Abstract
Intervertebral discs (IVDs) degeneration is one of the major causes of back pain. Upon degeneration, the IVDs tissue become inflamed, and this inflammatory microenvironment may cause discogenic pain. Cellular senescence is a state of stable cell cycle arrest in response to a variety of cellular stresses including oxidative stress and adverse load. The accumulation of senescent IVDs cells in the tissue suggest a crucial role in the initiation and development of painful IVD degeneration. Senescent cells secrete an array of cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP). The SASP promote matrix catabolism and inflammation in IVDs thereby accelerating the process of degeneration. In this study, we quantified the level of senescence in degenerate and non-degenerate IVDs and we evaluated the potential of two natural compounds to remove senescent cells and promote overall matrix production of the remaining cells.
Human IVDs were obtained from organ donors. Pellet or monolayer cultures were prepared from freshly isolated cells and cultured in the presence or absence of two natural compounds: Curcumin and its metabolite vanillin. Monolayer cultures were analyzed after four days and pellets after 21 days for the effect of senolysis. A cytotoxicity study was performed using Alamar blue assay. Following treatment, RNA was extracted, and gene expression of senescence and inflammatory markers was evaluated by real-time q-PCR using the comparative ΔΔCt method. Also, protein expression of p16, Ki-67 and Caspase-3 were evaluated in fixed pellets or monolayer cultures and total number of cells was counted on consecutive sections using DAPI and Hematoxylin. Proteoglycan content was evaluated using SafraninO staining or DMMB assay to measure sulfated glycosaminoglycan (sGAG) and antibodies were used to stain for collagen type II expression.
We observed 40% higher level of senescent cells in degenerate compare to the non-degenerate discs form unrelated individuals and a 10% increase when we compare degenerate compare to the non-degenerate discs of the same individual. Using the optimal effective and safe doses, curcumin and vanillin cleared 15% of the senescent cells in monolayer and up to 80% in pellet cultures. Also, they increased the number of proliferating and apoptotic cells in both monolayer and pellets cultures. The increase in apoptotic cell number and caspase-3/7 activity was specific to degenerate cells. Following treatment, mRNA expression levels of SASP factors were decreased by four to 32-fold compared to the untreated groups. Senescent cell clearance decreased, protein expression of MMP-3 and −13 by 15 and 50% and proinflammatory cytokines levels of IL-1, IL-6 and IL-8 by 42, 63 and 58 %. Overall matrix content was increased following treatment as validated by an increase in proteoglycan content in pellet cultures and surrounding culture media.
This work identifies novel senolytic drugs for the treatment of IVD degeneration. Senolytic drugs could provide therapeutic interventions that ultimately, decrease pain and provide a better quality of life of patients living with IVDs degeneration and low back pain.
