Peroxynitrite Induces Gene Expression in Intervertebral Disc Cells

In Brief Study Design. In vitro stimulation of human intervertebral disc (IVD) cells. Objective. To investigate the oxidative/nitrosative effects of peroxynitrite on human nucleus pulposus (NP) cells. Summary of Background Data. Peroxynitrite is an important tissue-damaging species generated at sites of inflammation and degeneration. The aim of this study was to examine the effects of oxidative/nitrosative stress caused by peroxynitrite and the peroxynitrite donor SIN-1 in human NP cells. Methods. Degenerated human IVD tissue was analyzed for nitrosylation by immunofluorescence. In addition, human NP cells were isolated from IVDs, expanded and stimulated either with peroxynitrite itself or a stable peroxynitrite donor (SIN-1). Nitrosylation, accumulation of intracellular reactive oxygen species, NF-κB nuclear translocation, and cell viability were analyzed by fluorescence. Gene expression of TNF-α, IL-1β, IL-6, IL-8, and IL-10 was quantified by real-time (RT)-PCR. Results. Degenerated IVD tissue showed strong nitrosylation, especially in the NP. Isolated human NP cells showed a strong signal for nitrosylation and intracellular reactive oxygen species on stimulation with peroxynitrite or SIN-1. NF-κB/p65 sustained nuclear translocation of NF-κB/p65 and stimulation of IL-1β, IL-6, and IL-8 expression was noted on treatment of cells with SIN-1. Conclusion. This study provides evidence that peroxynitrite may play a role in disc degeneration and discogenic back pain development by an increased synthesis of proinflammatory cytokines. Nuclear translocation of NF-κB was identified as the potential underlying pathway. Therefore, neutralizing peroxynitrite and its derivatives (e.g., via the use of antioxidants) may be a novel treatment option for discogenic back pain. The presence of nitrosylation in degenerated intervertebral discs indicated a role of reactive oxygen/nitrogen species in this process. In human nucleus pulposus cells, peroxynitrite and SIN-1 stimulated expression of proinflammatory cytokines, which correlated with sustained nuclear translocation of NF-κB. Therefore, peroxynitrite may play a primary role in the development of disc degeneration and possibly discogenic pain.