clinical signs, inflammatory cell infiltration, collagen content, and bronchoalveolar lavage fluid-soluble profibrotic mediators. Furthermore, we investigated fibrotic disease progression after 2 U/kg i.t. bleomycin administration at days 3, 7, and 14 by quantifying the expression of clinically relevant signaling molecules and pathways, epithelial mesenchymal transition (EMT) biomarkers, ECM components, and histopathology of the lung. A single bleomycin challenge resulted in a progressive fibrotic response in rat lung tissue over 14 days based on lung collagen content, histopathological changes, and modified Ashcroft score. The early fibrogenesis phase (days 3 to 7) is associated with an increase in profibrotic mediators including TGFβ1, IL6, TNFα, IL1β, CINC1, WISP1, VEGF, and TIMP1. In the mid and late fibrotic stages, the TGFβ/Smad and PDGF/AKT signaling pathways are involved, and clinically relevant proteins targeting galectin-3, LPA1, transglutaminase-2, and lysyl oxidase 2 are upregulated on days 7 and 14. Between days 7 and 14, the expressions of vimentin and α-SMA proteins increase, which is a sign of EMT activation. We confirmed ECM formation by increased expressions of procollagen-1Aα, procollagen-3Aα, fibronectin, and CTGF in the lung on days 7 and 14. Our data provide insights on a complex network of several soluble mediators, clinically relevant signaling pathways, and target proteins that contribute to drive the progressive fibrotic phenotype from the early to late phase (active) in the rat bleomycin model. The framework of endpoints of our study highlights the translational value for pharmacological interventions and mechanistic studies using this model.
特发性肺纤维化（IPF）是一种破坏性的间质性肺病，其特征是细胞外基质（ECM）的无情沉积，导致肺扭曲和功能障碍。人类特发性肺纤维化的动物模型可以深入了解疾病进展的机制途径，并为评估新的治疗方法提供手段。在这项研究中，我们描述了博来霉素浓度对经典大鼠博来霉素模型中疾病进展的影响。在剂量反应研究（1.5、2、2.5 U/kg 腹腔内注射）中，我们在博来霉素攻击后 14 天使用包括临床症状、炎症细胞浸润、胶原蛋白含量和支气管肺泡灌洗液可溶性促纤维化介质在内的终点来描述肺纤维化。此外，我们通过量化临床相关信号分子和途径、上皮间质转化（EMT）生物标志物、细胞外基质成分以及肺组织病理学，研究了 2 U/kg 腹腔内注射博来霉素后第 3、7 和 14 天的纤维化疾病进展。单次博来霉素挑战导致大鼠肺组织在 14 天内出现渐进性纤维化反应，这是基于肺胶原蛋白含量、组织病理学变化和改良 Ashcroft 评分得出的。早期纤维化阶段（第 3 至 7 天）与包括 TGFβ1、IL6、TNFα、IL1β、CINC1、WISP1、VEGF 和 TIMP1 在内的促纤维化介质的增加有关。在中晚期纤维化阶段，TGFβ/Smad 和 PDGF/AKT 信号通路参与其中，并且针对半乳糖凝集素-3、LPA1、转谷氨酰胺酶-2 和赖氨酰氧化酶 2 的临床相关蛋白在第 7 天和第 14 天上调。在第 7 天和第 14 天之间，波形蛋白和α-SMA 蛋白的表达增加，这是 EMT 激活的标志。我们通过在第 7 天和第 14 天肺组织中增加前胶原-1Aα、前胶原-3Aα、纤维连接蛋白和 CTGF 的表达来证实 ECM 的形成。我们的数据提供了关于几个可溶性介质、临床相关信号通路和靶蛋白的复杂网络的见解，这些介质、通路和靶蛋白有助于推动大鼠博来霉素模型中从早期到晚期（活跃）的进行性纤维化表型。我们研究终点的框架突出了使用该模型进行药理学干预和机制研究的转化价值。
