Simultaneously Blocking Necrosis And Apoptosis To Protect Tmj Chondrocytes From Tnf-Alpha Induced Death: A Preliminary Study

Purpose: To study the effect of protecting temporomandibular joint chondrocyte from TNF-alpha induced death by simultaneously blocking necrosis and apoptosis in hope to find a potential adjuvant therapy target for temporomandibular joint post-traumatic osteoarthritis. Methods: Primary human condylar chondrocytes were cultured in vitro and cell growth curve was tested by CCK-8 method. Since acute post-traumatic inflammation and high levels of cytokines especially TNF-alpha could induce chondrocyte apoptosis, 20 ng/mL TNF-alpha was added to induce cell death. Before the application of TNF-alpha, chondrocytes were pre-treated with pan-caspase inhibitor Z-VAD-FMK (ZT Group), or specific programmed necrosis inhibitors Nec-1 (NT group), or the combination of these two inhibitors (ZNT group) or neither (T group). Apoptotic and necrotic cell death rates were detected using flow cytometry. CM-H2DCFDA staining was used to determine Reactive Oxygen Species (ROS) levels. Cell death rates and ROS levels were respectively compared and statistically analyzed. Results: T group showed exacerbated cell death and increased ROS levels in the remaining living cells, while cell death and ROS levels of ZT, NT and ZNT groups were significantly alleviated compared to T group (P< 0.05). Amongst these three groups, ZNT group achieved the minimum cell death rates and ROS levels. Conclusion: Under the circumstances of a high TNF-alpha concentration, condylar cartilage cells could go through both apoptosis and necroptosis. Blocking both forms of programmed cell death pathways can significantly improve cell survival rate. TNF-alpha initiated apoptosis and necroptosis pathways might be a potential adjuvant therapy target for temporomandibular joint post-traumatic osteoarthritis.