ORIGINAL RESEARCH published: 26 March 2019 doi: 10.3389/fphar.2019.00283 Treatment With the Delta Opioid Agonist UFP-512 Alleviates Chronic Inflammatory and Neuropathic Pain: Mechanisms Implicated Sara Polo 1,2 , Andrés Felipe Díaz 1,2 , Núria Gallardo 1,2 , Sergi Leánez 1,2 , Gianfranco Balboni 3 and Olga Pol 1,2* 1 Grup de Neurofarmacologia Molecular, Institut d’Investigació Biomèdica Sant Pau, Barcelona, Spain, 2 Grup de Neurofarmacologia Molecular, Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, Cagliari, Italy 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 Edited by: pain. The possible mechanisms implicated were also assessed. We evaluated the Francisco Ciruela, University of Barcelona, Spain following effects in male C57BL/6J mice with inflammatory pain induced by complete Reviewed by: Freund’s adjuvant or neuropathic pain caused by the chronic constriction of sciatic Temugin Berta, nerve: (1) the antinociceptive effects of UFP-512; (2) the effects of UFP-512 on the University of Cincinnati, United States Mariana Spetea, expression of Nrf2, heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1, University of Innsbruck, Austria phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), inducible nitric oxide synthase, *Correspondence: DOR, and mitogen-activated protein kinases (MAPK) in the spinal cord of animals with Olga Pol inflammatory or neuropathic pain; (3) the antinociceptive effects of the coadministration opol@santpau.es of UFP-512 with the Nrf2 activator sulforaphane (SFN); and (4) the antidepressant effects Specialty section: of UFP-512 in animals with depressive-like behaviors associated with neuropathic pain. This article was submitted to Our results demonstrated that the intraperitoneal administration of UFP-512 inhibited Neuropharmacology, a section of the journal chronic inflammatory and neuropathic pain and reduced the depressive-like behaviors Frontiers in Pharmacology associated with persistent neuropathic pain. The antiallodynic effects of UFP-512 were Received: 10 December 2018 significantly augmented when it was coadministered with SFN in both types of chronic Accepted: 07 March 2019 Published: 26 March 2019 pain. The administration of UFP-512 increased/reestablished the spinal cord protein Citation: levels of Nrf2 and HO-1 in mice with inflammatory or neuropathic pain. However, while Polo S, Díaz AF, Gallardo N, during inflammatory pain UFP-512 inhibited spinal c-Jun N-terminal kinase (JNK) and Leánez S, Balboni G and Pol O (2019) extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation induced by peripheral Treatment With the Delta Opioid Agonist UFP-512 Alleviates Chronic inflammation. This DOR agonist blocked the spinal activated PI3K/Akt signaling pathway Inflammatory and Neuropathic Pain: under chronic neuropathic pain conditions, but it did not alter the enhanced protein Mechanisms Implicated. Front. Pharmacol. 10:283. levels of p-JNK or p-ERK1/2 induced by sciatic nerve injury. These results revealed the doi: 10.3389/fphar.2019.00283 antinociceptive and antidepressant effects of UFP-512 in animals with chronic pain and Frontiers in Pharmacology | www.frontiersin.org 1 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain 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: analgesia, depression, Nrf2 transcription factor, delta opioid receptors, oxidative stress, chronic pain, UFP-512 INTRODUCTION tolerance after chronic administration in contrast to other DOR agonists, for example, UFP-502 (Vergura et al., 2006, 2008; Chronic pain is a complex experience that is composed Aguila et al., 2007). of sensory and affective components. Therefore, when pain Several studies also showed the potential use of some becomes persistent or severe causes great suffering that presents DOR agonists, such as [D-Pen2 ,D-Pen5 ]-enkephalin (DPDPE), an important clinical problem with enormous economic and SNC-80, and UFP-512, as antidepressant drugs (Vergura et al., social burdens (Monti and Caporali, 2015). One factor that 2008; Lutz and Kieffer, 2013), and these effects were also contributes to the intractable nature of chronic pain is the confirmed by an increase in depressive-like behaviors in existence of comorbidities associated such as depression, which DOR-KO mice in different depressive animal models (Filliol also negatively influence the perception of pain and create et al., 2000). It is also well known that persistent neuropathic a vicious circle that eventually leads to unwanted results pain is associated with several comorbidities, such as depression, (Kawai et al., 2017). which is difficult to treat with typical antidepressants (La Chronic inflammatory and neuropathic pains are difficult to Porta et al., 2016). Notably, we recently demonstrated that treat because actual therapies, such as NSAIDs, antidepressants, treatment with the nuclear factor erythroid 2-related factor 2 and antiepileptics, have several side effects, and these agents do (Nrf2) inducer sulforaphane (SFN) inhibited the depressive-like not effectively alleviate pain and/or the associated emotional behaviors linked with chronic neuropathic pain (Ferreira- disorders. The opioid system plays a crucial role in pain control, Chamorro et al., 2018). In vitro studies also revealed that UFP-512 and activation of µ-(MOR), δ-(DOR), and κ-opioid receptors induced activation of Nrf2 (Cao S. et al., 2015). Therefore, produces analgesia (Pol, 2007; Cahill et al., 2014; Albert- we evaluated the possible antidepressant effects of UFP-512 in Vartanian et al., 2016; Bodnar, 2016). However, it is curious that animals with depressive-like behaviors associated with persistent while the administration of MOR agonists is highly effective for neuropathic pain. the treatment of inflammatory pain, these agents exhibit limited The contribution of oxidative stress to central and peripheral efficacy in neuropathic pain and produce several adverse side sensitization (Meeus et al., 2013; Riego et al., 2018) and effects, such as sedation, constipation, and respiratory depression. the protective role played by Nrf2 in reducing oxidative In contrast, and although the administration of several DOR stress via enhancing the expression of several antioxidant or agonists produce limited analgesic effects in acute pain, these detoxification enzymes, including heme-oxygenase 1 (HO-1) and agonists reduced chronic inflammatory and neuropathic pain NAD(P)H quinone oxidoreductase 1 (NQO1), is well recognized with similar efficacy (Pradhan et al., 2011). Moreover, DOR (Nguyen et al., 2009). Recent works demonstrated that the knockout (KO) mice exhibited increased inflammatory and administration of Nrf2 inducers reduced inflammatory (Redondo neuropathic pain responses when submitted to an inflammatory et al., 2017) and neuropathic pain (Negi et al., 2011; McDonnell or injury stimulus (Nadal et al., 2006), which supports the et al., 2017a; Wang and Wang, 2017) via activation of the hypothesis of DOR agonists as an interesting alternative Nrf2/HO–1/NQO1 signaling pathway and/or inhibition of the for the treatment of chronic inflammatory and neuropathic synthesis of pro-inflammatory mediators and mitogen-activated pain. However, comparisons of the antinociceptive effects of protein kinases (MAPK) activation (Negi et al., 2011; McDonnell several DOR agonists, such as [D-Ser2, Leu5, Thr6]-enkephalin et al., 2017b; Redondo et al., 2017; Wang and Wang, 2017). Based (DSLET) and (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1- on these findings and that UFP-512 exerts its cytoprotective piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC- effects in cell cultures via activation of the Nrf2/HO–1/NQO1 80), in inflammatory and neuropathic pain were only performed pathway (Cao S. et al., 2015), we evaluated whether the inhibitory in models of acute inflammatory pain (formalin test) or in effects of this DOR agonist during chronic inflammatory or the early stages of neuropathic pain (16 days after surgery) neuropathic pain were also produced via activation of this (Obara et al., 2009). Therefore, to the present study we antioxidant pathway and/or reduction of the inflammatory and evaluated the antinociceptive effects produced by another nociceptive responses implicated in the maintenance of chronic specific DOR agonist, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid pain. In addition and due to we previously demonstrated that (UFP-512), in animals with chronic inflammatory (14 days the activation of Nrf2 following SFN administration increased after induction) or neuropathic (28 days after surgery) pain. the antinociceptive effects of DOR and MOR agonists during We used UFP-512 because this agonist did not produce inflammatory and neuropathic pain (McDonnell et al., 2017a; convulsions or alter locomotor activity, and it exhibits Redondo et al., 2017; Ferreira-Chamorro et al., 2018), the anxiolytic and antidepressant properties without signs of potential improvement of the antinociceptive effects of UFP-512 Frontiers in Pharmacology | www.frontiersin.org 2 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain by its coadministration with SFN in animals with inflammatory the method used by our group (Hervera et al., 2013). Animals or neuropathic pain was also evaluated. were tested at 28 days after surgery. Sham-operated mice, whose We assessed the following effects in a mouse model of surgery was the same as described above without sciatic nerve inflammatory pain induced by the subplantar injection of ligation, were used as controls. complete Freund’s adjuvant (CFA) and a neuropathic pain model caused by chronic constriction of the sciatic nerve (CCI): (1) the Nociceptive Behavioral Tests antiallodynic and antihyperalgesic effects of the intraperitoneal Mechanical allodynia was evaluated by measuring the hind administration of UFP-512 in chronic inflammatory and paw withdrawal response to the stimulation with the von Frey neuropathic pain; (2) the reversal of the antinociceptive effects filament test. Mice were placed into methacrylate cylinders of UFP-512 by the specific DOR antagonist, naltrindole, and the (20 cm high × 9 cm in diameter) on a lifted wire grid across unspecific opioid antagonist, naloxone; (3) the antinociceptive which von Frey filaments (North Coast Medical, Inc., San Jose, effects of the coadministration of SFN with a low dose of UFP-512 CA, United States) were applied to each hind paw by using the in animals with chronic inflammatory and neuropathic pain; up/down paradigm described by Chaplan et al. (1994). After (4) the antidepressant effects of UFP-512 on the depressive-like 1 h of habituation, test was started with the filament of 0.4 g behaviors linked to persistent neuropathic pain; and (5) the and the strength of the following filament was decreased or effects of this DOR agonist on the expression of Nrf2, HO-1, increased in accordance with the response. The filament of 3.0 g NQO1, inducible nitric oxide synthase (NOS2), phosphoinositide was used as a cutoff. The threshold of response was calculated 3-kinase (PI3K)/protein kinase B (Akt), DOR, c-Jun N-terminal from the sequence of filament strength used during the up/down kinase (JNK), and extracellular signal regulated kinase 1/2 procedure utilizing an Excel program (Microsoft Iberia SRL, (ERK1/2) in the spinal cord of mice with inflammatory or Barcelona, Spain), which includes curve fitting of the data. A clear neuropathic pain. paw withdrawal, licking, or shaking the paw was considered to be a nociceptive-like response. Thermal hyperalgesia was evaluated by measuring hind paw MATERIALS AND METHODS withdrawal latency in response to radiant heat using the plantar test (Ugo Basile, Italy) as previously described by Hargreaves et al. Animals (1988). Animals were placed in methacrylate cylinders (20 cm The animals to carry out the experimental procedures were high × 9 cm diameter) on a glass surface and after 1 h of 7-week old male C57BL/6J mice, between 21 and 25 g of weight, habituation, the heat source was positioned under the plantar obtained from Envigo Laboratories (Barcelona, Spain). They were surface of the hind paw and activated with a light beam intensity. accommodated in a room with 12 h/12 h light/dark conditions, A cut-off or 12 s was used to avoid tissue damage. The mean paw under controlled temperature of 22◦ C and humidity of 66%. They withdrawal latencies of each hind paw were determined by the had free access to food and water. After a minimum of 7 days average of three separated trials, taken at 5 min intervals. from their arrival, animals were used for doing the experiments. In CCI-injured mice, thermal allodynia to a cold stimulus was All experiments were conducted between 9:00 a.m. and 5:00 p.m., also measured using the cold plate test (Ugo Basile, Italy) and and executed in accordance with the National Institute of according to the method described by Bennett and Xie (1988). Health Guide for the Care and Use of Laboratory Animals and Each animal was placed on the cold plate at 4 ± 0.5◦ C and the approved by the local Committee of Animal Use and Care of the number of elevations of each hind paw was recorded for 5 min. Autonomous University of Barcelona. All efforts were made to minimize the number of animals used and their suffering. Depression-Like Behavior Test Evaluation of depressive-like behaviors was performed using Induction of Inflammatory Pain the tail suspension test (TST), in which the total duration of Inflammatory pain was induced by the subplantar injection immobility of the animal was quantified according to the method of 30 µl of CFA (F5881, Sigma–Aldrich, St. Louis, MO, described by Steru et al. (1985), with some modifications. Briefly, United States) in the right hind paw under isoflurane anesthesia mice were suspended by the tail from a horizontal wooden bar (3% induction, 2% maintenance) according to the method used (35 cm above the floor) using adhesive tape (1 cm from the tip by our group (Leánez et al., 2009). Animals were tested at of the tail). The immobility time in seconds was recorded over 14 days after CFA injection. Contralateral hind paws were a total period of 6 min. All the behavioral experiments were used as controls. executed by an experimenter blinded to the treatment applied. Induction of Neuropathic Pain Western Blot Analysis Neuropathic pain was caused by the chronic constriction Mice were euthanized by cervical dislocation at 14 days after of sciatic nerve (CCI). Nerve ligation was performed under CFA injection (inflammatory pain) or 28 days after sciatic isoflurane anesthesia (3% induction, 2% maintenance). Blunt nerve ligation (neuropathic pain). Tissues from the ipsilateral dissection was made to segregate the biceps femoris from the side of the lumbar section of the spinal cord were extracted, gluteus superficialis on the right side. The nerve was tied by frozen in liquid nitrogen, and stored at −80◦ C until use. three ligatures (4/0 silk) around it, leaving 1 mm spacing and Samples of each tissue from three animals were pooled into taking care to preserve epineural circulation, in accordance to one experimental sample to obtain the necessary protein levels Frontiers in Pharmacology | www.frontiersin.org 3 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain to perform western blot. The protein levels of Nrf2, HO-1, as a control (n = 6 animals per dose), while in neuropathic NQO1, NOS2, PI3K, p-Akt, DOR, p-JNK, and p-ERK1/2 were pain model, sham-operated animals were used as control (n = 6 analyzed. Tissue homogenization was made in ice cold lysis animals per dose). buffer (50 mM Tris Base, 150 nM NaCl, 1% NP-40, 2 mM The reversibility of the antinociceptive effects of 30 mg/kg EDTA, 1 mM phenylmethylsulfonylfluoride, 0.5 Triton X-100, UFP-512, which produces the maximal antiallodynic and 0.1% sodium dodecyl sulfate, 1 mM Na3 VO4 , 25 mM NaF, antihyperalgesic effects after peripheral inflammation and sciatic 0.5% protease inhibitor cocktail, and 1% phosphatase inhibitor nerve injury, with the subcutaneous administration of the cocktail). All reagents were obtained from Sigma–Aldrich, specific DOR antagonist, naltrindole (4 mg/kg) and the except for NP-40, which was acquired from Calbiochem unspecific opioid antagonist, naloxone (1 mg/kg) was evaluated (Darmstadt, Germany). After crude homogenate solubilization 14 days after CFA injection or 28 days after surgery (n = 6 (1 h at 4◦ C), sonification (10 s), and centrifugation (15 min animals per group). The doses of all tested antagonists were at 4◦ C) at 700 g, the supernatant (60 µg of total protein) was selected according to previous works (Hervera et al., 2013; mixed with 4× laemmli loading buffer and loaded onto 4% Carcolé et al., 2014). stacking/10% separating sodium dodecyl sulfate polyacrylamide In order to study the possible enhancement of the analgesic gels. Afterward, the proteins were electrophoretically transferred effects of UFP-512 induced by SFN, in other set of experiments we onto PVDF membranes (120 min). Then, they were blocked evaluated the antiallodynic and antihyperalgesic effects produced (1 h and 15 min) with phosphate-buffered saline plus 5% by the intraperitoneal administration of a low dose of UFP-512 nonfat dry milk or Tris-buffered saline with Tween 20 plus (1 mg/kg) combined with 10 mg/kg of SFN at day 14 after 5% nonfat dry milk or 5% of bovine serum albumin. After CFA injection or 28 days after surgery. The dose of SFN that, an overnight incubation at 4◦ C was made with specific was selected in accordance to other studies (Redondo et al., rabbit primary antibody anti Nrf2 (1:160; ab62352, Abcam, 2017; Ferreira-Chamorro et al., 2018) and the dose of UFP-512 Cambridge, United Kingdom), NOS2 (1:160; ab204017, was chosen from the dose–response curves performed in this Abcam, Cambridge, United Kingdom), HO-1 (1:150; study, as the ones that produced minimal inhibitory effects ab137749, Abcam, Cambridge, United Kingdom), PI3K (1:200; in both pain models. SFN was intraperitoneally administrated ab232997, Abcam, Cambridge, United Kingdom) and DOR 2 h before UFP-512 injection and the nociceptive responses (1:300; AB1560 Merck, Billerica, MA, United States), anti-NQO1 were evaluated 1 h later, in accordance to our previous works (1:250, N5288, Sigma–Aldrich, St. Louis, MO, United States), (Redondo et al., 2017; Ferreira-Chamorro et al., 2018) (n = 6 p-Akt (1:200; 9271, Cell Signaling Technology, Danvers, MA, animals per group). United States), Akt (1:200; 9272, Cell Signaling Technology, In animals with neuropathic pain, at day 28 after CCI, the Danvers, MA, United States), p-JNK (1:250; 9251, Cell Signaling antidepressant effects produced by 1 mg/kg of UFP-512 in the Technology, Danvers, MA, United States), JNK (1:250; 9252, TST were also assessed. Sham-operated mice were used as control Cell Signaling Technology, Danvers, MA, United States), (n = 8 animals per group). p-ERK1/2 (1:250; 9101, Cell Signaling Technology, Danvers, Finally, the effects of UFP-512 on the expression of MA, United States), and ERK1/2 (1:250; 9102, Cell Signaling Nrf2, HO-1, NQO1, NOS2, PI3K, p-Akt, DOR, p-JNK, Technology, Danvers, MA, United States). Blots were and p-ERK1/2 in the ipsilateral site of spinal cords of incubated for 1 h at room temperature with a horseradish animals with chronic peripheral inflammation or sciatic nerve peroxidase-conjugated anti-rabbit secondary antibody (GE injury were evaluated by using western blot. Naive and Healthcare, Little Chalfont, United Kingdom) to detect proteins, sham-operated mice treated with vehicle were used as controls for which were then visualized by chemiluminescence reagents CFA-injected animals and CCI-injured mice, respectively (n = 4 (ECL kit; GE Healthcare, Little Chalfont, United Kingdom) samples per group). and exposition onto hyper film (GE Healthcare, Little Chalfont, United Kingdom). The intensity of the blot was quantified by Drugs using densitometry. A rabbit anti-glyceraldehyde-3-phosphate UFP-512 was synthetized by Balboni et al. (2002), SFN dehydrogenase (GAPDH) antibody (1:5000; ABS16, Merck, was acquired from Merck Chemicals and Life Science S.A.U Billerica, MA, United States) was used as a loading control. (Madrid, Spain), and naltrindole and naloxone were purchased in Sigma–Aldrich (St. Louis, MO, United States). UFP-512 Experimental Procedure was dissolved in saline solution (0.9%) and intraperitoneally First of all, in both inflammatory and neuropathic pain models, administered at 1, 3, 10, 20, and 30 mg/kg. SFN was behavioral baseline responses were measured in the following dissolved in dimethylsulfoxide (1% in saline solution 0.9%) order: von Frey filaments, plantar test, and/or cold plate test. and intraperitoneally administered at 10 mg/kg. Naltrindole At 14 days after CFA injection or at 28 days after surgery, the and naloxone were also diluted in saline solution (0.9%) and antinociceptive effects produced by 1, 3, 10, 20, and 30 mg/kg of subcutaneously administered at 4 and 1 mg/kg, respectively. All UFP-512 were tested according to the sequence described above. drugs were prepared daily just before use and administered in UFP-512 was injected intraperitoneally and the nociceptive a final volume of 10 ml/kg, at 1 h (UFP-512), 3 h (SFN), and responses were tested 1 h later in accordance to our previous 30 min (naltrindole and naloxone) before testing in accordance pilot studies and another work (Aguila et al., 2007). In the to our previous pilot studies and other works (Aguila et al., 2007; inflammatory pain model, the contralateral hind paw was used Hervera et al., 2013; Carcolé et al., 2014; Redondo et al., 2017; Frontiers in Pharmacology | www.frontiersin.org 4 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain Ferreira-Chamorro et al., 2018). For each group treated with a as well as the effects of these antagonists administered alone drug, the respective control group received the same volume of were analyzed by using a one-way ANOVA, followed by the corresponding vehicle. Student–Newman–Keuls test. In both pain models, the comparison of the antiallodynic and Statistical Analysis antihyperalgesic effects produced by the coadministration of SFN Data are expressed as mean ± standard error of the with UFP-512 was assessed by using a one-way ANOVA, followed mean (SEM). All the statistical analysis was carried out by the Student–Newman–Keuls test. using the SPSS program (version 17 for windows, IBM The analysis of the antidepressant actions of UFP-512 in España, Madrid, Spain). animals with neuropathic pain was performed by using a two-way In both pain models and for each nociceptive behavioral test, ANOVA, followed by the corresponding one-way ANOVA, and the comparison of the effects produced by the intraperitoneal the Student–Newman–Keuls test. administration of different doses of UFP-512 or saline was The effects of UFP-512 on the protein levels of Nrf2, evaluated by using a one-way ANOVA, followed by the HO-1, NQO1, NOS2, PI3K, p-Akt, DOR, p-JNK, and Student–Newman–Keuls test. p-ERK1/2 in the spinal cord of mice with inflammatory or In all experiments, the antiallodynic effects in the von neuropathic pain were analyzed by using a one-way ANOVA, Frey filaments and the antihyperalgesic effects in the plantar followed by Student–Newman–Keuls test. A P < 0.05 was test were expressed as the percentage of maximal possible considered significant. effect, where the test latencies predrug (baseline) and postdrug administration are compared and calculated according to the following equation: RESULTS Maximal possible effect (%) = [(drug–baseline)/(cut-off– Effects of UFP-512 on Mechanical baseline)] × 100. Allodynia and Thermal Hyperalgesia In the cold plate test, the inhibitory effects were calculated Induced by Inflammatory Pain according to the following equation: To study the antinociceptive actions of UPF-512 during inflammatory pain, we evaluated the effects of acute Inhibition (%) = [(paw elevations number at baseline–paw intraperitoneal administration of 1, 3, 10, 20, and 30 mg/kg elevations number after drug)/paw elevations number at UPF-512 on CFA-induced mechanical allodynia and thermal baseline] × 100. hyperalgesia 14 days after injection. Our results showed For each test, the reversal of the systemic antinociceptive that UFP-512 administration 1 h before testing inhibited effects produced by UFP-512 with naltrindole or naloxone CFA-induced mechanical allodynia (Figure 1A) and thermal 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. Frontiers in Pharmacology | www.frontiersin.org 5 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain hyperalgesia (Figure 1B) in a dose-dependent manner and and thermal allodynia induced by sciatic nerve injury 28 days reached a maximum effect at 30 mg/kg. after surgery. Our results indicated that UFP-512 administration The mechanical antiallodynic effects produced by 10, 20, 1 h before testing inhibited CCI-induced mechanical allodynia and 30 mg/kg UFP-512 in the ipsilateral paw of CFA-injected (Figure 2A), thermal hyperalgesia (Figure 2B), and thermal mice were significantly greater than the effects produced by allodynia (Figure 2C) in a dose-dependent manner, and 30 mg/kg each respective dose in the contralateral paw (P < 0.001, produced the maximum effect. one-way ANOVA, followed by the Student–Newman–Keuls Therefore, the mechanical antiallodynic effects produced by test). Moreover, the antiallodynic effects produced by the 10, 20, and 30 mg/kg of UFP-512 in CCI-injured mice were administration of high doses (10, 20, or 30 mg/kg) of UFP-512 greater than the effects produced by each respective dose in were significantly higher than the effects produced by lower sham-operated mice and the effects produced by 1 and 3 mg/kg doses (1 and/or 3 mg/kg) of this DOR agonist in the of this DOR agonist in CCI-injured mice (P < 0.001; one-way ipsilateral paw (P < 0.001, one-way ANOVA, followed by the ANOVA, followed by the Student–Newman–Keuls test). Student–Newman–Keuls test). The thermal antihyperalgesic effects produced by 1, 3, 10, The thermal antihyperalgesic effects produced by 3, 10, 20, 20, and 30 mg/kg of UFP-512 in CCI-injured animals were and 30 mg/kg UFP-512 in the ipsilateral paw of CFA-injected higher than the effects produced by each respective dose in mice were also greater than the effects produced by each sham-operated mice (P < 0.001; one-way ANOVA, followed by respective dose in the contralateral paw (P < 0.001, one-way the Student–Newman–Keuls test). The antihyperalgesic effects ANOVA, followed by the Student–Newman–Keuls test). The of 10, 20, and 30 mg/kg of UFP-512 were also higher than the antihyperalgesic actions induced by 10, 20, or 30 mg/kg UFP-512 effects of 1 and 3 mg/kg in CCI-injured mice (P < 0.001; one-way in the ipsilateral paw were also higher than the effects produced ANOVA, followed by the Student–Newman–Keuls test). by 1 and 3 mg/kg of UFP-512 in the same paw. Similar effects were observed for thermal allodynia. The The intraperitoneal administration of saline did not elicit any antiallodynic actions produced by 3, 10, 20, and 30 mg/kg of antinociceptive effects in the contralateral or ipsilateral paw of UFP-512 in CCI-injured mice were greater than the effects CFA-injected mice in either test (data not shown). produced by these doses in sham-operated mice (P < 0.001; one-way ANOVA, followed by the Student–Newman–Keuls test). Effects of UFP-512 on the Mechanical The antiallodynic effects produced by high doses (10, 20, and 30 mg/kg) of UFP-512 in CCI-injured mice were greater than the Allodynia, Thermal Hyperalgesia, and effects produced by 1 mg/kg of this drug in these animals. Thermal Allodynia Induced by Sciatic Treatment with UFP-512 did not affect the contralateral Nerve Injury paw of CCI-injured or sham-operated mice in any experiment To study the antinociceptive actions of UPF-512 during (data not shown). The intraperitoneal administration of saline neuropathic pain, we evaluated the effects of the acute did not elicit any antinociceptive effect in the contralateral intraperitoneal administration of UFP-512 at 1, 3, 10, 20, and or ipsilateral paw of CCI-injured or sham-operated mice 30 mg/kg on the mechanical allodynia, thermal hyperalgesia, (data not shown). 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. Frontiers in Pharmacology | www.frontiersin.org 6 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain Reversal of the Antinociceptive Effects intraperitoneally administered 1 h before testing, and naltrindole of UFP-512 in Animals With Inflammatory and naloxone were subcutaneously administered 30 min before or Neuropathic Pain testing. Our results showed that the mechanical antiallodynic To evaluate the specificity of the antinociceptive effects (Figure 3A) and thermal antihyperalgesic (Figure 3B) effects produced by a high dose of UFP-512 (30 mg/kg) during produced by UFP-512 in the ipsilateral paw of CFA-injected inflammatory and neuropathic pain, mice were administered animals and the mechanical antiallodynic (Figure 3C), thermal with the specific DOR antagonist, naltrindole (4 mg/kg) and antihyperalgesic (Figure 3D), and thermal antiallodynic the unspecific opioid antagonist, naloxone (1 mg/kg). UPF was (Figure 3E) effects produced by this drug in the ipsilateral paw 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). Frontiers in Pharmacology | www.frontiersin.org 7 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain of sciatic nerve-injured mice were reversed by the subcutaneous Effect of UFP-512 on the Expression of administration of naltrindole and naloxone (P < 0.005; one-way PI3K, p-Akt, DOR, p-JNK, and p-ERK1/2 ANOVA, followed by the Student–Newman–Keuls test). The subcutaneous administration of both antagonists in the Spinal Cords of Animals With administered alone in CFA-injected animals (Figures 3A,B) Inflammatory Pain and sciatic nerve-injured mice (Figures 3C–E) did not produce To evaluate the possible effects of UFP-512 treatment on any significant effect in the ipsilateral paw of these animals. PI3K/p-AKT, MAPK (JNK and ERK1/2), and DOR expression Naltrindole and naloxone administration alone also did not during inflammatory pain, the effects of this DOR agonist on produce any effect in the contralateral paw of CFA-injected the protein levels of PI3K (Figure 6A), p-Akt (Figure 6B), DOR mice or sciatic nerve-injured mice or in the ipsilateral and (Figure 6C), p-JNK (Figure 6E), and p-ERK1/2 (Figure 6F) contralateral paw of sham-operated mice (data not shown). in the spinal cords of CFA-injected mice treated with UFP-512 were also assessed. Our results did not show any significant alterations of PI3K, Effect of UFP-512 on the Expression of p-AKT, or DOR expression following inflammation or treatment. Nrf2, HO-1, NQO1, and NOS2 in the In contrast, the significant increase in spinal cord protein levels of Spinal Cords of Animals With p-JNK (P < 0.027, one-way ANOVA vs. naïve mice treated with Inflammatory Pain vehicle) and p-ERK1/2 (P < 0.024, one-way ANOVA vs. naïve To evaluate the possible mechanisms of the antinociceptive mice treated with vehicle) induced by peripheral inflammation effects of UFP-512 during inflammatory pain, the expression were reversed with UFP-512 treatment. of Nrf2, HO-1, NQO1, and NOS2 in the spinal cords of CFA-injected mice treated with 30 mg/kg UFP-512 or vehicle was Effect of UFP-512 on the Expression of assessed 14 days after CFA injection. We used naïve mice treated PI3K, p-Akt, DOR, p-JNK, and p-ERK1/2 with vehicle as controls in these experiments. Our results showed that peripheral inflammation did not in the Spinal Cords of Animals With alter the spinal cord expression of Nrf2, but Nrf2 protein Neuropathic Pain levels increased significantly in CFA-injected mice treated with To assess the possible effects of UFP-512 treatment on UFP-512 (P < 0.022, one-way ANOVA vs. CFA-injected mice PI3K/p-AKT, MAPK (JNK and ERK1/2), and DOR expression treated with vehicle) (Figure 4A). Peripheral inflammation did during neuropathic pain, the effects of this DOR agonist on the not alter the expression of HO-1 in the spinal cord compared protein levels of PI3K (Figure 7A), p-Akt (Figure 7B), DOR to naïve animals treated with vehicle, but HO-1 levels increased (Figure 7C), p-JNK (Figure 7E), and p-ERK1/2 (Figure 7F) in significantly in CFA-injected mice treated with UFP-512 the spinal cords of CCI-injured mice treated with UFP-512 were (P < 0.011, one-way ANOVA vs. naive and CFA-injected mice also investigated. treated with vehicle) (Figure 4B). No significant differences in Our data showed that, in contrast to the effects of peripheral NQO1 (Figure 4C) or NOS2 (Figure 4D) were observed between inflammation, sciatic nerve injury increased the spinal protein the three evaluated groups. levels of PI3K (P < 0.027, one-way ANOVA compared to sham-operated mice treated with vehicle), and UFP-512 inhibited Effect of UFP-512 on the Expression of this increase. The nerve injury-induced increased expression of p-Akt (P < 0.029, one-way ANOVA compared to sham-operated Nrf2, HO-1, NQO1, and NOS2 in the mice treated with vehicle) was also reversed by UFP-512 Spinal Cords of Animals With administration. No changes in the protein levels of DOR were Neuropathic Pain observed in the three groups tested. To assess the possible mechanisms of the antinociceptive effects Our results also demonstrated that, similar to inflammatory of UFP-512 during neuropathic pain, the expression of Nrf2, HO- pain, sciatic nerve injury augmented the expression of p-JNK 1, NQO1, and NOS2 in the spinal cords of CCI-injured mice (P < 0.007, one-way ANOVA vs. sham-operated mice treated treated with 30 mg/kg of this DOR agonist or vehicle was assessed with vehicle) and p-ERK1/2 (P < 0.026; one-way ANOVA vs. 28 days after surgery. sham-operated mice treated with vehicle) in the spinal cord. Reduction in Nrf2 protein levels in the spinal cord However, in this case, treatment with UFP-512 did not inhibit this following CCI (P < 0.007, one-way ANOVA, compared to increased expression. sham-operated mice treated with vehicle) was completely normalized with UFP-512 administration (Figure 5A). CCI Effects of the Coadministration of SFN did not alter the protein levels of HO-1, but treatment with UFP-512 significantly increased HO-1 expression in on the Antinociceptive Actions of CCI-injured mice (P < 0.008, one-way ANOVA compared UFP-512 in Animals With Inflammatory or to CCI-injured and sham-operated mice treated with vehicle) Neuropathic Pain (Figure 5B). Neither CCI nor UFP-512 treatment altered NQO1 To study the antinociceptive actions produced by the (Figure 5C) or NOS2 (Figure 5D) expression compared to administration of SFN plus UFP-512 during chronic sham-operated-mice. pain, the effects of the intraperitoneal administration of Frontiers in Pharmacology | www.frontiersin.org 8 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain 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. 10 mg/kg of SFN combined with a low dose (1 mg/kg) neuropathic pain (Figure 8B; P < 0.001, one-way ANOVA, of UFP-512 against the mechanical allodynia and thermal followed by the Student–Newman–Keuls test). hyperalgesia caused by peripheral inflammation or sciatic nerve Regarding thermal hyperalgesia, our data showed that injury were evaluated. the administration of SFN plus UFP-512 produced an For mechanical allodynia, the coadministration of SFN with antihyperalgesic effect in CFA-injected mice that was higher UFP-512 produced an antiallodynic effect that was significantly than these drugs administered alone (P < 0.001, one-way higher than either UFP-512 or SFN administration alone in ANOVA, followed by the Student–Newman–Keuls test) animals with inflammatory (Figure 8A; P < 0.001, one-way (Figure 8C). The combination of both drugs tended to increase ANOVA, followed by the Student–Newman–Keuls test) or the antihyperalgesic effects produced by each drug administered Frontiers in Pharmacology | www.frontiersin.org 9 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain 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. separately in CCI mice (Figure 8D), but the combination neuropathic pain, the effects of the intraperitoneal administration effects only differed significantly when compared to the effects of 1 mg/kg UFP-512 in CCI-injured mice were evaluated 28 days produced by SFN or vehicle administered alone (P < 0.001, after surgery using the TST 1 h after injection. one-way ANOVA, followed by the Student–Newman–Keuls test). Two-way ANOVA revealed a significant effect of the surgery (P < 0.049) and treatment (P < 0.001). The increased immobility time in CCI-injured animals (Figure 9; P < 0.001, The Effects of UFP-512 on the one-way ANOVA vs. sham-operated mice treated with saline) Depressive-Like Behaviors Associated was significantly reduced with UFP-512 administration. This With Neuropathic Pain treatment also reduced the immobility time in sham-operated To assess the possible antidepressant effects of UFP-512 in mice (P < 0.001, one-way ANOVA vs. sham-operated mice animals with depressive-like behaviors associated with persistent treated with saline). Therefore, our results confirmed the Frontiers in Pharmacology | www.frontiersin.org 10 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain 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. antidepressant effects of this DOR agonist in animals without induced by peripheral inflammation in CFA-injected mice, and pain and further demonstrated its antidepressant activity in this drug inhibited activation of PI3K/Akt in CCI animals, but mice with depressive-like behavior associated with persistent it did not alter CCI-induced JNK and ERK1/2 phosphorylation. neuropathic pain. UFP-512 inhibited the depressive-like behavior associated with persistent neuropathic pain, and its coadministration with SFN improved its antiallodynic effects in animals with inflammatory DISCUSSION or neuropathic pain. Our results revealed, for the first time, that treatment The present study revealed that the administration of UFP-512 with UFP-512 inhibited the allodynia and hyperalgesia caused diminished the allodynia and hyperalgesia caused by chronic by peripheral inflammation or sciatic nerve injury in a peripheral inflammation and increased Nrf2-HO-1 protein dose-dependent manner. These results are consistent with levels in the spinal cords of these animals. This DOR agonist the analgesic effects of other DOR agonists, such as DPDPE also inhibited the mechanical and thermal allodynia and or SNC-80, during inflammatory (Carcolé et al., 2014) or thermal hyperalgesia induced by sciatic nerve ligation and neuropathic pain caused by nerve injury (Hervera et al., 2010) normalized and/or increased the spinal cord expression of or diabetes (Castany et al., 2016; McDonnell et al., 2017a). Nrf2 and HO-1. The antinociceptive effects of UFP-512 were Our data also revealed that analogous doses of UFP-512 reversed by the administration of the specific DOR antagonist, produced comparable antiallodynic and antihyperalgesic effects naltrindole, and the unspecific opioid antagonist, naloxone. in animals with inflammatory or neuropathic pain. A dose UFP-512 blocked the phosphorylation of JNK and ERK1/2 of 30 mg/kg of UFP-512, for example, similarly inhibited the Frontiers in Pharmacology | www.frontiersin.org 11 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain 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. mechanical allodynia (65%) and thermal hyperalgesia (60%) in required in female rats to produce analogous anti-hyperalgesic both pain models. These results are consistent with the similar responses that achieved in male rats, revealing sex differences effectivity showed by other DOR agonists in the inhibition in the peripheral effects of this DOR agonist (Saloman et al., of acute inflammatory (3 h) and neuropathic pain (16 days 2011). Therefore, the potential peripheral antinociceptive effects after surgery) (Obara et al., 2009) and further demonstrated of UFP-512 in male and female rodents need to be investigated. that this analogous efficacy was also observed during chronic The reversion of the antiallodynic and antihyperalgesic inflammatory (14 days after CFA injection) and neuropathic effects of UFP-512 during inflammatory or neuropathic pain pain (28 days after CCI). Therefore, and considering the was evaluated by measuring its effects in the presence of few side effects of this DOR agonist compared to MOR the specific DOR antagonist, naltrindole, and the unspecific agonists (Aguila et al., 2007; Fernández-Dueñas et al., 2007; opioid antagonist, naloxone. Both antagonists reversed the Hervera et al., 2012; Bodnar, 2016), the present results support antinociceptive effects of UFP-512, which suggests that these the use of UFP-512 as an interesting treatment for chronic effects are mediated via DOR, although we cannot exclude a pain management. contribution of MOR to the antinociceptive effects of UFP-512 The present study only evaluated the antinociceptive effects in these experimental conditions. In accordance to our results, of systemic UFP-512 in male mice. However, the peripheral previous studies of Balboni et al. (2002, 2008) also reported administration of other DOR agonists, such as DPDPE, also that the antinociceptive effects of centrally administered UFP-512 inhibits inflammatory and neuropathic pain (Hervera et al., 2010, were DOR-mediated based on the reversal by the specific DOR 2013; Carcolé et al., 2014) and 10-fold higher dose of DPDPE is antagonist, naltrindole. Frontiers in Pharmacology | www.frontiersin.org 12 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain 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. Notably, previous studies in cell cultures showed that UFP-512 pain suggests that the coadministration of Nrf2 activators with stimulated the Nrf2 transcription factor (Cao S. et al., 2015), UFP-512 is another interesting approach for the treatment which is a key mechanism of defense against oxidative stress of chronic pain because the maximal antinociceptive effect and inflammatory processes. Our results further demonstrated (approx. 60%) was induced using a small dose of UFP-512, that UFP-512 also enhanced the expression of Nrf2 and HO- minimizing side effects. In accordance to our findings, other 1 in the spinal cords of mice with peripheral inflammation studies also demonstrated that SFN potentiated the analgesic and normalized the decreased expression of Nrf2 induced by effects of specific DOR agonists, such as DPDPE and SNC-80, in nerve injury or enhanced the protein levels of HO-1 in the animals with diabetic neuropathy (McDonnell et al., 2017a) and spinal cord of sciatic nerve-injured mice. These latter results improved the local antiallodynic and/or antihyperalgesic effects confirmed the long-lasting state of oxidative stress that is of MOR agonists, for example morphine, under inflammatory induced by sciatic nerve ligation in the spinal cord (Riego and neuropathic pain conditions (Redondo et al., 2017; et al., 2018) and revealed, for the first time, the ability of UFP- Ferreira-Chamorro et al., 2018). 512 to normalize this effect in vivo. Moreover and tacking The expression of HO-1, which is a relevant antioxidant account that several Nrf2 inducers produce antinociceptive enzyme, was also increased by UFP-512 in the spinal cord of effects during chronic pain (Negi et al., 2011; McDonnell et al., mice with inflammatory or neuropathic pain. This result suggests 2017a; Redondo et al., 2017), the augment or normalization that this isoenzyme plays a role in the antinociceptive effects of Nrf2 expression in the spinal cord following systemic UFP- produced by this DOR agonist. Others studies also demonstrated 512 administration supports the hypothesis that Nrf2 might that the administration of HO-1 inducer compounds, such be involved in the antinociceptive effects of UFP-512 in the as cobalt protoporphyrin IX, improved the antinociceptive presence of chronic inflammatory or neuropathic pain. The activity of other DOR agonists during inflammatory pain substantial improvement in the antiallodynic actions of UFP-512 (Carcolé et al., 2014) or diabetic neuropathy (Castany et al., induced by SFN in animals with inflammatory or neuropathic 2016). In summary, these findings suggest that the Nrf2/HO-1 Frontiers in Pharmacology | www.frontiersin.org 13 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain inflammatory or neuropathic pain, which indicates that this isoenzyme is not crucial for the effects of UFP-512. The participation of MAPKs in the development and maintenance of chronic pain and the differential activation of MAPK pathways in neurons and glia (microglia and astrocytes) during inflammatory and neuropathic pain (Ji et al., 2009; Edelmayer et al., 2014) are well documented. Thus, under neuropathic pain conditions the expression of p-P38 is increased in microglia, and JNK is activated in astrocytes (Cao J. et al., 2015). Our findings support the role of MAPK in the maintenance of chronic pain because increased expression of p-JNK and p-ERK1/2 was observed in the spinal cords of animals with inflammatory or neuropathic pain. But curiously, while UFP-512 blocked the activation of these proteins in FIGURE 9 | Effects of UFP-512 on the depressive-like behaviors associated CFA-injected mice, this DOR agonist did not alter the elevated with neuropathic pain. Immobility time in seconds (s) for CCI-injured and expression of p-JNK or p-ERK1/2 in the spinal cords of sham-operated mice treated with 1 mg/kg of UFP-512 or saline evaluated at sciatic nerve-injured animals. Perhaps, repeated administration 28 days after surgery in TST are represented. UFP-512 was administered 1 h of this compound may be necessary to counteract the spinal before testing. In all groups, ∗ indicates significant differences vs. sham-operated mice treated with saline and + indicates significant differences activation of JNK and ERK1/2 induced by sciatic nerve injury. vs. CCI-injured mice treated with saline (P < 0.05, one-way ANOVA, followed In summary, our results indicated that the alleviation of by the Student–Newman–Keuls test). Results are expressed as mean ± SEM; chronic inflammatory pain of UFP-512 was mainly produced n = 8 animals for each experimental group. via inhibition of JNK and ERK1/2 activation, and other mechanisms, such as the inhibition of PI3K/p-Akt, might be implicated in their antinociceptive effects under neuropathic pathway is involved in the analgesic actions of UFP-512 pain conditions. during chronic pain. In addition, no changes in the expression of DOR were Curiously, and in contrast to the in vitro effects (Cao S. et al., observed in the spinal cords of animals with inflammatory or 2015), UFP-512 treatment did not alter the unchanged expression neuropathic pain, which is consistent with a previous report of another important detoxifying enzyme, NQO1, in the spinal (Obara et al., 2009). UFP-512 did not alter DOR expression cords of animals with inflammatory or neuropathic pain. These during inflammatory or neuropathic pain. results suggest that NQO1 does not play a relevant role in the The antidepressant-like activity of DOR agonists is well mechanism of action of UFP-512 under chronic pain conditions. acknowledged. Indeed, DOR KO mice exhibit increased The important role of the PI3K/Akt signaling pathway in the depressive-like behavior (Filliol et al., 2000), and several development of chronic pain is well accepted. Recent studies works demonstrated antidepressant-like effects produced by demonstrated the different participation of diverse PI3K isoforms different DOR agonists in various behavioral paradigms (Lutz in inflammatory and neuropathic pain (Pritchard et al., 2016; and Kieffer, 2013). In this lane, the antidepressant properties Liu et al., 2018). PI3K-β is involved in the nerve injury-induced of UFP-512 in naïve animals were previously established in sensitization of the spinal cord and contributes to neuropathic the forced swimming test (Aguila et al., 2007; Vergura et al., pain (Liu et al., 2018), but this β isoform does not participate in 2008). However and given that persistent neuropathic pain the development of inflammatory pain (Pritchard et al., 2016). is generally associated with depressive-like behavior (Kawai In accordance, our findings demonstrated that sciatic nerve et al., 2017), we evaluated the possible antidepressant activity ligation activated the PI3Kβ/p-Akt signaling in spinal cords, but of UFP-512 in animals with neuropathic pain. Our results peripheral inflammation did not alter its expression. Notably, confirmed the antidepressant-like effects of this drug after UFP-512 inhibited the sciatic nerve injury-induced upregulation intraperitoneal administration in naïve animals (Aguila et al., of PI3K-β/p-Akt. The administration of specific inhibitors of 2007) and further demonstrated that UFP-512 inhibited the PI3K-β isoform also reduces neuropathic pain (Liu et al., depressive-like behavior associated with persistent neuropathic 2018). Therefore, the normalization of this pathway induced pain (28 days after surgery). These data reveal that UFP-512 by UFP-512 suggests that the antiallodynic and antihyperalgesic alleviates neuropathic pain and inhibits the depressive-like effects of this DOR agonist during neuropathic pain partially behavior associated with chronic pain. occur via the inhibition of this nociceptive pathway. The present study also evaluated the effect of UFP-512 on the expression of NOS2 during inflammatory and neuropathic pain. CONCLUSION Peripheral inflammation or sciatic nerve injury did not modify the spinal cord protein levels of NOS2 at 14 or 28 days after In conclusion, our study revealed the antinociceptive properties CFA-injection or CCI-induction, respectively, which is consistent of UFP-512 in chronic inflammatory and neuropathic pain. with a previous study (Redondo et al., 2017). The administration Activation of the Nrf2-HO-1 pathway and the inhibition of of UFP-512 did not alter NOS2 expression in animals with p-JNK and p-ERK1/2 in the spinal cord may explain these effects Frontiers in Pharmacology | www.frontiersin.org 14 March 2019 | Volume 10 | Article 283 Polo et al. UFP-512 Inhibits Inflammatory and Neuropathic Pain in animals with inflammatory paint. Induction of the Nrf2-HO-1 AUTHOR CONTRIBUTIONS pathway and blockade of PI3K/p-Akt signaling caused by nerve injury may be the principal reason for the antinociceptive SP performed the behavioral tests. SP, AD, NG, and SL performed effects of UFP-512 during neuropathic pain. Moreover, the western blot assays. SP and OP performed the statistical UFP-512 exhibited antidepressant properties in animals with analysis. GB contributed in new reagents or analytic tools. depressive-like behavior associated with neuropathic pain, and OP designed the study and wrote the manuscript. All authors its coadministration with SFN improved the antiallodynic effects contributed to manuscript revision, read, and approved the of this DOR agonist in chronic pain. Consequently, this study submitted version. suggests the administration of UFP-512 as a new alternative for the treatment of chronic inflammatory and neuropathic pain and the depressive-like behavior associated with neuropathic pain. FUNDING This work was supported by Ministerio de Economía y DATA AVAILABILITY Competitividad, Instituto de Salud Carlos III (Grant: PS0900968) and Ministerio de Ciencia, Innovación y Universidades, Instituto All datasets generated for this study are included in the de Salud Carlos III (Grant: PI1800645), and Fondo Europeo de manuscript and/or the supplementary files. Desarrollo Regional (FEDER), Unión Europea. REFERENCES of novel antinociceptive therapeutics. Biochem. Pharmacol. 87, 390–398. doi: 10.1016/j.bcp.2013.11.019 Aguila, B., Coulbault, L., Boulouard, M., Léveillé, F., Davis, A., Tóth, G., et al. Fernández-Dueñas, V., Pol, O., García-Nogales, P., Hernández, L., Planas, E., and (2007). In vitro and in vivo pharmacological profile of UFP-512, a novel Puig, M. M. (2007). Tolerance to the antinociceptive and antiexudative effects selective delta-opioid receptor agonist; correlations between desensitization of morphine in a murine model of peripheral inflammation. J. Pharmacol. 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