Sulforaphane Inhibited the Nociceptive Responses, Anxiety- and Depressive-Like Behaviors Associated With Neuropathic Pain and Improved the Anti-allodynic Effects of Morphine in Mice

Abstract

Chronic neuropathic pain is associated with anxiety- and depressive-like disorders. Its treatment remains a serious clinical problem due to the lack of efficacy of the available therapeutic modalities. We investigated if the activation of the transcription factor Nrf2 could modulate the nociceptive and emotional disorders associated with persistent neuropathic pain and potentiated the analgesic activity of morphine. The possible mechanisms implicated in these effects have been also evaluated. Therefore, in C57BL/6 mice with neuropathic pain induced by the chronic constriction of the sciatic nerve (CCI), we assessed the antinociceptive, anxiolytic, and anti-depressant effects of the repeated intraperitoneal administration of a Nrf2 inducer, sulforaphane (SFN), and the effects of this treatment on the local antinociceptive actions of morphine. The protein levels of Nrf2, heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase-1 (NQO1), CD11b/c (a microglial activator marker), mitogen-activated protein kinases (MAPK) and μ opioid receptors (MOR) in the spinal cord, prefrontal cortex and hippocampus from mice, at 28 days after CCI, were also evaluated. Our results showed that the repeated administration of SFN besides inhibiting nociceptive responses induced by sciatic nerve injury also diminished the anxiety- and depressive-like behaviors associated with persistent neuropathic pain. Moreover, SFN treatment normalized oxidative stress by inducing Nrf2/HO-1 signaling, reduced microglial activation and JNK, ERK1/2, p-38 phosphorylation induced by sciatic nerve injury in the spinal cord and/or hippocampus and prefrontal cortex. Interestingly, treatment with SFN also potentiated the antiallodynic effects of morphine in sciatic nerve-injured mice by regularizing the down regulation of MOR in the spinal cord and/or hippocampus. This study suggested that treatment with SFN might be an interesting approach for the management of persistent neuropathic pain and comorbidities associated as well as to improve the analgesic actions of morphine.

Keywords: Nrf2 transcription factor; analgesia; anxiety; chronic pain; depression; opioids; oxidative stress.

FIGURE 1

The antinociceptive effects of SFN during neuropathic pain. Mechanical antiallodynic (A), thermal antihyperalgesic (B), and thermal antiallodynic (C) effects produced by the repeated administration of 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve-injury (CCI) or Sham-operation (SHAM). In all panels, for each day and treatment evaluated, ^∗ indicates significant differences vs. Sham-operated animals treated with vehicle, + vs. Sham-operated animals treated with SFN and # vs. sciatic nerve-injured animals treated with SFN (P < 0.05, one way ANOVA followed by Student–Newman–Keuls test). Results are shown as mean values ± SEM; n = 6 animals per experimental group.

FIGURE 2

The anxiolytic and anti-depressant effects induced by SFN in animals with neuropathic pain. Effects of the repetitive administration of 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) or Sham-operation (SHAM) on the anxiety- (elevated plus maze, A–C) and depressive-liked (tail suspension test, D) behaviors associated with neuropathic pain. In all panels, ^∗ indicates significant differences vs. Sham-operated animals treated with vehicle, + vs. Sham-operated animals treated with SFN and # vs. sciatic nerve-injured animals treated with SFN (P < 0.05, one way ANOVA followed by Student–Newman–Keuls test). Results are shown as mean values ± SEM; n = 8 animals per experimental group.

FIGURE 3

Effects of SFN on the local antinociceptive effects of morphine during neuropathic pain. The mechanical antiallodynic (A), thermal antihyperalgesic (B), and thermal antiallodynic (C) effects produced by the acute administration of 10 mg/kg SFN or vehicle combined with a subanalgesic dose (50 μg) of morphine or saline administered on the ipsilateral paw of sciatic nerve-injured mice at 28 days after surgery were shown. In all panels,^∗ indicates significant differences vs. vehicle plus saline treated mice, + vs. vehicle plus morphine treated mice and # vs. SFN plus saline treated mice (P < 0.05, one way ANOVA followed by Student–Newman–Keuls test). Results are shown as mean values ± SEM; n = 6 animals per experimental group.

FIGURE 4

Effects of SFN on the expression of Nrf2, HO-1, NQO1, CD11b/c, and MOR in the spinal cord from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A), HO-1 (B), NQO1 (C), CD11b/c (D), and MOR (E) protein levels in the ipsilateral site of the spinal cord from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F). In all panels, ^∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice (P < 0.05, one-way ANOVA followed by Student–Newman–Keuls test). Data are expressed as mean values ± SEM; n = 4 samples per group.

FIGURE 5

Effects of SFN on the expression of Nrf2, HO-1, NQO1, CD11b/c, and MOR in the prefrontal cortex from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A), HO-1 (B), NQO1 (C), CD11b/c (D), and MOR (E) protein expression in the prefrontal cortex from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F). In all panels, ^∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice (P < 0.05, one way ANOVA followed by Student–Newman–Keuls test). Data are expressed as mean values ± SEM; n = 4 samples per group.

FIGURE 6

Effects of SFN on the expression of Nrf2, HO-1, NQO1, CD11b/c, and MOR in the hippocampus from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on Nrf2 (A), HO-1 (B), NQO1 (C), CD11b/c (D), and MOR (E) protein expression in the hippocampus from CCI-induced neuropathic pain in mice are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. Examples of western blots for Nrf2 (75 kDa), HO-1 (32 kDa), NQO1 (28 kDa), CD11b/c (160 kDa), and MOR (50 kDa) proteins in which GAPDH (36 kDa) was used as a loading control are also shown (F). In all panels, ^∗ indicates significant differences vs. Sham vehicle treated mice and # indicates significant differences vs. CCI plus SFN treated mice (P < 0.05, one way ANOVA followed by Student–Newman–Keuls test). Data are expressed as mean values ± SEM; n = 4 samples per group.

FIGURE 7

Effect of SFN on the expression of MAPK in the spinal cord from animals with neuropathic pain. Effects of repetitive treatment with 10 mg/kg SFN or vehicle from days 14 to 28 after sciatic nerve injury (CCI) on the protein levels of p-JNK (A), p-ERK ½ (B), and p-P38 (C) in the spinal cord are represented. The protein levels from Sham-operated (SHAM) mice treated with vehicle has been also represented as controls. In all panels, ^∗ indicates significant differences vs. Sham-operated vehicle treated mice and # indicates significant differences vs. CCI-SFN treated mice (P < 0.05, one way ANOVA followed by Student–Newman–Keuls test). Each column represents the mean and vertical bars indicate the standard error of the mean (SEM); n = 4 samples per group. Representative examples of western blots for p-JNK/total JNK protein (46–54 kDa), p-ERK ½/total ERK ½ (42–44 kDa) and p-P38/total P38 (38 kDa) are also shown.