title: Long-term treatment of clarithromycin at a low concentration improves hydrogen peroxide-induced oxidant/antioxidant imbalance in human small airway epithelial cells by increasing Nrf2 mRNA expression.
abstract: Clarithromycin (CAM), a representative macrolide antibiotic, has been used widely at low doses for long-term therapy of chronic inflammatory airway diseases. Anti-inflammatory effects of macrolide antibiotics were first discovered in clinical practice. Although oxidative stress is known as a key pathogenesis factor in chronic airway inflammatory diseases, the mechanism of action of low-dose, long-term CAM therapy remains unclear. We aimed to examine the cytoprotective action of CAM against hydrogen peroxide (H2O2)-induced cell dysfunction, focusing on CAM dose and treatment duration, and using human small airway epithelial cells (SAECs), the main cells involved in chronic airway inflammatory diseases.
  
  
  
  SAECs were pretreated with CAM (1, 5 or 10 μM) for 24, 48 or 72 h, and were subsequently exposed to H2O2for 0.5-4 h. Levels of interleukin (IL)-8, glutathione (GSH) and glutathione disulfide (GSSG), and the activities of nuclear factor (NF)-κB and γ-glutamylcysteine synthetase (γ-GCS) were assayed using specific methods. IL-8 mRNA and NF erythroid 2-related factor 2 (Nrf2) mRNA expression were measured using real-time reverse transcription polymerase chain reaction (RT-PCR). Tukey's multiple comparison test was used for analysis of statistical significance.
  
  
  
  Pretreatment with low-dose (1 or 5 μM), long-term (72 h) CAM inhibited H2O2-induced IL-8 levels, NF-κB activity, and IL-8 mRNA expression, and improved the GSH/GSSG ratio via the maintenance of γ-GCS expression levels. Similar to its enhancing effect on the GSH/GSSG ratio, pretreatment with low-dose CAM for 72 h significantly increased Nrf2 mRNA expression (p < 0.01 and p < 0.05). In contrast, these alterations were not observed after pretreatment with high-dose (10 μM) or short-term (24 and 48 h) CAM.
  
  
  
  CAM is efficacious against cell dysfunction caused by oxidative stress under low-dose, long-term treatment conditions. This effect depended on the suppression of NF-κB activation and improvement of the H2O2-induced oxidant/antioxidant imbalance that is achieved by increasing Nrf2 mRNA expression in SAECs. The present study may provide the first evidence of why low-dose, long-term administration of macrolides is effective for treating chronic inflammatory airway diseases.
authors.affiliation: Department of Pharmacology & Therapeutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-15-4-1 Maeda, Teine, Sapporo, Hokkaido, 006-8590, Japan.|Department of Hospital Pharmacy & Pharmacology, Asahikawa Medical University, Asahikawa, 078-8510, Japan.
authors.forename: Kuninori
authors.initials: K
authors.lastname: Iwayama
authors.affiliation: Department of Pharmacology & Therapeutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-15-4-1 Maeda, Teine, Sapporo, Hokkaido, 006-8590, Japan.|Department of Pharmacy, Shin-Sapporo Towakai Hospital, Sapporo, 004-0041, Japan.
authors.forename: Ayuko
authors.initials: A
authors.lastname: Kusakabe
authors.affiliation: Department of Pharmacology & Therapeutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-15-4-1 Maeda, Teine, Sapporo, Hokkaido, 006-8590, Japan.|Department of Dispensary, Rainbow Community Pharmacy, Sapporo, 062-0012, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ohtsu
authors.affiliation: Department of Pharmacology & Therapeutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-15-4-1 Maeda, Teine, Sapporo, Hokkaido, 006-8590, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nawano
authors.affiliation: Department of Public & Health, Hokkaido Pharmaceutical University School of Pharmacy, Sapporo, 006-8590, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Tatsunami
authors.affiliation: Department of Pharmacology & Therapeutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-15-4-1 Maeda, Teine, Sapporo, Hokkaido, 006-8590, Japan.|Department of Hospital Pharmacy & Pharmacology, Asahikawa Medical University, Asahikawa, 078-8510, Japan.
authors.forename: Ko-Ichi
authors.initials: KI
authors.lastname: Ohtaki
authors.affiliation: Department of Public & Health, Hokkaido Pharmaceutical University School of Pharmacy, Sapporo, 006-8590, Japan.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Tampo
authors.affiliation: Department of Pharmacology & Therapeutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-15-4-1 Maeda, Teine, Sapporo, Hokkaido, 006-8590, Japan. nh2767@hokuyakudai.ac.jp.
authors.forename: Nobumasa
authors.initials: N
authors.lastname: Hayase
mesh_terms: D000975:Antioxidants / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D017291:Clarithromycin / Q000008:administration & dosage*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics; D051267:NF-E2-Related Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D016877:Oxidants / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020545:Respiratory Mucosa / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D016896:Treatment Outcome
chemical_list: D000975:Antioxidants; D016209:Interleukin-8; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D016877:Oxidants; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; D017291:Clarithromycin
keywords: Anti-inflammatory effect; Clarithromycin; Human small airway epithelial cells; Interleukin-8; Long-term treatment; Low-dose; Nuclear factor erythroid 2-related factor 2; Nuclear factor-κB; Oxidant/antioxidant balance; γ-Glutamylcysteine synthetase