Analgesic and Antidepressant Effects of Oltipraz on Neuropathic Pain in Mice by Modulating Microglial Activation

Abstract

Nerve injury provokes microglial activation, contributing to the sensory and emotional disorders associated with neuropathic pain that do not completely resolve with treatment. In C57BL/6J mice with neuropathic pain induced by chronic constriction of the sciatic nerve (CCI), we evaluated the effects of oltipraz, an antioxidant and anticancer compound, on (1) allodynia and hyperalgesia, (2) microglial activation and pain signaling pathways, (3) oxidative stress, and (4) depressive-like behaviors. Twenty-eight days after surgery, we assessed the effects of oltipraz on the expression of CD11b/c (a microglial marker), phosphoinositide 3-kinase (PI3K)/ phosphorylated protein kinase B (p-Akt), nuclear factor-κB (NF-κB) transcription factor, and mitogen activated protein kinases (MAPK) in the spinal cord, hippocampus, and prefrontal cortex. Our results show that oltipraz alleviates neuropathic pain by inhibiting microglial activation and PI3K/p-Akt, phosphorylated inhibitor of κBα (p-IκBα), and MAPK overexpression, and by normalizing and/or enhancing the expression of antioxidant proteins, nuclear factor erythroid derived-2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and NAD(P)H:quinone oxidoreductase-1 (NQO1) in the spinal cord. The inhibition of microglial activation and induction of the Nrf2/HO-1/NQO1 signaling pathway in the hippocampus and/or prefrontal cortex may explain the antidepressant effects of oltipraz during neuropathic pain. These data demonstrate the analgesic and antidepressant effects of oltipraz and reveal its protective and antioxidant properties during chronic pain.

Keywords: analgesia; inflammation; microglia; neuropathic pain; oltipraz; oxidative stress.

Figure 1

Repeated treatment with oltipraz reduces mechanical allodynia, thermal hyperalgesia, and thermal allodynia in CCI-injured mice. The development of (A) mechanical allodynia, (B) thermal hyperalgesia, and (C) thermal allodynia in the ipsilateral paw of the CCI-injured or sham-operated (SHAM) mice treated with 10 mg/kg oltipraz (OLT) or vehicle for 11 consecutive days is shown. The effects of oltipraz were evaluated at days 18, 21, 25, and 28 after surgery. For each test and time evaluated, * denotes significant differences vs. sham-operated mice treated with vehicle, + denotes significant differences vs. sham-operated mice treated with oltipraz, and # denotes significant differences vs. CCI-injured mice treated with oltipraz (p < 0.05; one-way ANOVA followed by the Student-Newman-Keuls (SNK) test). Results are presented as the mean ± standard error of the mean (SEM); n = 6–8 animals per experimental group.

Figure 2

Repeated treatment with oltipraz reduces depressive-like behaviors in CCI-injured mice. Immobility time (s) evaluated by the (A) tail suspension test (TST) and (B) forced swimming test (FST) 28 days after surgery in the CCI-injured and sham-operated (SHAM) mice treated for 11 consecutive days with 10 mg/kg of oltipraz (OLT) or vehicle is shown. For each test evaluated, * denotes significant differences vs. sham-operated mice treated with vehicle, + denotes significant differences vs. sham-operated mice treated with oltipraz, and $ denotes significant differences vs. CCI-injured mice treated with vehicle (p < 0.05; one-way ANOVA followed by the SNK test). Results are presented as the mean ± SEM; n = 8 animals per experimental group.

Figure 3

Effects of oltipraz on the expression of CD11b/c, phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt), and phosphorylated inhibitor of κBα (p-IkBα) in the spinal cord of the CCI-injured mice. The relative protein levels of (A) CD11b/c, (B) PI3K, (D) p-Akt, and (E) p-IKBα on the ipsilateral side of the spinal cord in the CCI-injured mice treated with oltipraz (OLT) or vehicle are represented. The sham-operated mice (SHAM) treated with vehicle were used as controls. (C) Representative examples of blots for CD11b/c (160 kDa), PI3K (130 kDa), and GAPDH (37 kDa), and (F) for p-Akt (60 kDa), Akt (60 kDa), p-IKBα (40 kDa) and IKBα (40 kDa). CD11b/c and PI3K are expressed relative to GAPDH levels whereas phosphorylated proteins are expressed relative to their corresponding total proteins. In all panels, * denotes significant differences vs. sham-operated mice treated with vehicle (p < 0.05; one-way ANOVA followed by the SNK test). Results are presented as the mean ± SEM; n = 5 samples per experimental group.

Figure 4

Effects of oltipraz on the expression of nuclear factor erythroid derived-2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) in the spinal cord of the CCI-injured mice. The relative protein levels of (A) Nrf2, (B) HO-1, and (C) NQO1 on the ipsilateral side of the spinal cord in the CCI-injured mice treated with oltipraz (OLT) or vehicle are represented. The sham-operated mice (SHAM) treated with vehicle were used as controls. (D) Representative examples of blots for Nrf2 (100 kDa), HO-1 (32 kDa), NQO1 (28 kDa) and GAPDH (37 kDa). Protein levels are expressed relative to GAPDH levels. In all panels, * denotes significant differences vs. sham-operated mice treated with vehicle and + denotes significant differences vs. CCI-injured vehicle-treated mice (p < 0.05; one-way ANOVA followed by the SNK test). Results are presented as the mean ± SEM; n = 5 samples per experimental group.

Figure 5

Effects of oltipraz on the expression of phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated extracellular signal regulated kinase 1/2 (p-ERK 1/2), and p-P38 in the spinal cord of the CCI-injured mice. The relative protein levels of (A) p-JNK, (B) p-ERK 1/2, and (C) p-P38 on the ipsilateral side of the spinal cord of the CCI-injured mice treated with oltipraz (OLT) or vehicle are represented. The sham-operated mice (SHAM) treated with vehicle were used as controls. Phosphorylated proteins are expressed relative to their corresponding total protein levels. (D) Representative examples of blots for p-JNK/total JNK protein (46–54 kDa), p-ERK ½/total ERK ½ (42–44 kDa), and p-P38/total P38 (40 kDa). In all panels, * denotes significant differences vs. sham-operated mice treated with vehicle (p < 0.05; one-way ANOVA followed by the SNK test). Results are presented as the mean ± SEM; n = 5 samples per experimental group.

Figure 6

Effects of oltipraz on the expression of CD11b/c, PI3K, p-Akt, and p-IkBα in the hippocampus of the CCI-injured mice. The relative protein levels of (A) CD11b/c, (B) PI3K, (D) p-Akt, and (E) p-IKBα in the hippocampus of the CCI-injured mice treated with oltipraz (OLT) or vehicle. The sham-operated mice (SHAM) treated with vehicle were used as controls. Representative examples of blots for (C) CD11b/c (160 kDa), PI3K (130 kDa), and GAPDH (37 kDa); and (F) for p-Akt (60 kDa), Akt (60 kDa), p-IKBα (40 kDa), and IKBα (40 kDa). CD11b/c and PI3K are expressed relative to GAPDH levels whereas phosphorylated proteins are expressed relative to their corresponding total proteins. In all panels, * denotes significant differences vs. sham-operated mice treated with vehicle (p < 0.05; one-way ANOVA followed by the SNK test). Results are presented as the mean ± SEM; n = 5 samples per experimental group.

Figure 7

Effects of oltipraz on the expression of Nrf2, HO-1 and NQO1 in the hippocampus of the CCI-injured mice. The relative protein levels of (A) Nrf2, (B) HO-1, and (C) NQO1 in the hippocampus of the CCI-injured mice treated with oltipraz (OLT) or vehicle. The sham-operated mice (SHAM) treated with vehicle were used as controls. (D) Representative examples of blots for Nrf2 (100 kDa), HO-1 (32 kDa) and NQO1 (28 kDa) and GAPDH (37 kDa). Protein levels are expressed relative to GAPDH levels. In all panels, * denotes significant differences vs. sham-operated mice treated with vehicle (p < 0.05; one-way ANOVA followed by the SNK test). The results are presented as the mean ± SEM; n = 4–5 samples per experimental group.

Figure 8

Effects of oltipraz on the expression of CD11b/c, PI3K, p-Akt, and p-IkBα in the prefrontal cortex of the CCI-injured mice. The relative protein levels of (A) CD11b/c, (B) PI3K, (D) p-Akt, and (E) p-IKBα in the prefrontal cortex of the CCI-injured mice treated with oltipraz (OLT) or vehicle. The sham-operated mice (SHAM) treated with vehicle were used as controls. Representative examples of blots for (C) CD11b/c (160 kDa), PI3K (130 kDa) and GAPDH (37 kDa), and for (F) p-Akt (60 kDa), Akt (60 kDa), p-IKBα (40 kDa) and IKBα (40 kDa). CD11b/c and PI3K are expressed relative to GAPDH levels whereas phosphorylated proteins are expressed relative to their corresponding total proteins. In all panels, * denotes significant differences vs. sham-operated mice treated with vehicle (p < 0.05; one-way ANOVA followed by the SNK test). Results are presented as the mean ± SEM; n = 4-–5 samples per experimental group.

Figure 9

Effects of oltipraz on the expression of Nrf2, HO-1 and NQO1 in the in the prefrontal cortex of the CCI-injured mice. The relative protein levels of (A) Nrf2, (B) HO-1, and (C) NQO1 in the prefrontal cortex of the CCI-injured mice treated with oltipraz (OLT) or vehicle are represented. The sham-operated mice (SHAM) treated with vehicle were used as controls. (D) Representative examples of blots for Nrf2 (100 kDa), HO-1 (32 kDa) and NQO1 (28 kDa) and GAPDH (37 kDa). Protein levels are expressed relative to GAPDH levels. In all panels, * denotes significant differences vs. sham-operated mice treated with vehicle and + denotes significant differences vs. CCI-injured vehicle-treated mice (p < 0.05; one-way ANOVA followed by the SNK test). The results are presented as the mean ± SEM; n = 5 samples per experimental group.