Research On The Mechanism Of The Effects Of Hydrogen Peroxide And Interleukin 1β On Cultured Rabbit Joint Chondrocytes In Vitro

Osteoarthritis (OA) is a degenerative disease. It shows local, progressive destruction of articular cartilage and joint marginal osteophyte formation, and is accompanied by varying degrees of synovitis. The symptoms of OA are joint swell, ache, deformity, dysfunction and disability. The chondrocytes, which are the only cell type present in mature cartilage, are responsible for the synthesis and update of extracellular matrix and the maintenance of matrix integrity. The change of chondrocytes and extracellular matrix play a critical role in OA. Although the pathogenesis of OA is not yet clear, an increasing number of studies have found that reactive oxygen species (ROS) and cytokines play an important role.Objectives:In order to know the mechanism of ROS and cytokines in chondrocytes damage deeply and provide a theoretical basis with the discovery of new drugs and the OA therapy for further medical research, this experiment monolayer culture New Zealand rabbit articular chondrocytes in vitro and discuss the mechanism of ROS and interleukin-1β(IL-1β) induce rabbit articular chondrocytes apoptosis.Methods:1. Primary normal chondrocytes were separated from the articular cartilage of 6 weeks old New Zealand rabbits using the methods of mechanical separation and chemical digestion, and identified by toluidine blue staining.2. The chondrocytes were treated with different concentration of hydrogen peroxide (H2O2) and measured the cell viability by Cell Counting Kit (CCK-8). The experiment observed the cell viability of chondrocytes treated by H2O2 in different concentration and time, and chose appropriate concentration and time of H2O2for next experiments.3. Chondrocytes were treated with 1.5 mM of H2O2 only or combinating with cyclosporine A (CsA) or inhibitors of caspase 3,8,9, and then assessed cell viability by CCK-8.4. The apoptotic rate was evaluated by flow cytometry (FCM) using Annexin V-FITC/propidium iodide (PI) double staining. 5. We also used FCM to analyze mitochondrial membrane potential (△Ψm) and the dynamics of△Ψm in the single living cell stained by Rhodamin123 was monitored in real-time using confocal microscope.6. This research measured the cell viability of chondrocytes treated by IL-1βin different concentration and time CCK-8.Results:1. The chondrocytes, which biopsied from the cartilage tissue and then monolayer cultured by the methods of mechanical separation and chemical digestion, were confirmed by morphological observation and toluidine blue stainig.2. Exposure of chondrocytes to 1.5 mM of H2O2 can induce chondrocytes viability decreaseing. But the mitochondrial membrane stabilizing agent CsA and caspase3/8/9 inhibitors did not show any inhibitory effect on the H2O2h-induced viability decreaseing.3.1.5 mM of H2O2 treating chondrocytes for 2 h can induce a burst apoptosis and NAC can inhibit this effect to a certain extent.4. H2O2 treatment chondrocytes induced a marked reduction of mitochondrial membrane potential (ATm), and the△Ψm raised and maintained about 20 minutes before suddenly vanishing by monitored in real-time using confocal microscope.5. The concentration of 5-100ng/ml of IL-1βcan induce chondrocytes viability increasing.Conclusion:1. Monolayer culture of chondrocytes can get a large number of high purity, morphology and phenotypic stability of chondrocytes in a short time, and can meet the experimental needs.2. H2O2 induces chondroctye apoptosis by caspases-independent mitochondrial pathway.3. IL-1βcan make the chondrocytes proliferation.