antioxidants Article Hydrogen Sulfide Inhibits Inflammatory Pain and Enhances the Analgesic Properties of Delta Opioid Receptors Aina Porta 1,2,† , Laura Rodríguez 1,2,† , Xue Bai 1,2 , Gerard Batallé 1,2 , Gerad Roch 1,2 , Enric Pouso-Vázquez 1,2 , Gianfranco Balboni 3 and Olga Pol 1,2, * 1 Grup de Neurofarmacologia Molecular, Institut d’Investigació Biomèdica Sant Pau, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain; aina.porta@e-campus.uab.cat (A.P.); laura.rodriguezpe@e-campus.uab.cat (L.R.); xue.bai@e-campus.uab.cat (X.B.); gerard.batalle@e-campus.uab.cat (G.B.); gerard.roch@e-campus.uab.cat (G.R.); enrique.pousovazquez@e-campus.uab.cat (E.P.-V.) 2 Grup de Neurofarmacologia Molecular, Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, Department of Life and Environmental Sciences, University of Cagliari, 09042 Cagliari, Italy; gbalboni@unica.it * Correspondence: opol@santpau.es; Tel.: +34-619-757-054 † These authors contributed equally to this work. Abstract: Chronic inflammatory pain is present in many pathologies and diminishes the patient’s quality of life. Moreover, most current treatments have a low efficacy and significant side effects.  Recent studies demonstrate the analgesic properties of slow-releasing hydrogen sulfide (H2 S) donors  in animals with osteoarthritis or neuropathic pain, but their effects in inflammatory pain and related Citation: Porta, A.; Rodríguez, L.; pathways are not completely understood. Several treatments potentiate the analgesic actions of δ- Bai, X.; Batallé, G.; Roch, G.; opioid receptor (DOR) agonists, but the role of H2 S in modulating their effects and expression during Pouso-Vázquez, E.; Balboni, G.; Pol, inflammatory pain remains untested. In C57BL/6J male mice with inflammatory pain provoked O. Hydrogen Sulfide Inhibits by subplantar injection of complete Freund’s adjuvant, we evaluated: (1) the antiallodynic and Inflammatory Pain and Enhances the Analgesic Properties of Delta Opioid antihyperalgesic effects of different doses of two slow-releasing H2 S donors, i.e., diallyl disulfide Receptors. Antioxidants 2021, 10, 1977. (DADS) and phenyl isothiocyanate (P-ITC) and their mechanism of action; (2) the pain-relieving https://doi.org/10.3390/antiox effects of DOR agonists co-administered with H2 S donors; (3) the effects of DADS and P-ITC on the 10121977 oxidative stress and molecular changes caused by peripheral inflammation. Results demonstrate that both H2 S donors inhibited allodynia and hyperalgesia in a dose-dependent manner, potentiated Academic Editors: Emma Mitidieri the analgesic effects and expression of DOR, activated the antioxidant system, and reduced the and Vincenzo Brancaleone nociceptive and apoptotic pathways. The data further demonstrate the possible participation of potassium channels and the Nrf2 transcription factor signaling pathway in the pain-relieving activities Received: 30 October 2021 of DADS and P-ITC. This study suggests that the systemic administration of DADS and P-ITC and Accepted: 8 December 2021 local application of DOR agonists in combination with slow-releasing H2 S donors are two new Published: 11 December 2021 strategies for the treatment of inflammatory pain. Publisher’s Note: MDPI stays neutral Keywords: analgesia; antioxidants; delta opioid receptors; inflammation; oxidative stress; pain with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Chronic pain ranks among the 10 most prevalent diseases worldwide. It affects 18% Copyright: © 2021 by the authors. of the general population in developing countries and is thus a serious public health Licensee MDPI, Basel, Switzerland. problem [1]. Chronic inflammatory pain is characterized by multiple molecular, chemi- This article is an open access article cal, and physiological changes in the central and peripheral nervous system that cause distributed under the terms and important plasticity changes that perpetuate pain. The management of chronic pain is an conditions of the Creative Commons unmet medical need and a big challenge in pain research. That is to say, although different Attribution (CC BY) license (https:// therapeutic options are currently available most have limited efficacy and significant side creativecommons.org/licenses/by/ effects [2,3]. Consequently, research into novel treatments for pain management is of great 4.0/). global importance. Antioxidants 2021, 10, 1977. https://doi.org/10.3390/antiox10121977 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 1977 2 of 21 Hydrogen sulfide (H2 S) is a gaseous neurotransmitter that plays a key role in different physiological processes in the central and peripheral nervous system. The list of biological functions and signaling pathways modulated by the endogenous and exogenous adminis- tration of H2 S is extensive and includes several processes, such as energetic dysfunctions, apoptosis, inflammatory responses, and oxidative stress [4]. However, the role of this neu- rotransmitter in chronic pain modulation is controversial, i.e., it has protective functions when released slowly, but exerts pro-inflammatory effects when released quickly [5]. In this context, recent studies proved that the exogenous administration of slow-releasing H2 S donors, such as phenyl isothiocyanate (P-ITC) or allyl isothiocyanate (A-ITC), atten- uated osteoarthritis and neuropathic pain, thus turning these compounds into potential therapeutical targets for the treatment of inflammatory pain [6–8]. Other investigations also demonstrated the antinociceptive effects of other slow-releasing H2 S compounds, such as diallyl disulfide (DADS) and morpholin-4-ium 4-methoxyphenyl (morpholino) phosphinodithioate dichloromethane complex (GYY4137), in models of osteoarthritis or nerve injury-induced neuropathic pain [9,10]. These and other studies further revealed that the possible mechanisms involved in H2 S-mediated antinociceptive actions in nociceptive, osteoarthritis, and neuropathic pain might be produced by opening the voltage-gated potassium channels, such as the Kv7 [6,7,10] and the ATP-sensitive (KATP) potassium channels [11,12]. However, the participation of these potassium channels in the possible antinociceptive effects of slow-releasing H2 S donors during inflammatory pain has not been elucidated. Several studies have also demonstrated that some of the H2 S effects are mediated by triggering the Nrf2 transcription factor signaling pathway [5], which is highly implicated in defense mechanisms, redox homeostatic maintenance, and pain modulation [13,14]. The induction of Nrf2 activates the endogenous antioxidant system by promoting the tran- scription of various antioxidant enzymes, including heme oxygenase 1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), superoxide dismutase 1 (SOD-1), and glutathione S- transferase mu 1 (GSTM1), among others [15], that protect the organism against oxidative stress [16]. The contribution of the Nrf2/HO-1 signaling pathway in the analgesic actions of H2 S during neuropathic pain is evidenced by the fact that their effects are reversed as a result of specific Nrf2 and HO-1 inhibitors, such as ML-385 and tin protoporphyrin IX (SnPP) [5]. Moreover, in a neuropathic pain model, treatment with NAHS, an H2 S donor, upregulated the expression of Nrf2 and HO-1 in microglial cells of the spinal cord [17]. In this study, we examined the possible implication of the Nrf2/HO-1-NQO1 pathway in the pain-relieving effects of DADS and P-ITC during inflammatory pain. Oxidative stress plays an essential role in the progress and preservation of chronic pain. The imbalance between oxidative and antioxidative changes induced by inflammation leads to oxidative stress. In consequence, the inhibition of the production of pro-inflammatory cytokines and reactive oxygen species limits the development of inflammatory pain [18]. Different studies revealed the protective role of H2 S in oxidative stress in vitro [19] and in vivo [9]. Moreover, one study demonstrated that apoptosis precedes oxidative stress with the upregulation of Bcl2-associated X (BAX), an apoptosis-related protein [20]. Furthermore, in mice with acute liver injury, treatment with DADS inhibited apoptosis via the regulation of the PI3K/Akt signaling pathway [21]. In this study, the effects of DADS and P-ITC in the apoptotic and nociceptive reactions generated by peripheral inflammation were evaluated by measuring the expression of BAX and p-Akt in the paw. Opioid receptors are key targets in pain modulation. There are three different major opioid receptor types: µ (MOR), κ, and δ (DOR), which are endogenously activated by peptides, including endorphins and dynorphins [22]. Nevertheless, it is well known that certain opioids present low efficacy, particularly MOR agonists, in the management of neuropathic pain and are accompanied by significant side effects [23,24]. In contrast, treatment with DOR agonists has recently been proposed as a more secure alternative to MOR agonists as their activation is associated with fewer unwanted effects, especially when Antioxidants 2021, 10, 1977 3 of 21 administered locally [25]. However, DOR agonists, such as [D-Pen2,D-Pen5]-enkephalin (DPDPE) and H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), have been shown to be moderately effective in chronic pain [26,27]. Therefore, novel strategies, such as their co-administration with other types of drugs, have been shown to enhance the antinociceptive effects of DOR agonists. In this way, the administration of Nrf2 or HO-1 activators potentiated the pain-relieving actions of DOR agonists in animals with chronic inflammatory pain [27,28]. Nevertheless, the effects of H2 S inducers in terms of their palliative effects and the expression of DOR in animals with chronic inflammatory pain have not yet been evaluated. Therefore, in a model of inflammatory pain induced by the subplantar administration of complete Freund’s adjuvant (CFA), our objectives were to evaluate: (1) the analgesic effects induced by the intraperitoneal administration of DADS and P-ITC; (2) their mecha- nism of action and effects in the oxidative, nociceptive, and apoptotic responses provoked by peripheral inflammation and (3) the effects of DADS and P-ITC in the antinociceptive actions and expression of DOR in paw tissues. 2. Materials and Methods 2.1. Animals The experiments were carried out with 6–8-week-old male C57BL/6 mice (25–30 g), ac- quired at Envigo Laboratories (Barcelona, Spain), accommodated under 12/12 h light/dark conditions in a room with a controlled temperature of 22 ◦ C and humidity of 66% until use. The animals had free access to food and water and were used after 7 days of acclimatiza- tion to the housing conditions. All experiments were conducted between 9:00 a.m. and 5:00 p.m. and executed in accordance with the guidelines of the European Commission’s directive (2010/63/EC) and the Spanish Law (RD 53/2013) regulating animal research, and they were approved by the local Committee of Animal Use and Care of the Autonomous University of Barcelona (ethical code: 9863). All efforts were made to diminish the suffering and number of animals employed in this study. 2.2. Induction of Inflammatory Pain Inflammatory pain was induced by the subplantar injection of 30 µL of CFA (Sigma- Aldrich, St. Louis, MO, USA) in the right hind paw during short anesthetic procedures with isoflurane (3% induction, 2% maintenance), as used by our group [29]. Contralateral paws served as controls. Control animals were injected with the same volume of saline solution (NaCl 0.9%; SS). The development of mechanical allodynia and thermal hyperalgesia was evaluated by using the following sequence: von Frey filaments and plantar test. All animals were tested in each paradigm before and at 10 days after CFA-injection. 2.3. Mechanical Allodynia Mechanical allodynia was evaluated by measuring the hind paw withdrawal response after stimulation with the von Frey filaments of different bending forces (ranging between 0.4 g and 3.0 g). Mice were placed in transparent tubes (20 cm high × 9 cm diameter) on a raised wire grid bottom, across which von Frey filaments (North Coast Medical, Inc., San Jose, CA, USA) were applied using the up/down paradigm described in [30]. The test started with the low bending force filament (0.4 g) and the strength of the next filaments was increased or decreased depending on the animal’s response. The threshold of the response was calculated from the sequence of filament strength used during the up/down procedure and using an Excel program (Microsoft Iberia SRL, Barcelona, Spain), which included curve fitting of the data. The 3.0 g filament was used as a cut-off. A clear paw withdrawal, licking, or shaking the paw was considered as a positive response. Both ipsilateral and contralateral hind paws were assessed. Antioxidants 2021, 10, 1977 4 of 21 2.4. Thermal Hyperalgesia Thermal hyperalgesia was evaluated by assessing the paw withdrawal latency in response to beaming heat in the plantar test apparatus (Ugo Basile, Varese, Italy) [31]. Animals were placed in Plexiglas cylinders (20 cm high × 9 cm diameter) situated on a glass surface. The heat source was positioned under the plantar surface of the hind paws and activated with a light beam intensity until paw withdrawal, or the cut-off time of 12 s was reached. Mean paw withdrawal latencies were determined from the mean of three separate measurements of both the ipsilateral and contralateral hind paws. In both tests, animals were habituated to the environment for 1 h before the test, so that they were quiet at the time of testing. All these experiments were performed by experimenters blinded to the experimental conditions. 2.5. Preparation of Whole-Cell Extracts and Western Blot Analysis Animals were euthanized by cervical dislocation at 10 days after CFA or SS injection. The subplantar tissue samples of the hind paws on the ipsilateral side were extracted, quickly frozen, and maintained at −80 ◦ C until use. The protein levels of 4-HNE, HO- 1, NQO1, SOD1, GSTM1, p-Akt, BAX and DOR were analyzed. Tissue samples were homogenized in ice-cold lysis buffer (50 mM Tris-Base, 150 nM NaCl, 1% NP-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 Triton X-100, 0.1% sodium dodecyl sulfate, 1 mM Na3 VO4 , 25 mM NaF, 0.5% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail). All reagents were acquired from Sigma-Aldrich (St. Louis, MO, USA), except NP- 40, which was obtained from Calbiochem (Darmstadt, Germany). After solubilization of crude homogenate for 1 h at 4 ◦ C, samples were sonicated for 10 s and centrifuged at 4 ◦ C for 20 min at 700× g. The supernatants (60 µg of total protein) were mixed with 4 × Laemmli loading buffer and loaded onto 4% stacking/12% separating sodium dodecyl sulfate polyacrylamide gels. Subsequently, the proteins were electrophoretically transferred onto a polyvinylidene fluoride membrane for 120 min and blocked with phosphate-buffered saline plus 5% nonfat dry milk or Tris-buffered saline with Tween 20 plus 5% nonfat dry milk or 5% bovine serum albumin for 75 min. Thereafter, the protein samples were incubated overnight at 4 ◦ C with specific rabbit primary antibodies anti 4-HNE (1:150; Abcam, Cambridge, UK); HO-1 (1:150; Abcam, Cambridge, UK); NQO1 (1:200; Abcam, Cambridge, UK); GSTM1 (1:150; Novus Biologic, Littleton, CO, USA); SOD-1 (1:150; Novus Biologic, Littleton, CO, USA); phospho-Akt (1:200; Cell Signaling Technology, Danvers, MA, USA); total Akt (1:250; Cell Signaling Technology, Danvers, MA, USA); BAX (1:250; Cell Signaling Technology, Danvers, MA, USA); and DOR (1: 300; Abcam, Cambridge, UK). Blots were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Little Chalfont, UK). Proteins were detected using the chemiluminescence reagents (ECL kit; GE Healthcare, Little Chalfont, Buckinghamshire, UK). Lastly, immunoblots were quantified using a densitometric analysis with Image- J program (National Institutes of Health, Bethesda, MD, USA). We used a rabbit anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5000; Merck, Billerica, MA, USA) as a loading control. 2.6. Experimental Procedures In the first set of experiments, the antinociceptive effects produced by the acute administration of different doses of DADS (25–200 µmols/kg), P-ITC (18.6–29 µmols/kg), or vehicle at 10 days after CFA-injection were evaluated (n = 6 animals per dose). Mice were tested at 1 h after DADS or P-ITC injection in accordance with our previous studies [8,10]. In other groups of animals, we evaluated the reversion of the antinociceptive effects produced by a high dose of DADS (200 µmols/kg) or P-ITC (29 µmols/kg) with the selective Kv7 potassium channel blocker XE-991 (12 µmols/kg), the KATP channel blocker (glibenclamide, 10 mg/kg), and the Nrf2 (ML-385, 25 mg/kg), HO-1 (SnPP, 10 mg/kg), or NQO1 (dicoumarol, 10 mg/kg) inhibitors (n = 6 animals per group). In all experiments, vehicle plus vehicle-treated animals were used as controls. The doses of DADS and P-ITC Antioxidants 2021, 10, 1977 5 of 21 were selected according to the dose–response curves produced in this study and the doses of XE-991, glibenclamide, ML-385, SnPP, and dicoumarol were selected in accordance with previous studies [11,32–34]. CFA-injected mice treated with 200 µmols/kg of DADS, 29 µmols/kg of P-ITC, or vehicle were euthanized by cervical dislocation, and the protein levels of 4-HNE, HO-1, NQO1, GSTM1, SOD-1, p-AKT, BAX and DOR in the ipsilateral paw from mice with peripheral inflammation were evaluated by Western blot assay. In these experiments, saline-vehicle-treated mice were used as controls (n = 3–4 samples per group). In other experiments, we studied the analgesic effects produced by the acute sub- plantar administration of different doses (50–300 µg) of two DOR agonists, DPDPE and UFP-512, at 30 min after their local administration (n = 6 animals per group). These doses were selected in accordance with other studies [27,28]. To analyze the potential enhancement of the analgesic effects of DOR agonists induced by H2 S, we measured the antinociceptive actions generated by the intraperitoneal adminis- tration of low doses of DADS (25 µmol/kg) or P-ITC (18.6 µmol/kg) in combination with the subplantar administration of a low dose (50 µg) of DPDPE or UFP-512. The doses of DPDPE and UFP-512 were extracted from the corresponding dose–response curves. DADS and P-ITC were administered 30 min before the subplantar injection of DPDPE or UFP-512, and tests were performed 30 min later (n = 6 animals per group). 2.7. Drugs P-ITC, DADS, DPDPE, and glibenclamide, obtained from Sigma-Aldrich (St. Louis, MO, USA), and UFP-512, synthesized by Balboni et al., (2002) [35], were dissolved in SS. XE- 991, purchased in Tocris Bioscience (Ellisville, MO, USA), ML-385 and dicoumarol, obtained from Eurodiagnostico S.L, (Madrid, Spain) and SnPP, purchased in Frontier scientific (Livchem GmbH & Co, Frankfurt, Germany), were dissolved in dimethyl sulfoxide (1% SS). P-ITC and DADS were intraperitoneally administered at 10 mL/kg 1 h before the tests, while XE-991, glibenclamide, ML-385, SnPP, and dicoumarol were also intraperi- toneally administered at 10 mL/kg 30 min before the tests, in accordance with previous studies [10,11,32,34]. DPDPE and UFP-512 were administered via subplantar injection in a final volume of 30 µL 30 min before the behavioral tests, in accordance with our previous studies [27,28]. All drugs were freshly prepared just before being administered. For each group treated with a drug, the respective control group received the same volume of vehicle. 2.8. Statistical Analyses Data are expressed as mean values ± standard error of the mean (SEM). We used the Graph Pad Prism program (version 8 for Windows) for the statistical analysis. The analysis of the effects in each behavioral test produced by different doses of DADS, P-ITC, DPDPE, UFP-512, or vehicle was conducted using a one-way ANOVA and a Student–Newman–Keuls test. The ED50 of the drugs was calculated by linear regression analysis using GraphPad Prism (version 8 for Windows). The antinociceptive actions of DADS or P-ITC administered alone and in combination with DPDPE or UFP-512 were also analyzed using a one-way ANOVA followed by a Student–Newman–Keuls test. The effects produced by the co-administration of H2 S donors with specific potas- sium channel blockers or Nrf2/HO-1/NQO1 antagonists were analyzed using a one-way ANOVA followed by a Student–Newman–Keuls test. In these experiments, antinociception in von Frey filaments and plantar tests is ex- pressed as the percentage of the maximal possible effect, in which the test latencies pre (baseline) and post drug administration were compared and calculated according to the following equation: Maximal possible effect (%) = [(drug − baseline)/(cut-off − baseline)] × 100 channel blockers or Nrf2/HO-1/NQO1 antagonists were analyzed using a one-way ANOVA followed by a Student–Newman–Keuls test. In these experiments, antinociception in von Frey filaments and plantar tests is ex- pressed as the percentage of the maximal possible effect, in which the test latencies pre Antioxidants 2021, 10, 1977 (baseline) and post drug administration were compared and calculated according to6 the of 21 following equation: Maximal possible effect (%) = [(drug– baseline)/(cut-off—baseline)] × 100 Changes in the protein levels of 4-HNE, HO-1, NQO1, SOD1, GSTM1, p-AKT, BAX and DORChanges in thein pawthetissues proteinwere levels of examined also 4-HNE, HO-1, usingNQO1, SOD1, a one-way GSTM1, ANOVA p-AKT, and BAX the corre- and DOR in the paw tissues were also examined using a one-way ANOVA sponding Student–Newman–Keuls test. A p < 0.05 was considered statistically significant. and the corre- sponding Student–Newman–Keuls test. A p < 0.05 was considered statistically significant. 3. Results 3. Results 3.1. Effects of the Acute Administration of DADS and P-ITC in the Mechanical Allodynia and 3.1. Effects of the Acute Administration of DADS and P-ITC in the Mechanical Allodynia and Thermal ThermalHyperalgesia HyperalgesiaInduced Inducedby byCFA CFA We evaluated the effects We evaluated the effects of the of theacute acuteintraperitoneal intraperitonealadministration administration of of 25,25, 70, 70, 100,100, and and 200 µ mols/kg of DADS or 18.6, 23.2, and 29 µ mols/kg of P-ITC or 200 µmols/kg of DADS or 18.6, 23.2, and 29 µmols/kg of P-ITC or their corresponding their corresponding vehicles vehicleson onCFA-induced CFA-inducedmechanical mechanicalallodynia allodyniaandandthermal thermalhyperalgesia hyperalgesiaatat10 10days daysafter after inducing inducinginflammation. inflammation.Our Ourresults resultsshowed showedthat thatthe theadministration administrationofofDADS DADSinhibited inhibited CFA-induced CFA-inducedmechanical mechanicalallodynia allodynia(Figure (Figure1A)1A)and andthermal thermalhyperalgesia hyperalgesia(Figure (Figure1B)1B)inin aadose-dependent dose-dependentmanner mannerand andreached reachedthe themaximum maximumeffecteffectatatthe thedose doseofof200 200µµmols/kg. mols/kg. Similar Similarresults resultswere wereobserved observed with P-ITC, with which P-ITC, also also which inhibited mechanical inhibited allodynia mechanical (Fig- allodynia ure 1A) and thermal hyperalgesia (Figure 1B) in a dose-dependent manner, (Figure 1A) and thermal hyperalgesia (Figure 1B) in a dose-dependent manner, having a having a max- imal effecteffect maximal at a dose at a of 29 of dose µ mols/kg. 29 µmols/kg. Figure 1. Effects of the acute intraperitoneal administration of DADS and P-ITC on the mechanical allodynia Figure and thermal 1. Effects hyperalgesia of the acute inducedadministration intraperitoneal by peripheral of inflammation. Effects DADS and P-ITC onofthe different doses mechanical allodynia and thermal hyperalgesia induced by peripheral inflammation. Effects of different (logarithmic axis) of DADS and P-ITC (µmols/kg) on mechanical allodynia (A) and thermal hyperal- doses gesia (B) induced by CFA in the ipsilateral paw of mice. For each test, * denotes significant differences vs. mice administered with vehicle, + denotes significant differences vs. mice administered with 25 µmols/kg of DADS or 18.6 µmols/kg of P-ITC, respectively, and # indicates significant differences vs. mice treated with 70 µmols/kg of DADS or 23.2 µmols/kg of P-ITC, respectively (p < 0.05; one-way ANOVA followed by Student–Newman–Keuls test). The results are shown as mean values of the maximal possible effect (%) ± SEM; n = 6 animals per dose and treatment. The mechanical antiallodynic and thermal antihyperalgesic effects produced by differ- ent doses of DADS, i.e., 70, 100, and 200 µmols/kg, in mice with inflammatory pain were Antioxidants 2021, 10, 1977 7 of 21 greater than those produced by the vehicle (p < 0.0001; one-way ANOVA, followed by the Student–Newman–Keuls test). In both tests, the effects of DADS at 100 and 200 µmol/kg were greater than those produced by 25 and/or 70 µmols/kg. Similarly, the antiallodynic and antihyperalgesic effects generated by 23.2 and 29 µmols/kg of P-ITC in mice with CFA-induced peripheral inflammation were greater than those produced by vehicle (p < 0.0001; one-way ANOVA, followed by the Student–Newman– Keuls test). In addition, in both tests, the effects of 29 µmol/kg P-ITC were greater than those produced by 18.6 and 23.2 µmol/kg (p < 0.001; one-way ANOVA, followed by the Student–Newman–Keuls test) and the effects of 23.2 µmol/kg P-ITC were also greater than those produced by 18.6 µmols/kg (p < 0.001; one-way ANOVA, followed by the Student–Newman–Keuls test). Neither the vehicle nor DADS or P-ITC exhibited any antiallodynic or antihyperalgesic effect in the contralateral paws (data not shown). Moreover, by analyzing the ED50 values of DADS and P-ITC, the data revealed that the potency of P-ITC in the inhibition of the mechanical and thermal sensitivity induced by peripheral inflammation was between 3.9 and 3.8 times greater than that of DADS (Table 1), indicating that the synthetic H2 S donor (P-ITC) is more potent than the natural (DADS) donor in inhibiting inflammatory pain. Table 1. Comparison of the potencies (ED50 ) of DADS and P-ITC in the inhibition of mechanical allodynia and thermal hyperalgesia induced by peripheral inflammation. Mechanical Allodynia Thermal Hyperalgesia DADS 87.3 ± 19.8 82.9 ± 9.6 P-ITC 22.3 ± 5.6 21.6 ± 4.5 Ratio (DADS/P-ITC) 3.9 3.8 Data are expressed as ED50 values (µmol/kg) ± SEM (n = 6 animals per dose). For each test, the ratio of the ED50 values between drugs is indicated. 3.2. Reversion of the Antiallodynic and Antihyperalgesic Effects Induced by High Doses of DADS or P-ITC with Their Co-Administration with Two Potassium Channels Inhibitors To evaluate the possible mechanisms implicated in the antinociceptive effects of DADS and P-ITC during inflammatory pain, the effects of the acute intraperitoneal administration of 200 µmols/kg of DADS or 29 µmols/kg of P-ITC in combination with a selective Kv7 potassium channel blocker (XE-991) or a KATP channel blocker (glibenclamide) on the mechanical allodynia and thermal hyperalgesia caused by peripheral inflammation were assessed. Our results showed that both XE-991 and glibenclamide reversed the antiallodynic effects produced by 200 µmol/kg of DADS (p < 0.001, one-way ANOVA vs. vehicle + vehicle-treated mice) (Figure 2A) and 29 µmol/kg of P-ITC (p < 0.001, one-way ANOVA vs. vehicle + vehicle-treated mice) (Figure 2C), and they also reversed the thermal anti- hyperalgesic effects of DADS (p < 0.001, one-way ANOVA vs. vehicle + vehicle-treated mice) (Figure 2B) and P-ITC (p < 0.001, one-way ANOVA vs. vehicle + vehicle-treated mice) (Figure 2D) during inflammatory pain. The administration of XE-991 and glibenclamide alone did not produce any significant effect in the ipsilateral paws of CFA-injected animals. Moreover, XE-991 and glibenclamide administered alone or in combination with DADS or P-ITC did not produce any effect in the contralateral paws of CFA-injected mice (data not shown). Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 21 Moreover, XE-991 and glibenclamide administered alone or in combination with DADS Antioxidants 2021, 10, 1977 or P-ITC did not produce any effect in the contralateral paws of CFA-injected mice8 (data of 21 not shown). Figure 2. Effects of the administration of DADS or P-ITC alone or in combination with XE-991 or glibenclamide on the mechanical allodynia Figure 2. Effects andadministration of the thermal hyperalgesia of DADSinduced by CFA. or P-ITC aloneThe or antiallodynic in combination (A,C) withand antihyperalgesic XE-991 (B,D) effects or glibenclamide on the produced mechanical by allodynia DADS at and200 thermal µmols/kg or P-ITC atinduced hyperalgesia 29 µmols/kg by CFA.in The the ipsilateral paws antiallodynic of mice (A,C) with inflammatory and antihyperalgesic pain (B,D) ef- co-treated with a by fects produced Kv7 potassium DADS at 200channel blocker µ mols/kg (XE-991, or P-ITC at 2912 µmols/kg), µ mols/kg a KATP in the channel ipsilateral blocker paws (glibenclamide, of mice 10 mg/kg), with inflammatory pain orco-treated vehicle arewith a Kv7 shown. Forpotassium channel each test and blocker treatment (XE-991, evaluated 12 µ mols/kg), & indicates a KATP significant channel differences vs. blocker the other(glibenclamide, groups (p < 0.05,10 mg/kg), or one-way vehiclefollowed ANOVA are shown.byFor each test and treatment evaluated Student–Newman–Keuls & indicates test). Results significant are represented asdifferences mean values vs. of thethe other groups maximal (p < 0.05, possible one-way effect (%) ± ANOVA followed SEM values; by Student–Newman–Keuls n = 6 animals per experimental group. test). Results are represented as mean values of the maximal possible effect (%) ± SEM values; n = 6 animals per experimental group. 3.3. Reversion of the Antiallodynic and Antihyperalgesic Effects Induced by High Doses of DADS or3.3. Reversion P-ITC of theCo-Administration with Their Antiallodynic and Antihyperalgesic EffectsofInduced with Specific Inhibitors by High Doses of DADS the Nrf2/HO-1-NQO1 Signaling or P-ITC Pathway with Their Co-Administration with Specific Inhibitors of the Nrf2/HO-1-NQO1 Signal- ing Pathway To study the probable participation of the Nrf2/HO-1/NQO1 signaling pathway in the analgesic actionsthe To study of probable DADS and P-ITC, the effects participation of theofNrf2/HO-1/NQO1 the co-administration of 200 pathway signaling µmols/kgin of theDADS or 29 µmols/kg analgesic actions of of P-ITCand DADS withP-ITC, a selective Nrf2 (ML-385, the effects 25 mg/kg), HO-1 (SnPP, of the co-administration of 200 10 mg/kg), or NQO1 (dicoumarol, 10 mg/kg) inhibitor on the mechanical µ mols/kg of DADS or 29 µ mols/kg of P-ITC with a selective Nrf2 (ML-385, 25 mg/kg), allodynia and thermal hyperalgesia caused by peripheral inflammation were evaluated. HO-1 (SnPP, 10 mg/kg), or NQO1 (dicoumarol, 10 mg/kg) inhibitor on the mechanical Our results allodynia demonstrated and thermal the reversion hyperalgesia caused of bythe antiallodynic peripheral (Figure 3A,C) inflammation were and antihy- evaluated. peralgesic effects (Figure 3B,D) induced by DADS or P-ITC in the ipsilateral paw of mice with inflammatory pain after their co-administration with ML-385, SnPP, or dicoumarol (p < 0.0001, one-way ANOVA followed by Student–Newman–Keuls). In both tests, the Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 21 Our results demonstrated the reversion of the antiallodynic (Figure 3A,C) and anti- Antioxidants 2021, 10, 1977 hyperalgesic effects (Figure 3B,D) induced by DADS or P-ITC in the ipsilateral paw 9 of 21 of mice with inflammatory pain after their co-administration with ML-385, SnPP, or dicou- marol (p < 0.0001, one-way ANOVA followed by Student–Newman–Keuls). In both tests, the administration administration of ML-385, of ML-385, SnPP,SnPP, or dicoumarol or dicoumarol alone alone did did not not produce produce any significant any significant effect effect in the ipsilateral paws of CFA-injected in the ipsilateral paws of CFA-injected animals.animals. Figure 3. Effects of DADS and P-ITC administered alone or in combination with inhibitors of the Nrf2/HO-1-NQO1 Figure 3. Effects of DADS and P-ITC administered alone or in combination with inhibitors of the Nrf2/HO-1-NQO1 sig- signaling pathway naling pathway onon thethemechanical mechanicalallodynia allodyniaandandthermal thermalhyperalgesia hyperalgesiainduced inducedby by CFA. CFA. The The effects effects on on the themechanical mechanical allodynia allodynia (A,C) and thermal hyperalgesia (B,D) in the ipsilateral paws of mice with inflammatory pain treatedwith (A,C) and thermal hyperalgesia (B,D) in the ipsilateral paws of mice with inflammatory pain treated withDADS DADS at at200 µ mols/kg or 200µmols/kg or P-ITC P-ITC atat29 µ mols/kg alone 29µmols/kg alone or or co-administered co-administered with with aaNrf2 Nrf2inhibitor inhibitor(ML-385, (ML-385,25 25mg/kg), mg/kg), an an HO-1 HO-1 inhibitor (SnPP, inhibitor (SnPP, 10 10 mg/kg), mg/kg), or oraaNQO1 NQO1inhibitor inhibitor(dicoumarol, (dicoumarol, DC, DC,1010 mg/kg) areare mg/kg) shown. For For shown. eacheach test and test treatment eval- and treatment uated & indicates significant differences vs. the other groups (p < 0.05, one-way ANOVA followed by Student–Newman– evaluated & indicates significant differences vs. the other groups (p < 0.05, one-way ANOVA followed by Student–Newman– Keuls test). Keuls test). Results Results are are represented represented asas mean meanvalues valuesofofthe themaximal maximalpossible possibleeffect effect(%) (%)±± SEM SEM values; values; nn == 66 animals animals per per experimental group. experimental group. In addition, In addition, treatment treatment with with ML-385, ML-385, SnPP, SnPP, or or dicoumarol dicoumarol administered administered alone alone or or in in combination with DADS or P-ITC did not induce any effect in the contralateral paws combination with DADS or P-ITC did not induce any effect in the contralateral paws of of CFA-injectedmice CFA-injected mice(data (datanot notshown). shown). 3.4. 3.4. Effects Effects of of Treatment Treatmentwith withDADS DADSandandP-ITC P-ITCon onthe theExpression Expressionofof4-HNE 4-HNEininthe thePaws Pawsofof Ani- Animals with Peripheral Inflammation mals with Peripheral Inflammation To To measure measure thethe effects effects of of DADS DADS and and P-ITC P-ITC on on the the oxidative oxidative stress stress induced induced by by CFA, CFA, we we evaluated their actions in the expression of the oxidative marker 4-HNE. Ourresults evaluated their actions in the expression of the oxidative marker 4-HNE. Our results showed showed an an increase increase in in the the expression expression of of 4-HNE 4-HNE in in the the paw paw of of CFA-injected CFA-injectedmicemicetreated treated with vehicle (p < 0.0001, one-way ANOVA as compared with saline plus vehicle treated mice), which was completely reversed in DADS- or P-ITC-treated mice (Figure 4). Antioxidants 2021, 10, x FOR PEER REVIEW 10 of 21 Antioxidants 2021, 10, 1977 with vehicle (p < 0.0001, one-way ANOVA as compared with saline plus vehicle treated 10 of 21 mice), which was completely reversed in DADS- or P-ITC-treated mice (Figure 4). Figure 4. Effects of treatment with DADS and P-ITC on the expression of 4-HNE in the ipsilateral paws of animals with Figure 4. Effects peripheral of treatment inflammation. Thewith DADS protein andofP-ITC levels 4-HNEoninthe theexpression ipsilateralofpaws 4-HNE in the ipsilateral of CFA-injected micepaws of animals treated with with vehicle peripheral inflammation. The protein levels of 4-HNE in the ipsilateral paws of CFA-injected mice treated (CFA-VEHI), DADS (CFA-DADS), or P-ITC (CFA-P-ITC) are shown (A). The expression of this protein in the ipsilateral with vehicle (CFA-VEHI), DADS (CFA-DADS), or P-ITC (CFA-P-ITC) are shown (A). The expression of this protein in the ipsilateral paws of mice treated with saline plus vehicle (SS-VEHI) is also represented as controls (A). Representative blots for 4- paws of mice treated with saline plus vehicle (SS-VEHI) is also represented as controls (A). Representative blots for 4-HNE HNE and GAPDH are shown (B). For each column, * indicates significant differences when compared with saline plus and GAPDH are shown (B). For each column, * indicates significant differences when compared with saline plus vehicle- vehicle-treated treated animals,animals, # indicates # indicates differences differences when compared when compared with CFA-injected with CFA-injected mice mice treated treated with with DADS, DADS, and and & & indicates indicates sig- significant differences nificant differences when when compared compared with with CFA-injectedmice CFA-injected micetreated treatedwith withP-ITC P-ITC(p(p<<0.05, 0.05,one-way one-wayANOVA ANOVA followed followed by by Student–Newman–Keuls test). Data are expressed as mean ± SEM;n= ± SEM; n =3–4 3–4samples. samples. 3.5. Effects Effects of of Treatment Treatment with DADS and P-ITC on the Expression of HO-1, NQO1, SOD-1, and GSTM1 in the Paws of Mice with Peripheral Inflammation To assess the implication of the antioxidant system in the antinociceptive effects of slow-releasing slow-releasing H H2S 2 Sdonors donorsduring during inflammatory inflammatory pain, pain, we we evaluated evaluated the expression the expression of HO-of HO-1, NQO1, SOD-1, and GSTM1 in the ipsilateral paw of CFA-injected 1, NQO1, SOD-1, and GSTM1 in the ipsilateral paw of CFA-injected mice treated with mice treated with DADS DADS or P-ITC. or P-ITC. WhileWhile peripheral peripheral inflammation inflammation enhanced enhanced the paw the paw HO-1HO-1 (p < 0.0022, (p < 0.0022, one- one-way way ANOVA,ANOVA, as compared as compared with saline with saline + vehicle-treated + vehicle-treated animals)animals) (Figure(Figure 5A) and 5A) and SOD-1 SOD-1 expression (p < 0.0036, one-way ANOVA, as compared with saline expression (p < 0.0036, one-way ANOVA, as compared with saline + vehicle-treated ani- + vehicle-treated animals) mals) (Figure (Figure 5C), 5C), it it decreasedthe decreased thepawpawexpression expressionofofGSTM1 GSTM1 (p (p << 0.0313, 0.0313, one-way one-way ANOVA, as compared with saline + vehicle-treated animals) (Figure ANOVA, as compared with saline + vehicle-treated animals) (Figure 5D). Non-changes 5D). Non-changes in in the the NQO1 NQO1 levels levels werewere detected detected ininthe thepaws pawsofofCFA CFAinjected injected mice mice treated treated with with vehicle vehicle (Figure 5B). (Figure 5B). Moreover, Moreover, while while treatment treatment with with DADS DADS and and P-ITC P-ITC maintained maintained the the high high levels levels of HO-1 (p < 0.0022, one-way ANOVA, as compared with saline + vehicle-treated of HO-1 (p < 0.0022, one-way ANOVA, as compared with saline + vehicle-treated animals) animals) (Figure 5A), (Figure 5A), both both treatments treatments enhanced enhanced the the expression expression ofof NQO1 NQO1 (p (p << 0.0043, 0.0043, one-way one-way ANOVA, as compared with saline + vehicle and CFA + vehicle treated animals) (Figure 5B). ANOVA, as compared with saline + vehicle and CFA + vehicle treated animals) (Figure Regarding the SOD-1 levels, only P-ITC treatment maintained the high levels of this 5B). Regarding the SOD-1 levels, only P-ITC treatment maintained the high levels of this protein (p < 0.0036, one-way ANOVA, as compared with saline + vehicle-treated animals) protein (p < 0.0036, one-way ANOVA, as compared with saline + vehicle-treated animals) (Figure 5C) and both treatments normalized the decreased expression of GSTM1 provoked (Figure 5C) and both treatments normalized the decreased expression of GSTM1 pro- by CFA (p < 0.0313, one-way ANOVA, as compared with CFA plus vehicle-treated animals) voked by CFA (p < 0.0313, one-way ANOVA, as compared with CFA plus vehicle-treated (Figure 5D). animals) (Figure 5D). 3.6. Effects of Treatment with DADS and P-ITC on the Expression of p-AKT, BAX, and DOR in the Paws of Animals with Peripheral Inflammation To assess the role played by DADS and P-ITC in the nociceptive and apoptotic reac- tions induced by CFA, we evaluated the expression of p-AKT and BAX in the ipsilateral paw of CFA-injected mice treated with these H2 S donors. CFA increased the paw expres- sion of p-AKT/AKT (p < 0.0006, one-way ANOVA) (Figure 6A) and BAX (p < 0.0096, one-way ANOVA) (Figure 6B) as compared with their respective saline + vehicle-treated animals. Treatment with DADS and P-ITC normalized the upregulation of both p-AKT and BAX induced by peripheral inflammation. Antioxidants 2021, 10, x FOR PEER REVIEW 11 of 21 Antioxidants 2021, 10, 1977 11 of 21 Figure 5. Effects of treatment with DADS and P-ITC on the expression of HO-1, NQO1, SOD-1, and GSTM1 in the paw Figure 5. Effects of treatment with DADS and P-ITC on the expression of HO-1, NQO1, SOD-1, and GSTM1 in the paw tissues of animals with peripheral inflammation. The protein levels of HO-1 (A), NQO1 (B), SOD-1 (C), and GSTM1 (D) in tissues of animals with peripheral inflammation. The protein levels of HO-1 (A), NQO1 (B), SOD-1 (C), and GSTM1 (D) the ipsilateral in the paw ipsilateral of CFA-injected paw of CFA-injected mice treated mice with treated vehicle with (CFA-VEHI), vehicle (CFA-VEHI), DADSDADS(CFA-DADS) (CFA-DADS) or P-ITC (CFA-P-ITC) or P-ITC (CFA-P-ITC)are shown. The expression of these proteins in the paw tissues of mice treated with saline + vehicle (SS-VEHI) as are shown. The expression of these proteins in the paw tissues of mice treated with saline + vehicle (SS-VEHI) as controls controls is also shown. For each is also shown. protein, For * indicates each protein, significant * indicates differences significant when compared differences with SS plus when compared withvehicle-treated mice, + indicates SS plus vehicle-treated mice, + significant differences indicates significant when compared differences with CFA when compared plus with CFAvehicle-treated animals,animals, plus vehicle-treated # indicates significant # indicates differences significant when differences when compared compared withplus with CFA CFADADS-treated plus DADS-treated mice, mice, and &and & indicates indicates significant significant differences differences whenwhen compared compared with with CFA CFAplus plus P-ITC-treated P-ITC-treated mice mice (p (p