Effect Of TNFR₁Gene Silencing On Early Apoptosis Of Ofloxacin-and Marbofloxacin-Treated Chondrocytes From Juvenile Dogs

Quinolones (QNs) are a family of broad-spectrum antibacterial synthetic drugs which possess the characteristics such as high activities, high bioavailability, low price, and low probability of drug-resistant mutation as well as few cross reactions with other therapeutic agents. Therefore, QNs are widely used in clinical practice for their distinct effects and some other advantages. However, it is found by reserchers that QNs might make juvenile animals especially dogs show up the symptoms of cartilage toxic such as claudication, joint damage and so on. For the above reasons, clinical applications of QNs are restricted. The reason why QNs result in cartilage toxic remains unclear. The clinic research has proved that the relative gene of TNFR signal pathway among the death receptor ones provides expressions in different levels in QNs-treated chondrocytes in the early days. Therefore, whether the TNFR signaling pathway participates in the QNs induced chondrocyte apoptosis still need further researches to support. In this study, we use siRNA silencing the TNFR1gene to explore if the TNFR signaling pathway is involving in early apoptosis of QNs-treated chondrocytes.Taking the cartilage of juvenile dogs, we used the collagenase II isolated chondrocytes cultured until the third generation.We processed the experiment by using100μg/mL Marbofloxacin and Ofloxacin treated chondrocytes for2h,8h, and24h. Following that, we used MTT assay and Flow cytometry to detect the chondrocyte activity and apoptosis rate at each time point; we used RT-PCR method to detect the mRNA expression level of TNF-a, TNFR1, TRADD, FADD and Caspase-8, which are the critical factor of TNFR signaling pathway, at each time point; Then, we used the Western Blot to detect TNF-a and TNFR1protein expression levels at each time point. According to the results of the RT-PCR, we used the siRNA interference to silence TNFR1gene and detect the apoptosis rate of chondrocyte and the gene expression levels of the gene of downstream of TNFR1.Within24h, the Marbofloxacin and Ofloxacin treated chondrocytes chondrocyte apoptosis rate increased significantly (p<0.01), cell viability was decreased significantly (p<0.01), and it showed a time-dependent relationship. The mRNA levels of factors TNFa, TNFR1, TRADD, FADD and Caspase-8, which are related to the TNFR signaling pathway, peaked majority at8h. The death receptor iconic TNF-a and TNFR1protein expression levels increased significantly during2-24h (p<0.01). The silence of the TNFR1gene could inhibit (p<0.01) Marbofloxacin or Ofloxacin induced chondrocyte apoptosis rate and reduce the expression levels of the gene of downstream.We researched on the expressions of death receptor pathway of the relevant factors in cartilage of QN-treated within24h.And we proved that TNFR signaling pathway could be activated by Marbofloxacin and Ofloxacin and played a dominant role in apoptosis.