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. 2020 Oct 28;26:100330. doi: 10.1016/j.jbo.2020.100330

Fig. 3.

Fig. 3

Role of RAGE and TLR4 in BCABP. (A) Immunofluorochemical analysis of RAGE and TLR4 expression on peripherin+ SNs innervating bone. Bone sections were incubated with rabbit anti-peripherin (1:100), mouse anti-RAGE (1:100) and mouse anti-TLR4 (1:100) antibody at 4 C overnight as primary antibodies, followed by Alexa Fluor 488 anti-rabbit IgG (1:1000) and Alexa Fluor 647 anti-mouse IgG (1:1000) as secondary antibodies. Both RAGE and TLR4 were expressed on peripherin+ SNs innervating bone. White asterisks in the figures indicate cortical bone. Scale bar 100 µm. (B) Effects of pharmacological antagonist of RAGE and TLR4 on the progression of BCABP in 4T1 mice. One single intra-plantar injection of the RAGE antagonist, FPS-ZM1 (10 mg/kg/mouse), the TLR4 antagonist, TAK-242 (2 mg/kg/mouse), or the neutralizing antibody to HMGB1 (2 mg/kg/mouse) was given to 4T1 mice with BCABP at day 14 (arrow), and changes in hind-paw mechanical hypersensitivity of 4T1 mice were monitored by von Frey test 3, 6, 12, 18 and 24 h after antagonist and HMGB1 antibody injection. FPS-ZM1 reduced hind-paw mechanical hypersensitivity as early as 3 h after administration from 3.56 ± 0.39 g to 5.18 ± 0.43 g (Mean ± SD), which lasted until 12 h (untreated vs FPS-ZM1 = 3.27 ± 0.19 g vs 5.22 ± 0.21 g) (Mean ± SD) and disappeared after 18 h. HMGB1 antibody also reduced hind-paw mechanical hypersensitivity 12 h after injection (untreated vs HMGB1 antibody = 3.27 ± 0.19 g vs 6.92 ± 0.65 g) (Mean ± SD), which lasted until 24 h (untreated vs HMGB1 Antibody = 3.33 ± 0.15 g vs 6.38 ± 0.64 g) (Mean ± SD). Overall HMGB1 antibody appeared more effective than FPS-ZM1 at relieving BCABP under this experimental conditions. TAK-242 given on this experimental protocol showed no effects on BCABP. Data are shown as mean ± SD (n = 8 for each group).* Significantly different from 4T1 mice (p < 0.01). # Significantly different from 4T1 mice (p < 0.05). (C) Role of RAGE and TLR4 in the activation of SNs in DRGs of 4T1 mice. DRGs were harvested from 4T1 mice after 12 h treatment with vehicle, FPS-ZM1, TAK-242 and HMGB1 antibody, immediately lysed, and subjected to Western analysis. FPS-ZM1 and HMGB1 antibody decreased pERK1/2 and pCREB in DRGs of 4T1 mice, while TAK-242 had no effects.