Division of Physiology, School of Nursing and Rehabilitation Sciences, Showa University, Yokohama, Japan.
J Inflamm (Lond). 2010 Nov 23;7:56. doi: 10.1186/1476-9255-7-56.
Low-dose and long-term administration of 14-membered macrolide antibiotics, so called macrolide therapy, has been reported to favorably modify the clinical conditions of chronic airway diseases. Since there is growing evidence that macrolide antibiotic-resistant bacteria's spreaders in the populations received macrolide therapy, it is strongly desired to develop macrolide antibiotics, which showed only anti-inflammatory action. The present study was designed to examine the influence of clarithromycin (CAM) and its metabolized materials, M-1, M-4 and M-5, on free radical generation from nasal polyp fibroblasts (NPFs) through the choice of nitric oxide (NO), which is one of important effector molecule in the development of airway inflammatory disease in vitro.
NPFs (5 × 105 cells/ml) were stimulated with 1.0 μg/ml lipopolysaccharide (LPS) in the presence of agents for 24 hours. NO levels in culture supernatants were examined by the Griess method. We also examined the influence of agents on the phosphorylation of MAPKs, NF-κB activation, iNOS mRNA expression and iNOS production in NPFs cultured for 2, 4, 8, and 12 hours, respectively.
The addition of CAM (> 0.4 μg/ml) and M-4 (> 0.04 μg/ml) could suppress NO production from NPFs after LPS stimulation through the suppression of iNOS mRNA expression and NF-κB activation. CAM and M-4 also suppressed phosphorylation of MAPKs, ERK and p38 MAPK, but not JNK, which are increased LPS stimulation. On the other hand, M-1 and M-5 could not inhibit the NO generation, even when 0.1 μg/ml of the agent was added to cell cultures.
The present results may suggest that M-4 will be a good candidate for the agent in the treatment of chronic airway inflammatory diseases, since M-4 did not have antimicribiological effects on gram positive and negative bacteria.
低剂量和长期使用 14 元大环内酯类抗生素，即所谓的大环内酯类疗法，已被报道可改善慢性气道疾病的临床状况。由于越来越多的证据表明，接受大环内酯类治疗的人群中，大环内酯类抗生素耐药菌的传播者，因此强烈需要开发仅具有抗炎作用的大环内酯类抗生素。本研究旨在通过选择一氧化氮（NO）来研究克拉霉素（CAM）及其代谢产物 M-1、M-4 和 M-5 对鼻息肉成纤维细胞（NPF）自由基生成的影响，NO 是气道炎症性疾病体外发展的重要效应分子之一。
用 1.0μg/ml 脂多糖（LPS）刺激 NPF（5×105 细胞/ml）24 小时。用格里斯法检测培养上清液中的 NO 水平。我们还分别检测了药物对 NPF 培养 2、4、8 和 12 小时后 MAPKs 磷酸化、NF-κB 激活、iNOS mRNA 表达和 iNOS 产生的影响。
CAM（>0.4μg/ml）和 M-4（>0.04μg/ml）的加入可以通过抑制 iNOS mRNA 表达和 NF-κB 激活来抑制 LPS 刺激后 NPF 产生的 NO。CAM 和 M-4 还抑制了 MAPKs、ERK 和 p38 MAPK 的磷酸化，但不抑制 JNK 的磷酸化，JNK 是 LPS 刺激增加的。另一方面，即使在细胞培养物中加入 0.1μg/ml 的药物，M-1 和 M-5 也不能抑制 NO 的产生。
本研究结果表明，M-4 将是治疗慢性气道炎症性疾病的候选药物，因为 M-4 对革兰氏阳性和阴性菌没有抗菌作用。
