Abstract
Introduction
The intervertebral discs (IVDs) are the largest avascular tissues in the body characterized by an abundant extracellular matrix and low cell density, with innervation limited at the most peripheral annular layers. During IVD degeneration, the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), can stimulate nerve growth and hyperinnervation of the inner annulus fibrosus (AF) and nucleus pulposus (NP), which further could lead to back pain. Back pain can potentially be suppressed by BMP4 supplementation to decrease the density of innervation. Link N is a naturally occurring peptide that can stimulate BMP4 in human IVDs. The aim of the present study was to determine the effect of Link N on NGF, BDNF, and substance P (SP) in human AF cells stimulated with cytokines or punctured bovine discs and determine the possible mechanisms related to its effect.
Material and Methods
Human lumbar spines were obtained through Héma-Québec harvesting organ donation within 24 hour after death. Annulus fibrosus (AF) cells were isolated from AF region of human IVDs with Thompson grade 2 of degeneration, cultured in monolayers and stimulated with TNFα (100 ng/mL) and IL1β (10 ng/mL) in the presence or absence of Link N (1 µg/mL) for 48 hours. Total RNA was isolated and gene expression of neurotrophins, substance P, and their receptors was measured using RT PCR. AF cells isolated from human IVDs with Thompson grade 4 were cultured for 48 hours with or without Link N (1 µg/mL) and the NGF release in the media was measured by Western blotting. Coccygeal IVDs from the tails of adult bovine steers (20–25 months) were isolated with the cartilaginous endplates. After 24 hours preconditioning in complete DMEM, the IVDs were treated (control, capsaicin [1.5 µg/mL), punctured by 16 G needle, punctured by 16 G needle and incubated with Link N [10 µg/mL]) and the disc culture media was collected at different time points for analysis. SP in the media was concentrated by solid phase extraction and was assayed by Elisa.
Results
Proinflammatory cytokines IL1β and TNFα have been shown to trigger the expression of NGF and SP, as well as promoting the degradation of the extracellular matrix in human IVD cells. Our results demonstrate that gene expression of NGF, BDNF, TrkA, and TrkB significantly decreased in human disc cells stimulated with either IL-1β or TNFα supplemented with Link N when compared with the control. Additionally, Link N was also able to inhibit NGF release in the media when AF grade 4 was stimulated with IL-1β. Link N supplementation also led to a significant decrease in SP after 24 hour in injured discs, when compared with controls.
Conclusion
Link N can suppress the stimulation of NGF, BDNF, and their receptors TrkA and TrkB, respectively, as well as the neurotransmitter SP in degenerate discs. This suggests that Link N has the potential to inhibit pain induced by neuronal innervation caused by disc degeneration. Thus, administration of Link N has the potential to not only repair the discs in early stages of the disease but also suppress pain.