Involvement of CXCL12/CXCR4 in CB2 receptor agonist-attenuated morphine tolerance in Walker 256 tumor-bearing rats with cancer pain

Abstract

While low-dose cannabinoid 2 (CB2) receptor agonists attenuate morphine tolerance in cancer pain models, chemokine ligand 12 (CXCL12)/chemokine receptor 4 (CXCR4) expression induces morphine tolerance. Whether CB2 receptor agonists attenuate morphine tolerance by modulating CXCL12/CXCR4 signaling or whether CXCL12/CXCR4 signaling affects the mu opioid receptor (MOR) in the development of morphine tolerance in cancer pain remains unclear. In this study, we investigated the attenuation of morphine tolerance by a non-analgesic dose of the CB2 receptor agonist AM1241, focusing specifically on the modulation of CXCL12/CXCR4 signaling and its effect on the MOR. Rats received intrathecal Walker 256 tumor cell implantations and were treated with morphine combined with the intrathecal injection of AM1241 or the CB2 receptor antagonists AM630 and AM1241, or a CXCL12-neutralizing antibody, exogenous CXCL12, or the CXCR4 antagonist AMD3100. Our results show that CXCL12 and CXCR4 levels increased significantly in morphine-tolerant rats and were reduced by AM1241 pretreatment, which was reversed by AM630. CXCL12/CXCR4 expression accelerated the development of morphine tolerance and downregulated MOR expression. CXCR4 colocalized with MOR and CB2. Therefore, a non-analgesic dose of AM1241 attenuated morphine tolerance via CXCL12/CXCR4 signaling, whereas CXCL12/CXCR4 signaling participated in the development of morphine tolerance, potentially by modulating MOR expression in Walker 256 tumor-bearing rats.

Keywords: Cancer pain; Cannabinoid 2; Chemokine ligand 12/chemokine receptor 4; Morphine tolerance; Mu opioid receptor.

Fig. 1

Establishment of morphine tolerance in Walker 256 tumor-bearing rats. A von Frey values (n = 15). B Hot plate values (n = 15). Data are presented as mean ± standard deviation (SD) of three or four independent experiments, each performed in triplicate. Controls received saline + vehicle (SV). Tests were performed day before administration and 30 min after daily morphine injection. Day 0 = day before first administration. Days 1–8 = eight consecutive days of administration. Repeated measures two-way ANOVA was used to examine the effects of the treatment (P < 0.0001), time (P < 0.0001), and their interaction (P < 0.0001). Tukey’s multiple comparison test showed significant within-group and between-group differences in PWT and PWL (P < 0.05). #P < 0.05 compared with the SV group, ※P < 0.05 compared with day 1 in the corresponding group. PWT, pain withdrawal threshold; PWL, pain withdrawal latency

Fig. 2

AM1241 attenuated morphine tolerance by decreasing CXCL12 and CXCR4 levels in Walker 256 rats. Von Frey (A) and hot plate (B) values in MA, MAA, MV, and SV groups (n = 15). Controls received saline + vehicle. Repeated measures two-way ANOVA was used to examine the effects of the treatment (P < 0.0001), time (P < 0.0001), and their interaction (P < 0.0001). Tukey’s multiple comparison test revealed significant within-group and between-group differences in PWT and PWL. *P < 0.05 compared with MV group, #P < 0.05 compared with SV group, ※P < 0.05 compared with day 1 in the corresponding group. C, E Immunohistochemical images of CXCL12 and CXCR4 in MA, MAA, MV, and SV groups (n = 5); scale bar = 50 μm. D, F AOD of CXCL12 and CXCR4 in slices of spinal cord and DRG for each group. G, J Western blotting of CXCL12 and CXCR4 levels in MA, MAA, MV, and SV groups (n = 5). H, K Analysis of CXCL12 and CXCR4 levels in MA, MAA, MV, and SV groups; I, L Fold change in Cxcl12 and Cxcr4 mRNA levels compared to that of the control for each group (n = 5). Data are presented as mean ± SD of three or four independent experiments, each performed in triplicate. One-way ANOVA was used to analyze data, followed by Tukey’s multiple comparison test. CXCL12 chemokine ligand 12, CXCR4 chemokine receptor 4, AM1241 cannabinoid type 2 (CB2) receptor agonist, AM630 CB2 antagonist, GAPDH glyceraldehyde-3-phosphate dehydrogenase (loading control), MA morphine + AM1241, MAA morphine + AM1241 + AM630, MV morphine + vehicle, SV saline + vehicle, AOD average optical density, PWT pain withdrawal threshold, PWL pain withdrawal latency

Fig. 3

CXCL12/CXCR4 signaling is implicated in development of morphine tolerance due to MOR inhibition. Von Frey (A) and hot plate (B) values in MAb, MD, MC, MV, and SV groups (n = 15). Control received saline + vehicle. Repeated measures two-way ANOVA was used to examine effects of treatment (P < 0.0001), time (P < 0.0001), and their interaction (P < 0.0001). Tukey’s multiple comparison test showed within-group and between-group differences in PWT and PWL (P < 0.05). *P < 0.05 compared with MV group, #P < 0.05 compared with the SV group, ※P < 0.05 compared with day 1 in corresponding group. C Immunohistochemical images of MOR in MAb, MC, MV, and SV groups (n = 5); scale bar = 50 μm. D AOD of MOR in slices of spinal cord and DRG for each group. One-way ANOVA was used to analyze data, followed by Tukey’s multiple comparison test. Data are presented as mean ± SD of three or four independent experiments, each performed in triplicate. MOR, mu opioid receptor; CXCL12-Ab, chemokine ligand 12-neutralizing antibody; AMD3100, chemokine receptor 4 antagonist; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (loading control), MAb morphine + CXCL12-Ab; MC, morphine + CXCL12, MD morphine + AMD3100, MV morphine + vehicle, SV saline + vehicle, SV saline + vehicle, AOD average optical density, DRG dorsal root ganglion

Fig. 4

MOR detected using western blotting and qRT-PCR in MAb, MC, MD MV, and SV groups. A, B Western blotting of MOR level in MAb, MC, MD, MV, and SV groups (n = 5); C Changes in MOR mRNA levels compared to that of control for each group. Data are presented as the mean ± SD of three or four independent experiments (n = 5), each performed in triplicate. One-way ANOVA was used to analyze data, followed by Tukey’s multiple comparison test. MOR, mu opioid receptor; CXCL12-Ab, chemokine ligand 12-neutralizing antibody; AMD3100, chemokine receptor 4 antagonist; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (loading control). MAb morphine + CXCL12-Ab, MC morphine + CXCL12, MD morphine + AMD3100, MV morphine + vehicle, SV saline + vehicle

Fig. 5

Colocalization of CXCR4 with CB2 and MOR in spinal cord. A, B Immunofluorescence staining showing co-expression of CXCR4 (green) with CB2 and MOR (red) in MA, MD, MV, and SV groups (n = 5). C Colocalization of cell AODs in slices of spinal cord for each group. Data are presented as mean ± SD of three independent experiments, each performed in triplicate. One-way ANOVA was used to analyze the data, followed by Tukey’s multiple comparison test. CXCR4 chemokine receptor 4, MOR mu opioid receptor, AMD3100 CXCR4 antagonist, AM1241 cannabinoid type 2 (CB2) receptor agonist, AOD average optical density. Scale bar, 50 μm. MA morphine + AM1241; MD morphine + AMD3100, MV morphine + vehicle, SV saline + vehicle

Fig. 6

Colocalization of CXCR4 with CB2 and MOR in the DRG. A, B Immunofluorescence staining images showing co-expression of CXCR4 (green) with CB2 and MOR (red) in the MA, MD, MV, and SV groups (n = 5). C Colocalization of cell AODs in slices of DRG for each group. Data are presented as mean ± SD of three independent experiments, each performed in triplicate. One-way ANOVA was used to analyze data, followed by Tukey’s multiple comparison test. CXCR4 chemokine receptor 4, MOR mu opioid receptor, AMD3100 CXCR4 antagonist, AM1241 cannabinoid type 2 (CB2) receptor agonist, AOD average optical density, SV saline + vehicle, DRG dorsal root ganglion. Scale bar, 50 μm. MA morphine + AM1241, MD morphine + AMD3100, MV morphine + vehicle, SV saline + vehicle