Abstract
Premature senescence of nucleus pulposus (NP) cells and inflammation are two common features of degenerated discs. This study investigated the effects of the inflammatory cytokine TNF-α on the premature senescence of NP cells and the molecular mechanism behind this process. Rat NP cells were cultured with or without different concentrations of TNF-α for 1 and 3 days. The inhibitor LY294002 was used to determine the role of the PI3K/Akt pathway. NP cells that were incubated with TNF-α for 3 days followed by 3 days of recovery in the control medium were used to analyze cellular senescence. Results showed that TNF-α promoted premature senescence of NP cells, as indicated by decreased cell proliferation, decreased telomerase activity, increased SA-β-gal staining, the fraction of cells arrested in the G1 phase of the cell cycle, the attenuated ability to synthesize matrix proteins and the up-regulated expression of the senescence marker p16 and p53. Moreover, a high TNF-α concentration produced greater effects than a low TNF-α concentration on day 3 of the experiment. Further analysis indicated that the inhibition of the PI3K/Akt pathway attenuated the TNF-α-induced premature senescence of NP cells. Additionally, TNF-α-induced NP cell senescence did not recover after TNF-α was withdrawn. In conclusion, TNF-α promotes the premature senescence of NP cells, and activation of the PI3K/Akt pathway is involved in this process.
Highlights
Intervertebral disc degeneration (IDD) is frequently associated with low back pain (LBP), which leads to patient disability and considerable financial ruin[1]
The range of 0–40 ng/mL TNF-αdid not significantly affect nucleus pulposus (NP) cell viability on days 1, 3 and 5 (Fig. 1C). These results indicate that TNF-αinhibits NP cell proliferation in a dose-dependent manner
LY294002 down-regulated the expression of senescence markers (p16 and p53) (Fig. 6E,F) and promoted matrix biosynthesis (Supplementary Figure S1C–E) in NP cells treated with TNF-α.Together, these findings indicate that the premature senescence of NP cells caused by TNF-αis mediated partly by the PI3K/Akt signaling pathway
Summary
Intervertebral disc degeneration (IDD) is frequently associated with low back pain (LBP), which leads to patient disability and considerable financial ruin[1]. The extracellular matrix within the nucleus pulposus (NP) undergoes dramatic molecular changes, such as decreased hydration, decreased proteoglycan content and alterations in collagen content[3]. These matrix changes directly reflect NP cell biology, which is indicated by the finding that NP cells display an altered gene or protein expression profile during disc degeneration degeneration[4]. Apart from the increase in senescent cells during disc degeneration, the accompanying inflammation within NP is a common phenomenon during disc degeneration[11] Many inflammatory cytokines, such as TNF-α, IL-1βand IL-17, are up-regulated in degenerated discs[12,13,14,15]. Glycosaminoglycan (GAG) content, gene expression and protein expression of matrix macromolecules (aggrecan and collagen II) were measured to assess the matrix homeostatic phenotype of these cells
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