Fig. 4

o-Vanillin reduced the protein expression of SASP factors (IL-1β, NGF, IL-8, and TNF-α) following TLR-2/6 activation of IVD cells from patients with back pain and IVD degeneration. Disc cells from degenerate IVD were cultured for 48 h with TLR-2/6 with o-vanillin (TLR-2/6 + VAN) or without o-vanillin (TLR-2/6) treatment for 6 h or no induction with TLR-2/6 (CTRL). a–d Using Immunocytochemistry, untreated and treated IVD cells were stained for DAPI and either a IL-1β, b NGF, c IL-8, and d TNF-α, respectively. Scale Bars = 25 μm. e Quantification of the percentage of the cells that stained positive for IL-1β, NGF, IL-8, and TNF-α when non-induced, treated with TLR-2/6 agonist or treated with TLR-2/6 agonist and o-vanillin; n = 5. f ELISAs were performed to measure the concentration of TNF-α, IL-1β, and IL-8 in cell media and NGF from cell lysate in non-induced, TLR-2/6 agonist treated or the combined treatment TLR-2/6 agonist and o-vanillin treated samples. Percentage of positive cells in e were the average for n = 5, . ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 indicate significant difference between the TLR-2/6 agonist treated to the non-induced control and *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant difference between the tested groups. e, f Mean ± SD, statistical analysis was done using paired t-test