Fig 2: The fractures induce a spinal increase in fractalkine and CX3CR1, and microgliosis is reduced following repetitive injections with glabridin. (A–C) The ELISA experiments detected the dynamic alternations of spinal fractalkine, CX3CR1, and Iba-1 proteins following the fractures and orthopedic surgeries (n = 5). Intrathecal glabridin (50 µg) was delivered daily for 4 consecutive days on days 3, 4, 5, and 6 following the fractures. (D–F) The ELISA experiments revealed that the up-modulation of spinal fractalkine, CX3CR1, and Iba-1 proteins after the fractures and orthopedic surgeries was inhibited by the glabridin pretreatment (n = 5). (G) The immunofluorescence staining showed representative photomicrographs of Iba-1 in the dorsal horn following the fractures and glabridin exposure (the scale bar is 50 µm). All data are expressed as means ± SEM and analyzed by a one-way or two-way ANOVA with Bonferroni post hoc comparisons.

Fig 3: Spinal exposure to exogenous fractalkine abolishes glabridin-induced anti-nociceptive effects on chronic allodynia. Glabridin (50 µg) and recombinant fractalkine (50 ng) were intrathecally injected on days 3, 4, 5, and 6 after the fracture surgeries. The behavioral phenotypes of mechanical allodynia (A) and cold allodynia (B) were recorded. (C,D) The ELISA experiments identified changes in the spinal CX3CR1 and Iba-1 proteins after the fractures and the glabridin and fractalkine interventions. (E,F) Representative photomicrographs and statistical results of the spine morphology following the fractures and glabridin and fractalkine treatments (the scale bar is 5 µm). All behavioral results (n = 6) and biochemical results (n = 3–5) are shown as means ± SEM and analyzed by a one-way or two-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05 vs. group sham + vehicle; # p < 0.05 vs. group fracture + vehicle.

Fig 4: Acute nociceptive phenotypes are initiated by exogenous fractalkine and ameliorated by co-applications of minocycline. The microglial inhibitor minocycline (i.t.; 20 µg) and anti-CX3CR1 (i.t.; 20 µg) were given one hour prior to the delivery of the recombinant fractalkine (i.t.; 50 ng). (A,B) The exogenous fractalkine-evoked acute nociceptive phenotypes were ameliorated following the pre-applications of minocycline and anti-CX3CR1, respectively. All behavioral results are shown as means ± SEM (n = 6) and analyzed by a two-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05 vs. baseline; # p < 0.05 vs. group recombinant fractalkine (50 ng). The biochemical experiments were performed 3 hours following the fractalkine injections. (C) The ELISA experiments showed that anti-CX3CR1 decreased the overexpression of the Iba-1 proteins in the dorsal horn following the exogenous fractalkine injections. All biochemical results are expressed as means ± SEM (n = 5) and analyzed by a one-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05 vs. naïve; # p < 0.05 vs. group recombinant fractalkine.

Fig 5: CX3CL1 promotes the translocation of c-Jun and promoter binding of AP-1 formation in human oral squamous cell carcinoma (OSCC) cells. (A) Immunofluorescent staining showed that c-Jun translocated into the nucleus after cell were treated with CX3CL1; nuclear translocation was prevented when the cells were pre-treated with CX3CR1 mAb, U73122, GF109203 or PP2. (B) Quantified analysis of c-Jun activation with CX3CL1 using Western blot. (C) CX3CL1 treatment upregulated AP-1 luciferase activity. (D) Quantified analysis of AP-1 luciferase activity with specific inhibitors and CX3CL1 recombinant protein (30 ng/mL) treatment. (E) Chromatin immunoprecipitation analysis of c-Jun and AP-1 promoter binding site with neutralizing antibody and specific inhibitors. Results are expressed as the mean ± SD of four independent experiments. *, p < 0.05 and #, p < 0.05 as compared to control and CX3CL1 treatment.