Toxic effects of levofloxacin on rat annulus fibrosus cells: an in-vitro study.
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.


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.
Department of Spine Surgery, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China (mainland).
Zhi-Long
ZL
Bai
Department of Spine Surgery, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China (mainland).
Qian
Q
Chen
Department of Spine Surgery, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China (mainland).
Si-Dong
SD
Yang
Department of Rehabilitation Medicine, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China (mainland).
Feng
F
Zhang
Department of Spine Surgery, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China (mainland).
Hai-Ying
HY
Wang
Department of Spine Surgery, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China (mainland).
Da-Long
DL
Yang
Department of Spinal Surgery, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China (mainland).
Wen-Yuan
WY
Ding
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D007403:Intervertebral Disc / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology*; D064704:Levofloxacin / Q000633:toxicity*; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011419:Propidium / Q000378:metabolism; D017207:Rats, Sprague-Dawley
D017304:Annexin A5; D011419:Propidium; D064704:Levofloxacin; D053148:Caspase 3; D019278:Matrix Metalloproteinase 3; D016650:Fluorescein-5-isothiocyanate