Kimura Toru, Nojiri Takashi, Hino Jun, Hosoda Hiroshi, Miura Koichi, Shintani Yasushi, Inoue Masayoshi, Zenitani Masahiro, Takabatake Hiroyuki, Miyazato Mikiya, Okumura Meinoshin, Kangawa Kenji
Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka, 565-8565, Japan.
Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Suita-City, Osaka, Japan.
Respir Res. 2016 Feb 19;17:19. doi: 10.1186/s12931-016-0335-6.
Pulmonary fibrosis has high rates of mortality and morbidity; however, no effective pharmacological therapy has been established. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, selectively binds to the transmembrane guanylyl cyclase (GC)-B receptor and exerts anti-inflammatory and anti-fibrotic effects in various organs through vascular endothelial cells and fibroblasts that have a cell-surface GC-B receptor. Given the pathophysiological importance of fibroblast activation in pulmonary fibrosis, we hypothesized that the anti-fibrotic and anti-inflammatory effects of exogenous CNP against bleomycin (BLM)-induced pulmonary fibrosis were exerted in part by the effect of CNP on pulmonary fibroblasts.
C57BL/6 mice were divided into two groups, CNP-treated (2.5 μg/kg/min) and vehicle, to evaluate BLM-induced (1 mg/kg) pulmonary fibrosis and inflammation. A periostin-CNP transgenic mouse model exhibiting CNP overexpression in fibroblasts was generated and examined for the anti-inflammatory and anti-fibrotic effects of CNP via fibroblasts in vivo. Additionally, we assessed CNP attenuation of TGF-β-induced differentiation into myofibroblasts by using immortalized human lung fibroblasts stably expressing GC-B receptors. Furthermore, to investigate whether CNP acts on human lung fibroblasts in a clinical setting, we obtained primary-cultured fibroblasts from surgically resected lungs of patients with lung cancer and analyzed levels of GC-B mRNA transcription.
CNP reduced mRNA levels of the profibrotic cytokines interleukin (IL)-1β and IL-6, as well as collagen deposition and the fibrotic area in lungs of mice with bleomycin-induced pulmonary fibrosis. Furthermore, similar CNP effects were observed in transgenic mice exhibiting fibroblast-specific CNP overexpression. In cultured-lung fibroblasts, CNP treatment attenuated TGF-β-induced phosphorylation of Smad2 and increased mRNA and protein expression of α-smooth muscle actin and SM22α, indicating that CNP suppresses fibroblast differentiation into myofibroblasts. Furthermore, human lung fibroblasts from patients with or without interstitial lung disease substantially expressed GC-B receptor mRNA.
These data suggest that CNP ameliorates bleomycin-induced pulmonary fibrosis by suppressing TGF-β signaling and myofibroblastic differentiation in lung fibroblasts. Therefore, we propose consideration of CNP for clinical application to pulmonary fibrosis treatment.
肺纤维化具有较高的死亡率和发病率；然而，尚未确立有效的药物治疗方法。C型利钠肽（CNP）是利钠肽家族的一员，它选择性地与跨膜鸟苷酸环化酶（GC）-B受体结合，并通过具有细胞表面GC-B受体的血管内皮细胞和成纤维细胞在各个器官中发挥抗炎和抗纤维化作用。鉴于成纤维细胞活化在肺纤维化中的病理生理重要性，我们推测外源性CNP对博来霉素（BLM）诱导的肺纤维化的抗纤维化和抗炎作用部分是由CNP对肺成纤维细胞的作用所介导的。
将C57BL/6小鼠分为两组，即CNP治疗组（2.5μg/kg/min）和对照组，以评估BLM诱导（1mg/kg）的肺纤维化和炎症。构建了在成纤维细胞中过表达CNP的骨膜蛋白-CNP转基因小鼠模型，并在体内通过成纤维细胞检测CNP的抗炎和抗纤维化作用。此外，我们使用稳定表达GC-B受体的永生化人肺成纤维细胞评估了CNP对转化生长因子-β（TGF-β）诱导的向肌成纤维细胞分化的抑制作用。此外，为了研究CNP在临床环境中是否作用于人肺成纤维细胞，我们从肺癌患者手术切除的肺中获取原代培养的成纤维细胞，并分析GC-B mRNA转录水平。
CNP降低了博来霉素诱导的肺纤维化小鼠肺中促纤维化细胞因子白细胞介素（IL）-1β和IL-6的mRNA水平，以及胶原蛋白沉积和纤维化面积。此外，在成纤维细胞特异性过表达CNP的转基因小鼠中也观察到了类似的CNP作用。在培养的肺成纤维细胞中，CNP处理减弱了TGF-β诱导的Smad2磷酸化，并增加了α-平滑肌肌动蛋白和SM22α的mRNA和蛋白表达，表明CNP抑制成纤维细胞向肌成纤维细胞的分化。此外，患有或未患有间质性肺病的患者的人肺成纤维细胞均大量表达GC-B受体mRNA。
这些数据表明，CNP通过抑制肺成纤维细胞中的TGF-β信号传导和肌成纤维细胞分化来改善博来霉素诱导的肺纤维化。因此，我们建议考虑将CNP用于肺纤维化治疗的临床应用。
