NF-kB Mediates Cartilage Degradation induced by Trauma Injury and IL-1

Abstract

BACKGROUND: IL-1 is one of the major pro-inflammatory cytokines responsible for cartilage degradation. Several studies have shown that IL-1 mediates the upregulation of tissue degradation through the NF-κB and Mitogen-activated protein kinase (MAPK, p38) pathways, but its role in cartilage degradation after blunt trauma injury is not clear. The objective of this study was to determine the roles of NF-κB and p38 in IL-1- induced cell death, proteoglycan (PG) degradation, nitric oxide (NO) production, and related gene upregulation in cartilage after blunt injury. METHODS: Full-thickness cartilage plugs were obtained from mature bovine knees (>18 mo) and pre-cultured in DMEM. The signaling pathways (p38 and NF-kB) were inhibited by pretreatment with 10μM SB202190 for p38 (p38i) and 50μM BAY117085 for NF-κB (IκBi) for 1 hour. Samples in Injury and Injury+IL-1 groups received impact injury with impact energy of 15J/cm2. IL-1 and Injury+IL-1 groups were treated with 1 ng/ml IL-1. Cell viability was assessed using fluorescein diacetate and propidium iodine. The mRNA from cartilage was isolated using Trizol and RNeasy Mini kit (Qiagen), reverse transcribed, and analyzed using qPCR to determine pro-inflammatory cytokine and tissue remodeling genes (IL- 6, MMP-3, TIMP-3). All gene expression was normalized to GAPDH. The medium was analyzed for proteoglycan (PG) release/loss and nitric oxide (NO) production using dimethylmethylene blue (DMMB) and Greiss assays, respectively. RESULTS: Increased cell death was found in the Injury and Injuy+IL-1 groups. Increase of PG loss was found in IL-1, Injury and Injury+IL-1 treated groups (37%, 104% and 126%, respectively). Significant decreases (69-73%) of PG loss were found in all IκBi treated groups, while little or no changes were found in the p38 groups. Results from the qPCR analysis supported the findings. IκBi treatment reduced MMP-3 upregulation induced by IL-1 and Injury, while there was minimal change with the p38 inhibitor. Similarly, NO production was also decreased in the IκBi treated groups. DISCUSSION and CONCLUSION: Our study suggests that the NF-κB signaling pathway plays a greater role than p38 in IL-1 mediated PG loss and NO production in cartilage after trauma injury. Future studies are needed to determine the time-course response and specific NF-κB mediators for downstream regulation, as well as the effects in long-term therapeutic treatment to ameliorate the progress of post traumatic osteoarthritis.