The Effects Of TNF/TNFR1Signaling Pathway And Endocytopalsmic Reticulum Stress (ERs) On The Apoptosis Mechanism Of Ofloxacin And Marbofloxacin In Juvenile Dog Joint Chondrocytes

Quinolones (QNs) are widely used for their broad antibacterial spectrum and good antibacterial activities, desirable pharmacokinetic characteristics and few cross reactions with other therapeutic agents. However, QNs have adverse effects including chondrotoxicity in juvenile animals, so the use of QNs is contraindicated in children and adolescents. Previous studies have demonstrated that QNs induce chondrocyte apoptosis mainly through disturbance of β1integrin functions and injurys of oxiditive stress. Those hypotheses are all cause chondrocytes demage through mitochondrial pathway, but the exact mechanism still remains unclear. Apoptosis could be initiated through the stimulation of death receptors and through an intrinsic pathway from mitochondria. In addition endocytoplasmic reticulum (ER) can also participate in the initiation of apoptosis. This study was undertaken to investigate whether other apoptosis pathways were involved in QNs induced arthropathy and further elucidate the mechanisms of its adverse effects.In this study, third generation of chondrocytes were treated with ofloxacin and marbofloxacin at final concentrations of20μg/mL,50μg/mL and100μg/mL respectively in vitro for2h,8h and24h. Cell survival rate, cell apoptosis rate and death receptor pathway factors TNFa (intracellular tumor necrosis factor-alpha), TNFR1(TNF receptor-1), TRADD (TNF receptor1associated via death domain), FADD (Fas-associated protein with death domain), Caspase-8and ERs mediated apoptosis factors Caspase-12, GADD153(CHOP or DDIT3), GRP78(Bip), Calpain, and anti-apoptosis factors Bcl-2(B-cell lymphoma2), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) gene expression levels were measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) analysis to determine the dose-response relationship. Furthermore, expression of death receptor pathway representative proteins TNFa/TNFR1and endocytoplasmic reticulum (ER) pathway representative protein Caspase-12were confirmed using Western Blot.We have found that mRNA expression of TNFa, TNFR1, TRADD, FADD and Caspase-8were initiated at2h, highly expressed at8h (ofloxacin treated group were at24h), and trend to fall at24h. Expression of GADD153, GRP78, Calpain and Caspase-12mRNA were upregulated treated with ofloxacin and marbofloxacin. In ofloxcain group, Caspase-12, GADD153and Calpain mRNA expression were initiated at2h, peak at8h (Calpain was at24h), and GRP78was not significantly increased before24h. In marbofloxacin group, mRNA expression of Caspase-12, GADD153and GRP78were started at2h, but peaked at24, and Calpain was not increased before24h. It seemed Ofloxacin had a highter toxotity. Anti-apoptosis factors NF-κB and Bcl-2were also raised after2h, all in a dose-dependent fashion. The expressions of Bcl-2in marbofloxcin group were highter than those in ofloxcin group, but NF-κB were lower. TNFa and TNFR1proteins were expressed at8h and Caspase-12proteins were expressed at24h.Our research has established the method of joint chondrocytes of juvenile dog. Our results indicate that death receptor pathway TNF/TNFR1and ERs mediated apoptosis factors are involved in ofloxacin and marbofloxacin-induced apoptosis of in vitro cultured juvenile dog joint chondrocytes within24h.