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 ha...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. In regarding to the chondrotoxicity of QNs,numerous studies have been done. The current hypothesis suggests that QNs compete with the β1 integrin receptors residing on chondrocyte surface for extracellular Mg ions,which leads to alternation in β1 integrin expression,or function and eventually results in chondrocyte death. Stupack et al (2001)demonstrated that caspase-8 could be recruited to unligated integrins in adherent cells and further initiate apoptosis in a death receptor-independent manner. Sheng et al (2008) found that ofloxacin induced rabbit's chondrocyte apoptosis by causing disturbance of β1 integrin functions and subsequently through caspase-8-dependent mitochondrial pathway.Apoptosis could be initiated through the stimulation of death receptors and through an intrinsic pathway from mitochondria. Santangelo and Bertone (2011) and Wu et al (2011) found that TNFα could be expressed in human primary condrocytes inducing by interleukin-1beta (IL-1) or lipopolysaccharide (LPS). However,to date there have not been sufficient results to support that signaling from the death receptors was involved in QNs-induced chondrocyte apoptosis.In addition,dilated cisternae of rough endoplasmic reticulum (ER) have been noticed in QNs-induced arthropathy.ER can participate in the initiation of apoptosis. Varieties of harmful cellular stimuli could lead to ERs (ER stress). Elevated ERs results in cellular apoptosis. But whether ERs mediates apoptosis in QNs-treated chondrocytes is not clear yet.In this study,we chose two QNs agents and chondrocytes were treated with ofloxacin and marbofloxacin at final concentrations of 20 μg / mL,50 μg / mL and 100 μg / mL respectively in vitro for 2 h,8 h and 24 h. Cell survival rate,cell apoptosis rate and death receptor pathway factors TNFα (intracellular tumor necrosis factor-alpha),TNFR1 (TNF receptor-1),TRADD (TNF receptor 1 associated via death domain),FADD (Fas-associated protein with death domain),caspase-8 and ERs mediated apoptosis factors caspase-12,GADD153 (CHOP or DDIT3),GRP78 (Bip),calpain,and anti-apoptosis factors Bcl-2 (B-cell lymphoma 2),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. We further silenced the expression of TNFR1 successfully by transferring TNFR1-siRNA to chondrocytes to confirm whether TNFα / TNFR1 signaling pathways are involved ofloxacin and marbofloxacin-induced apoptosis. Furthermore,expression of death receptor pathway representative proteins TNFα / TNFR1 and endocytoplasmic reticulum (ER) pathway representative protein caspase-12 were confirmed using Western Blot.We have found that ofloxacin and marbofloxacin could induce apoptosis of chondrocytes in a time-and dose-dependent fashion within 24 h. mRNA of TNFα,TNFR1,TRADD,FADD and caspase-8 (caspase-8 of ofloxacin treated group were at 24 h) were highly expressed at 8 h,and GADD153,GRP78,calpain and caspase-12 at 8 h or 24 h,and antiapoptosis factors NF-κB and Bcl-2 were also raised after 2 h,all in a dose-dependent fashion. Expression of caspase-8was downregulated after silenced TNFR1. TNFα and TNFR1 proteins were expressed at 8 h and caspase-12 proteins were expressed at 24 h. In addition,ofloxacin showed a higher toxicity.Our results indicate that death receptor pathway TNF / TNFR1 and ERs mediated apoptosis factors are involved in ofloxacin and marbofloxacin-induced apoptosis of in vitro cultured juvenile dog joint chondrocytes within 24 h.展开更多
文摘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. In regarding to the chondrotoxicity of QNs,numerous studies have been done. The current hypothesis suggests that QNs compete with the β1 integrin receptors residing on chondrocyte surface for extracellular Mg ions,which leads to alternation in β1 integrin expression,or function and eventually results in chondrocyte death. Stupack et al (2001)demonstrated that caspase-8 could be recruited to unligated integrins in adherent cells and further initiate apoptosis in a death receptor-independent manner. Sheng et al (2008) found that ofloxacin induced rabbit's chondrocyte apoptosis by causing disturbance of β1 integrin functions and subsequently through caspase-8-dependent mitochondrial pathway.Apoptosis could be initiated through the stimulation of death receptors and through an intrinsic pathway from mitochondria. Santangelo and Bertone (2011) and Wu et al (2011) found that TNFα could be expressed in human primary condrocytes inducing by interleukin-1beta (IL-1) or lipopolysaccharide (LPS). However,to date there have not been sufficient results to support that signaling from the death receptors was involved in QNs-induced chondrocyte apoptosis.In addition,dilated cisternae of rough endoplasmic reticulum (ER) have been noticed in QNs-induced arthropathy.ER can participate in the initiation of apoptosis. Varieties of harmful cellular stimuli could lead to ERs (ER stress). Elevated ERs results in cellular apoptosis. But whether ERs mediates apoptosis in QNs-treated chondrocytes is not clear yet.In this study,we chose two QNs agents and chondrocytes were treated with ofloxacin and marbofloxacin at final concentrations of 20 μg / mL,50 μg / mL and 100 μg / mL respectively in vitro for 2 h,8 h and 24 h. Cell survival rate,cell apoptosis rate and death receptor pathway factors TNFα (intracellular tumor necrosis factor-alpha),TNFR1 (TNF receptor-1),TRADD (TNF receptor 1 associated via death domain),FADD (Fas-associated protein with death domain),caspase-8 and ERs mediated apoptosis factors caspase-12,GADD153 (CHOP or DDIT3),GRP78 (Bip),calpain,and anti-apoptosis factors Bcl-2 (B-cell lymphoma 2),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. We further silenced the expression of TNFR1 successfully by transferring TNFR1-siRNA to chondrocytes to confirm whether TNFα / TNFR1 signaling pathways are involved ofloxacin and marbofloxacin-induced apoptosis. Furthermore,expression of death receptor pathway representative proteins TNFα / TNFR1 and endocytoplasmic reticulum (ER) pathway representative protein caspase-12 were confirmed using Western Blot.We have found that ofloxacin and marbofloxacin could induce apoptosis of chondrocytes in a time-and dose-dependent fashion within 24 h. mRNA of TNFα,TNFR1,TRADD,FADD and caspase-8 (caspase-8 of ofloxacin treated group were at 24 h) were highly expressed at 8 h,and GADD153,GRP78,calpain and caspase-12 at 8 h or 24 h,and antiapoptosis factors NF-κB and Bcl-2 were also raised after 2 h,all in a dose-dependent fashion. Expression of caspase-8was downregulated after silenced TNFR1. TNFα and TNFR1 proteins were expressed at 8 h and caspase-12 proteins were expressed at 24 h. In addition,ofloxacin showed a higher toxicity.Our results indicate that death receptor pathway TNF / TNFR1 and ERs mediated apoptosis factors are involved in ofloxacin and marbofloxacin-induced apoptosis of in vitro cultured juvenile dog joint chondrocytes within 24 h.
