Mechanism Of H₂O₂-induceds Apoptosis Of Rabbit Chondrocytes

ObjectiveOsteoarthritis （OA）, one of the most common joint diseases with unknown etiology, ischaracterized by the progressive destruction of articular cartilage and the apoptosis ofchondrocytes. Although reactive oxygen species （ROS） is known to be the immediate cause ofchondrocytes apoptosis and metabolic derangement, the detailed function remains unclear. Thepurpose of this study is to elucidate the molecular mechanisms of H₂O₂-mediated rabbitchondrocytes apoptosis.Methods（1）Primary normal chondrocytes were separated from the articular cartilage of6weeksold New Zealand rabbits.（2）Take advantage of treating chondrocytes with different concentration of hydrogenperoxide （H₂O₂） and measuring the cell viability by Cell Counting Kit （CCK-8） to chooseappropriate concentration and time of H₂O₂for next experiments.（3）FCM analysis of apoptosis with Annexin/PI double staining method or with PIstaining was to determine the fashion of the H₂O₂-induced cell death.（4）FCM method and confocal fluorescence imaging were used to examine the effect ofH₂O₂-induced loss of mitochondrial membrane potential.（5）Confocal fluorescence imaging was utilized to examine the lysosomal membranepermeabilization （LMP）.（6）Fluorogenic substrates were utilized to measure the activation of caspases.（7）FRET technique was used to measure the activation of caspases-9.（8）Detecting the translocation of BID and Bax in chondrocytes from cytoplasm tomitochondria.（9）Western blotting was used to detect the expression ofAIF, caspase, Cyt.c and FasL. Results（1）Different concentrations of resveratrol induced a dose-dependent cytotoxicity. Thestatistically significant concentrations0.3mM H₂O₂were chose for the subsequent experiments.（2）A variety of apoptotic detecting methods had confirmed that the fashion by whichH₂O₂-induced cell death was apoptosis，which can be inhibited by NAC.（3）H₂O₂treatment chondrocytes induced a significant reduction of mitochondrialmembrane potential （Δ Ψm）.（4）The lysosomal structure seems to be kept intact during the apoptotic process whenanalyzed by the laser fluorescence confocal microscopy.（5）H₂O₂treatment chondrocytes induced a remarkably increased expression of caspase-3and caspase-8but not caspase-9.（6）H₂O₂induced no translocation of BID and Bax from cytoplasm to mitochondria.（7）AIF but not Cyt.c was released from the mitochondria after H₂O₂treatment.（8）H₂O₂increased the expression of FasL.ConclusionH₂O₂induces apoptosis via both AIF-mediated caspase-independent and deathreceptor-mediate multiply signaling pathway in rabbit chondrocytes.