Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin

Abstract

Background There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16INK4a, SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

Cellular Senescence in Intervertebral Disc Aging and Degeneration: Molecular Mechanisms and Potential Therapeutic Opportunities

“…Although several TLRs are detected in isolated human IVD cells, expression of TLR-1/2/3/4 and 6 was found to be dependent on degree of IDD [ 58 ]. Activation of human IVD cells with TLR-2/6 agonists for up to 48 h can induce senescence [ 59 ]. In addition, stimulation with the TLR-4 ligand lipopolysaccharide (LPS) for 24 h increases the expression of p16 INK4a and p21 to induce senescence in human NP cells from IDD patients [ 60 ].…”

“…Both compounds reduced inflammatory cytokines, chemokines, and growth and neutrophilic factors in pellet culture and in the intact human IVDs [ 167 ]. In addition, o-vanillin reduced the number of SnCs in cell pellet culture from degenerated discs and inhibited the TLR-2-induced senescence of disc cells isolated from patients with back pain and IVD degeneration [ 59 ]. Mechanistically, o-vanillin reduced TLR-2 and p16 INK4a co-expressing cells, suggesting that blockage of TLR-2 by o-vanillin reduces the detrimental effect of SnCs [ 59 ].…”

Cellular senescence and the tumour microenvironment

“…COX‐2 upregulation and the overproduction of PGE 2 have also been observed in human nonalcoholic steatohepatitis‐associated liver cancer [15]. Moreover, TLR2 [47,49] and TLR4 [50] are also reportedly activated in senescent cells, indicating their potential involvement in the extrinsic control of SASP induction [50].…”

Senolytics: Eliminating Senescent Cells and Alleviating Intervertebral Disc Degeneration

“…The latest study has found that the grape seed extract PCC1 also has senolytic properties, which may be stronger than existing compounds ( Xu Q. et al, 2021 ). Among them, curcumin and its metabolite o-Vanillin have been proven to eliminate senescent cells in the intervertebral disc, reducing SASP secretion and alleviating IVDD ( Cherif et al, 2019 ; Mannarino et al, 2021 ). Our previous study has elucidated that quercetin could significantly reduce the proportion of senescent cells in the intervertebral disc through the Nrf-2/NF-κB pathway and slow down the deterioration of IVDD ( Shao et al, 2021 ).…”