title: Levofloxacin increases apoptosis of rat annulus fibrosus cells via the mechanism of upregulating MMP-2 and MMP-13.
abstract: Levofloxacin was previously reported to induce apoptosis of rat annulus fibrosus (AF) cells by upregulating active caspase-3 and matrix metalloproteinase (MMP)-3 expression in vitro. However, the effects of levofloxacin on rat AF cells, as well as the related mechanism, have not been revealed completely. The purpose of this study was to further explore the changes in extracellular matrix and MMPs of rat AF cells based on levofloxacin-induced apoptosis. AF cells isolated from rat AF regions were cultured in monolayers and treated with levofloxacin in a dose- and time-dependent manner. To determine the cytotoxic effects of levofloxacin, inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells. The mRNA expression levels of MMP-2, -9 and -13 were quantified by reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR). Protein level of MMP-2 and MMP-13 were determined by western blot. The results showed that levofloxacin induced marked AF cell apoptosis, which was observed by inverted phase-contrast microscopy, and indicated by the increased expression of active caspase-3. Both RT-qPCR and western blot revealed that MMP-2 and MMP-13 expression were upregulated by levofloxacin treatment in a time- and dose-dependent manner. Moreover, cellular binding to type I collagen was found to be decreased by levofloxacin. In conclusion, the results above suggest that the possible cytotoxic effects of levofloxacin on AF cells in vitro may be attributed to the decreased cell binding to type I collagen and up-regulated expression of MMP-2 and MMP-13.
authors.affiliation: Department of Spinal Surgery, The Third Hospital of Hebei Medical University Shijiazhuang 050051, China.
authors.forename: Hai-Kun
authors.initials: HK
authors.lastname: Wei
authors.affiliation: Department of Spinal Surgery, The Third Hospital of Hebei Medical University Shijiazhuang 050051, China.
authors.forename: Si-Dong
authors.initials: SD
authors.lastname: Yang
authors.affiliation: Department of Spinal Surgery, The Third Hospital of Hebei Medical University Shijiazhuang 050051, China.
authors.forename: Zhi-Long
authors.initials: ZL
authors.lastname: Bai
authors.affiliation: Department of Spinal Surgery, The Third Hospital of Hebei Medical University Shijiazhuang 050051, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Spinal Surgery, The Third Hospital of Hebei Medical University Shijiazhuang 050051, China.
authors.forename: Da-Long
authors.initials: DL
authors.lastname: Yang
authors.affiliation: Department of Spinal Surgery, The Third Hospital of Hebei Medical UniversityShijiazhuang 050051, China; Hebei Provincial Key Laboratory of Orthopedic BiomechanicsShijiazhuang 050051, China.
authors.forename: Wen-Yuan
authors.initials: WY
authors.lastname: Ding
keywords: Levofloxacin; annulus fibrosus; apoptosis; intervertebral disc degeneration; matrix metalloproteinase