CXCR1 participates in bone cancer pain induced by Walker 256 breast cancer cells in female rats

Abstract

Bone cancer pain (BCP) is a clinically intractable mixed pain, involving inflammation and neuropathic pain, and its mechanisms remain unclear. CXC chemokine receptor 1 (CXCR1, IL-8RA) and 2 (CXCR2, IL-8RB) are high-affinity receptors for interleukin 8 (IL8). According to previous studies, CXCR2 plays a crucial role in BCP between astrocytes and neurons, while the role of CXCR1 remains unclear. The objective of this study was to investigate the role of CXCR1 in BCP. We found that CXCR1 expression increased in the spinal dorsal horn. Intrathecal injection of CXCR1 siRNA effectively attenuated mechanical allodynia and pain-related behaviors in rats. It was found that CXCR1 was predominantly co-localized with neurons. Intrathecal injection of CXCR1-siRNA reduced phosphorylated JAK2/STAT3 protein levels and the NLRP3 inflammasome (NLRP3, caspase1, and IL-1β) levels. Furthermore, in vitro cytological experiments confirmed this conclusion. The study results suggest that the spinal chemokine receptor CXCR1 activation mediates BCP through JAK2/STAT3 signaling pathway and NLRP3 inflammasome (NLRP3, caspase1, and IL-1β).

Keywords: CXCR1; JAK2/STAT3 signaling pathway; NLRP3 inflammasome; bone cancer pain; spinal cord.

Figure 1.

Experimental paradigms. CT: computed tomography; HE: Hematoxylin and eosin; IF: Immunofluorescence; RT-PCR: Quantitative Real-Time Polymerase Chain Reaction; IL-8: interleukin eight; JAK2: janus-activated kinase two; STAT3: signal transducer and activator of transcription three; PWT: paw withdrawal threshold; WB: Western blotting; ELISA: Enzyme-linked immunosorbent assay; PC12: PC12 cell lines.

Figure 2.

Intratibial inoculation of Walker 256 cells produces bone destruction and progressive hyperalgesia. (a)The paw withdrawal threshold (PWT) of the ipsilateral hind paw was significantly decreased on BCP from 6 to 18 days after surgery. **p < 0.01 versus sham; ***p < 0.001 versus sham; n = 8, two-way repeated-measures ANOVA. (b) Three-dimensional (3D) reconstruction of CT scan showing marked bone destruction of the left tibia 18 days after tumor cell injection. Arrows point to cortical destruction sites. N = 4. (c) Representative images of hematoxylin-eosin staining showing that the bone marrow cavity of rats in the sham-operated group was filled with lymphocytes and macrophages. Trabecular surface cancer cell infiltration and bone resorption pits appeared in the bone marrow cavity of the tibia 18 days after tumor cell inoculation. N = 4. Scale bar: 50 μm.

Figure 3.

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.

Figure 4.

Distribution and cellular localization of CXCR1 in the dorsal horn of the spinal cord. (a–o) Immunofluorescence data show that CXCR1 (green) was predominantly expressed in neurons (red), but not in astrocytes (red) or microglia (red). All sections were counterstained with DAPI (blue) to show nuclei. White arrows indicate possible colocalization sites. NeuN (neuronal nucleus, neuron-specific marker); GFAP (glial fibrillary acidic protein, astrocyte-specific marker); Iba-1 (ionized calcium-binding adaptor molecule 1, microglia-specific mark); n = 4. Scale bar: 50 μm.

Figure 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.

Figure 6.

BCP-induced activation of spinal JAK2/STAT3 signaling is dependent on CXCR1 in vivo and in vitro. (a–c) The phosphorylated proteins of the JAK2/STAT3 pathway were time-dependently increased in the spinal cord of rats with BCP. *p < 0.05, **p < 0.01, ***p < 0.001, compared to sham group, one-way ANOVA. (d–f) Phosphorylated proteins of the JAK2/STAT3 pathway were reduced after intrathecal injection of CXCR1-siRNA. *p < 0.05, ***p < 0.001, compared to sham group, one-way ANOVA. (g–i) Phosphorylated protein of JAK2/STAT3 was dependent on CXCR1 activation in PC12 cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.

BCP-induced activation of the NLRP3 inflammasome is dependent on CXCR1 in vivo and in vitro. (a–c) Elevated NLRP3, caspase1, and IL-1β 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-1β was dependent on CXCR1 in PC12 cells. ***p < 0.001, n = 3.