Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine 5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Abstract

Opioids, such as morphine, are the most potent drugs used to treat pain. Long-term use results in high tolerance to morphine. High mobility group box-1 (HMGB1) has been shown to participate in neuropathic or inflammatory pain, but its role in morphine tolerance is unclear. In this study, we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days. We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1. HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4 receptor expression in microglia, thereby inducing morphine tolerance. Glycyrrhizin, an HMGB1 inhibitor, markedly attenuated chronic morphine tolerance in the mouse model. Finally, compound C (adenosine 5'-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin (heme oxygenase-1 inhibitor) alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tolerance, and alleviated morphine tolerance in the mouse model. These findings suggest that morphine induces HMGB1 release via the adenosine 5'-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway, and that inhibiting this signaling pathway can effectively reduce morphine tolerance.

Keywords: Toll-like receptor 4; adenosine 5′-monophosphate-activated protein kinase; heme oxygenase-1; high mobility group box-1; interleukin-1β; microglia; morphine tolerance; neuroinflammation; neuron; nuclear factor-κB p65.

Figure 1

Morphine induces HMGB1 release. (A, B) Rats were intrathecally injected with morphine (20 μg/10 μL) for 7 consecutive days. Morphine induced HMGB1 release into the CSF. CSF was collected 1 hour after the last morphine injection. (A) HMGB1 expression in the CSF was verified by western blot (n = 6/group). (B) Immunostaining for HMGB1 and specific markers showed that HMGB1 (green, Alexa Fluor 488) co-localized with neurons (NeuN, red, Cy3) but not with astrocytes (GFAP, red, Cy3) or microglia (Iba1, red, Cy3), and that HMGB1 also co-localized with nuclei (DAPI) in the dorsal horn of the spinal cord in control mice. Double-positive staining for HMGB1 and NeuN and co-localization of HMGB1 with DAPI were decreased in morphine-treated mice compared with control mice. Scale bars: 100 μm. (C) HMGB1 expression in SH-SY5Y cells induced by morphine (50, 100, and 200 μM). Data are expressed as mean ± SD. *P < 0.05, ***P < 0.001, vs. vehicle group (one-sample t-test). (D) Morphine (200 μM, 12 hours) induced HMGB1 release (green, Alexa Fluor 488) from SH-SY5Y cells into the extracellular environment. Scale bars: 10 μm. CSF: Cerebral spinal fluid; DAPI: 4′,6-diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; HMGB1: high mobility group box 1; Iba1: ionized calcium-binding adaptor molecule 1.

Figure 2

Glycyrrhizin attenuates chronic morphine tolerance. The effect of glycyrrhizin on morphine tolerance was evaluated by tail-flick assay. (A) Glycyrrhizin did not affect acute morphine-mediated analgesia (n = 8/group). (B) Glycyrrhizin alleviated chronic morphine tolerance in mice (n = 8/group). (C) Immunofluorescence analysis showed that glycyrrhizin markedly inhibited c-fos and CGRP (both green, Alexa Fluor 488) activation in the spinal cord after morphine administration (n = 4/group). Scale bars: 75 μm. Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001, vs. vehicle group; #P < 0.05, ###P < 0.001, vs. morphine group (A, B: two-way analysis of variance followed by Tukey’s multiple-comparison test; C: one-way analysis of variance followed by Tukey’s multiple-comparison test). CGRP: Calcitonin gene-related peptide; MPE: maximal possible effect.

Figure 3

Extracellular HMGB1 triggers an inflammatory response that is dependent on TLR4 expression in microglia. (A) Glycyrrhizin (25, 50, 100 mg/kg) inhibited morphine-induced upregulation of p-p65 in the spinal cord (n = 4/group). (B) Glycyrrhizin (25, 50, 100 mg/kg) markedly reduced the level of mature IL-1β expression in the spinal cord (n = 4/group). (C) Immunofluorescence analysis showed that glycyrrhizin suppressed Iba1 (green, Alexa Fluor 488) activation in the spinal cord (n = 4/group). **P < 0.01, ***P < 0.001, vs. vehicle group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. morphine group (one-way analysis of variance followed by Tukey’s multiple-comparison test). (D) The level of NF-κB p65 phosphorylation induced by treatment with HMGB1 in the presence or absence of TLR4 antagonist was measured in BV-2 cells. The cells were pretreated with TLR4 antagonist (TAK242, 10 μM) for 15 minutes before the addition of recombinant HMGB1 (25 nM). Cell extracts were collected after 12 hours of HMGB1 treatment. (E, F) BV-2 cells were incubated with conditioned medium collected from morphine-treated (200 μM, 12 hours) SH-SY5Y cells for 12 hours in presence of anti-HMGB1 antibody (2 μg/mL) or normal IgG (2 μg/mL), and then the cell extracts were collected. *P < 0.05, **P < 0.01, vs. control group; #P < 0.05, ##P < 0.01, vs. HMGB1 group (one-way analysis of variance followed by Tukey’s multiple-comparison test). Data are expressed as mean ± SD. The experiment was repeated three times. Ab: Antibody; CM: conditional medium; GA: glycyrrhizin; HMGB1: high mobility group box 1; Iba1: ionized calcium binding adapter molecule 1; IL-1β: interleukin-1β; NF-κB: nuclear factor-κB; TLR4: Toll-like receptor 4.

Figure 4

Morphine-induced HMGB1 release requires HO-1. (A) Morphine increased HO-1 expression in the supernatants of SH-SY5Y cells (50, 100, 200 μM), as assessed by western blot. (B) Znpp downregulated HMGB1 expression by SH-SY5Y cells. The cells were treated with Znpp (HO-1 inhibitor, 2 μM) for 12 hours before morphine administration, and the results were analyzed by western blot. (C) Transfection with HO-1 siRNA downregulated HO-1 expression in SH-SY5Y cells. (D) HO-1 siRNA decreased morphine-induced HMGB1 expression, as assessed by western blot. Data were normalized to the control group and are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. morphine group (A: one-sample t-test; B–D: one-way analysis of variance followed by Tukey’s multiple-comparison test). HMGB1: High mobility group box 1; HO-1: heme oxygenase-1; siRNA: small interfering RNA; Znpp: Zinc protoporphyrin IX.

Figure 5

Morphine-induced AMPK phosphorylation increases HMGB1 release by upregulating HO-1. (A) Morphine promoted AMPK phosphorylation in SH-SY5Y cells, as assessed by western blot. (B) Compound C inhibited morphine-induced heme oxygenase-1 (HO-1) expression in SH-SY5Y cells. The cells were treated with Compound C (AMPK inhibitor, 10 μM) for 2 hours before morphine (200 μM, 12 hours) administration. (C) Compound C suppressed the HMGB1 expression induced by morphine (200 μM) in SH-SY5Y cells. (D) Transfection with AMPK siRNA decreased AMPK expression and phosphorylation in SH-SY5Y cells. (E) AMPK siRNA markedly decreased morphine-induced HO-1 expression. (F) AMPK siRNA inhibited the HMGB1 expression induced by morphine (200 μM), as assessed by western blot. Data were normalized to the control group and are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. morphine group (A: one-sample t-test; B–F: one-way analysis of variance followed by Tukey’s multiple-comparison test). AMPK: 5′-Monophosphate-activated protein kinase; CC: compound C; HMGB1: high mobility group box 1; siRNA: small interfering RNA.

Figure 6

Compound C potentiates acute morphine analgesia and suppresses chronic morphine tolerance. (A, B) The effect of Compound C on morphine tolerance was evaluated by tail-flick assay. (A) Compound C potentiated acute morphine analgesia (n = 8/group). Mice were pretreated with different doses of Compound C (1, 3, or 10 μg/10 μL) before morphine injection, and MPE was evaluated after morphine (10 μg/10 μL) administration on the first day. (B) Compound C alleviated chronic morphine tolerance in mice (n = 8/group). Morphine (10 μg/10 μL) was injected intrathecally with different doses of Compound C (1, 3, or 10 μg/10 μL) once daily. (C) Administration of Compound C (10 μg/10 μL) for 7 consecutive days suppressed HMGB1 release into the CSF, as assessed by western blot (n = 4/group). (D–F) Administration of Compound C (10 μg/10 μL) for 7 consecutive days downregulated the AMPK phosphorylation, HO-1 expression, and p-p65 expression induced by morphine (10 μg/10 μL) in the spinal cord (n = 4/group). Data were normalized to the control in C–F. Data are expressed as mean ± SD. *P < 0.05, ***P < 0.001, vs. vehicle group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. morphine group (A, B: two-way analysis of variance followed by Tukey’s multiple-comparison test; C–F: one-way analysis of variance followed by Tukey’s multiple-comparison test). AMPK: 5′-Monophosphate-activated protein kinase; CC: Compound C; CSF: cerebrospinal fluid; HMGB1: high mobility group box 1; HO-1: heme oxygenase-1; MPE: maximal possible effect.