antioxidants Article Novel Heme Oxygenase-1 Inducers Palliate Inflammatory Pain and Emotional Disorders by Regulating NLRP3 Inflammasome and Activating the Antioxidant Pathway Montse Pérez-Fernández 1,2 , Irene Suárez-Rojas 1,2 , Xue Bai 1,2 , Ignacio Martínez-Martel 1,2 , Valeria Ciaffaglione 3 , Valeria Pittalà 4 , Loredana Salerno 4 and Olga Pol 1,2, * 1 Grup de Neurofarmacologia Molecular, Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain 2 Grup de Neurofarmacologia Molecular, Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Institute of Crystallography, National Council of Research (CNR), 95126 Catania, Italy 4 Department of Drug and Health Sciences, University of Catania, 95125 Catania, Italy * Correspondence: opol@santpau.es; Tel.: +34-619-757-054 Abstract: Chronic pain caused by persistent inflammation is current in multiple diseases and has a strong negative impact on society. It is commonly associated with several mental illnesses, which can exert a negative influence on pain perception, and needs to be eradicated. Nevertheless, actual therapies are not sufficiently safe and effective. Recent reports demonstrate that the induction of heme oxygenase-1 (HO-1) enzyme produces analgesic effects in animals with osteoarthritis pain and reverses the grip strength loss caused by sciatic nerve crush. In this research, we evaluated the potential use of three new HO-1 inducers, 1m, 1a, and 1b, as well as dimethyl fumarate (DMF), for treating persistent inflammatory pain induced by the subplantar injection of complete Freud’s adjuvant and the functional deficits and emotional sickness associated. The modulator role of these Citation: Pérez-Fernández, M.; treatments on the inflammatory and antioxidant pathways were also assessed. Our findings revealed Suárez-Rojas, I.; Bai, X.; that repeated treatment, for four days, with 1m, 1a, 1b, or DMF inhibited inflammatory pain, reversed Martínez-Martel, I.; Ciaffaglione, V.; grip strength deficits, and reversed the linked anxious- and depressive-like behaviors, with 1m being Pittalà, V.; Salerno, L.; Pol, O. Novel the most effective. These treatments also suppressed the up-regulation of the inflammasome NLRP3 Heme Oxygenase-1 Inducers Palliate and activated the expression of the Nrf2 transcription factor and the HO-1 and superoxide dismutase Inflammatory Pain and Emotional 1 enzymes in the paw and/or amygdala, thus revealing the anti-inflammatory and antioxidant Disorders by Regulating NLRP3 capacity of these compounds during inflammatory pain. Results suggest the use of 1m, 1a, 1b, Inflammasome and Activating the and DMF, particularly 1m, as promising therapies for inflammatory pain and the accompanying Antioxidant Pathway. Antioxidants functional disabilities and emotional diseases. 2023, 12, 1794. https://doi.org/ 10.3390/antiox12101794 Keywords: analgesia; anxiety; depression; heme oxygenase-1; inflammasome; inflammatory pain; Academic Editor: Maria-Jose Alcaraz Nrf2 transcription factor; oxidative stress Received: 25 August 2023 Revised: 18 September 2023 Accepted: 21 September 2023 Published: 23 September 2023 1. Introduction Chronic pain is defined by the International Association of the Study of Pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage” [1]. Chronic pain is a complex disease highly Copyright: © 2023 by the authors. manifested in the adult population, being an important social and public health problem Licensee MDPI, Basel, Switzerland. with a strong impact on society [2]. Chronic pain is related to an increased sensitivity to This article is an open access article painful stimulus (hyperalgesia) and the perception of pain after a non-painful stimulus distributed under the terms and (allodynia), and also includes a maladjustment in physical functioning that negatively conditions of the Creative Commons Attribution (CC BY) license (https:// affects patients with inflammatory diseases. Therefore, and due to the positive correlation creativecommons.org/licenses/by/ between pain and physical functioning, an evaluation of the effects of treatments on physical 4.0/). function is highly recommended [3]. In addition, it is well recognized that chronic pain is Antioxidants 2023, 12, 1794. https://doi.org/10.3390/antiox12101794 https://www.mdpi.com/journal/antioxidants Antioxidants 2023, 12, 1794 2 of 16 associated with significant emotional disorders, such as anxiety and/or depression, which are not only related to the development of chronic pain, but can also increase the sensitivity to pain [4,5]. The current approaches to palliate chronic inflammatory pain mainly rely on the use of opioids and non-steroidal anti-inflammatory drugs (NSAIDs). However, despite all efforts, these therapies are not fully effective for treating the functional and affective disorders linked with inflammatory pain, and, further, they carry important side effects [6]. Therefore, further investigations are necessary for developing better treatments for chronic inflammatory pain. It has been known that, during the inflammatory process, the production of mitochon- drial reactive oxygen species (ROS) can be potentiated. The increase in ROS induces the activation of several inflammasomes, especially NLRP3 [7]. This inflammasome consists of the sensor molecule NOD-like receptor protein 3 (NLRP3), the apoptosis-associated speck-like protein (ASC), and a precursor caspase-1 (pro-caspase-1). This complex trig- gers the maturation of several pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and IL-18 [7]. Consequently, the NLRP3 activation is related to several inflammatory dis- eases, for instance inflammatory pain, and with the depressive-like behaviors provoked by lipopolysaccharides [8,9]. Therefore, the inactivation of NLRP3 inflammasome could be a strategy for treating inflammatory pain. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcriptional factor that plays a crucial role in the regulation of many antioxidant, anti-inflammatory, and cell sur- vival genes [10]. Its activation inhibits chronic pain, including inflammatory and neu- ropathic [11–13]. These effects are mainly related to the fact that, under normal conditions, Nrf2 binds to Kelch-like ECH associated-protein 1 (Keap1) in the cytoplasm and forms the Nrf2-Keap1 complex. However, under oxidative stress situations, it is dissociated and the Nrf2 migrates to the nucleus and initiates the transcription of multiple genes related to the antioxidant responses, such as heme oxygenase-1 (HO-1) or NAD(P)H: quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GSTM1), and superoxide dismutase 1 (SOD-1), among others [14]. Consequently, targeting this pathway can be beneficial for the treatment of chronic pain. There are several types of Nrf2 activators; one of them is dimethyl fumarate (DMF), a derivate of fumaric acid [15] that is approved by the FDA for the treatment of multiple sclerosis [16]. DMF has multiple properties, for instance anti-inflammatory, antioxidant, and neuroprotective properties [17]. DMF exerts a positive impact on the antioxidant system by modifying Keap-1 and preventing its binding to Nrf2, thus permitting that Nrf2 to be translocated into the nucleus and activating the antioxidant genes’ transcription [18]. The analgesic properties of DMF in animals with osteoarthritis [19,20], neuropathic [21], and post-operative pain [22] have been recently revealed. It also has been proven that DMF improves the grip strength deficits observed after a crush of the sciatic nerve [23] and that produced by multiple sclerosis [24]. Furthermore, it has been seen that DMF atten- uates gouty arthritis and colitis by suppressing NLRP3 inflammasome activation [25,26]. However, the effects of DMF on the expression of NLRP3 from animals with allodynia, hyperalgesia, and grip strength deficits provoked by a peripheral inflammation have not yet been assessed. One of the principal genes regulated by Nrf2 is HO-1, a potent cytoprotective and anti-inflammatory enzyme that degrades heme into carbon monoxide, free iron, biliverdin, and bilirubin. In mammals, three isoforms of HO had been discovered. HO-1 is an isoform that is induced during different pathophysiology conditions and has anti-inflammatory and antinociceptive effects, in part by decreasing the expression of pro-inflammatory cytokines (IL-6, IL-1 and TNF-α) [27]. On the contrary, the HO-2 isoform has been more associated with pronociceptive properties [28] and HO-3 is still an elusive and poorly understood isoform. Recently, the new DMF derivatives 1m, 1a, and 1b (Figure 1) were synthesized at the University of Catania with the objective of producing more potent HO-1 inducers [29,30]. Each of these derivatives was designed maintaining the α, β-unsaturated dicarbonyl Recently, the new DMF derivatives 1m, 1a, and 1b (Figure 1) were synthesized at the University of Catania with the objective of producing more potent HO-1 inducers [29,30]. Antioxidants 2023, 12, 1794 3 of 16 Each of these derivatives was designed maintaining the α, β-unsaturated dicarbonyl func- tion of DMF as the central chain crucial for HO-1 induction (blue) and introducing differ- ent symmetric substituted or unsubstituted phenyl rings by means of an ester or amide function of DMF as the central chain crucial for HO-1 induction (blue) and introducing linkage. In particular, compound different symmetric1msubstituted carried an or unsubstituted benzyl unsubstituted phenyl ringsamide moiety by means of an (ma- ester or genta), whereas 1a amidecontained linkage.aInphenethyl ester (green), particular, compound andan1b 1m carried a 4-chloro unsubstituted phenyl benzyl amideester moiety (orange). (magenta), whereas 1a contained a phenethyl ester (green), and 1b a 4-chloro phenyl ester (orange). Figure 1. Chemical structure of novelstructure Figure 1. Chemical HO-1 inducers based of novel HO-1 on the inducers DMF based structure, on the 1m, 1b, DMF structure, 1m,and 1a,1a, 1b, and synthetized synthetized by [29,30]. by [29,30]. In these studies, the authors demonstrated in vitro that 1b and 1m both increase the In these studies, theexpression HO-1 authors evendemonstrated in than more effectively vitro thatand DMF, 1b1mand 1mmost is the both increase potent the in compound HO-1 expression even more effectively than DMF, and 1m is the most potent compound of increasing HO-1 activation. The authors also proved that these molecules are devoid cytotoxic activity and can reduce ROS production caused by palmitic acid. However, their in increasing HO-1 activation. The authors also proved that these molecules are devoid of possible antinociceptive, anxiolytic, and/or antidepressant effects during inflammatory cytotoxic activity and can pain andreduce ROS production the mechanisms implied are caused by palmitic acid. However, their unidentified. possible antinociceptive, anxiolytic, Therefore, and/or in a male mice antidepressant model of complete effects Freund’sduring adjuvantinflammatory (CFA)-induced in- flammatory pain, our objectives pain and the mechanisms implied are unidentified. are to evaluate the effects of treatment with DMF, 1m, 1a, and 1b, on: (1) the mechanical allodynia and thermal hyperalgesia provoked by peripheral Therefore, in ainflammation; male mice (2) model of complete Freund’s adjuvant (CFA)-induced in- the grip strength deficits caused by paw inflammation; (3) the anxiety and flammatory pain, our objectives behaviors depressive-like are to evaluate related tothe effectsinflammatory persistent of treatment with pain; andDMF, (4) the 1m, 1a, expression and 1b, on: (1) the mechanical allodynia and of NLRP3 inflammasome andthermal hyperalgesia the antioxidant provoked proteins Nrf2, by peripheral HO-1, SOD-1, NQO1, and GSTM1 in paw tissues and amygdala. inflammation; (2) the grip strength deficits caused by paw inflammation; (3) the anxiety and depressive-like2.behaviors related Materials and Methodto persistent inflammatory pain; and (4) the expres- sion of NLRP3 inflammasome 2.1. Animals and the antioxidant proteins Nrf2, HO-1, SOD-1, NQO1, and GSTM1 in paw tissues Maleand amygdala. C57BL/6 mice purchased from Envigo Laboratories (Barcelona, Spain) were used. These animals were under 12/12 h light/dark conditions and controlled temperature (22 ± 1 ◦ C) and humidity (55 ± 10%) with able access to food and water. The experiments 2. Materials and Method were carried out after 7 days of acclimatation to the housing conditions and between 2.1. Animals 9:00 a.m. and 5:00 p.m. In accordance with the guidelines stablished by the European Com- mission’s directive and Spanish Law (RD 53/2013) and approved by the local Committee Male C57BL/6 ofmice purchased Animal from Use and Care Envigo of the Laboratories Autonomous University (Barcelona, Spain) of Barcelona (ethical were code 9863), used. These animalsevery wereattempt underwas 12/12 h light/dark conditions and controlled temperature made to minimize the suffering and the number of animals used. (22 ± 1 °C) and humidity (55 ± 10%) with able access to food and water. The experiments 2.2. Generation of Inflammatory Pain were carried out after 7To days of acclimatation to the housing conditions and between 9:00 induce the inflammatory pain, animals were subplantarly injected with 30 µL a.m. and 5:00 p.m. Inofaccordance with the CFA (Sigma-Aldrich, St.guidelines Louis, MO, stablished by the USA) in the right European hind paw. ThisCommis- process was sion’s directive and Spanish Law (RD 53/2013) and approved by the local Committee performed under short anesthetic conditions with isoflurane (3% induction and of 3% mainte- nance) [11]. Animal Use and Care of the Autonomous University of Barcelona (ethical code 9863), every attempt was made to minimize the suffering and the number of animals used. 2.2. Generation of Inflammatory Pain To induce the inflammatory pain, animals were subplantarly injected with 30 µL of CFA (Sigma-Aldrich, St. Louis, MO, USA) in the right hind paw. This process was per- Antioxidants 2023, 12, 1794 4 of 16 2.3. Allodynia, Hyperalgesia, and Grip Strength Measurements Mechanical allodynia was assessed using the von Frey filaments. Mice were located on a wire grid through where the von Frey filaments of different strength (North Coast Medical, Inc., San Jose, CA, USA) were applied following the up–down paradigm proposed by [31]. The filaments had between 0.4 to 3.0 g of bending force. Withdrawing, shaking, or licking the hind paw was considered a positive reaction. Based on the animal’s answer to a filament, the following filament applied was of higher or lower strength. Thermal hyperalgesia was evaluated using the plantar test (Ugo Basile, Varese, Italy) as described by [32]. The mice were positioned on a glass surface inside Plexiglas tubes (20 cm high × 9 cm diameter). The heat source was applied under the hind paws. The paw withdrawal latencies were obtained from the mean of three separate trials. To prevent paw tissue damage, mice were exposed to the head for a maximum of 12 s. The hind paws grip strength was measured using a computerized grip strength meter (Model 47200. Ugo Basile, Varese, Italy) [33]. The animal had to grasp with both hind legs a metal bar connected to the grip strength meter that recorded the maximum force of each measurement (g). Between 3 to 4 measures were performed for each animal. 2.4. Emotional-like Behaviors The assessment of the anxiety-like behaviors was conducted using the elevated plus maze (EPM) [34] and open field (OF) [35] tests. In the EPM, we used a maze that was elevated at a height of 45 cm from the floor and consisted of four arms, two closed and two opened. The closed arms were enclosed by walls measuring 15 cm. In the initiation of the test, mice were placed in the central square of the maze, always facing one open arm. We recorded the mouse’s behavior for 5 min, allowing, and quantified the number of entries into the close and open arms and the percentage of time spent in the open arms. The second test conducted to evaluate anxiety-like behaviors was the OF, which consisted of putting the animal in the center of a square box measuring 44 cm × 44 cm, with walls standing at a height of 30 cm. They were allowed to explore the box freely for 5 min and their behavior was recorded. Then, the number of squares crossed and entries into the central area, and the time passed in it, were quantified. To assess the depressive-like behaviors of the animals, we performed the tail suspen- sion test (TST) [36] and the force swimming test (FST) [37]. In the FST, mice were suspended from an elevated surface of approximately 35 cm from the ground. The animal was attached to the surface with an adhesive tape attached to the tail. We recorded 8 min for each animal and measured its immobility time from the last 6 min. In the FST test, the animals were collocated in a cylinder with 10 cm of water at 24 ± 1 ◦ C. Each animal was recorded for 6 min, and its inactivity time was gauged for the last 4 min. All these experiments were performed by investigators blinded to the experimental conditions and animals were habituated to the environment for 1 h before the experiment. 2.5. Western Blot Mice were euthanized by cervical dislocation at 16 days after CFA or saline solution (SS) injection. The ipsilateral subplantar paw and the contralateral amygdala were dissected and quickly preserved at −80 ◦ C for later analysis. To evaluate the levels of proteins NLRP3, Nrf2, HO-1, SOD-1, NQO1, and GSTM1, the extracted tissues were homogenized using an ice-cold lysis buffer consisting of RIPA buffer, 0.5% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The homogenate tissues stayed at 4 ◦ C for 1 h, after which they were sonicated (10 s) and centrifugated at 4 ◦ C for 20 min at 1000 rpm. The supernatant (60 µg of total protein) was mixed with 4× Laemmli loading buffer and loaded onto a 12% separating sodium dodecyl sulphate polyacrylamide gel. Once the electrophoresis was done, proteins were transferred onto a polyvinylidene Antioxidants 2023, 12, 1794 5 of 16 fluoride membrane for 120 min. Membranes were blocked with phosphate-buffered saline plus Tween 20 or Tris-buffered saline plus Tween 20 and different percentages of non-fat dry milk or bovine serum albumin. After 75 min, the blocking solution was discarded, and membranes were incubated overnight at 4 ◦ C with a specific primary antibody, anti NLRP3 (1:200; Adipogen Life Sciences, Epalinges, Switzerland); Nrf2 (1:115; Abcam, Cambridge, UK); HO-1 (1:100; Enzo Life Science, New York, NY, USA); SOD-1 (1:150; Novus Biologic, Littleton, CO, USA); NQO1 (1:200; Sigma-Aldrich, St. Louis, MO, USA); or GSTM1 (1:100; Novus Biologic, Littleton, CO, USA), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000; Merck, Billerica, MA, USA) as a loading control. Then, membranes were incubated with a blocking solution together with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies for 1 h at room temperature. Furthermore, using ChemiDoc and chemiluminescent reagents, proteins were de- tected. After capturing an image of the membrane, each protein band was analyzed by densitometry employing the Image-J program (National Institutes of Health, Bethesda, MD, USA). 2.6. Drugs DMF (Sigma–Aldrich, St. Louis, MO, USA) and 1m, 1b, and 1a synthesized by [29,30] were dissolved in 0.05% of Cremophor EL (CMC)/dimethyl sulfoxide/ethanol (Sigma- Aldrich, St. Louis, MO, USA) in a mixture of 12:2:2 in accordance with another study [21]. All drugs were prepared immediately before use in a final volume of 10 mL/kg. For each group injected with a drug, the corresponding control group was given an identical volume of vehicle (0.05% CMC/dimethyl sulfoxide/ethanol in a mixture of 12:2:2; VEH). 2.7. Experimental Design In the first experiment, at 13 days of SS or CFA injection, mice were administered with 300 mg/kg of DMF, 1m, 1a, 1b, or VEH given orally for 4 consecutive days. In these animals, we assessed the mechanical allodynia, thermal hyperalgesia, and grip strength at day 0 (before SS or CFA injection), at day 12 (pre-treatment), and every day of treatment until day 16 after SS or CFA injection, after 3 h of drug administration. We used 6 animals for each group of SS-injected mice (SS-VEH, SS-DMF, SS-1m, SS-1a, and SS-1b) and 6 animals for each group of CFA-injected mice (CFA-VEH, CFA-DMF, CFA-1m, CFA-1a, and CFA-1b), 60 mice in total. In other groups of animals, we studied the anxiolytic and antidepressant effects produced by the same drugs, DMF, 1m, 1a, and 1b, also orally administered, at 300 mg/kg, during 4 consecutive days (from day 13 to 16 after CFA injection). Tests were performed on the last day of administration (day 16) and at 3 h after the oral administration of DMF, 1m, 1a, and 1b. We used 8 animals per group (SS-VEH, CFA-VEH, CFA-DMF, CFA-1m, CFA-1a, and CFA-1b); that is a total of 48 mice. In total, 108 mice were used. Lastly, the protein levels of NLRP3, Nrf2, HO-1, SOD-1, NQO1, and GSTM1 in the paw and amygdala of CFA-injected animals treated with DMF, 1m, 1a, and 1b were analyzed. Controls were SS-injected mice treated with VEH (n = 3 samples per group). 2.8. Statistical Analyses The GraphPad Prism 9.0 software (La Jolla, CA, USA) and SPSS version 28 (IBM, Madrid, Spain) were employed for the statistical analysis. The normal distribution of the data was determined using the Kolmogorov–Smirnov test. A three-way analysis of variance (ANOVA) with repeated measures was used to evaluate the effects of inflammation, treatment, and time of treatment and their possible interactions in animals treated with DMF, 1m, 1a, and 1b or VEH. For each day of treatment, the possible differences between groups were evaluated by using a one-way ANOVA followed by the Tukey post-test. The one-way and Tukey post-test was also employed to evaluate the possible differ- ences between DMF, 1m, 1a, and 1b or VEH treatments on the anxiety- and depressive-like Antioxidants 2023, 12, 1794 6 of 16 behaviors as well as in their effects on the expression of different proteins in the paw and amygdala. In all cases, p < 0.05 was considered statistically significant. Results are plotted as the mean ± standard error of the mean (SEM). 3. Results 3.1. Effects of the Treatment with DMF, 1m, 1a, and 1b on the Mechanical Allodynia, Thermal Hyperalgesia, and Grip Strength Loss Caused by CFA We evaluated the effects of the administration of DMF, 1m, 1a, and 1b, for four repetitive days, on the mechanical allodynia, thermal hyperalgesia, and grip strength deficits induced by peripheral inflammation from day 13 to day 16 after CFA injection (Figure 2). In the case of mechanical allodynia, the three-way repeated measures ANOVA revealed significant effects of inflammation (p < 0.003), time (p < 0.000), and treatment (p < 0.05). It also showed significant interactions between inflammation and treatment (p < 0.05), inflammation and time (p < 0.000), and treatment and time (p < 0.001) and a triple interaction between inflammation, treatment, and time (p < 0.000). The results indicated that the decreased threshold of the ipsilateral hind paw withdrawal to von Frey filaments stimulus caused by CFA (p < 0.001, one-way ANOVA vs. SS-injected mice treated with VEH) was completely reverted after two days of treatment with 1m and these effects were higher than those produced by DMF, 1a, and 1b at the same day (p < 0.001, one-way ANOVA) (Figure 2A). Moreover, three days of treatment with DMF, 1a, and 1b were required to block the allodynia. For thermal hyperalgesia, the three-way repeated measures ANOVA revealed signifi- cant effects of inflammation (p < 0.000), treatment (p < 0.000), and time (p < 0.000) as well as interactions between inflammation and treatment (p < 0.000), inflammation and time (p < 0.000), treatment and time (p < 0.000), and between inflammation, treatment, and time (p < 0.000) (Figure 2B). In this case, while only two days of treatment with 1m and 1b were required to completely reverse the reduced paw withdrawal provoked by CFA (p < 0.001, one-way ANOVA vs. SS-injected mice treated with VEH), three days of treatment with DMF and 1a were necessary. Our results also revealed that, at two days of treatment, the antihyperalgesic effects produced by 1 m and 1b were higher than those produced by DMF and/or 1a at the same day. Concerning the grip strength deficits provoked by CFA, the three-way repeated mea- sures ANOVA revealed significant effects of inflammation (p < 0.027), treatment (p < 0.001), and time (p < 0.000). It also showed significant interactions among inflammation and treatment (p < 0.002), inflammation and time (p < 0.000), treatment and time (p < 0.000), and between the three of them (p < 0.000). After a strong loss of grip strength at 12 days from CFA injection, a similar recovery in VEH- and DMF-treated mice was observed in the following 3 days, but, at 16 days post-CFA injection, the recovery of grip strength in DMF-treated mice was significantly improved compared with VEH-treated mice (p < 0.001, one-way ANOVA) (Figure 2C). In contrast, a completed reversion of the grip strength deficits induced by CFA (p < 0.001, one-way ANOVA vs. SS-injected mice treated with VEH) was observed after two days of treatment with 1m, 1a, and 1b. In all tests, no significant effects of the repetitive administration of DMF, 1m, 1a, and 1b were detected in the ipsilateral paws of SS-injected mice (Figure 2), nor in the contralateral paws of CFA- or SS-injected mice. Antioxidants 2023, 12, 1794 Antioxidants 2023, 12, x FOR PEER REVIEW 7 of 16 7 of 16 The Figure2.2.The Figure inhibition inhibition of the of the mechanical mechanical allodynia, allodynia, thermalthermal hyperalgesia, hyperalgesia, and gripand grip strength strength deficits deficits provoked provokedbybyCFA CFA with thethe with repetitive administration repetitive of DMF, administration 1m, 1a, of DMF, 1m,and1a, 1b.and Effects 1b. of the repetitive Effects of the repetitive administration administration of of DMF, DMF,1m,1m,1a, 1a, 1b, 1b, or VEH on the or VEH onreduced von Frey the reduced vonfilaments strengthstrength Frey filaments (g) (A), (g) (A), withdrawal latency (s) (B) and grip strength (g) (C) generated by CFA in the ipsilateral hind paws. withdrawal latency (s) (B) and grip strength (g) (C) generated by CFA in the ipsilateral hind paws. For each test, symbols represent significant differences vs. * their respective SS group, + CFA plus For each DMF test, treated symbols animals, represent & CFA plus 1msignificant differences treated animals, $ CFAvs. * their plus respective 1b treated SS group, animals, and # CFA+ CFA plus DMF treated animals, & CFA plus 1m treated animals, $ CFA plus 1b treated animals, and # CFA plus 1a treated animals (p < 0.05, one-way ANOVA followed by Tuckey test). Data are presented as mean values ± SEM; n = 6 animals for group. To study if repetitive oral administration with DMF, 1m, 1b, and 1a for four days can inhibit the anxiety-like behaviors accompanying inflammatory pain, we used the EPM and TST. The results obtained from the EPM showed that the decreased number of entries into the open arms observed in CFA-plus-VEH-treated mice as compared with SS–VEH- Antioxidants 2023, 12, 1794 8 of 16 treated animals (p < 0.003, one-way ANOVA) (Figure 3A) was reversed with DMF, 1m, 1a, and 1b treatments (Figure 3A). No changes between groups were observed, nor in the amount of time that 3.2. theEffects animals spentwith of Treatment in the DMF,open arms 1m, 1a, (Figure and 1b 3B), norBehaviours on the Anxiety-like in the number Associatedofwith entries into the closed arms (Figure 3C). Inflammatory Pain To study Regarding the OF test, dataif repetitive oral administration also revealed with DMF, percentage that the decreased 1m, 1b, and 1aoffortime four days thatcan inhibit the anxiety-like behaviors accompanying inflammatory pain, we used the EPM and CFA-injected animals treated with VEH spent in the central area (p < 0.000, one-way TST. The results obtained from the EPM showed that the decreased number of entries into ANOVA vs. SS-plus-VEH-treated mice)inwas the open arms observed normalized with CFA-plus-VEH-treated theasfour mice treatments compared (Figure with SS–VEH-treated 3E). No alterations between animals (p groups were detected, < 0.003, one-way nor in ANOVA) (Figure 3A)the wasnumber of entries reversed with DMF, 1m, into 1a, the and 1b treatments (Figure 3A). No changes between central area (Figure 3D) or in the number of squares crossed (Figure 3F).groups were observed, nor in the amount of time that the animals spent in the open arms (Figure 3B), nor in the number of entries into the closed arms (Figure 3C). Figure 3. Inhibition of anxiety-like behaviors induced by CFA with the repetitive administration of Figure 3. Inhibition of DMF, anxiety-like behaviors 1m, 1a, and induced 1b. The effects by CFA of the oral with theofrepetitive administration DMF, 1m, 1a, administration and 1b in the EPM ofand DMF, 1m, 1a, and 1b. The effects of the oral administration of DMF, 1m, 1a, and 1b in the EPM OF tests, during four consecutive days, at 16 days after CFA injection, are shown. The behaviors and OF tests, during four consecutive evaluated in thedays, at the EPM are 16number days after CFA of entries intoinjection, are the open arms (A), shown. Theofbehaviors percentage time spent (B), evaluated in the EPM are the number of entries into open arms (A), the percentage of time and the number of entries into closed arms (C). In the OF test, the number of entries spent in the central (B), and the number of entries into closed arms (C). In the OF test, the number of entries in the (F) area (D), the percentage of time spent in the central area (E), and the number of squares crossed are represented. The effects of VEH in SS-injected mice are also displayed. For each test evaluated, central area (D), the percentage of time spent in the central area (E), and the number of squares different symbols represent significant differences vs.: € all the groups except CFA-injected mice crossed (F) are represented. The effects of VEH in SS-injected mice are also displayed. For each test treated with 1a, and @ the rest of groups, (p < 0.05; one-way ANOVA and Tukey test). Data are evaluated, different symbols represent significant differences vs.: € all the groups except CFA-in- presented as mean values ± SEM; n = 8 animals for group. jected mice treated with 1a, and @ the rest of groups, (p < 0.05; one-way ANOVA and Tukey test). Data are presented as meanRegarding values ± the SEM; OFntest, data alsofor = 8 animals revealed group.that the decreased percentage of time that CFA-injected animals treated with VEH spent in the central area (p < 0.000, one- way ANOVA vs. SS-plus-VEH-treated mice) was normalized with the four treatments 3.3. Effects of Treatment with DMF, 1m, 1a, and 1b on the Depressive-like Behaviours Linked (Figure 3E). No alterations between groups were detected, nor in the number of entries into with Inflammatory Pain the central area (Figure 3D) or in the number of squares crossed (Figure 3F). To study if repetitive oral 3.3. Effects administration of Treatment with with DMF, 1m, 1a, DMF, and 1b on1m, 1a, and 1b,Behaviours the Depressive-like for fourLinked days,with could inhibit the depressive-like Inflammatory Pain behaviors accompanying inflammatory pain, we utilized the TST (Figure 4A) and To FST study(Figure 4B). if repetitive Inadministration oral both tests, with the increased DMF, 1m, 1a,immobility time and 1b, for four days, could inhibit the depressive-like behaviors accompanying caused by CFA (p < 0.005, one-way ANOVA vs. their corresponding SS-plus-VEH-treated inflammatory pain, we utilized the TST (Figure 4A) and FST (Figure 4B). In both tests, the increased immobility time caused animals) was inhibited after four days of treatment with DMF, 1m, 1a, or 1b. Antioxidants 2023, 12, 1794 9 of 16 OR PEER REVIEW 9 of 16 by CFA (p < 0.005, one-way ANOVA vs. their corresponding SS-plus-VEH-treated animals) was inhibited after four days of treatment with DMF, 1m, 1a, or 1b. Figure 4. Inhibition of the depressive-like behaviors accompanying inflammatory pain with the Figure 4. Inhibition ofrepetitive administration of DMF, 1m, 1a, and 1b. The effects of the oral administration of DMF, 1m, the depressive-like behaviors accompanying inflammatory pain with the re- 1a, and 1b in the TST and FST during four consecutive days, at 16 days after CFA injection, are shown. petitive administration of DMF, 1m, 1a, and 1b. The effects of the oral administration of DMF, 1m, Pannels (A,B) represent the immobility time(s) in the TST and FST, respectively. The effects of VEH in 1a, and 1b in the TSTSS-injected and FSTanimals during fourdisplayed. are also consecutive For eachdays, test, @at 16 days represents after CFA significant injection, differences are vs. the rest shown. Pannels (A) and (B)groups of the represent the (p < 0.05, immobility one-way ANOVA time(s) and Tuckey intest). the Data TSTare and FST, shown as respectively. mean values ± SEM; The effects of VEH in SS-injected animals are also displayed. For each test, @ represents significant dif- n = 8 animals for group. ferences vs. the rest of3.4. theEffects groups (p <1m, of DMF, 0.05, 1a, one-way ANOVA and 1b Treatments and on the Tuckey Expression of test). NLRP3, Data Nrf2,are shown HO-1, as SOD-1, mean values ± SEM; nNQO1,= 8 animals for group. and GSTM1 in the Paw and Amygdala from CFA-Injected Mice The actions of the repetitive administration of DMF, 1m, 1a, and 1b during four con- 3.4. Effects of DMF, 1m, 1a, days secutive and on 1b the levels of protein Treatments on therelated to the inflammatory Expression of NLRP3, (NLRP3) Nrf2,and antioxidant HO-1, (Nrf2, HO-1, SOD-1, NQO1, and GSTM1) pathways were evaluated. SOD-1, NQO1, and GSTM1 In theinpawthetissues Paw of and Amygdala CFA-injected from mice, CFA-Injected results indicated thatMice the over-expression of the NLRP3 inflammasome (p < 0.002, one-way ANOVA The actions of the repetitive administration of DMF, 1m, 1a, and 1b during vs. SS plus VEH) was normalized four con- with the four assessed treatments (Figure 5A). It was also seen that the administration of secutive days on theDMF, levels of and 1m, 1a, protein related enhanced 1b significantly to the inflammatory the expression of (NLRP3) and antioxi- Nrf2 when compared with dant (Nrf2, HO-1, SOD-1, animals NQO1, and GSTM1) with and without peripheralpathways inflammation were evaluated. treated with VEH (p < 0.000, one-way ANOVA) In the paw tissues (Figure 5B). Ourmice, of CFA-injected results results also showed that the protein indicated that thelevels of HO-1 (Figure 5D) over-expression of were significantly up-regulated in CFA-injected animals treated with VEH (p < 0.000, one- the NLRP3 inflammasome way ANOVA (p