Fig 2: Ingredients in GXNI that bind to CXCR1. Structural-based binding prediction of GXNI ingredients to CXCR1 by molecular docking. (A) The score of -CDOCKER ENERGY of ingredients in GXNI combined with CXCRI. CETSA and ITDRFCETSA were utilized to assess the binding between GXNI or DSS and CXCR1 in thermodynamic levels. (B, C) WB blots of CETSA and ITDRFCETSA. (D, E) The curve of CETSA and ITDRFCETSA. (F) Danshensu-CXCRI interaction is depicted by active binding site (3D & 2D model).

Fig 3: BCP-induced activation of the NLRP3 inflammasome is dependent on CXCR1 in vivo and in vitro. (a–c) Elevated NLRP3, caspase1, and IL-1ß proteins in BCP rats were reversed after intrathecal injection of CXCR1-siRNA. **p < 0.01, ***p < 0.001, n = 4. (d–f) Increased protein level in NLRP3, caspase-1, and IL-1ß was dependent on CXCR1 in PC12 cells. ***p < 0.001, n = 3.

Fig 4: Increased CXCR1 protein and mRNA expression in the spinal dorsal horn of BCP rats. (a–d) Immunolabeled CXCR1 (green) in the spinal cord of BCP rats. Pre-incubation of CXCR1 antibody with excessive CXCR1 blocking peptide served as the specificity control of CXCR1 antibody. Ipsilateral (Ipsi), contralateral (Contra), nucleus (blue). (e) The mRNA expression of CXCR1 was increased in the BCP group compared with the sham group. *p < 0.05, n = 7, compared to sham, Student’s t-test. (f) The protein expression of CXCR1 increased in a time-dependent manner in the BCP group compared with the sham-operated group. **p < 0.01; n = 4, one-way ANOVA.

Fig 5: Spinal cord blockade of CXCR1 attenuates abnormal gait and mechanical hyperalgesia in BCP rats. (a-b) Relative expression of CXCR1-mRNA and CXCR2-mRNA in PC12 cells after CXCR1-siRNA transfection. ***P < 0.001. n = 3, Student's t-test. (c) Western blot results showing the reduction of CXCR1 protein levels after CXCR1-siRNA treatment. **p < 0.01. n = 4, Student’s t-test. (d) Intrathecal injection of CXCR1-siRNA relieved tumor-induced mechanical allodynia 10 days after BCP. *p < 0.05, **p < 0.01, compared to sham, two-way repeated measures ANOVA. (e-g) Representative CatWalk gaits, including print view, timing view, and print intensity, in sham (e), BCP+siControl (f), BCP + si-CXCR1 (g) groups. (h-j) Intrathecal injection of CXCR1-siRNA significantly attenuated tumor-induced reductions in maximum contact area (h), maximum contact maximum intensity (j), and mean intensity (i) in tumor-bearing rats. Starting on day 7 after tumor inoculation, CXCR1-siRNA was intrathecally injected daily for six consecutive days. Behavioral testing was performed 4 h after the last injection. Data were calculated as percentages of ipsilateral (left)/contralateral (right) hind paws. Data are presented as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. LH, left rear; RH, right rear.