u-0126 and Intervertebral-Disc-Degeneration

Intervertebral disc (IVD) degeneration is a spinal disorder that triggers an inflammatory response and subsequent development of spinal pseudoarthrosis. The aim of the present study is to elucidate the role of the extracellular signal-regulated kinase (ERK) pathway in inflammation-induced IVD cells. Inflammatory human nucleus pulposus (NP) cells (NPCs) were induced using tumor necrosis factor-α and the ERK pathway was blocked using a selective molecule-based inhibitor U0126. Gene expression of catabolic and anabolic markers, proinflammatory, and NPCs markers was investigated. The enzymatic activity of matrix metalloproteinases (MMP)2/MMP9 was determined by gelatin zymography and nitrite production was assessed by Griess reaction. The NPC metabolic activity and viability were assessed using resazurin sodium-salt and live/dead assays, and subsequently, the specificity of U0126 on ERK1/2 signaling was determined. The catabolic enzyme MMP3 (p = 0.0001) and proinflammatory cytokine interleukin 6 (p = 0.036) were downregulated by U0126 in NPCs under inflammatory conditions. A significant increase of the cytokeratin 19 (p = 0.0031) was observed, suggesting a partial and possible recovery of the NP phenotype. U0126 does not seem to have an effect on prostaglandin production, aggrecanases, or other anabolic genes. We confirmed that U0126 selectively blocks the ERK phosphorylation and only affects the cell metabolic activity without the reduction of viable cells. Inhibition of ERK signaling downregulates important metalloproteinases and proinflammatory cytokines, and upregulates some NP markers, suggesting its potential to treat IVD degeneration.

Topics: Butadienes; Cytokines; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Gelatin; Humans; Interleukin-6; Intervertebral Disc; Intervertebral Disc Degeneration; Keratin-19; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Nitriles; Nitrites; Nucleus Pulposus; Prostaglandins; Sodium; Tumor Necrosis Factor-alpha

The loss of intervertebral disc (IVD) cells due to excessive apoptosis induced by inflammatory cytokines is a major cause of IVD degeneration. This study aims to explore the mechanism of interleukin-1β (IL-1β)-induced apoptosis of annulus fibrosus cells (AFCs). It's hypothesized that IL-1β induces apoptosis through the extracellular signal-regulated kinase (ERK) pathway in AFCs.. The mRNA and protein expression levels of apoptosis-associated genes were analyzed by quantitative real-time PCR and Western blotting. The apoptotic rate was measured by flow cytometry. Three experimental groups were established, including Control, IL-1β, and IL-1β+U0126 groups, respectively.. Increase in the expression of apoptosis-associated genes including B-cell lymphoma-2 associated X (Bax), caspase-3, and caspase-9, and meanwhile, decrease in the expression of B-cell lymphoma-2 (Bcl-2) gene were found in patients with degenerative IVDs. In in vitro tests, both apoptosis and phosphorylated ERK expression in rat AFCs decreased in the IL-1β+U0126 group compared with the IL-1β group. The expression levels of Bax, caspase-3, and caspase-9 in AFCs decreased significantly in the IL-1β+U0126 group compared with those in the IL-1β group. The expression level of Bcl-2, on the other hand, significantly increased.. Findings from this study suggest that IL-1β induces apoptosis in AFCs through the ERK pathway, and therefore, ERK inhibition may provide certain protection against the adverse effects of IL-1β.

Topics: Adult; Aged; Annulus Fibrosus; Apoptosis; Butadienes; Female; Gene Expression Regulation; Humans; Interleukin-1beta; Intervertebral Disc Degeneration; Male; MAP Kinase Signaling System; Middle Aged; Nitriles