title: Excessive reactive oxygen species are therapeutic targets for intervertebral disc degeneration.
abstract: BACKGROUND
  Oxidative stress has been reported to be involved in numerous human diseases, including musculoskeletal disorders such as osteoarthritis. However, the interaction between intervertebral disc (IVD) degeneration and oxidative stress is not well understood. The purpose of the present study was to elucidate the contribution of oxidative stress to IVD degeneration and the efficacy of antioxidant treatment for degenerative discs.
  
  
  METHODS
  The expression level of an oxidative stress marker, nitrotyrosine, was assessed by immunohistochemistry and Western blotting. For evaluating intracellular reactive oxygen species (ROS) levels and oxidative stress in rat annulus fibrosus (AF) cells, flow cytometry and luciferase assay with an OKD48 construct were performed. The grade of IVD degeneration was assessed by magnetic resonance imaging and histological analysis.
  
  
  RESULTS
  A high frequency of nitrotyrosine-positive cells was observed in rat and human degenerative discs. mRNA expression of catabolic factors such as tumor necrosis factor-alpha (TNF-alpha), matrix metalloprotease-3 (MMP-3), and cyclooxygenase-2 (COX-2) was significantly induced by treatment with H2O2 or buthionine sulfoximine, whereas that of aggrecan, an important chondrogenic proteoglycan, was reduced in a dose-dependent manner. Treatment with mitogen-activated protein kinase (MAPK) inhibitors blocked the inductive effect of excessive ROS on COX-2 mRNA expression. Western blotting confirmed the phosphorylation of MAPKs in H2O2 and BSO-treated AF cells. Conversely, we showed that TNF-α induced oxidative stress with increased intracellular ROS levels in AF cells. Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated the catabolic effect of excessive ROS and TNF-alpha in vitro. Finally, we showed that oral administration of NAC prevented IVD degeneration in rat degenerative model.
  
  
  CONCLUSIONS
  A positive feedback loop was formed between excessive ROS and TNF-alpha in AF cells. Thus, oxidative stress contributes to the progression of IVD degeneration and NAC can be a therapeutic option for IVD degeneration.
authors.affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. ssatosea@yahoo.co.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Suzuki
authors.affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. nfujita2007@yahoo.co.jp.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Fujita
authors.affiliation: Department of Orthopaedic Surgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan. hosonao@outlook.com.
authors.forename: Naobumi
authors.initials: N
authors.lastname: Hosogane
authors.affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. watakota@gmail.com.
authors.forename: Kota
authors.initials: K
authors.lastname: Watanabe
authors.affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. keni8888@z7.keio.jp.
authors.forename: Ken
authors.initials: K
authors.lastname: Ishii
authors.affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. toyama@z6.keio.jp.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Toyama
authors.affiliation: Department of Stem Cell Biology, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo, 162-8655, Japan. keiyot@gmail.com.
authors.forename: Keiyo
authors.initials: K
authors.lastname: Takubo
authors.affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. keisukehoriuchi@gmail.com.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Horiuchi
authors.affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. miyamoto@z5.keio.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyamoto
authors.affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. masa@a8.keio.jp.
authors.forename: Masaya
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. morio@a5.keio.jp.
authors.forename: Morio
authors.initials: M
authors.lastname: Matsumoto
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000328:Adult; D000368:Aged; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D015153:Blotting, Western; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007403:Intervertebral Disc / Q000187:drug effects / Q000378:metabolism; D055959:Intervertebral Disc Degeneration / Q000473:pathology*; D008279:Magnetic Resonance Imaging; D008297:Male; D008875:Middle Aged; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species; D000111:Acetylcysteine