TLR4 expression increases in the bone cancer pain model. (B) Real-time quantitative RT-PCR analyses of mRNA temporal expression of TLR4 in rat lumbar spinal cord. The relative levels of spinal TLR4 mRNA significantly increased on days 6, 12 and18 (n = 4, ANOVA1w, P < 0.01, post hoc Dunnett testing, df = 3, F = 1.17) after inoculation (AI) in bone cancer pain rats compared with sham-injected controls and normal rats. The level of gene expression was calculated after normalizing against β-actin in each sample and is presented as relative mRNA expression units. (A, C) The protein level of TLR4 detected by western blotting analysis in spinal cord. A significant up-regulation of TLR4 protein level (n = 4, ANOVA1w, P < 0.01, post hoc Dunnett testing, df = 3, F = 1.06) on the sixth day AI. The protein levels further increased at 12 and 18 days (P < 0.01) after inoculation compared with sham-injected controls and normal rats, respectively. Each data point represents at least three independent experiments. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01 vs. normal group; ▲P < 0.05, ▲▲P < 0.01 vs. sham group; #P < 0.05, ##P < 0.01 vs. 6 day AI; ◆P < 0.05, ◆◆P < 0.01 vs. 12 day AI.