Studies On The Mechanisms Of Oxidative Damage Of Juvenile Rabbit Joint Chondrocytes Induced By Ofloxacin

Quinolones (QNs) belong to chemosynthesis antibacterials, which possess the elementary structure of 4-quinolone, and can selectively inhibit the DNA gyrase of bacterium. Due to its broad antibacterial spectrum, good antibacterial activity, low incidence of cross tolerance with other antibacterial agents, convenient administration and so on,there are 4 generation of QNs to have been developed in the near 50 years. Recently, FQNs is being widely used clinically because of its double bactericidal activity to G^-and G⁺. However, all kinds of drug adverse reaction (ADR) has been observed in clinical therapy of QNs, such as ADR in photoxicity, heart toxicity, chondrotoxicity and so on. Especially, people gradually pay close attention to the chondrotoxicity of QNs since accumulating evidences have demonstrated that QNs can generate toxicity on articular cartilage of juvenile animals,which have suggested that QNs possess the same toxicity on people's articular cartilage. In addition, lots of clinical data have shown that QNs can cause swelling and pain of joint after QNs has been administrated to children, and injuries of chondrocytes is observed in aborted fetus. So, QNs has been restricted to the application to the children, teenagers, pregnancy woman and lactation women. But the mechanism of ofloxacin-induced chondrotoxicity is not clear, especially, ofloxacin-induced oxidative damage. In the present study, we use the typical QNs, ofloxacin as the objective drug and the juvenile rabbit articular chondrocytes as experimental model to investigate the possible mechanism of QNs chondrotoxicity focusing on oxidative damage, DNA and protein damage, mitochondrion and the apoptosis.1 Ofloxacin induced oxidative damage to joint chondrotoxicity of juvenile rabbitChondrocytes from juvenile rabbit joint are incubated with ofloxacin at concentrations of 0, 5, 10, 20, 40 and 80μg/ml, respectively. Cell viability assay was performed essentially by a modified MTT assay, and activity of lactate dehydrogenase (LDH) was determined. The results showed ofloxacin decreases the viability of chondrocytes in a time and concentration-dependent manner. And ofloxacin can increase LDH activity in culture medium, and injury cell membrane resulted in LDH leakage. The extent of oxidative stress is assessed by measuring reactive oxygen species (ROS), nitrogen oxide (NO) and antioxidant defense system including reduced glutathione (GSH) and avtivity of antioxidant enzymes. It is observed that ofloxacin induced a concentration-dependent increase in intracellular ROS production, which may be an early mediator of ofloxacin cytotoxicity. Furthermore, antioxidant enzyme activities, such as glutathione peroxidase (GSH-px), catalase (CAT) and superoxide dismutase (SOD), were rapid decreased after treatment with ofloxacin.In addition, SOD decline and ROS production were strongly inhibited, and the loss in cell viability was partly abated by additional glutathione (GSH), N-acetylcysteine (NAC) and dithiothreitol (DTT). In conclusion, these results clearly demonstrate that oxidative stress plays an important role in ofloxacin-induced injuries of Juvenile rabbit joint chondrocytes.It is well known that ROS may injure intracellular biomacromolecule, such as lipid, DNA and protein. Use chromatometry of TBA, comet assay and western blot to measure the content of MDA, the extent of DNA damage and the content of protein carbonylation. It was observed that ofloxacin resulted in a significant lipid peroxidation, revealed by a concentration-dependent increase in the level of thiobarbituric acid reactive substances (TBARS). The study indicated that ofloxacin can increase tail length, tail DNA% and tail moment resulted in DNA damage. Western blot analysis succeeded to detect specifically increased protein carbonylation by ofloxacin in chondrocytes.2 Role of oxidative stress in ofloxacin-induced mitochondrion damage and apoptosis to chondrocytesChondrocytes from juvenile rabbit joint are incubated with ofloxacin at concentrations of 0, 5, 10, 20, 40 and 80μg/ml, respectively. Use different fluorescent probe to measure concentration of Ca²⁺, transmembrane potential of mitochondrion and cell apoptosis. The study showed ofloxacin increased concentration of Ca²⁺, decreased transmembrane potential of mitochondrion, and induced cell apoptosis to chondrocytes. And we also found scavenger of ROS could inhibit mitochondrial injury and suppress ofloxacin-induced apoptosis. In addition, cyclosporine (Cys), inhibitor of PTP, could inhibit cytotoxicity by ofloxacin to chondrocytes.The results indicated ofloxacin could induce increase of ROS, increase the level of MDA and damage cell membranc, protein and DNA, and resulted in cell apoptosis. Ofloxacin also induce cell apoptosis by increasing concentration of Ca²⁺, decreasing transmembrane potential of mitochondrion and opening PTP. In addition, the study showed antioxidant may inhibit ofloxacin-induced damage to chondrocytes.In conclusion, oxidative damage is one of the important mechanism in ofloxacin-induced damage to chongdrocytes. Oxidative stress increase concentration of Ca²⁺, decrease transmembrane potential of mitochondrion and open PTP. Lipid peroxidation damage induce chondrocytes apoptosis and antioxidants may prevent the damage of chondrocytes by ofloxacin.