Treatment With the Delta Opioid Agonist UFP-512 Alleviates Chronic Inflammatory and Neuropathic Pain: Mechanisms Implicated

Abstract

We investigated whether administration of the δ-opioid receptor (DOR) agonist H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), which also activates nuclear factor erythroid 2-related factor 2 (Nrf2), alleviated chronic inflammatory and/or neuropathic pain and inhibited the depressive-like behaviors associated with persistent neuropathic pain. The possible mechanisms implicated were also assessed. We evaluated the following effects in male C57BL/6J mice with inflammatory pain induced by complete Freund's adjuvant or neuropathic pain caused by the chronic constriction of sciatic nerve: (1) the antinociceptive effects of UFP-512; (2) the effects of UFP-512 on the expression of Nrf2, heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1, phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), inducible nitric oxide synthase, DOR, and mitogen-activated protein kinases (MAPK) in the spinal cord of animals with inflammatory or neuropathic pain; (3) the antinociceptive effects of the coadministration of UFP-512 with the Nrf2 activator sulforaphane (SFN); and (4) the antidepressant effects of UFP-512 in animals with depressive-like behaviors associated with neuropathic pain. Our results demonstrated that the intraperitoneal administration of UFP-512 inhibited chronic inflammatory and neuropathic pain and reduced the depressive-like behaviors associated with persistent neuropathic pain. The antiallodynic effects of UFP-512 were significantly augmented when it was coadministered with SFN in both types of chronic pain. The administration of UFP-512 increased/reestablished the spinal cord protein levels of Nrf2 and HO-1 in mice with inflammatory or neuropathic pain. However, while during inflammatory pain UFP-512 inhibited spinal c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation induced by peripheral inflammation. This DOR agonist blocked the spinal activated PI3K/Akt signaling pathway under chronic neuropathic pain conditions, but it did not alter the enhanced protein levels of p-JNK or p-ERK1/2 induced by sciatic nerve injury. These results revealed the antinociceptive and antidepressant effects of UFP-512 in animals with chronic pain and the different mechanism of action of this DOR agonist in the presence of inflammatory or neuropathic pain. Our data also suggest the administration of UFP-512 as an alternative for the treatment of chronic pain and the depressive-like behaviors associated with neuropathic pain.

Keywords: Nrf2 transcription factor; UFP-512; analgesia; chronic pain; delta opioid receptors; depression; oxidative stress.

FIGURE 1

Effects of UFP-512 on the mechanical allodynia and thermal hyperalgesia induced by CFA. The effects of acute intraperitoneal administration of different doses (logarithmic axis) of UFP-512 on the mechanical allodynia (A) and thermal hyperalgesia (B) induced by CFA in ipsilateral (continuous lines) and contralateral hind paws (discontinuous lines) are shown. UFP-512 was administered 1 h before testing. In both panels and for each dose assessed, ^∗ indicates significant differences vs. their respective effects in the contralateral paw, ⁺ indicates significant differences vs. the effect produced by 1 mg/kg of UFP-512 in the ipsilateral paw and ^# indicates significant differences vs. the effect produced by 3 mg/kg of UFP-512 in the ipsilateral paw (P < 0.05; one-way ANOVA, followed by the Student–Newman–Keuls test). In both tests, data are expressed as mean values of maximal possible effect (%) ± SEM; n = 6 animals per dose.

FIGURE 2

Effects of UFP-512 on the mechanical allodynia, thermal hyperalgesia, and thermal allodynia induced by CCI. The effects of the intraperitoneal acute administration of different doses (logarithmic axis) of UFP-512 on the mechanical allodynia (A), thermal hyperalgesia (B), and thermal allodynia (C) induced by sciatic nerve injury in the ipsilateral hind paw of CCI-injured (continuous lines) or sham-operated mice (discontinuous lines) are represented. UFP-512 was administered 1 h before testing. For each test and dose evaluated, ^∗ indicates significant differences vs. their respective effects in sham-operated mice, ⁺ indicates significant differences vs. the effects produced by 1 mg/kg of UFP-512 in CCI-injured mice, and ^# indicates significant differences vs. the effects produced by 3 mg/kg of UFP-512 in CCI-injured mice (P < 0.05; one-way ANOVA, followed by the Student–Newman–Keuls test). Data are expressed as mean values of maximal possible effect (%) for mechanical allodynia and thermal hyperalgesia, and inhibition (%) for thermal allodynia ± SEM; n = 6 animals per dose.

FIGURE 3

Reversion of the antiallodynic and antihyperalgesic effects of UFP-512. Reversal of the effects induced by 30 mg/kg UFP-512 on the mechanical allodynia (A) and thermal hyperalgesia (B) induced by CFA, and on the mechanical allodynia (C), thermal hyperalgesia (D), and thermal allodynia (E) caused by CCI, in ipsilateral paws, with the administration of the specific DOR antagonist, naltrindole (4 mg/kg), and the unspecific opioid antagonist, naloxone (1 mg/kg) are shown. The effects of vehicle, naltrindole (4 mg/kg), or naloxone (1 mg/kg) administered alone are also represented. UFP-512 was intraperitoneally injected 1 h before testing while both naltrindole and naloxone were subcutaneously injected 30 min before testing. For each test, ^∗ represents significant differences compared to the other groups (P < 0.05; one-way ANOVA, followed by the Student–Newman–Keuls test). Data are expressed as mean values of maximal possible effect (%) for mechanical allodynia and thermal hyperalgesia, and inhibition (%) for thermal allodynia ± SEM (six animals for each group).

FIGURE 4

Effects of UFP-512 on the expression of Nrf2, HO-1, NQO1, and NOS2 in the spinal cords of animals with peripheral inflammation. Protein levels of Nrf2 (A), HO-1 (B), NQO1 (C), and NOS2 (D) in the ipsilateral site of the spinal cords of CFA-injected mice treated with UFP-512 (CFA-UFP-512) or vehicle (CFA-vehicle) are represented. Controls corresponding to naive mice treated with vehicle (naive-vehicle) are also shown. For each protein, ^∗ indicates significant differences vs. naïve mice treated with vehicle and + indicates significant differences vs. CFA-injected mice treated with vehicle (P < 0.05, one-way ANOVA, followed by the Student–Newman–Keuls test). Representative examples of western blots for Nrf2 (100 kDa), HO-1 (32 kDa), NQO1 (28 kDa), and NOS2 (100 kDa) proteins in which GAPDH (37 kDa) was used as a loading control are also shown (E). Results are expressed as mean ± SEM; n = 4 samples per group.

FIGURE 5

Effects of UFP-512 on the expression of Nrf2, HO-1, NQO1, and NOS2 in spinal cords of animals with neuropathic pain. Protein levels of Nrf2 (A), HO-1 (B), NQO1 (C), and NOS2 (D) in the ipsilateral site of spinal cords of CCI-injured mice treated with UFP-512 (CCI-UFP-512) or vehicle (CCI-vehicle) are represented. Controls corresponding to sham-operated mice treated with vehicle (sham-vehicle) are also shown. For each protein, ^∗ indicates significant differences vs. sham-operated mice treated with vehicle, ⁺ indicates significant differences vs. CCI-injured mice treated with vehicle, and ^# indicates significant differences vs. CCI-injured mice treated with UFP-512 (P < 0.05, one-way ANOVA, followed by the Student–Newman–Keuls test). Representative examples of western blots for Nrf2 (100 kDa), HO-1 (32 kDa), NQO1 (28 kDa), and NOS2 (100 kDa) proteins in which GAPDH (37 kDa) was used as a loading control are also shown (E). Results are expressed as mean ± SEM; n = 4 samples per group.

FIGURE 6

Effects of UFP-512 on the expression of PI3K, p-Akt, DOR, p-JNK, and p-ERK 1/2 in the spinal cords of animals with peripheral inflammation. Protein levels of PI3K (A), p-Akt (B), DOR (C), p-JNK (E), and p-ERK 1/2 (F) in the ipsilateral site of spinal cords of CFA-injected mice treated with UFP-512 (CFA-UFP-512) or vehicle (CFA-vehicle) are represented. Controls corresponding to naive mice treated with vehicle (naive-vehicle) are also shown. For each protein, ^∗ indicates significant differences vs. naive-vehicle treated mice and ^# indicates significant differences vs. CFA-injected mice treated with UFP-512 (P < 0.05, one-way ANOVA, followed by the Student–Newman–Keuls test). Representative examples of western blots for PI3K (130 kDa), p-Akt (60 kDa), Akt (60 kDa), DOR (36 kDa), and GAPDH (37 kDa) are shown in (D) and for p-JNK/total JNK protein (46–54 kDa) and p-ERK 1/2/total ERK 1/2 (42–44 kDa) in (G). Phosphorylated proteins are expressed relative to their corresponding total proteins while the rest are relative to GAPDH. Results are expressed as mean ± SEM; n = 4 samples per group.

FIGURE 7

Effects of UFP-512 on the expression of PI3K, p-Akt, DOR, p-JNK, and p-ERK 1/2 in the spinal cords of animals with neuropathic pain. Protein levels of PI3K (A), p-Akt (B), DOR (C), p-JNK (E), and p-ERK 1/2 (F) in the ipsilateral site of the spinal cord from CCI-injured mice treated with UFP-512 (CCI-UFP-512) or vehicle (CCI-vehicle) are represented. Controls corresponding to sham-operated mice treated with vehicle (sham-vehicle) are also shown. For each protein,^∗ indicates significant differences vs. sham-operated mice treated with vehicle and ^# indicates significant differences vs. CCI-injured mice treated with UFP-512 (P < 0.05, one-way ANOVA, followed by the Student–Newman–Keuls test). Representative examples of western blots for PI3K (130 kDa), p-Akt (60 kDa), Akt (60 kDa), DOR (36 kDa), and GAPDH (37 kDa) are shown in (D) and for p-JNK/total JNK protein (46–54 kDa) and p-ERK 1/2/total ERK 1/2 (42–44 kDa) in (G). Phosphorylated proteins are expressed relative to their corresponding total proteins while the rest are relative to GAPDH. Results are expressed as mean ± SEM; n = 4 samples per group.

FIGURE 8

Effects of the coadministration of UFP-512 with SFN on the mechanical allodynia and thermal hyperalgesia induced by peripheral inflammation or sciatic nerve injury. Mechanical antiallodynic and thermal antihyperalgesic effects produced by 10 mg/kg SFN and 1 mg/kg UFP-512 intraperitoneally administered alone or combined in animals with inflammatory (A, C) or neuropathic pain (B, D) are shown. SFN and UFP-512 were administered at 3 and 1 h before testing, respectively. For each test, ^∗ indicates significant differences vs. its respective animals treated with vehicle plus saline, ⁺ indicates significant differences vs. its respective animals treated with vehicle plus UFP-512, and ^# indicates significant differences vs. its respective animals treated with SFN plus saline (P < 0.05, one-way ANOVA, followed by the Student–Newman–Keuls test). Data are expressed as mean values of maximal possible effect (%) for mechanical allodynia and thermal hyperalgesia ± SEM; n = 6 animals per group.

FIGURE 9

Effects of UFP-512 on the depressive-like behaviors associated with neuropathic pain. Immobility time in seconds (s) for CCI-injured and sham-operated mice treated with 1 mg/kg of UFP-512 or saline evaluated at 28 days after surgery in TST are represented. UFP-512 was administered 1 h before testing. In all groups, ^∗ indicates significant differences vs. sham-operated mice treated with saline and ⁺ indicates significant differences vs. CCI-injured mice treated with saline (P < 0.05, one-way ANOVA, followed by the Student–Newman–Keuls test). Results are expressed as mean ± SEM; n = 8 animals for each experimental group.