Abstract
Introduction
Intervertebral disk (IVD) degeneration is a major cause of lower back pain. Evidence suggests that inflammatory cytokines produced by chondrocyte-like cells within the IVD during degeneration are integral in the pathogenesis of this disease, with a particular role for IL-1 hypothesized. Targeting intracellular signaling mechanisms of IL-1 could provide a mechanism to inhibit multiple catabolic cytokines simultaneously. Many cytokines share intracellular signaling pathways and thus inhibiting signaling rather than direct receptors could prevent compensatory actions occurring. Here we investigated signaling pathways activated by IL-1 together with an anabolic factor to identify differential pathways.
Materials and Methods
Human nucleus pulposus (NP) cells were extracted via collagenase digestion and expanded in monolayer culture, prior to transfer to alginate bead culture for 2 weeks to enable redifferentiation. Following redifferentiation cells were treated with either IL-1 (10 ng/mL) or CDMP-1 (10 ng/ml) for 30 minutes, protein extracted and R&D proteome array deployed to investigate potential differential signaling molecules. In addition, NFkB activation was investigated using immuno-fluorescence and activation of pERK, pP38 MAPK, and AKT were investigated using BD phosflow techniques. In addition, cells were pretreated with inhibitors of signaling pathways: SB203580 (p38 MAPK) and Helenalin (NFkB) to investigate effects on matrix and matrix degrading enzyme gene expression following stimulation with CDMP and IL-1.
Results
R&D proteome array enabled the investigation of 46 signaling molecules simultaneously following and identified a number of differentially activated pathways, including p38MAPK (Fig. 1). IL-1 induced p38MAPK, NFkB, AKT, and ERK pathways were confirmed using BD phosflow and immunofluorescence techniques. Inhibition of NFkB pathway using helenine reduced the inhibitory effect on aggrecan induced by IL-1, but helenine alone also induced an inhibition of aggrecan induction by CDMP.
Conclusion
The catabolic cytokine IL-1 has been implicated in the pathogenesis of IVD degeneration which leads to low back pain. The identification of differential intracellular signaling pathways between anabolic and catabolic factors could provide new methods for modulating the catabolic response, and preventing further IVD degeneration.
Yes
None declared