antioxidants Article The Antinociceptive, Antioxidant and Anti-Inflammatory Effects of 5-Fluoro-2-Oxindole during Inflammatory Pain Alejandro Redondo 1,2,† , Gabriela Riego 1,2,† 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; josealejandro.redondo@e-campus.uab.cat (A.R.); gabrielaalejan.riego@e-campus.uab.cat (G.R.) 2 Grup de Neurofarmacologia Molecular, Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain * Correspondence: opol@santpau.es; Tel.: +34-619-757-054 † These authors contributed equally to this work.  Received: 4 October 2020; Accepted: 7 December 2020; Published: 9 December 2020  Abstract: Recent studies demonstrate that 5-fluoro-2-oxindole inhibits neuropathic pain but the antinociceptive actions of this drug and its effects on the plasticity, oxidative and inflammatory changes induced by peripheral inflammation as well as on the effects and expression of µ-opioid receptors (MOR) have not been evaluated. In C57BL/6 male mice with inflammatory pain provoked by the subplantar administration of complete Freund’s adjuvant (CFA), we evaluated: (1) the antinociceptive actions of 5-fluoro-2-oxindole and its reversion with the HO-1 inhibitor, tin protoporphyrin IX (SnPP); (2) the effects of 5-fluoro-2-oxindole in the protein levels of mitogen-activated protein kinase (MAPK), Nrf2, NADPH quinone oxidoreductase1 (NQO1), heme oxygenase 1 (HO-1), oxidative stress marker (4-hydroxy-2-nonenal; 4-HNE), inducible nitric oxide synthase (NOS2), microglial markers (CD11b/c and IBA-1), and MOR in the spinal cord and/or paw of animals with inflammatory pain; (3) the antinociceptive effects of morphine in 5-fluoro-2-oxindole pre-treated animals. Treatment with 5 and 10 mg/kg of 5-fluoro-2-oxindole inhibited the allodynia and hyperalgesia induced by CFA in a different, time-dependent manner. These effects were reversed by SnPP. Treatment with 5-fluoro-2-oxindole increased the expression of NQO1, HO-1 and MOR and inhibited the CFA-induced upregulation of phosphorylated MAPK, 4-HNE, NOS2, CD11b/c and IBA-1 in spinal cords and/or paws. The local effects of morphine were improved with 5-fluoro-2-oxindole. This work reveals that 5-fluoro-2-oxindole inhibits the plasticity, oxidative and inflammatory responses provoked by peripheral inflammation and potentiates the antinociceptive effects of morphine. Thus, treatment with 5-fluoro-2-oxindole alone and/or combined with morphine are two remarkable new procedures for chronic inflammatory pain management. Keywords: analgesia; inflammation; morphine; opioids; oxidative stress; oxindole; pain 1. Introduction Several studies showed the protective role played by numerous oxindole derivates such as isorhynchophylline (IRN) and rhynchophylline (RIN), versus diabetes, thrombosis, bacterial infection, asthma, cancer and inflammation [1–4]. The antinociceptive actions of different oxindole alkaloids during acute inflammation or visceral pain have also been demonstrated [5–7]. A recent study further revealed the antinociceptive effects of 5-fluoro-2-oxindole in animals with chronic neuropathic pain [8]. Nonetheless, the potential pain-relieving action of 5-fluoro-2-oxindole in animals with chronic inflammatory pain has not yet been evaluated. Antioxidants 2020, 9, 1249; doi:10.3390/antiox9121249 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 1249 2 of 17 Microglial cells are decisive for the progress and preservation of inflammatory pain [9,10]. Microglial activation initiates the synthesis of inflammatory mediators, for instance tumor necrosis factor α (TNFα), several interleukins and inducible nitric oxide synthase (NOS2), which promote inflammatory pain. Peripheral inflammation also induced the activation of mitogen-activated protein kinase (MAPK)/NF-κB signaling pathways in the spinal cord and paw [11], whose inhibition is a mechanism of action of the oxindoles, RIN and IRN, for attenuating the inflammatory responses induced by lipopolysaccharide (LPS) in cell cultures [12,13]. The administration of 5-fluoro-2-oxindole also inhibits the spinal cord and hippocampal activation of microglia provoked by nerve injury [8]. Nonetheless, the regulatory effects of 5-fluoro-2-oxindole in MAPK phosphorylation, microglial activation and NOS2 overexpression in the spinal cord and/or paw tissues of mice with inflammatory pain have not been evaluated. The antioxidant enzymes, NADPH quinone oxidoreductase1 (NQO1) and/or heme oxygenase 1 (HO-1), participate in the analgesic actions of several compounds such as sulforaphane, oltipraz and carbon monoxide-releasing molecules (CORM’s) during inflammatory and neuropathic pain [11,14–17]. Other works also proved that the analgesic effects of 5-fluoro-2-oxindole in animals with sciatic nerve injury-induced neuropathic pain are mediated via augmenting the Nrf2 transcription factor, NQO1 and HO-1 protein levels [8] and that the anti-inflammatory effects of IRN were produced via avoiding the down-regulation of the antioxidant proteins superoxide dismutase, glutathione peroxidase 1 and catalase [3]. The probable contribution of the Nrf2 transcription factor, NQO1 and HO-1 in the pain-relieving actions of 5-fluoro-2-oxindole, and the effects of this treatment in the levels of 4-hydroxy-2-nonenal (4-HNE)-positive proteins, an oxidative stress marker, during inflammatory pain were not identified. Several works demonstrated that different drugs are able to potentiate the analgesic effects of µ-opioid receptor (MOR) agonists in animals with neuropathic or inflammatory pain. Thus, the effectiveness of morphine increased in animals with inflammatory or neuropathic pain pre-treated with activators of Nrf2 (sulforaphane) or HO-1 (cobalt protoporphyrin IX (CoPP)) as well as with CORM’s [11,14,15,18,19]. 5-fluoro-2-oxindole also enhanced the antiallodynic and antihyperalgesic effects of morphine in sciatic nerve-injured animals [8], but its role in modulating the analgesic properties of morphine and MOR expression during inflammatory pain remains untested. In mice with chronic inflammatory pain generated with the subplantar injection of complete Freund’s adjuvant (CFA), we assessed the effects of 5-fluoro-2-oxindole in: (a) the mechanical allodynia and thermal hyperalgesia provoked by peripheral inflammation and the reversion of its effects with the administration of the HO-1 inhibitor, tin protoporphyrin IX (SnPP); (b) the protein levels of MAPK, Nrf2, HO-1 and NQO1, the oxidative stress (4-HNE) and inflammatory (NOS2, CD11b/c and IBA-1) markers as well as of MOR in the spinal cord and/or paw tissues; (c) the antinociceptive actions of locally administered morphine. 2. Materials and Methods 2.1. Animals Male C57BL/6 mice (21–25 g), acquired at Envigo Laboratories (Barcelona, Spain), were accommodated under 12/12 h light/dark conditions in a room with controlled temperature of 22 ◦ C and humidity of 66% until use. These animals with free access to food and water were used after 7 days acclimatization to the housing conditions. All the planned experiments were performed between 9:00 a.m. and 5:00 p.m. and carried out in conformity with the guidelines of the European Commission’s directive (2010/63/EC), the Spanish Law (RD 53/2013) regulating animal research and approved by the local Committee of Animal Use and Care of the Autonomous University of Barcelona (the ethical code is 1325R5). Maximal exertions to diminish animal suffering and the number of animals used were made. Antioxidants 2020, 9, 1249 3 of 17 2.2. Induction of Inflammatory Pain Chronic inflammatory pain was incited with the subplantar injection of CFA (30 µL; Sigma-Aldrich, St. Louis, MO, USA) into the right hindpaw under brief anesthetic conditions with isoflurane in accordance with our previous work [20]. 2.3. Mechanical Allodynia Mechanical allodynia was evaluated by measuring the hindpaw withdrawal response to von Frey filament stimulation. Mice were sited in methacrylate cylinders (20 cm high × 9 cm in diameter; Servei Estació, Barcelona, Spain) on a wire grid bottom, across which von Frey filaments (North Coast Medical, Inc., San Jose, CA, USA) with bending force in the range 0.008–3.5 g were applied by using the up/down paradigm [21]. The test was imitated with the 0.4 g filament, and the strength of the following filament was increased or decreased according to the response. The threshold of response was calculated from the sequence of filament strength used during the up/down procedure using an Excel program (Microsoft Iberia SRL, Barcelona, Spain), which includes the adjustment to the data curve. A clear paw withdrawal, licking the paw, or shaking the paw was considered a nociceptive like response. 2.4. Thermal Hyperalgesia Thermal hyperalgesia was evaluated by assessing the paw withdrawal latency in response to radiant heat in the plantar test (Ugo Basile, Varese, Italy) [22]. Animals were positioned in Plexiglas tubes (20 cm high × 9 cm diameter) placed on a glass surface. The heat source was situated under the plantar surface of the hind paws and activated with a light beam intensity until paw withdrawal. The cut-off time is 12 s. Paw withdrawal latencies were determined from the mean of three separate assays. In both tests, animals were familiarized to the environment for 1 h before the test so that they were quiet. Both ipsilateral and contralateral hind paws were tested. 2.5. Western Blot Analysis Naïve and CFA-injected mice treated with 5 mg/kg 5-fluoro-2-oxindole or vehicle for 11 days were euthanized by cervical dislocation at 0 and 14 days after CFA injection. The spinal cord of the lumbar section and the subplantar tissue of the hind legs of the ipsilateral side were extracted, frozen and kept at −80 ◦ C until use. Protein levels of p-JNK/JNK, p-ERK 1/2/ERK 1/2, p-P38/P38, Nrf2, HO-1, NQO1, 4-HNE, NOS2, CD11b/c, IBA-1 and MOR, were analyzed. The homogenization of tissues was done 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, and 1% phosphatase inhibitor cocktail). NP-40 was purchased from Calbiochem (Darmstadt, Germany) and all other reagents were acquired from Sigma-Aldrich. After solubilization of crude homogenate for 1 h at 4 ◦ C, it was sonicated for 10 s and centrifuged at 4 ◦ C for 15 min at 700× g. Then, the supernatant (60 µg of total protein) was mixed with 4× Laemmli loading buffer and loaded onto 4% stacking/10–12% separating sodium dodecyl sulfate polyacrylamide gels. After that, proteins were electrophoretically transferred onto a polyvinylidene fluoride membrane for 120 min and blocked with phosphate-buffered saline with Tween 20 plus 5% nonfat dry milk or Tris-buffered saline with Tween 20 plus 5% nonfat dry milk or 5% bovine serum albumin for 1 h and 15 min, and then incubated with specific rabbit primary antibody anti-phospho-JNK, total JNK, phospho-ERK 1/2, total ERK 1/2, and total P38 (1:250; Cell Signaling Technology, Danvers, MA, USA), and phospho-P38 (1:200; Cell Signaling Technology), Nrf2 (1:160; Abcam, Cambridge, UK), HO-1 (1:200; Abcam), NQO1 (1:333; Sigma-Aldrich), 4-HNE (1:150, Abcam), NOS2 (1:150; Abcam), CD11b/c (1:200; Novus Biologic, Littleton, CO, USA), IBA-1 (1:150; Thermo Fisher Scientific, Waltham, MA, USA) and (MOR (1:333; Merck, Billerica, MA, USA) overnight at 4 ◦ C. A horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Antioxidants 2020, 9, x FOR PEER REVIEW 4 of 17 Antioxidants 2020, 9, 1249 4 of 17 GE Healthcare, Little Chalfont, UK) and exposure to Kodak film. Blot intensity was quantified by densitometry Little using Chalfont, UK)Image-J was used program (National to detect Institutes proteins, of Health, which were Bethesda, visualized with MD, USA). A rabbit chemiluminescence anti-glyceraldehyde-3-phosphate reagents dehydrogenase (ECL kit; GE Healthcare, Little Chalfont, UK)(GAPDH) antibody and exposure (1:5000;film. to Kodak Merck, BlotBillerica, intensity MA, was USA) was used as a loading control. quantified by densitometry using Image-J program (National Institutes of Health, Bethesda, MD, USA). A rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5000; Merck, Billerica, 2.6. Experimental MA, Procedures USA) was used as a loading control. At first, we investigated the mechanical antiallodynic and thermal antihyperalgesic effects of the 2.6. Experimental Procedures daily intraperitoneal administration of 5 and 10 mg/kg of 5-fluoro-2-oxindole or vehicle At first, we investigated (dimethylsulfoxide 1% solutionthe in mechanical saline) fromantiallodynic day 4 to 14 afterand CFA thermal antihyperalgesic injection (n = 6 animals effects per of the daily group). intraperitoneal We used contralateraladministration paws as controls. of 5 and 10 mg/kg of 5-fluoro-2-oxindole or vehicle (dimethylsulfoxide Therefore, we1%evaluated solution inthe saline) from day reversion of4 the to 14antiallodynic after CFA injection and (n = 6 animals per group). antihyperalgesic effects We used contralateral paws as controls. generated by 5 mg/kg 5-fluoro-2-oxindole intraperitoneally administered during 11 consecutive days with Therefore, we evaluated the intraperitoneal the reversionofof 5the administration antiallodynic mg/kg SnPP, aand antihyperalgesic selective effects SnPP HO-1 inhibitor. generated was by 5 mg/kg 5-fluoro-2-oxindole administered intraperitoneally 30 min after 5-fluoro-2-oxindole administered injection duringwere and animals 11 consecutive tested 30 mindays with after the SnPP intraperitoneal administration of 5 mg/kg SnPP, a selective HO-1 inhibitor. SnPP injection (n = 6 animals per group). The dose of SnPP was selected in accordance with other studies was administered 30 min after 5-fluoro-2-oxindole injection and animals were tested 30 min after SnPP injection (n = 6 [18,23]. animals Theper group). protein Theof levels dose of SnPP was p-JNK/JNK, selected p-ERK in accordance 1/2/ERK with other 1/2, p-P38/P38, Nrf2,studies HO-1, [18,23]. NQO1, 4-HNE, NOS2, The protein IBA-1 CD11b/c, levels and of p-JNK/JNK, MOR in thep-ERK 1/2/ERK ipsilateral 1/2, site of thep-P38/P38, spinal cords Nrf2, andHO-1, paws NQO1, from mice4-HNE, with NOS2, CD11b/c, peripheral IBA-1 and inflammation MORwith treated in the ipsilateral 5 mg/kg site of the spinalor 5-fluoro-2-oxindole cords andduring vehicle paws from mice with 11 consecutive peripheral inflammation days were evaluated treated with by Western 5 mg/kg blot assay. We5-fluoro-2-oxindole used naive mice treated or vehicle during as with vehicle 11 controls consecutive (n = days were evaluated 3–4 samples per group).by Western blot assay. We used naive mice treated with vehicle as controls (n = 3–4 samples In other per group). groups, we investigated the antiallodynic and antihyperalgesic actions induced by co- In other groups, we investigated(5the administration of 5-fluoro-2-oxindole antiallodynic mg/kg, and antihyperalgesic intraperitoneal) or vehicle withactions morphineinduced by (50 µg, co-administration subplantar) or saline of 5-fluoro-2-oxindole during inflammatory (5 mg/kg, pain.intraperitoneal) Mice were tested or vehicle at 30with minmorphine (50 µg, after morphine subplantar) or saline administration (n = 6during animals inflammatory per group).pain. Mice of The dose were tested atwas morphine 30 min after morphine selected in accord administration with previous (n = 6 animals studies [11,19].per group). The dose of morphine was selected in accord with previous studies [11,19]. The researcher researcher whowho executed executed thesethese tests tests was was not not aware aware of of the the treatments treatmentsused.used. 2.7. Drugs 2.7. Drugs 5-fluoro-2-oxindole 5-fluoro-2-oxindole (Figure (Figure 1) 1) with with aa 97% 97% ofof purity purity was was purchased purchased in in Sigma-Aldrich Sigma-Aldrich (St. Louis, (St. Louis, MO, USA), dissolved in dimethylsulfoxide (1% in 0.9% of saline solution) and MO, USA), dissolved in dimethylsulfoxide (1% in 0.9% of saline solution) and intraperitoneally intraperitoneally administered administeredin inaafinal finalvolume volumeofof1010mL/kg, mL/kg,1 h1hbefore testing, before in in testing, conformity conformitywith a preceding with study a preceding [8]. study Morphine hydrochloride [8]. Morphine hydrochlorideacquired fromfrom acquired Alcaiber S.A. (Madrid, Alcaiber Spain)Spain) S.A. (Madrid, was dissolved in 0.9%insaline was dissolved 0.9% solution saline solution and subplantarly administered, in a final volume of 30 µL, 30 min beforebehavioral and subplantarly administered, in a final volume of 30 µL, 30 min before doing the doing the tests [11,19].tests behavioral All[11,19]. drugs were freshly All drugs prepared were freshlybefore use.before prepared For each use.group treated For each groupwith a drug, treated thea with respective control group received the same volume of corresponding vehicle. drug, the respective control group received the same volume of corresponding vehicle. Figure 1. Chemical structure of 5-fluoro-2-oxindole. 2.8. Statistical Analyses 2.8. Statistical Analyses All data are expressed as the mean values ± standard error of the mean (SEM). We used All data are expressed as the mean values ± standard error of the mean (SEM). We used the SPSS the SPSS program (version 13 for Windows, IBM, Madrid, Spain) for the statistical analysis. program (version 13 for Windows, IBM, Madrid, Spain) for the statistical analysis. A three-way A three-way repeated-measures ANOVA with paw, treatment and time as the factors of variation with repeated-measures ANOVA with paw, treatment and time as the factors of variation with the the corresponding one-way ANOVA and Student Newman Keuls test was used to analyze the effects corresponding one-way ANOVA and Student Newman Keuls test was used to analyze the effects of of 5-fluoro-2-oxindole on nociception. A one-way ANOVA followed by the Student Newman Keuls 5-fluoro-2-oxindole on nociception. A one-way ANOVA followed by the Student Newman Keuls test test was utilized for evaluating the effects of 5-fluoro-2-oxindole combined with SnPP or morphine. was utilized for evaluating the effects of 5-fluoro-2-oxindole combined with SnPP or morphine. The Antioxidants 2020, 9, 1249 5 of 17 The effects of 5-fluoro-2-oxindole in the expression of several proteins were analyzed using a one-way ANOVA and the Student Newman Keuls test. A value of p < 0.05 was considered significant. In the von Frey filaments and plantar tests, antinociception is expressed as the percentage of maximal possible effect, where the test latencies pre-drug (baseline) and post-drug administration are compared and calculated in accordance with this equation Maximal possible effect (%) = [(drug-baseline)/(cut-off-baseline)] × 100 Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 17 3. Results effects of 5-fluoro-2-oxindole in the expression of several proteins were analyzed using a one-way 3.1. ANOVAThe Antinociceptive and the Student Effects of 5-Fluoro-2-Oxindole Newman during Keuls test. A value of pPeripheral < 0.05 wasInflammation considered significant. In theshowed Data von Frey filaments that and plantar the repetitive tests, antinociception administration is expressed of 5-fluoro-2-oxindole as the percentage reduced the allodyniaof (Figure 2A). Significant effects of paw (p < 0.001), treatment (p < 0.001) and time (p < 0.001), and maximal possible effect, where the test latencies pre-drug (baseline) and post-drug administration are compared interactions and calculated between paw × treatment (p < 0.001), in accordance withpaw this × time (p < 0.011), treatment × time (p < 0.001) as equation well as among paw × treatment Maximal possible × effect time (p (%)< 0.001) were revealed by the three-way×repeated-measures = [(drug-baseline)/(cut-off-baseline)] 100 ANOVA. A gradual enhancement of the ipsilateral hind paw withdrawal threshold in response to von Frey filaments since days 1 to 11 of 5-fluoro-2-oxindole treatment was demonstrated (p < 0.001; 3. Results one-way ANOVA vs. The respective ipsilateral paws of mice treated with vehicle). Treatment with 5-fluoro-2-oxindole at 10 mg/kg completely inhibited the allodynia induced by peripheral inflammation 3.1. The Antinociceptive Effects of 5-Fluoro-2-Oxindole during Peripheral Inflammation after 7 days of treatment, while 11 days of treatment are required to block the allodynia with the Data showed administration of 5 that mg/kg theofrepetitive this drug.administration of 5-fluoro-2-oxindole reduced the allodynia (Figure For2A). Significant thermal effects the hyperalgesia, of paw (p < 0.001), three-way treatment repeated measures(p