Cannabinoid receptor 2‑selective agonist JWH015 attenuates bone cancer pain through the amelioration of impaired autophagy flux induced by inflammatory mediators in the spinal cord

Abstract

Bone cancer pain (BCP) is a severe complication of advanced bone cancer. Although cannabinoid receptor 2 (CB2) agonists may have an analgesic effect, the underlying mechanism remains unclear. CB2 serves a protective role in various pathological states through the activation of autophagy. Therefore, the present study aimed to determine whether the analgesic effects of the selective CB2 agonist JWH015 was mediated by the activation of autophagy in BCP. BCP was induced by the intra‑femur implantation of NCTC2472 fibrosarcoma cells in C3H/HeN mice. The pain behaviors were assessed on the following postoperative days. The selective CB2 agonist JWH015 (1 and 2 µg) was intrathecally administered on day 14 following implantation. AM630 (1 µg), a CB2 antagonist, was injected 30 min before JWH015 administration. Lipopolysaccharide (LPS; 100 nM)‑stimulated primary neurons were treated with JWH015 (1 µM) and AM630 (1 µM) to further verify the mechanism by which CB2 affects autophagy. The results demonstrated that autophagy flux was impaired in spinal neurons during BCP, as indicated by the increased ratio of microtubule‑associated protein 1 light chain 3β (LC3B)‑II/LC3B‑I and increased expression of p62. Intrathecal administration of JWH015 attenuated BCP, which was accompanied by the amelioration of impaired autophagy flux (decreased LC3B‑II/LC3B‑I ratio and decreased p62expression). In addition, the activation of glia cells and upregulation of the glia‑derived inflammatory mediators, interleukin (IL)‑1β and IL‑6 were suppressed by JWH015. In LPS‑stimulated primary neurons, IL‑1β and IL‑6 were increased, and autophagy flux was impaired; whereas treatment with JWH015 decreased the expression of IL‑1β and IL‑6, LC3B‑II/LC3B‑I ratio and expression of p62. These effects were by pretreatment with the CB2‑selective antagonist AM630. The results of the present study suggested that the impairment of autophagy flux was induced by glia‑derived inflammatory mediators in spinal neurons. Intrathecal administration of the selective CB2 agonist JWH015 ameliorated autophagy flux through the downregulation of IL‑1β and IL‑6 and attenuated BCP.

Figures

Figure 1.

Intrathecal administration of JWH015 attenuates bone cancer pain. Intra-femur implantation of NCTC 2476 cells induced mechanical hypersensitivity in the ipsilateral hind paw. The (A) PWMT and (B) NSF were measured on days 0, 4, 7, 10, 14, 21 and 28 after operation in Control, Sham and Tumor group mice. JWH015 and AM630 were intrathecally injected on day 14 after the operation. (C) PWMT and (D) NSF were tested before administration (baseline, 0 h) and at 4, 8, 12, 24, 48 and 72 h after administration of JWH015. Data are expressed as the mean ± SD; n=8; *P#P<0.05 vs. Sham at each time point. PWMT, paw withdrawal mechanical threshold; NSF, Number of spontaneous flinches.

Figure 2.

Autophagy flux is impaired in bone cancer pain mice. (A) Representative western blotting images of LC3BI/II and p62 protein expression in the spinal cord of mice in the Sham and Tumor groups on day 0, 4, 7, 10, 14, 21 and 28 post-operation. (B) Ratio of LC3B-II/LC3B-I and (C) quantification of p62 in the spinal cord in BCP. *P

Figure 3.

Autophagy flux is impaired in neurons in the spinal cord. (A-C) LC3B-immunoreactive cells (green) in the spinal cord were (A) NeuN-positive (red), (B) Iba1-negative (red), and (C) GFAP-negative (red). Scale bar, 50 µm; magnification, ×400. GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adaptor molecule 1; LC3B, microtubule-associated protein 1 light chain 3β; NeuN, neuronal nuclei antigen.

Figure 4.

Intrathecal administration of JWH015 ameliorates impaired autophagy flux in bone cancer pain. (A) Representative western blotting images of LC3B and p62 in the spinal cord 12 h after treatment with JWH015. (B) Ratio of LC3B-II/LC3B-I and (C) quantification of p62 protein expression levels. #P<0.05 vs. Sham + vehicle group; *P<0.05 vs. Tumor + vehicle group. (D) Representative western blots of LC3B and p62 protein expression in the spinal cord at 0, 4, 8, 12, 24, 48 and 72 h after JWH015 (2 µg) injection. (E) Ratio of LC3B-II/LC3B-I and (F) quantification of p62. *P<0.05 vs. 0 h. (G) Representative blots of LC3B and p62 in the spinal cord with treatment of vehicle, Baf-A1 (10 nM), JWH015 (2 µg), and Baf-A1 (10 nM) + JWH015 (2 µg) in BCP mice on day 14. (H) Ratio of LC3B-II/LC3B-I. (I) Quantification of p62. *P<0.05 vs. tumor + vehicle group. For all western blots, β-actin was used as a loading control; data are expressed as the mean ± SD n=3 per group. LC3B, microtubule-associated protein 1 light chain 3β.

Figure 5.

Intrathecal administration of JWH015 inhibits activation of astrocytes and microglia induced by bone cancer pain. (A) Images and quantification of immunostaining for GFAP (red) and Iba1 (green) in lumbar spinal dorsal horn were captured on day 14 after operation in mice in the Sham and Tumor groups. n=3; *P#P<0.05 vs. Sham + vehicle group; *P<0.05 vs. Tumor + vehicle group; n=3 per group. Scale bar, 50 µm; magnification, × 200. GFAP, glial fibrillary acidic protein; Iba1, ionized calcium binding adaptor molecule 1.

Figure 6.

Intrathecal administration of JWH015 downregulated the expression of pro-inflammatory mediators IL-1β and IL-6. (A) Representative western blotting images and (B) quantification of IL-1β and IL-6 protein expression in the spinal cord in Sham and Tumor mice on day 0, 4, 7, 10, 14, 21 and 28 after the operation. *P#P<0.05 vs. Sham + vehicle group; *P<0.05 vs. Tumor + vehicle. (E) Representative blots and (F) quantification of IL-1β and IL-6 in the spinal cord at 0, 4, 8, 12, 24, 48 and 72 h after JWH015 (2 µg) injection. *P<0.05 vs. 0 h. For all blots, β-actin was used as a loading control; data are expressed as the mean ± SD; n=3. IL, interleukin.

Figure 7.

Effects of JWH015 on inflammatory factors and autophagy in primary neurons. Primary neurons were stimulated with LPS (100 nM) for 0, 3, 6, 9, 12 and 24 h. (A) Western blotting demonstrated the upregulation of IL-1β and IL-6 after LPS-stimulation. (B) Quantification of IL-1β and IL-6 in the primary neurons after LPS-stimulation. *P#P<0.05 vs. LPS group; $P<0.05 vs. LPS + JWH015 group. (G) Decreased ratio of LC3B-II/LC3B-I and decreased expression of p62 after treatment with JWH015 in LPS-stimulated primary neurons. (H) Quantification of LC3B-II/LC3B-I and p62 in different groups. *P<0.05 vs. control group; #P<0.05 vs. LPS group; $P<0.05 vs. LPS + JWH015 group. In all western blots, β-actin was used as a loading control; data are expressed as the mean ± SD; n=3. IL, interleukin; LC3B, microtubule-associated protein 1 light chain 3β; LPS, lipopolysaccharide.