Bone Abstracts

Background: The activation of Wnt/β-catenin signaling promotes cellular senescence and induces matrix metalloproteinases (MMPs) expression in intervertebral disc (IVD). However, little is known about the effects of hyperbaric oxygen (HBO) on the Wnt/β-catenin signaling and MMPs expression in degenerated human IVDs.

Materials and methods: Nucleus pulposus cells (NPCs) were separated from the degenerated disc nucleus tissue by performing sequential enzymatic digestion. Control cells were maintained in 5% CO₂/95% air throughout the experiment. The hyperoxic cells were exposed to 100% O₂ for 25 min and then to 5% CO₂/95% air for 5 min at 2.5 ATA in a hyperbaric chamber. The mRNA or protein levels of Wnt3a, LRP6, β-catenin, GSK-3β, aggrecan, type II collagen as well as MMP-3, 9, and 13 were analyzed after HBO treatment. The translocation of β-catenin from cytosol to nucleus after HBO treatment was detected by western blot. To determine the β-catenin-Tcf/Lef transcriptional activity, we measured the activity of Tcf/Lef reporter gene Topflash (optimal Tcf-binding site) or Fopflash (mutated Tcf-binding site) in NPC.

Results: The mRNA level of Wnt3a was down-regulated while that of aggrecan and type II collagen were up-regulated after HBO treatment. The relative density ratio (phosho-protein/protein) for LRP6 and GSK-3β were down-regulated after HBO treatment. Our western blot analysis showed decreased levels of β-catenin and translocated β-catenin after HBO treatment. There was decreased TOP flash activity following HBO stimulation, whereas the FOP flash activity was not affected. HBO decreased Tcf-dependent transcription and suppressed the expression of MMP-9.

Conclusion: HBO treatment suppresses Wnt/β-catenin signaling and MMPs expression in degenerated human IVDs.