Abstract

BACKGROUND: Fluoroquinolones are in wide clinical use as safe and effective antibiotics. Articular cartilage, tendons, and epiphyseal growth plates have been recognized as targets of fluoroquinolone-induced connective tissue toxicity. However, the effects of fluoroquinolones on annulus fibrosus (AF) cells are still unknown.

MATERIAL AND METHODS: The main objective of this study was to investigate the effects of levofloxacin, a typical fluoroquinolone antibiotic drug, on rat AF cells in vitro. Rat annulus fibrosus (RAF) cells were treated with levofloxacin at different concentrations (0, 10, 20, 30, 40, 60, 80, and 90 μg/ml) and were assessed to determine the possible cytotoxic effects of levofloxacin. Inverted phase-contrast microscopy was used to accomplish the morphological observation of apoptosis of treated cells. Western blot and real-time quantitative RT-PCR (qPCR) was used to explore the expression of active caspase-3 and MMP-3. Flow cytometry was used to measure the apoptotic incidences.

RESULTS: Our study showed that levofloxacin, with concentrations at 30, 60, and 90 μg/ml, induced dose-dependent RAF cell apoptosis and higher expression of caspase-3 and MMP-3. More apoptotic cells were observed by inverted phase-contrast microscopy. Moreover, levofloxacin increased the activity of caspase-3, and it also reduced cell viability with different concentrations ranging from 10 to 80 μg/ml.

CONCLUSIONS: Our study results suggest that levofloxacin has cytotoxic effects on RAF cells, characterized by enhancing apoptosis and reducing cell viability, and indicate a potential toxic effect of fluoroquinolones on RAF cells.

Keywords: Annexin A5 - metabolism, Blotting, Western, Cell Shape - drug effects, Cell Survival - drug effects, Fluorescein-5-isothiocyanate - metabolism, Intervertebral Disc - pathology, Levofloxacin - toxicity, Matrix Metalloproteinase 3 - metabolism, Polymerase Chain Reaction, Propidium - metabolism, Rats, Sprague-Dawley