Fig 1: Intrathecal pre-administration of artesunate reduces fracture-associated spinal over-expressions of CCL21, TREM2, and DAP12 and microglia activation. Intrathecal artesunate (100 µg) was administered daily for three consecutive days on days 4, 5, and 6 after tibial fracture with orthopedic surgery. All biochemical data were collected on day 7 after sham and fracture surgeries. (A–C) ELISA identified that pretreatment with artesunate downregulated the increased levels of spinal CCL21, TREM2, and DAP12 proteins after tibial fracture. (D,E) Immunohistochemistry staining showed representative photomicrographs of the marker of microglia activation (Iba1) in the spinal dorsal horn after fracture intervention and artesunate treatment (scale bar, 50 µm). All biochemical results are expressed as mean ± SEM (n = 3–5) and analyzed by one-way ANOVA with Bonferroni post hoc comparisons. # p < 0.05 vs. group sham + DMSO, *p < 0.05 vs. group fracture + DMSO.
Fig 2: Fracture-associated behavioral allodynia and spinal over-expressions of CCL21, TREM2, and DAP12 after orthopedic surgery in mice. The development of mechanical allodynia was assessed by paw withdrawal mechanical threshold (A) and paw withdrawal mechanical frequency to 0.16 g filament (B) in the von Frey test after sham surgery and tibial fracture (n = 8). The development of cold allodynia was assessed by cold response scoring (C) in acetone test after sham surgery and tibial fracture (n = 8). The spinal dorsal horn L4-5 segments were collected for biochemical experiments. (D–F) ELISA identified the increased levels of spinal CCL21, TREM2, and DAP12 proteins after tibial fracture (n = 5). All the data are expressed as mean ± SEM and analyzed by two-way ANOVA with Bonferroni post hoc comparisons. *p < 0.05, **p < 0.01, ***p < 0.001 vs. group sham surgery.
Fig 3: Induction of acute pain by exogenous CCL21 and attenuation of allodynia by artesunate. Artesunate (i.t., 100 µg, indicated by a red arrow) was injected 60 min prior to recombinant CCL21 (i.t., 0.1 µg) application. (A,B) Exogenous CCL21-induced acute inflammatory pain was alleviated by the pre-application of artesunate. All behavioral results are mean ± SEM (n = 8) and analyzed by two-way ANOVA with Bonferroni post hoc comparisons. All biochemical data were collected at hour 12 after intrathecal injection. (C) ELISA identified that pretreatment with artesunate downregulated the increased levels of spinal TREM2 and DAP12 proteins after exogenous CCL21 exposure. All biochemical results are expressed as mean ± SEM (n = 5) and analyzed by one-way ANOVA with Bonferroni post hoc comparisons. # p < 0.05 vs. group DMSO, *p < 0.05 vs. group recombinant CCL21 (0.1 µg) + DMSO.
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