Naringenin Inhibits Superoxide Anion-Induced Inflammatory Pain: Role of Oxidative Stress, Cytokines, Nrf-2 and the NO−cGMP−PKG−KATPChannel Signaling Pathway

In the present study, the effect and mechanism of action of the flavonoid naringenin were evaluated in superoxide anion donor (KO2)-induced inflammatory pain in mice. Naringenin reduced KO2-induced overt-pain like behavior, mechanical hyperalgesia, and thermal hyperalgesia. The analgesic effect of naringenin depended on the activation of the NO−cGMP−PKG−ATP-sensitive potassium channel (KATP) signaling pathway. Naringenin also reduced KO2-induced neutrophil recruitment (myeloperoxidase activity), tissue oxidative stress, and cytokine production. Furthermore, naringenin downregulated KO2-induced mRNA expression of gp91phox, cyclooxygenase (COX)-2, and preproendothelin-1. Besides, naringenin upregulated KO2-reduced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) mRNA expression coupled with enhanced heme oxygenase (HO-1) mRNA expression. In conclusion, the present study demonstrates that the use of naringenin represents a potential therapeutic approach reducing superoxide anion-driven inflammatory pain. The antinociceptive, anti-inflammatory and antioxidant effects are mediated via activation of the NO−cGMP−PKG−KATP channel signaling involving the induction of Nrf2/HO-1 pathway.


Introduction
Pain is an unpleasant sensory and emotional experience, generally in association with tissue injury. During inflammation, pro-inflammatory mediators activate resident cells, recruited cells and nociceptors, thereby driving pain signaling. Nociceptive neurons do not express receptors for all inflammatory molecules, suggesting both direct and indirect activation and sensitization of nociceptors [1]. Increased levels of oxidative stress during the inflammatory response also contribute to nociception. For instance, reactive oxygen species (ROS) and reactive nitrogen species (RNS) can act directly and indirectly to induce nociceptor sensitization and activation [2][3][4][5]. The superoxide anion (O 2 − ) is a common form of ROS that can drive nociception [5,6]. O 2 − reacts with nitric oxide (NO) generating peroxynitrite, which also contributes to nociception [3]. Superoxide dismutase (SOD), an enzymatic antioxidant, transforms superoxide anion in hydrogen peroxide, which may also induce nociception [2]. Therefore, O 2 − is a crucial ROS to the biological underpinnings driving nociception. O 2 − increases other pro-inflammatory effects, including increasing vascular permeability [7], inducing cytokine release [8,9] and increasing neutrophil recruitment [9,10], as well as provoking overt pain-like behavior and hyperalgesia [2][3][4][5]. In a physiological state, O 2 − levels remain under control by the action of the endogenous antioxidant systems, including SOD, and the endogenous antioxidant reduced glutathione (GSH) [9]. However, the imbalance between oxidants and antioxidants during inflammation leads to oxidative stress. This is important, as inhibiting the production of pro-inflammatory cytokines and ROS limit the development of inflammatory pain [6,[11][12][13]. Naringenin (4',5,7-tryhidroxy-flavonone) is a flavonoid which belongs to flavonones class found in citric fruits, including lemon, orange, tangerine and grapefruit [14]. Naringenin inhibits the nociceptive responses in models of formalin-, acetic acid-and capsaicin-induced overt pain, as well as neuropathic pain [15][16][17]. Naringenin also inhibits inflammation by targeting cyclooxygenase (COX)-2 in ethanol-induced liver injury [18] and in vitro [19]. Moreover, naringenin inhibits phosphorylation of nuclear factor kappaB (NFκB) subunit p65 and mitogenactivated protein kinases (MAPK) in daunorubicin-induced nephrotoxicity [20] as well as inhibiting the EGFR-PI3K-Akt/ERK MAPK signaling pathway in human airway epithelial cells [21]. Naringenin also inhibits a number of aspects of oxidative stress, including lipid peroxidation and O 2 − production, as well as restoring GSH levels in UVB-induced oxidative stress in the skin of Hairless mice [22]. Furthermore, naringenin increases SOD in an experimental stroke model, highlighting its wide-acting induction of endogenous antioxidants [23]. In agreement with such antioxidant effects, naringenin also induces nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ heme oxygenase (HO)-1 in CCl 4 -induced hepatic inflammation [24]. Some flavonoids can induce antinociception by activating the NO−cGMP−PKG−ATP-sensitive potassium channel (K ATP ) signaling pathway [25][26][27][28]. Activating this signaling pathway is an important mechanism of action of a number of clinical analgesics, such as opioids [25], and some non-steroidal anti-inflammatory drugs including dipyrone [26], diclofenac [27], and indomethacin [28]. Given the above, the current study addresses the analgesic effects of naringenin in a model of O 2 − -triggered inflammatory pain. It was also investigated as to whether naringenin's mechanism of action involves the NO−cGMP−PKG−K ATP channel signaling pathway, the regulation of inflammatory mediators/enzymes and oxidative stress as well as the transcription factor Nrf2, and its downstream target, HO-1.

Animals
Male Swiss mice (25 ± 5 g) from Londrina State University were housed in standard plastic cages with free access to food and water, with a light/dark cycle of 12:12 h, at 21°C. All behavioral testing was performed between 9 a.m. and 5 p.m. in a temperature-controlled room. At the end of experiments, mice were anesthetized with isoflurane 3% to minimize suffering (Abbott Park, IL, USA) and killed by cervical dislocation followed by decapitation.

Overt pain-like behavioral tests
Abdominal writhing was induced by ip injection of 1 mg of KO 2 [6]. Immediately after stimulus injection, each mouse was placed individually in a large glass cylinder, and the intensity of nociceptive behavior was quantified by counting the total number of writhings occurring between 0 and 20 min after stimulus injection. The writhing response consists of a contraction of the abdominal muscle together with a stretching of hind limbs, and the intensity was expressed as the cumulative number of abdominal contortions over 20 min. The number of paw flinches and the time spent licking the paw were determined between 0-30 min after ipl injection of 30 μg of KO 2 . Each mouse was placed in a large glass cylinder immediately after stimulus injection. The intensity of nociceptive behavior was quantified by counting the total number of paw flinches and the time (seconds) spent licking the ipsilateral paw [6].

Mechanical hyperalgesia test
Mechanical hyperalgesia was evaluated by the electronic version of von Frey's test, as reported previously [29]. In a quiet, temperature controlled room, mice were placed in acrylic cages (12 x 10 x 17 cm) with wire grid floors, 15-30 min before the start of testing. The test consisted of evoking a hind paw flexion reflex with a handheld force transducer (electronic anesthesiometer, IITC Life Science, Woodland Hills, CA) adapted with a 0.5 mm 2 polypropylene tip. The investigator was trained to apply the tip perpendicularly to the central area of the plantar hind paw with a gradual increase in pressure. The gradual increase in pressure was manually performed in blinded experiments. The upper limit pressure was 15 g. The end-point was characterized by the removal of the paw followed by clear flinching movements. After paw withdrawal, the intensity of the pressure was automatically recorded, and the final value for the response was obtained by averaging three measurements. The animals were tested before and after treatments. The results are expressed by delta (Δ) withdrawal threshold (in grams) calculated by subtracting the mean measurements 0.5, 1, 3, 5 and 7 h after stimulus from the zerotime mean measurements [6].

Thermal hyperalgesia test
Heat thermal hyperalgesia was performed using a hot plate at 55 ± 1°C [6]. The end-point was characterized by the removal of the paw followed by clear paw flinching or licking movements. The upper time was 15 s to avoid possible injury. The results are expressed by total withdrawal latency (in seconds) of measurements obtained 0.5, 1, 3, 5 and 7 h after stimulus [30].

MPO assay
Neutrophil migration to the hind paw skin tissue was evaluated using an MPO kineticcolorimetric assay, as described previously [6]. Samples of paw skin tissue were collected 7 h after stimulus in ice-cold 50 mM K 2 HPO 4 buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) and kept at -80°C until use. Samples were homogenized, centrifuged (16,100g x 2 min, 4°C), and the resulting supernatant was assayed for MPO activity spectrophotometrically at 450 nm (Multiskan GO Microplate Spectrophotometer, Thermo Scientific, Vantaa, Finland), with three readings in 1 min. The MPO activity of samples was compared to a standard curve of neutrophils. Briefly, 15 μL of sample was mixed with 200 μL of 50 mM phosphate buffer, pH 6.0, containing 0.167 mg/mL o-dianisidine dihydrochloride and 0.015% hydrogen peroxide. The results are presented as MPO activity (number of neutrophils X 10 10 per g of tissue).

GSH measurement
Paw skin sample was collected and maintained at -80°C for at least 48 h. Sample was homogenized with 200 μL of 0.02 M EDTA. The homogenate was mixed with 25 μL of 50% trichloroacetic acid and was homogenized three times during 15 min. The mixture was centrifuged (15 min x 1500 g x 4°C). The supernatant was added to 200 μL of 0,2 M TRIS buffer, pH 8.2, and 10 μL of 0,01M DTNB. After 5 min, the absorbance was measured at 412 nm against a reagent blank with no supernatant. A standard curve was performed with standard GSH. The results are expressed as GSH nmol per mg paw [31].

FRAP assay
Paw skin sample was collected and immediately homogenized with 500 μL of 1.15% KCl, and was centrifuged (10 min x 200 g x 4°C). The ability of the sample to resist oxidative damage was determined using FRAP assays [32]. For the FRAP assay, 50 μL of supernatant was mixed with 150 μL of deionized water and 1.5 mL of freshly prepared FRAP reagent. The reaction mixture was incubated at 37°C for 30 min and the absorbance was measured at 595 nm. The results were equated against a Trolox standard curve (1.5-30 μmol/L, final concentrations). The results are expressed as Trolox equivalents per mg of paw.

Superoxide anion production
The quantitation of O 2 − production in tissue homogenates was performed using the NBT assay, as described previously [33]. Skin samples were collected 3h after the stimulus. Briefly, 50 μL of the homogenate was incubated with 100 μL of NBT (1 mg/mL) in 96-well plates at 37°C for 1h. The supernatant was carefully removed and the reduced formazan solubilized by adding 120 μL of 2M KOH and 140 μL of DMSO. The NBT reduction was measured at 600 nm using a microplate spectrophotometer reader (Multiskan GO, Thermo Scientific). The tissue weight was used for data normalization; thus the results are expressed as NBT reduction (OD/mg of paw).

Lipid Peroxidation
Tissue lipid peroxidation was assessed by the levels of thiobarbituric acid reactive substances (TBARS) [34]. For this assay, TCA 10% was added to the homogenate and the mixture was centrifuged (1000 g, 3 min, 4°C) to precipitate proteins. The protein-free supernatant was then separated and mixed with TBA (0.67%). The mixture was kept in water bath (15 min, 100°C). Malondialdehyde (MDA), an intermediate product of lipid peroxidation, was determined by difference between absorbances at 535 and 572 nm using a microplate spectrophotometer reader. The results were presented as nmol of MDA per mg of paw [34].

Cytokine measurement
Paw skin samples were collected 3 h after the injection of KO 2 , homogenized in 500 μL of icecold buffer containing protease inhibitors, centrifuged (3000 rpm x 10 min x 4°C) and the supernatants used to measure, TNFα and IL-10 levels, by an enzyme-linked immunosorbent assay (ELISA) using eBioscience kits. As a control, the concentrations of these cytokines were determined in animals injected with saline. The results are expressed as picograms (pg) of cytokine per mg of paw.

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was performed as previously described [33]. Paw skin samples were collected 3 h after stimulus and homogenized in trizol reagent, with the total RNA being isolated according to manufacturer's directions. The purity of total RNA was measured with a spectrophotometer with the wavelength absorption ratio (260/280 nm) being between 1.8 and 2.0 for all preparations. Reverse transcription of total RNA to cDNA, and qPCR were carried out using GoTaq 1 2- Step RT-qPCR System (Promega) following the manufacturer's instructions. The relative gene expression was measured using the comparative 2 −(ΔΔCq) method. The primers used were gp 91phox , sense:

Statistical analysis
Results are presented as means ± SEM of measurements made on six mice in each group per experiment, and are representative of two independent experiments. Two-way analysis of variance (ANOVA) was used to compare the groups and doses at all times (curves), when the hyperalgesic responses were measured at different times after the administration or enforcement of the stimuli. The factors analyzed were treatment, time, and time versus treatment interaction. When there was a significant time versus treatment interaction, one-way ANOVA followed by Tukey's post hoc was performed on each occasion. On the other hand, when the hyperalgesic responses were measured once after the administration or enforcement of the stimuli, the difference between responses were evaluated by one-way ANOVA followed by Tukey's post hoc. Statistical differences were considered to be significant at p <0.05.

Naringenin inhibits KO 2 -induced overt pain-like behavior
Mice received naringenin (16.7, 50,150 mg/kg, po) treatment 1h before injection of 1 mg of KO 2 ip for the assessment of the total number of writhings, or received 30 μg of KO 2 ipl for the evaluation of the total number of paw flinches and time spent licking the paw. The naringenin doses of 50 and 150 mg/kg inhibited KO 2 -induced writhing response at a similar magnitude of effect, with the 3 mg/kg dose showing no statistically significant effect (Fig 1a). The dose of 50 mg/kg of naringenin was therefore selected for the next experiments. Naringenin also inhibited KO 2 -induced paw flinches (Fig 1b) and time spent licking the paw (Fig 1c).

Naringenin inhibits KO 2 -induced cytokine production and mRNA expression
The protocol of this section was the same as for Fig 4, with samples analyzed by ELISA and RT-qPCR. Naringenin treatment inhibited KO 2 -induced TNFα (Fig 5a) and IL-10 (Fig 5b) production, and IL-33 mRNA expression (Fig 5c).

Naringenin inhibits KO 2 -induced COX-2 mRNA expression
The protocol of this section was the same as for Fig 4, with samples analyzed by RT-qPCR. Naringenin treatment inhibited KO 2 -induced COX-2 mRNA expression (Fig 6).

Naringenin inhibits KO 2 -induced preproET-1 mRNA expression
The protocol of this section was the same as for Fig 4, and samples analyzed by RT-qPCR. Naringenin treatment inhibited KO 2 -induced preproET-1 mRNA expression (Fig 7).

Naringenin increases Nrf2/HO-1 mRNA expression
The protocol of this section was the same as for Fig 4, with samples analyzed by RT-qPCR. Naringenin inhibited the KO 2 -induced decrease in Nrf2 mRNA expression (Fig 8a), whilst enhancing KO 2 -induced HO-1 mRNA expression (Fig 8b).

Discussion
Pain is a multisensory experience, which occurs in response to stimulation of the channels/ receptors that depolarizes nociceptor terminals, thereby generating an action potential, which activates synaptic transmission in the dorsal horn of spinal cord. Such processes induce a characteristic set of responses, including hyperalgesia (an increased response to a stimulus that normally provoke pain) and allodynia (pain due to a stimulus that does not normally provoke pain) [1]. Free radicals in biological systems are natural products during interactions between cells, as well as between tissues and organs. However, an imbalance between endogenous oxidants and antioxidants can alter cell homeostasis. Oxidants target proteins, thereby inducing cell damage and contributing to cell death [35]. Free radicals also cause inflammation [36]. Consequently, treatment with antioxidants is a promising approach to control a wide array of pathophysiological events. KO    overt pain-like behavior, as well as mechanical and thermal hyperalgesia, which can be inhibited by analgesics [6]. Data indicates that O 2 − -induced pain depends on both direct superoxide anion effects, and indirect effects, partly via the production of peroxynitrite [37]. O 2 − injection triggers inflammatory pain by mechanisms involving cytokine, COX-2 and ET-1 synthesis [6,38,39]. Oxidative stress is involved in the pain of varied inflammatory diseases such as rheumatoid arthritis [40,41], gout [42], delayed onset muscle soreness [43] and diabetes [44].  Therefore, KO 2 -induced inflammatory pain is a useful model to study the action of analgesics that target oxidative stress-dependent events [12]. The therapeutic properties of naringenin include antinociception [15][16][17], as well as antioxidant [22,23,[44][45][46][47][48] and anti-inflammatory activities [18,19,21,22]. However, the analgesic effect and mechanisms of action of naringenin in a model o oxidative stress-triggered inflammatory pain remained to be determined. Herein, we provide the first evidence that naringenin inhibits KO 2 -induced overt-pain like behavior, MPO activity, mechanical hyperalgesia and thermal hyperalgesia via reduction of cytokine production and oxidative stress.
The activation of the NO−cGMP−PKG−K ATP channel signaling pathway leads to nociceptor hyperpolarization, thereby reducing nociceptor neuronal transmission [25][26][27][28]. The present results show that naringenin inhibits the KO 2 -induced mechanical and thermal hyperalgesia, at least in part, by activating the NO−cGMP−PKG−K ATP channel signaling pathway. Free radical damage to membrane components leads to cellular dysfunction [49], which, by exposing neoepitopes, will contribute to an autoimmune response, which is receiving increasing interest as to its role in longer-term nociception. GSH is an important redox cellular system, with efficacy partly driven by the direct interactions of sulfhydryl groups (-SH) with ROS, thereby promoting a direct detox reaction [50]. Naringenin was able to restore GSH levels, as well as inhibiting other indicants of oxidative stress, such as FRAP, TBARS, and NBT in the paw skin.  [38]. Not all cytokines are nociceptive, with IL-10 having anti-hyperalgesic effects [43]. IL-33 is a hyperalgesic cytokine that regulates the production of TNFα and IL-10 [52-55] Naringenin inhibited O 2 − -induced production of TNFα and IL-10, as well as IL-33 mRNA expression. Cytokine-induced inflammatory hyperalgesia in turn, depends, at least in part, on COX-2-dependent production of prostanoids such as prostaglandin E 2 and prostacyclin [1]. O 2 − also induces COX-2 mRNA expression, with celecoxib (a selective COX-2 inhibitor) diminishing O 2 − -induced pain [6]. Naringenin inhibited O 2 − -induced COX-2 mRNA expression. There is also a close relationship between cytokines and ET-1. Endothelin receptor antagonists inhibit cytokine-induced hyperalgesia and cytokines induce preproET-1 mRNA expression and ET-1 production [56,57]. Bosentan, an endothelin receptor antagonist, inhibits O 2 − -induced hyperalgesia, and O 2 − induces preproET-1 mRNA expression [39]. Naringenin inhibited O 2 −induced prepro-ET-1 mRNA expression. Furthermore, activation of the ET receptors promotes oxidative stress and reduces the free radical scavenging ability [58]. Therefore, it is likely that ET-1 also contributes to the regulation of oxidative stress. In fact, bosentan inhibited O 2 −induced oxidative stress [39]. Consistent with the naringenin inhibition of cytokine production as well as COX-2 and pre-proET-1 mRNA expression, naringenin inhibits the activation of the pro-inflammatory transcription factor, NFκB, in several models of inflammation. For instance, naringenin inhibits NFκB activation in dextran sulphate sodium-induced colitis [59], ethanol-induced liver injury [18], streptozotocin-induced diabetes in mice [60] and rats [61], and experimental stroke [23]. Furthermore, naringenin also inhibits NFκB DNA-binding activity in ovalbumin-induced asthma [62] as well as in in vitro studies [63,64]. As such, it is not unlikely that naringenin inhibits KO 2 -induced cytokines production, as well as COX-2 and preproET-1, by inhibiting NFκB induction and activity. Nrf2 is an important transcriptional regulator of the antioxidant response. In fact, Nrf2 activation is essential to the production of endogenous antioxidants such as GSH, thioredoxin system, HO-1, and NQO1 (NAD(P)H dehydrogenase, quinone 1) [65]. Keap-1 detects cellular environment changes such as oxidative stress, resulting in the activation of Nrf2, which readily translocates to the nucleus and upregulates downstream targets such as HO-1 [66]. Furthermore, the activation of the Nrf2/HO-1 pathway inhibits the production of inflammatory molecules such as TNFα [67][68][69], IL-6 [68,69] and IL-1β [67,68]. Nrf2 indirectly modulates NFκB activity [70], given that Nrf2 and NFκB compete for binding to the nuclear complex, coactivator p300/CBP (E1A binding protein p300/CREB-binding protein) [71]. Importantly, naringenin activates Nrf2 and is an agonist of the aryl hydrocarbon receptor, contributing to reducing the production of reactive oxygen species and inflammatory mediators, as well as modulating specific immune cell activity [47,72]. In the present study, naringenin inhibited the KO 2 -induced decrease in Nrf2 mRNA expression and increased HO-1 mRNA expression. This mechanism might account for the analgesic effect of naringenin in KO 2 -induced inflammation, by inhibiting pro-hyperalgesic cytokine production, and gp91 phox as well as inducing antioxidant molecules.
The data presented here suggest that naringenin inhibits O 2 − -induced inflammatory overt pain-like behavior, hyperalgesia, and neutrophil recruitment by activating the NO−cGMP −PKG−K ATP channel signaling pathway, as well as inhibiting oxidative stress and cytokine production, coupled to decreasing the mRNA of gp91 phox , IL-33, COX-2, and preproET-1, and increasing Nrf2 and HO-1 mRNA expression. These data show for the first time that naringenin inhibits inflammatory pain triggered by O 2 − and its mechanisms. Therefore, suggesting that naringenin is a promising therapeutic approach as an analgesic, antioxidant and anti-inflammatory compound, requiring further investigation in nociception, as well as in the array of other medical conditions where these pathophysiological changes are also evident [73].