CBI7256S0009-2797(15)00017-410.1016/j.cbi.2015.01.011Elsevier Ireland LtdFig. 1Chemical structures of hesperidin and its derivative hesperidin methyl chalcone.Fig. 2Effects of HMC in the pain-like behavior induced by acetic acid and PBQ. Mice were treated with HMC (3–100mg/kg, 150μL, i.p.), Indo (2.5mg/kg, 150μL i.p. diluted in Tris/HCl buffer, pH 8.0) or vehicle (saline or Tris/HCl buffer, 150μL, i.p.; data for vehicles were pooled for all experiments since there was no difference between vehicles) 1h before i.p. stimulus with acetic acid (panel A; 0.8% in saline, 10μL/mg), PBQ (panel B; 2% DMSO in saline, 1890μg/kg), or the respective vehicles. The cumulative number of abdominal writhings was evaluated for 20min. Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle), (ANOVA followed by Tukey’s t test)].Fig. 3HMC inhibits pain-like behaviors induced by formalin, capsaicin, and CFA. Mice were treated with HMC (30mg/kg, 150μL, i.p.), Indo (2.5mg/kg, 150μL i.p.) or vehicle 1h before the injection of formalin (panels A and B; 1.5% formalin in saline, 25μL, i.pl.), capsaicin (panels C and D; 1.6μg, 20μL, i.pl.), or CFA (panels E and F; 10μL, i.pl.). The total number of flinches (panels A, C and E), and the time spent licking the paw (panels B, D and F) were evaluated for 30min during formalin and CFA tests, and for 5min during capsaicin test. Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle), (ANOVA followed by Tukey’s t test)].Fig. 4HMC inhibits carrageenan-induced mechanical and thermal hyperalgesia, paw edema, and neutrophil recruitment. Mice were treated with HMC (3–100mg/kg, 150μL, i.p.), Indo (2.5mg/kg, 150μL i.p.) or vehicle 1h before carrageenan (300μg, 25μL, i.pl.) or saline (25μL, i.pl.) injection. The intensity of hyperalgesia to mechanical (panel A) and thermal (panel B) stimuli, and the paw edema formation (panel C) were measured 1–5h after carrageenan injection. Neutrophil recruitment (5h) was assessed by MPO activity assay. Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle); ∗∗p<0.05 vs. HMC 100mg/kg, (ANOVA followed by Tukey’s t test)].Fig. 5Orally administered HMC inhibits carrageenan-induced mechanical and thermal hyperalgesia, paw edema, and neutrophil recruitment. Mice were treated with HMC (10–100mg/kg, 100μL, p.o.) or vehicle (saline, 100μL, p.o.), 1h before carrageenan (300μg, 25μL, i.pl.) or saline (25μL, i.pl.) injection. The intensity of hyperalgesia to mechanical (panel A) and thermal (panel B) stimuli, and the paw edema (panel C) were measured 1–5h after carrageenan injection. Neutrophil recruitment was assessed by MPO activity assay at 5h. Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle), (ANOVA followed by Tukey’s t test)].Fig. 6HMC inhibits capsaicin (TRPV1 agonist)-induced mechanical and thermal hyperalgesia, paw edema, and neutrophil recruitment. Mice were treated with HMC (30mg/kg, 150μL, i.p.), Indo (2.5mg/kg, 150μL i.p.) or vehicle 1h before capsaicin (1.6μg, 20μL, i.pl.) or saline (20μL, i.pl.) injection. The intensity of hyperalgesia to mechanical (panel A) and thermal (panel B) stimuli, and the paw edema formation (panel C) were measured 1–5h after capsaicin injection. Neutrophil recruitment (5h) was assessed by MPO activity assay. Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle), (ANOVA followed by Tukey’s t test)].Fig. 7Effects of HMC in CFA-induced persistent inflammation and hyperalgesia. Mice were treated daily with HMC (30mg/kg, 150μL, i.p.), Indo (2.5mg/kg, 150μL i.p.) or vehicle for 7days, starting 24h after CFA (10μL, i.pl.) stimulus. The intensity of hyperalgesia to mechanical (panel A) and thermal (panel B) stimuli, and the paw edema (panel C) formation were assessed 1–7days after CFA injection. After all the measurements, plantar tissue samples were collected, and neutrophil (panel C) and macrophage recruitment (panel D) were assessed by determining MPO and NAG activity in samples, respectively. Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle), (ANOVA followed by Tukey’s t test)].Fig. 8HMC inhibits carrageenan-induced oxidative stress. Mice were treated with HMC (30mg/kg, i.p.), Indo (2.5mg/kg, 150μL i.p.) or vehicle 1h before carrageenan (300μg, 25μL, i.pl.) or saline (25μL, i.pl.) injection. Samples from the plantar tissue were collected 3h after stimulus injection, and oxidative stress was assessed by determining the ABTS radical scavenging ability (panel A; ABTS assay), ferric reducing antioxidant power (panel B; FRAP assay), reduced GSH levels (panel C), and superoxide anion production (panel D; NBT assay). Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle), (ANOVA followed by Tukey’s t test)].Fig. 9Inhibitory effect of HMC in carrageenan-induced cytokine production. Mice were treated with HMC (30mg/kg, 150μL, i.p.), Indo (2.5mg/kg, 150μL i.p.) or vehicle 1h before carrageenan (300μg, 25μL, i.pl.) or saline (25μL, i.pl.) injection. Samples from the plantar tissue were collected 3h after stimulus. TNF-α (panel A), IL-1β (panel B), IL-6 (panel C), and IL-10 (panel D) levels were determined by ELISA. Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle), (ANOVA followed by Tukey’s t test)].Fig. 10HMC inhibits carrageenan-induced NF-κB activation. Mice were treated with HMC (30mg/kg, 150μL, i.p.), Indo (2.5mg/kg, 150μL i.p.) or vehicle 1h before carrageenan (300μg, 25μL, i.pl.) or saline (25μL, i.pl.) injection. Samples from the plantar tissue were collected 3h after the stimulus, and the NF-κB activity was determined by measuring the levels of total and phospho-NF-κB p65 subunit by ELISA. Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle), (ANOVA followed by Tukey’s t test)].Fig. 11HMC does not induce liver damage or stomach lesion. Mice were treated daily with HMC (30mg/kg, 150μL, i.p.), Indo (2.5mg/kg, 150μL, i.p.) or vehicle for 7days. Plasma and stomach tissue samples were collected. Liver (panels A and B) and stomach (panel C) damage was assessed by determining plasmatic levels of AST and ALT, or by MPO activity assay, respectively. Results are expressed as means±SEM, n=6 mice per group [∗p<0.05 vs. saline control; #p<0.05 vs. 0mg/kg (vehicle), (ANOVA followed by Tukey’s t test)].Fig. 12Proposed targets of hesperidin methyl chalcone (HMC) in inflammation and inflammatory pain. Inflammatory stimuli can activate resident inflammatory cells to produce receptor-dependent NF-κB activation {a} and consequent production of inflammatory molecules (TNFα, IL-1β, IL-6) {b}, oxidative stress (superoxide anion production, diminished levels of reduced glutathione (GSH), reduced ability to reduce iron and scavenge free radicals) {c} and even anti-inflammatory molecules (IL-10) important to limit the inflammatory response {b} [15–20]. The inflammatory molecules are released and induce inflammation (edema {d}, leukocyte recruitment {e; myeloperoxidase and N-acetyl-β-d-glucosaminidase activity} and further activation of inflammatory cells {f}) and nociceptive responses (mechanical and thermal hyperalgesia {g} and overt pain-like behaviors {h}) [15–24]. Some stimuli such as formalin and capsaicin directly activate nociceptors. Formalin induces nociception by activating TRPA1 (transient receptor potential ankyrin type 1) {i} in the first phase and by inducing the production of inflammatory molecules in the second phase {a} [45–46]. Capsaicin activates TRPV1 (transient receptor potential vanilloid type 1) {j} in nociceptors, being able to directly activate nociceptors and induce pain [49]. The depolarization of neurons also induces the local production of neuropeptides, which stimulate resident cells {l} to produce cytokines and reactive oxygen species that, in turn, facilitate the neuronal depolarization {m} [49–52]. The present study demonstrates that (HMC) targets the steps {a–h} and {j–m} during inflammation and inflammatory pain.Table 1Summary of experimental approaches, results section and figure of experiments.Experimental approachData presented atDose–response of hesperidin methyl chalcone (HMC) in acetic acid-induced abdominal writhingsResults Section 3.1., Fig. 2AEffect of HMC (30mg/kg) in phenyl-p-benzoquinone (PBQ)-induced abdominal writhingsResults Section 3.1., Fig. 2BEfficacy of HMC (30mg/kg) in reducing paw flinches and licking induced by formalin, capsaicin, and Complete Freund’s Adjuvant (CFA)Results Section 3.2., Fig. 3Dose–response of HMC in carrageenan-induced mechanical hyperalgesia, thermal hyperalgesia, edema, and myeloperoxidase (MPO) activityResults Section 3.3., Fig. 4Evaluation of orally given HMC in carrageenan-induced mechanical hyperalgesia, thermal hyperalgesia, edema, and MPO activityResults Section 3.3., Fig. 5Effects of HMC (30mg/kg) in capsaicin-induced mechanical hyperalgesia, thermal hyperalgesia, edema, and MPO activityResults Section 3.4., Fig. 6Effects of post-treatment with HMC (30mg/kg) in CFA-induced mechanical hyperalgesia, thermal hyperalgesia, edema, and N-acetyl-β-d-glucosaminidase activityResults Section 3.5., Fig. 7Efficacy of HMC (30mg/kg) in inhibiting carrageenan-induced oxidative stressResults Section 3.6., Fig. 8Effects of HMC (30mg/kg) in carrageenan-induced cytokine productionResults Section 3.7., Fig. 9Effects of HMC (30mg/kg) in carrageenan-induced NF-κB activationResults Section 3.7., Fig. 10Evaluation of possible hepatic and gastric toxicity effects of HMC (30mg/kg) after 7 daily treatmentsResults Section 3.8., Fig. 11Protective effects of the flavonoid hesperidin methyl chalcone in inflammation and pain in mice: Role of TRPV1, oxidative stress, cytokines and NF-κBFelipe A.Pinho-RibeiroaMiriam S.N.HohmannaSergio M.BorghiaAna C.ZarpelonaCarla F.S.GuazelliaMarilia F.ManchopeaRubiaCasagrandebWaldiceu A.VerriJr.a⁎waldiceujr@yahoo.com.brwaverri@uel.braDepartamento de Ciências Patológicas, Universidade Estadual de Londrina-UEL, Rod. Celso Garcia Cid, Km 380, PR445, 86057-970, Cx. Postal 10.011, Londrina, Paraná, BrazilDepartamento de Ciências PatológicasUniversidade Estadual de Londrina-UELRod. Celso Garcia CidKm 380, PR445, 86057-970Cx. Postal 10.011LondrinaParanáBrazilbDepartamento de Ciências Farmacêuticas, Universidade Estadual de Londrina Londrina-UEL, Avenida Robert Koch, 60, Hospital Universitário, 86038-350 Londrina, Paraná, BrazilDepartamento de Ciências FarmacêuticasUniversidade Estadual de Londrina Londrina-UELAvenida Robert Koch60, Hospital Universitário86038-350 LondrinaParanáBrazil⁎Corresponding author at: Department of Pathology, Biological Sciences Centre, Londrina State University, Rod. Celso Garcia Cid, Km 380, PR445, CEP 86057-970, Cx. Postal 10.011, Londrina, Parana, Brazil. Tel.: +55 43 33714979; fax: +55 43 33714387.Highlights•Hesperidin methyl chalcone (HMC) inhibited inflammation and pain.•HMC inhibited TRPV1 agonist-induced inflammation and pain.•HMC inhibited oxidative stress, cytokine production and NF-κB activation.•HMC did not present hepatic or gastric toxic effect during 7days of treatment.AbstractCytokines and reactive oxygen species are inflammatory mediators that lead to increased sensitivity to painful stimuli, and their inhibition represents a therapeutic approach in controlling acute and chronic pain. The water-soluble flavonone hesperidin methyl chalcone (HMC) is used in the treatment of venous diseases, but its bioactivity as anti-inflammatory and analgesic is poorly understood. The present study evaluated the protective effects of HMC in widely used mouse models of acute and prolonged inflammation and pain. Male Swiss mice were treated with HMC (3–100 or 30mg/kg, intraperitoneally) or vehicle (saline) 1h before inflammatory stimuli. In overt pain-like behavior tests, HMC inhibited acetic acid- and phenyl-p-benzoquinone-induced writhing, and capsaicin-, Complete Freund’s Adjuvant (CFA)- and formalin-induced paw flinching and licking. HMC also inhibited carrageenan-, capsaicin- and CFA-induced mechanical and thermal hyperalgesia. Mechanistically, HMC inhibited carrageenan-induced cytokine (TNF-α, IL-1β, IL-6, and IL-10) production, oxidative stress and NF-κB activation. Furthermore, HMC did not cause gastric or hepatic injury in a 7days treatment protocol. Thus, this is the first report that HMC reduces inflammation and inflammatory pain by targeting TRPV1 (transient receptor potential vanilloid type 1) receptor activity, oxidative stress, cytokine production, and NF-κB activity, which suggests its potential applicability in inflammatory diseases.KeywordsHesperidin methyl chalconePainInflammationCytokineOxidative stressNuclear factor-κB1IntroductionFlavonoids are natural compounds widely distributed in vegetables and fruits constituting one of the largest groups of natural products known [1]. Flavonoids present biological properties with possible medical applications such as antihepatotoxic, antitumoral, antimicrobial, antioxidant, anti-allergic, anti-inflammatory and analgesic [1,2]. The flavonoid class of flavonones is found almost exclusively in citrus fruits, and therefore, citrus fruits and juices are the major sources of flavonones intake by humans [3]. Many of the beneficial effects obtained through the consumption of orange juice have been attributed to the high contents of flavonone hesperidin (ranging from 200 to 590mg/L of juice) [4].Hesperidin exhibits anti-inflammatory properties [5], but also presents low water solubility and is poorly absorbed in the small intestine as many other flavonoids. Therefore, the improvement of water solubility by methylation enhances the bioavailability, metabolic stability and tissue distribution [6]. The methylation of hesperidin under alkaline conditions produces hesperidin methyl chalcone (HMC) (Fig. 1), which presents higher water solubility than hesperidin [7].HMC exhibits vasoprotective activity and thus is found in drugs used to treat acute hemorrhoid and chronic venous insufficiency, such as Cyclo 3 Fort and Cirkan [8–10]. Interleukin (IL)-1β and tumor necrosis factor (TNF)-α induce vascular changes that lead to increased blood stasis and enhance the endothelial permeability. On the other hand, disturbed blood flow triggers inflammation and leads to the vicious circle of chronic inflammation observed in chronic venous insufficiency [11,12]. This condition results in redness, heat, edema, and pain. Importantly, HMC improves the quality of life in patients with chronic venous insufficiency [13,14]. Despite the possible anti-inflammatory and analgesic effect of flavonoids [1,2] and the clinical use of HMC in clinical inflammatory conditions [8–10], it remains to be determined the underlying anti-inflammatory mechanisms of HMC and its role as analgesic.Inflammatory mediators such as cytokines (IL-1β, IL-6, and TNF-α) and reactive oxygen species (ROS) sensitize and/or activate nociceptive neurons [15–17]. The sensitization occurs initially in nociceptive neurons near the injured tissue and leads to an increase in local pain sensation [18]. In the early stages of acute inflammation, these mediators are produced by resident cells (macrophages and mast cells) and by recruited neutrophils [15,16]. For instance, TNF-α triggers the release of IL-1β and IL-6 that, in turn, lead to the production of PGE2 by cyclooxygenase (COX)-2 during carrageenan inflammation [16,19]. The activation of nicotinamide adenine dinucleotide phosphate oxidase (NOX) by TNF-α and IL-1β is an essential step to upregulate COX-2 expression and, consequently, the PGE2 production [20].Neutrophils produce superoxide anion via NOX2 during inflammation at rates that overwhelm endogenous reduced glutathione (GSH) and other antioxidant mechanisms. The excessive superoxide anion switches endothelial cells to a pro-inflammatory phenotype and increases the vascular permeability [21,22]. Moreover, superoxide anion also induces the peripheral sensitization-related pathway of nuclear factor kappa B (NF-κB) [23,24]. Accordingly, reducing cytokine and ROS production is an important therapeutic approach for treating inflammatory diseases.Considering the vasoprotective activity of HMC in inflammatory chronic venous insufficiency and the potential anti-inflammatory and analgesic mechanisms of flavonoids, the aim of this study was to investigate the effects of HMC in models of inflammatory pain as well as the role of oxidative stress, cytokine and NF-κB in the protective effects of HMC.2Materials and methods2.1MaterialsThe following materials were obtained from the sources indicated: NaCl 0.9% (Fresenius Kabi Brasil Ltda. Aquiraz, CE, Brazil), dimethyl sulfoxide (DMSO), HMC and carrageenan (Santa Cruz Biotechnology, Santa Cruz, CA, USA); enzyme-linked immunosorbent assay (ELISA) Ready-SET-Go! (eBioscience, San Diego, CA, USA) and PathScan® (Cell Signaling, Beverly, MA, USA) kits; alanine amino transferase (ALT) and aspartate amino transferase (AST) diagnostic Labtest® kits (Lagoa Santa, MG, Brazil); capsaicin, Complete Freund’s Adjuvant, phenyl-p-benzoquinone (PBQ), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and Nitroblue Tetrazolium (NBT) (Sigma–Aldrich, St. Louis, MO, USA); acetic acid and formaldehyde (Mallinckrodt Baker, S.A., Mexico City, Mexico); and Indomethacin (Prodome, Campinas, SP, Brazil).2.2General experimental proceduresMice (n=6 per group per experiment, representative of two separated experiments) received intraperitoneal (i.p.) treatment with HMC (3, 10, 30, and 100mg/kg, 150μL), indomethacin (Indo) (2.5mg/kg in 150μL of Tris/HCl buffer, pH 8.0, i.p.), or vehicle (sterile saline, 150μL) 1h before inflammatory stimulus or in post-treatment protocols with CFA-stimulated animals, in which mice received daily treatments for 7days, starting 24h after intraplantar (i.pl.) CFA stimulus (10μL). For comparison purposes, HMC (10, 30, and 100mg/kg, 100μL) was also administrated by gavage (p.o.), 1h before carrageenan (300μg, 25μL) i.pl. injection. For hepatotoxicity and gastric injury tests, mice were treated daily with HMC (30mg/kg, 150μL, i.p.) or Indo (2.5mg/kg, 150μL, i.p.) during 7days. The doses of inflammatory stimuli and the dose of Indo were determined previously in our laboratory in pilot studies and in previous works [25–30]. The writhing response was induced by i.p. injection of acetic acid (0.8% in saline, volume/volume, 10μL/mg of body weight) or PBQ (1890μg/kg, diluted in 2% DMSO/saline solution, volume/volume). Paw flinching and licking were induced by i.pl. injection of formalin (1.5% in saline, volume/volume, 25μL), CFA (10μL), or capsaicin (1.6μg, diluted in saline, 25μL). Mechanical and thermal hyperalgesia, and paw edema were evaluated 1–5h after carrageenan (300μg, 25μL), capsaicin (1.6μg, 25μL), or saline (25μL) i.pl. injection, and 0–7days after CFA (10μL) or saline (10μL) i.pl. injection. All i.pl. stimuli induced hyperalgesia and edema only in the injected paw. Cytokine (TNFα, IL-1β, IL-6, and IL-10) levels, NF-κB activity, and oxidative stress were determined 3h (peak of hyperalgesia) after carrageenan (300μg, 25μL, i.pl.) injection. Oxidative stress was assessed by GSH level, ABTS radical scavenger activity, ferric reducing antioxidant power (FRAP), and nitroblue tetrazolium (NBT) reduction assays. Neutrophil migration to plantar tissue was evaluated by myeloperoxidase (MPO) assay 5h after carrageenan (300μg, i.pl.) or capsaicin (1.6μg, i.pl.) injection, or 7days after CFA (10μL, i.pl.) injection. Macrophage accumulation in the plantar tissue was evaluated by N-acetyl-β-d-glucosaminidase (NAG) assay 7days after CFA (10μL, i.pl.) injection. MPO activity in the stomach, and AST and ALT blood levels were determined in mice treated with daily i.p. injections of vehicle (saline, 150μL), HMC (30mg/kg, 150μL), or Indo (2.5mg/kg, 150μL) during 7days. The vehicle data are representative of both HMC and Indo vehicles as no difference occurred in the results when they were compared by statistical analysis. The individuals evaluating the responses were unaware of the treatments. Table 1 summarizes the experimental approaches described above, results section and figure for each experiment.2.3AnimalsMale Swiss mice (25g), from the Londrina State University, Londrina, Parana, Brazil, were used in this study. Mice were housed in standard clear plastic cages with free access to water and food, and temperature of 23°C±2. A 12/12h light/dark cycle was used with lights on at 6h and off at 18h. All behavioral tests were performed between 9a.m. and 5p.m. in a temperature-controlled room (23°C±2). Animal care and handling procedures were in accordance with the International Association for Study of Pain (IASP) guidelines, and approved by the Ethics Committee of the Londrina State University (OF. CIRC. CEUA 096/2013, process number 7739.2013.96).2.4Writhing behavior testsAcetic acid- and PBQ-induced abdominal writhings were performed as described previously [26]. Acetic acid (0.8% in saline, volume/volume, 10μL/mg), PBQ (1890μg/kg, diluted in 2% DMSO/saline solution, volume/volume), or the respective vehicles were injected into the peritoneal cavities of mice. Each mouse was placed individually in a large glass cylinder, and the intensity of nociceptive response was quantified by counting the total number of writhings between 0 and 20min after stimulus.2.5Paw flinching and licking testsIn the formalin test, the number of paw flinches and the time spent licking the ipsilateral paw were determined between 0 and 30min after i.pl. injection of formalin [31]. The 30min period was divided into intervals of 5min, and clearly demonstrated the presence of first (0–5min) and second (10–30min) phases. Results are expressed as the cumulative number of paw flinches and the time spent licking the paw observed in each phase.In CFA- and capsaicin-induced overt pain tests, the number of paw flinches and the time spent licking the ipsilateral paw were observed for 30 and 5min after stimulus, respectively. Results are expressed as cumulative time spent licking the paw or total number of paw flinches.2.6Electronic pressure-meter testMechanical hyperalgesia was assessed as reported previously [32]. In a quiet room, mice were placed in acrylic cages (12×10×17cm) with wire grid floors, 30min before testing. The test consisted of evoking a hind paw reflex with a hand-held force transducer (electronic anesthesiometer; Insight, Ribeirao Preto, SP, Brazil) adapted with a 0.5mm2 polypropylene tip. The investigator was trained to apply the tip perpendicularly to the central area of the hind paw with a gradual increase in pressure. The end point was characterized by the removal of the paw followed by clear flinching movements, and the intensity of pressure was recorded automatically. The animals were tested before (basal) and after stimulus injection, and the value for each interval was an average of three measurements. The results are expressed by delta (Δ) withdrawal threshold (in g), calculated by subtracting the basal mean measurements from the mean measurements obtained at 1, 3 or 5h after i.pl. stimuli with the hyperalgesic agents.2.7Hot plate testMice were placed in the hot plate apparatus (EFF 361; Insight, Ribeirao Preto, SP, Brazil) maintained at 55°C. The first ipsilateral hind paw flexion reflex (nociceptive behavior) was registered. Two basal latencies at least 10min apart were determined for each mouse and the maximum latency (cut-off) was set at 20s to avoid tissue damage [33].2.8GSH levelsThe plantar tissue samples were collected 3h after stimulus injection, and the levels of GSH were determined using a spectrophotometric method [34]. Samples were homogenized using Tissue-Tearor (Biospec®) in ice-cold EDTA buffer (4mL, 0.02M). Homogenates (2.5mL) were treated with ddH2O (2mL) and trichloroacetic acid (0.5mL, 50% weight/volume). After 15min, the homogenates were centrifuged at 1500g for 15min, and the supernatant (1mL) was mixed with 2mL of a solution containing Tris 0.4M (pH 8.9) plus 50μL of DTNB. After 5min, the measurements were performed in 412nm (Shimadzu UV–Vis Spectrophotometer UV-1650, Shimadzu Corporation, Kyoto, Japan). The results are expressed as nmol of GSH per mg of tissue using a standard GSH curve (0.09–100nmol).2.9ABTS and FRAP assaysThe ability of samples to resist oxidative damage was determined by its free radical scavenging (ABTS assay) and ferric reducing (FRAP assay) properties. The tests were adapted to a 96-well microplate format from previously described assays [36]. Plantar tissue samples were collected 3h after i.pl. stimulus with carrageenan (300μg, 25μL) and homogenized immediately in ice-cold KCl buffer (500μL, 1.15% weight/volume). The homogenates were centrifuged (200g×10min×4°C), and the supernatants were used in both assays. Diluted ABTS solution (200μL) was mixed with 10μL of sample in each well. After 6min of incubation (25°C), the absorbance was measured at 730nm (Multiskan GO Microplate Spectrophotometer, ThermoScientific, Vantaa, Finland). For FRAP assay, the supernatants (10μL) were mixed with the freshly prepared FRAP reagent (150μL). The reaction mixture was incubated at 37°C for 30min, and the absorbance was measured at 595nm (Multiskan GO ThermoScientific). The results of ABTS and FRAP assays were equated against a standard Trolox curve (0.02–20nmol).2.10MPO activityThe neutrophil recruitment to the paw skin was evaluated by the MPO kinetic-colorimetric assay [36,37]. Samples were homogenized using a Tissue-Tearor (Biospec®) in ice-cold K2HPO4 buffer (400μL, 50mM, pH 6.0) containing HTAB (0.5% weight/volume), and the homogenates were centrifuged (16,100g×2min×4°C). The supernatants (30μL) were mixed with K2HPO4 buffer (200μL, 50mM, pH 6.0) containing o-dianisidine dihydrochloride (0.0167%, weight/volume) and hydrogen peroxide (0.05%, volume/volume). The absorbance was determined after 5min at 450nm (Multiskan GO ThermoScientific). The results of MPO activity are expressed as the number of neutrophils per mg of tissue by using a standard curve of neutrophils (196–400,000 cells).2.11NAG activityNAG (N-acetyl-β-d-glucosaminidase) activity was determined by an adapted colorimetric method previously described [38]. Briefly, the supernatants (20μL), obtained in the MPO activity assay, were placed in a 96-well plate and mixed with K2HPO4 buffer (80μL, 50mM, pH 6.0). The reaction was initiated by the addition of K2HPO4 buffer (100μL, 50mM, pH 6.0) containing 4-nitrophenyl N-acetyl-β-d-glucosaminide substrate (2.24mM). The plate was incubated at 37°C for 10min, and glycine buffer (100μL, 0.2M pH 10.6) was added. The enzymatic activity was determined spectrophotometrically at 400nm (Multiskan GO ThermoScientific). The results of NAG activity are expressed as the number of macrophages per mg of tissue by using a standard curve of macrophages (196–400,000 cells).2.12NBT assayThe superoxide anion production was performed in tissue homogenates as previously described [38]. Briefly, the homogenates (50μL) were mixed with NBT solution (100μL, 0.1% in ddH2O, weight/volume) and incubated in 96-well plates at 37°C for 1h. The aqueous mixture was removed carefully from the wells, and the formazan precipitate was solubilized by adding KOH (120μL, 2M) and DMSO (140μL) solutions in each well. The absorbance was measured at 600nm (Multiskan GO ThermoScientific). The weight of each sample was used for data normalization, and the results are expressed as NBT reduction (OD at 600nm).2.13Cytokine measurementThe paw skin samples were removed and homogenized in 500μL of ice-cold PBS containing protease inhibitors. Concentrations of TNF-α, IL-1β, IL-6, and IL-10 levels were determined using ELISA Ready-SET-Go! kits (eBioscience) according to the manufacturer’s directions. The results were obtained by comparing the optical density of samples at 450nm (Multiskan GO ThermoScientific) with standard curves, and expressed as picograms (pg) of cytokine per 100mg of tissue.2.14NF-κB activityThe paw skin samples were collected and homogenized in ice-cold lysis buffer (Cell Signaling). The homogenates were centrifuged (200g×10min×4°C), and the supernatants were used to assess the levels of phosphorylated and total NF-κB p65 subunit by ELISA using PathScan® kits (Cell Signaling) according to the manufacturer’s directions. The results are expressed as OD of samples (total p65/phospho-p65) at 450nm (Multiskan GO ThermoScientific).2.15Motor performanceIn order to discard possible muscle relaxant or sedative effects of HMC, motor performance was evaluated using the rota-rod test [39]. The apparatus consisted of a bar with a diameter of 2.5cm, subdivided into four compartments (EFF 411; Insight, Ribeirao Preto, SP, Brazil). The bar rotated at a constant speed of 22 rotations per min. The animals were selected previously by eliminating those mice who did not remain on the bar for two consecutive periods of 180 s. Mice were treated with vehicle (sterile saline, 150μL, i.p.) or HMC (30mg/kg, 150μL, i.p.) and exposed to the test (2, 4, and 6h after the treatment). The cut-off time used was 180s.2.16Hepatic and gastric toxicityPlasma levels of AST and ALT were used as indicators of hepatotoxicity [40]. These assays were performed using a diagnostic kit from Labtest® according to the manufacturer’s directions. The MPO activity was evaluated in stomach samples as indicative of gastric injury. Indo (2.5mg/kg in 150μL of Tris/HCl buffer, pH 8.0, i.p.), a non-selective COX inhibitor, was used as a positive control of toxicity.2.17Data analysesResults are presented as means±SEM of 6 mice per group per experiment, each experiment was performed twice. Two-way ANOVA was used to compare the groups and doses at all times when the parameters were measured at different times after the stimulus injection. The analyzed factors were treatments, time and time versus treatment interaction. One-way ANOVA followed by Tukey’s t-test was performed for each time-point. P<0.05 was considered significant.3Results3.1HMC inhibits the writhing response induced by acetic acid and PBQThe antinociceptive effect of HMC was tested initially in the models of overt pain-like behavior of writhings induced by i.p. injection of acetic acid or PBQ (Fig. 2). Firstly, mice were treated with HMC (3–100mg/kg, i.p., diluted in saline), indomethacin (Indo; 2.5mg/kg, i.p., diluted in Tris/HCl buffer), or vehicle (data for the vehicles were pooled for all experiments since there was no difference between vehicles) 1h before i.p. stimulus with acetic acid (0.8% volume/volume, 10μL/mg). Treatment with HMC showed a dose-dependent inhibition of writhing response induced by acetic acid with maximal effect at the doses of 30 and 100mg/kg compared to the vehicle group (Fig. 2A). No statistical difference was found between 30 and 100mg/kg treatments, indicating that a maximal effect of HMC was achieved at the dose of 30mg/kg (Fig. 2A). Thus, the dose of 30mg/kg was chosen for the following experiments using overt pain models. Indo (2.5mg/kg) also reduced the number of writhings induced by acetic acid (Fig. 2A). In another experiment, mice were treated with HMC (30mg/kg, i.p.), Indo (2.5mg/kg, i.p.), or vehicle, 1h before i.p. stimulus with PBQ (1890μg/kg, 2% DMSO in saline). The treatment with HMC and Indo also inhibited the writhing response induced by PBQ (Fig. 2B).3.2HMC inhibits paw flinching and licking behaviors induced by formalin, capsaicin, and CFAMice were treated with HMC (30mg/kg, i.p.), Indo (2.5mg/kg, i.p.) or vehicle 1h before i.pl. stimuli. Treatment with HMC and Indo inhibited the second phase of formalin (1.5%, 25μL)-induced paw flinching (Fig. 3A) and licking (Fig. 3B), but not the first phase. In the following experiment, mice received HMC, Indo (2.5mg/kg, i.p.) or vehicle 1h before capsaicin, a transient receptor potential vanilloid type 1 (TRPV1) agonist, (1.6μg, 20μL, i.pl.) injection. The number of paw flinches (Fig. 3C) and the time spent licking the paw (Fig. 3D) were significantly inhibited by HMC, but not by Indo. In CFA-induced (10μL) paw flinching and licking behaviors, the treatment with HMC also inhibited both the number of paw flinches (Fig. 3E) and the time spent licking the paw (Fig. 3F), while no inhibition was observed in Indo-treated mice.3.3HMC inhibits carrageenan-induced mechanical and thermal hyperalgesia, paw edema, and neutrophil recruitmentThe antinociceptive effect of HMC (3–100mg/kg, i.p., 1h) was assessed in the carrageenan (300μg, 25μL, i.pl.)-induced paw inflammation model. HMC inhibited carrageenan-induced mechanical (Fig. 4A) and thermal (Fig. 4B) hyperalgesia in a dose-dependent manner. HMC inhibited carrageenan-induced mechanical hyperalgesia at 3 and 5h after the stimulus at the doses of 30 and 100mg/kg and the dose of 10mg/kg inhibited mechanical hyperalgesia only 5h after stimulus (Fig. 4A). No significant effect was observed at the dose of 3mg/kg of HMC. Indo (2.5mg/kg, i.p.) inhibited mechanical hyperalgesia at 3 and 5h after stimulus. In the hot plate test, treatment with HMC at the doses of 30 and 100mg/kg and with Indo inhibited carrageenan-induced thermal hyperalgesia between 1 and 5h after stimulus with significant difference between the two doses of HMC at the last time point of measurement (5h). The doses of 3 and 10mg/kg had no effect in thermal hyperalgesia (Fig. 4B).Paw edema and neutrophil recruitment induced by carrageenan were also evaluated on the same animals. The treatment with HMC also inhibited carrageenan-induced paw edema at the doses of 30 and 100mg/kg at 1, 3 and 5h after stimulus, and Indo treatment reduced paw edema between 1 and 5h as well (Fig. 4C). After the last measurement (5h), mice were terminally anesthetized and samples of plantar tissue were collected for quantification of neutrophil recruitment by MPO activity. The treatment with HMC reduced MPO activity in the paw skin in a dose-dependent manner (Fig. 4D). The efficacy of HMC in reducing neutrophil migration was observed at the doses of 30 and 100mg/kg, without significant difference between these two doses. No significant effect was observed with the doses of 3 and 10mg/kg of HMC or with Indo. Similar results on mechanical hyperalgesia (Fig. 5A), thermal hyperalgesia (Fig. 5B), paw edema (Fig. 5C) and MPO activity (Fig. 5D) were obtained when HMC was given orally (Fig. 5), indicating HMC can also be used by oral route. Thus, the dose of 30mg/kg of HMC was chosen for all following experiments.HMC (30mg/kg) did not alter the motor response of the animals in the rota-rod test at 2, 4, and 6h after treatment (data not shown). These time points were based on the 1h treatment plus 1, 3 and 5h to measure carrageenan-induced hyperalgesia (Figs. 4 and 5). Therefore, HMC exerts its biological properties by reducing inflammatory pain and not by inducing muscle-relaxing or sedative effects.3.4HMC inhibits capsaicin-induced mechanical and thermal hyperalgesia, paw edema, and neutrophil recruitmentMice were treated with HMC at the dose of 30mg/kg or with Indo (2.5mg/kg, i.p.) (Fig. 6), 1h before stimulus with capsaicin (1.6μg, 20μL, i.pl). HMC inhibited capsaicin-induced mechanical (Fig. 6A) and thermal hyperalgesia (Fig. 6B) between 1 and 5h after stimulus injection. Indo inhibited mechanical hyperalgesia between 1 and 5h (Fig. 6A) and thermal hyperalgesia (Fig. 6B) between 3 and 5h after capsaicin injection. HMC also inhibited capsaicin-induced paw edema at 1 and 3h after stimulus without effect at 5h (Fig. 6C). The capsaicin-induced MPO activity was also reduced by HMC treatment (Fig. 6D). Indo did not affect capsaicin-induced paw edema (Fig. 6C) and MPO activity (Fig. 6D).3.5Post-treatment with HMC inhibits CFA-induced mechanical and thermal hyperalgesia, paw edema, and neutrophil and macrophage recruitmentThe protective effect of post-treatment with HMC was evaluated in CFA-induced paw inflammation. Mice were treated daily with HMC (30mg/kg) or Indo (2.5mg/kg, i.p.) for 7days, starting 24h after a single i.pl. injection of CFA (10μL). The treatment with HMC inhibited CFA-induced mechanical hyperalgesia (Fig. 7A), thermal hyperalgesia (Fig. 7B) and paw edema (Fig. 7C) at all time-points of measurement (1–7days after stimulus). Indo treatment also inhibited CFA-induced mechanical hyperalgesia (Fig. 7A), thermal hyperalgesia (Fig. 7B) and paw edema (Fig. 7C), except thermal hyperalgesia at the day 2 (Fig. 7B). After all measurements, mice were terminally anesthetized and the plantar tissues were collected for MPO and NAG activity assays. HMC inhibited CFA-induced MPO (neutrophil; Fig. 7D) and NAG (macrophage; Fig. 7E) activity compared to the vehicle group. Indo did not alter significantly CFA-induced MPO and NAG activity (Fig. 7D and E).3.6HMC inhibits the carrageenan-induced oxidative stressThe protective effect of HMC in carrageenan-induced oxidative stress was assessed. Mice were treated with HMC (30mg/kg) or Indo (2.5mg/kg) 1h before i.pl. injection of carrageenan (300μg, 25μL, i.pl.). Mice were terminally anesthetized 3h after the stimulus and samples from the plantar skin were collected to evaluate free radical scavenging ability (ABTS), ferric reducing antioxidant power (FRAP), reduced glutathione (GSH) levels, and superoxide anion levels (NBT reduction) (Fig. 8). Carrageenan-induced the reduction of ABTS scavenging ability (Fig. 8A), FRAP (Fig. 8B), and GSH levels (Fig. 8C), which were prevented by HMC treatment. In agreement, treatment with HMC also inhibited the carrageenan-induced increase of superoxide production (NBT assay, Fig. 8D) in plantar skin. Indo exhibited no antioxidant effect (Fig. 8).3.7HMC inhibits inflammation by reducing cytokine production and NF-κB activationThe levels of TNF-α, IL-1β, IL-6, and IL-10 were evaluated in samples from plantar tissue. Mice were treated with HMC (30mg/kg) or Indo (2.5mg/kg) followed by i.pl. injection with carrageenan, and plantar tissue samples collection at 3h after stimulus as described before. Treatment with HMC (30mg/kg, i.p.), but not with Indo, reduced carrageenan-induced increases in TNF-α, IL-1β, IL-6, and IL-10 levels (Fig. 9A–D, respectively).Considering the crucial role of NF-κB activation in inflammation and cytokine production, we also investigated if HMC modulates the activation of NF-κB by the levels of total and phosphorylated NF-κB p65 subunit. In agreement with the inhibition of cytokine production by HMC, it also inhibited carrageenan-induced NF-κB p65 activation (Fig. 10), while Indo was not capable of such inhibition.3.8HMC does not induce liver or stomach injuryPlasma levels of AST and ALT, and the MPO activity in the stomach were measured in order to evaluate whether HMC treatment during 7days induces liver damage or gastric injury, respectively. Mice were treated with vehicle, HMC (30mg/kg), or Indo (2.5mg/kg) daily during 7days. Treatment with HMC (Fig. 11A and B) did not alter the plasmatic AST and ALT levels compared to the vehicle group, indicating that the compound does not induce liver damage. The positive control Indo (non-selective COX inhibitor) induced significant increases in AST and ALT plasma levels (Fig. 11A and B). Some anti-inflammatory drugs, such as Indo, can induce gastric lesion due to their activity as inhibitors of COX-1 even if administrated by i.p. route. Moreover, the use of these anti-inflammatory drugs for 7days is a common practice, and in this period (Fig. 11C) HMC-treated mice showed no alteration of MPO activity in stomach samples compared to the vehicle group. As expected, treatment with Indo during 7days induced a significant increase of MPO activity in stomach samples, indicating the gastric lesion (Fig. 11C). Supporting previous reports [41], the treatment with HMC over 7days is safe regarding liver damage and gastric mucosal injuries.4DiscussionThe treatment with HMC improves vascular tonus and reduces symptoms of vascular diseases such as acute hemorrhoid and chronic venous disease [14,42], but its mechanisms of action remain poorly understood. In this study, we investigated the role of HMC in pain and inflammation. HMC (pre- or post-treatment) inhibited pain-like behavior induced by varied stimuli, which include acetic acid, PBQ, formalin, capsaicin, CFA and carrageenan, and also the biomarkers of tissue inflammation such as oxidative stress, leukocyte recruitment, cytokine production and NF-κB activation. Although these stimuli are different in their chemical nature and in the way they induce the inflammatory response, they share some mechanisms that may help explain the broad protective activity of HMC observed in the present study. In fact, most of them depend on cytokine production and oxidative stress to induce inflammation and pain as discussed below and at Fig. 12 that summarizes the inflammatory mechanisms investigated in this study and targets of HMC.Formalin induces a biphasic nociceptive response. The first phase of formalin test (neurogenic phase, 0–5min) depends on TRPA1 (transient receptor potential ankyrin type 1) channels activation in primary nociceptive neurons [44,45], while the second phase (10–30min) depends on cytokine production [46]. In agreement with the HMC inhibition of the second phase of formalin test, it also inhibited the acetic acid- and PBQ-induced writhings, which are also dependent on the release of inflammatory cytokines [26,47]. Thus, it is plausible that HMC exhibits its protective effect in the acetic acid- and PBQ-induced writhings and second phase of formalin test by targeting cytokine production without affecting TRPA1 channels. HMC inhibited capsaicin-induced overt pain-like behavior, suggesting that HMC could also target the redox-sensitive and neuron-selective TRPV1 (transient receptor potential vanilloid type 1) channel activity (Fig. 12) as observed with other flavonoids such as eriodictyol, baicalin, and vitexin [25,47,48].Corroborating the protective roles of HMC on cytokine- and TRPV1-mediated inflammation and pain, we found that HMC inhibited the hyperalgesia induced by several inflammatory stimuli that depend on these mechanisms, including the capsaicin-induced neurogenic inflammation. The direct activation of TRPV1-expressing primary neurons by capsaicin induces pain and lead to peripheral release of pro-inflammatory neuropeptides that, in turn, produce neurogenic inflammation (Fig. 12) [49]. Thus, the inhibition of TRPV1 activation also represents an important anti-inflammatory mechanism. On the other hand, during carrageenan- and CFA-induced inflammation, the expression of TRPV1 channels is upregulated in primary nociceptive neurons and its antagonism represents an important analgesic approach to treat inflammatory pain (Fig. 12) [50–52].Treatment with HMC inhibited the mechanical and thermal hyperalgesia induced by carrageenan and CFA, and the inflammation induced by both stimuli are mediated by a cytokine cascade which, in turn, induces edema and the recruitment of neutrophils and macrophages [19,53]. Recruited neutrophils and macrophages produce more hyperalgesic mediators, such as TNF-α, IL-1β, prostaglandin E2 and superoxide anion (Fig. 12) [54,55]. Thus, while inflammatory cytokines contribute by upregulating TRPV1 channels, redox active molecules that are produced by recruited cells increase the activity of TRPV1 channels by modifying its cysteine residues leading to hyperalgesia [56,57]. Moreover, superoxide anion and its derivatives are produced during inflammation and released in rates that overwhelm endogenous antioxidant defenses. Such an imbalance leads to depletion of GSH levels, oxidation of cysteine groups, and maladaptive activation of the NF-κB pathway [58] that, in turn, induces more superoxide production and contributes to edema [59], leukocyte recruitment [60], inflammatory cytokine production, and pain [61]. These cytokines induce cyclooxygenase (COX) expression and activation resulting in prostaglandin E2 production that induces nociceptor sensitization observed as hyperalgesia [19]. In line with this cascade of events, indomethacin (Indo; non-selective COX inhibitor) reduced inflammatory pain, but not cytokine production, oxidative stress, and NF-κB activation.Interestingly, the anti-inflammatory effects of HMC are not dependent on increased levels of IL-10, which corroborates the hypothesis that HMC targets upstream mechanisms that provide the link between these different stimuli to induce cytokine production, such as ROS- and TLR-related NF-κB activation [62]. In this sense, by targeting NF-κB activation, HMC inhibits the subsequent events of inflammation, such as oxidative stress, cytokine production, and pain, as well as its persistence. To our knowledge, this is the first study to demonstrate that HMC inhibits NF-κB activation in vivo.The inhibition of NF-κB by HMC lines up well with the protective effects that have been reported elsewhere, such as the reduction of capillary filtration rate and permeability during heat stress or in chronic venous disease [63,64], and the suppression of leukocyte adhesion to endothelial cells [65]. However, the in vivo anti-inflammatory activity of HMC has never been demonstrated until now. Here, we found that HMC treatment inhibits paw edema, neutrophil and macrophage recruitment in plantar tissue, and that this inhibition was related to suppression of oxidative stress, cytokine production, and NF-κB activation.Importantly, no signs of toxicity were found by HMC treatment during 7days, which is a regular time of treatment with anti-inflammatory drugs. Corroborating, it has been reported that HMC is safe in patients receiving 150mg daily for 90days [66]. HMC seems to be safe even when administrated in massive doses as much as 15g daily [41], which highlights its possible medical application in several diseases. On the other hand, Indo induced hepatic and gastric lesion when administered during 7days demonstrating HMC presents better safety profile than a standard COX inhibitor.5ConclusionsThe present study suggests that HMC exhibits anti-inflammatory and analgesic activities by reducing: (a) TRPV1 channel agonist-induced inflammation and pain as observed in capsaicin model; (b) leukocyte recruitment to the inflammatory foci, which is a contributing analgesic mechanism; (c) post-treatment with HMC inhibited CFA-induced prolonged inflammation and pain; (d) oxidative stress, as observed by prevention of carrageenan-induced superoxide anion production and depletion of the antioxidant capacity; and (e) NF-κB activation and NF-κB-related cytokine production in carrageenan-induced inflammation. Fig. 12 summarizes the anti-inflammatory and analgesic mechanisms of HMC. Moreover, no toxic or side effects that are induced by the anti-inflammatory COX inhibitor Indo were observed in HMC-treated animals. Taken together, these results suggest that HMC is a promising analgesic and anti-inflammatory compound that deserves to be further investigated.Conflict of interestThe authors declare that there are no conflicts of interest.Authors’ contributionsF.A.P.R, M.S.N.H., S.M.B., A.C.Z., C.F.S.G. and M.F.M. performed experiments and contributed to acquisition of data. F.A.P.R., M.S.N.H., S.M.B., A.C.Z., C.F.S.G., M.F.M., R.C. and W.A.V.J. analyzed and interpreted the data. F.A.P.R., M.S.N.H., R.C. and W.A.V.J. drafted and revised manuscript. F.A.P.R., RC and W.A.V.J. contributed to experimental design. R.C. and W.A.V.J. contributed to the conception of the study. All authors read and approved the final version of the manuscript.Transparency DocumentThe Transparency document associated with this article can be found in the online version.Transparency documentAcknowledgmentsThis work was supported by Brazilian Grants from Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministério da Ciência, Tecnologia e Inovação (MCTI), Secretaria da Ciência, Tecnologia e Ensino Superior (SETI)/Fundação Araucária and Governo do Estado do Paraná.References[1]W.A.VerriJr.F.T.M.C.VicentiniM.M.BaracatS.R.GeorgettiR.D.R.CardosoT.M.CunhaChapter 9 – flavonoids as anti-inflammatory and analgesic drugs: mechanisms of action and perspectives in the development of pharmaceutical formsR.Atta urStud. Nat. Prod. 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