Marília F. Manchope

Protective effects of the flavonoid hesperidin methyl chalcone in inflammation and pain in mice: role of TRPV1, oxidative stress, cytokines and NF-κB.

Cytokines 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 30 mg/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 Freunds 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 7 days 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.

The superoxide anion donor, potassium superoxide, induces pain and inflammation in mice through production of reactive oxygen species and cyclooxygenase-2

It is currently accepted that superoxide anion (O2 •−) is an important mediator in pain and inflammation. The role of superoxide anion in pain and inflammation has been mainly determined indirectly by modulating its production and inactivation. Direct evidence using potassium superoxide (KO2), a superoxide anion donor, demonstrated that it induced thermal hyperalgesia, as assessed by the Hargreaves method. However, it remains to be determined whether KO2 is capable of inducing other inflammatory and nociceptive responses attributed to superoxide anion. Therefore, in the present study, we investigated the nociceptive and inflammatory effects of KO2. The KO2-induced inflammatory responses evaluated in mice were: mechanical hyperalgesia (electronic version of von Frey filaments), thermal hyperalgesia (hot plate), edema (caliper rule), myeloperoxidase activity (colorimetric assay), overt pain-like behaviors (flinches, time spent licking and writhing score), leukocyte recruitment, oxidative stress, and cyclooxygenase-2 mRNA expression (quantitative PCR). Administration of KO2 induced mechanical hyperalgesia, thermal hyperalgesia, paw edema, leukocyte recruitment, the writhing response, paw flinching, and paw licking in a dose-dependent manner. KO2 also induced time-dependent cyclooxygenase-2 mRNA expression in the paw skin. The nociceptive, inflammatory, and oxidative stress components of KO2-induced responses were responsive to morphine (analgesic opioid), quercetin (antioxidant flavonoid), and/or celecoxib (anti-inflammatory cyclooxygenase-2 inhibitor) treatment. In conclusion, the well-established superoxide anion donor KO2 is a valuable tool for studying the mechanisms and pharmacological susceptibilities of superoxide anion-triggered nociceptive and inflammatory responses ranging from mechanical and thermal hyperalgesia to overt pain-like behaviors, edema, and leukocyte recruitment.

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.

Vanillic Acid Inhibits Inflammatory Pain by Inhibiting Neutrophil Recruitment, Oxidative Stress, Cytokine Production, and NFκB Activation in Mice

Vanillic acid (1) is a flavoring agent found in edible plants and fruits. It is an oxidized form of vanillin. Phenolic compounds form a substantial part of plant foods used as antioxidants with beneficial biological activities. These compounds have received considerable attention because of their role in preventing human diseases. Especially, 1 presents antibacterial, antimicrobial, and chemopreventive effects. However, the mechanisms by which 1 exerts its anti-inflammatory effects in vivo are incompletely understood. Thus, the effect of 1 was evaluated in murine models of inflammatory pain. Treatment with 1 inhibited the overt pain-like behavior induced by acetic acid, phenyl-p-benzoquinone, the second phase of the formalin test, and complete Freunds adjuvant (CFA). Treatment with 1 also inhibited carrageenan- and CFA-induced mechanical hyperalgesia, paw edema, myeloperoxidase activity, and N-acetyl-β-D-glucosaminidase activity. The anti-inflammatory mechanisms of 1 involved the inhibition of oxidative stress, pro-inflammatory cytokine production, and NFκB activation in the carrageenan model. The present study demonstrated 1 presents analgesic and anti-inflammatory effects in a wide range of murine inflammation models, and its mechanisms of action involves antioxidant effects and NFκB-related inhibition of pro-inflammatory cytokine production.

Naringenin: an analgesic and anti-inflammatory citrus flavanone

In this editorial, we discuss recent evidence from our research group on the analgesic and antiinflammatory mechanisms of the flavonoid naringenin (4’,5,7-tryhidroxy-flavanone). Flavonoids are polyphenolic compounds found in human diet [1]. Naringenin belongs to flavanone class of flavonoids, and it is mainly found in citrus fruits including, lemon, orange, tangerine and grapefruit [1-5]. The antioxidant activity is the most recognized effect of flavonoids, which depends, for instance, on hydrogen donation and electron stabilization in the phenolic rings [1]. Naringenin presents therapeutic effect in several models of inflammatory pain [2, 3, 5]. Naringenin inhibits the pain-like behavior induced by inflammatory stimuli such as phenyl-p-benzoquinone, acetic acid, formalin, complete Freund ́s adjuvant, capsaicin, carrageenan [2], superoxide anion [3], and LPS [5]. Moreover, naringenin inhibits UVB irradiation-induced skin inflammatory edema, cytokine production, myeloperoxidase activity, matrix metalloproteinase-9 activity, and oxidative stress [4]. Pathogen (PAMPs) and damage (DAMPs) associated molecular patterns and inflammatory mediators activate resident macrophages. Resident macrophages produce chemotactic molecules to recruit leukocytes to the inflammatory foci, mainly neutrophils in the early events of inflammation. Activated macrophages and neutrophils induce oxidative stress by producing superoxide anion and other reactive oxygen (ROS) and nitrogen species. Naringenin inhibits leukocyte recruitment [2-5] and production of superoxide anion [3-5], whilst increases GSH [2-4], and antioxidant capacity [3-5]. Naringenin also acts on macrophages inducing Nrf2 activation, a nuclear factor that induces antioxidant and anti-inflammatory responses, inducing HO-1 expression [3]. PAMPs, DAMPs and ROS induce NFκB activation in macrophages resulting in the production of pro-hyperalgesic cytokine such as IL-33, TNFα, IL-1β and IL-6. Pro-hyperalgesic cytokines induce the production of lipid mediators such as PGE2 that sensitize the nociceptor neurons. Naringenin inhibits LPSand carrageenan-induced NFκB activation in vivo [2] and in vitro [5], which contributes to naringenin inhibition of IL-33 [2], TNFα [3-5], IL-1β [2, 4, 5] and IL-6 [4,5] production and expression of cyclooxygenase-2 mRNA [3] (Figure 1).

Jararhagin-induced mechanical hyperalgesia depends on TNF-α, IL-1β and NFκB in mice

Jararhagin is a hemorrhagic metalloprotease from Bothrops jararaca snake venom. The hyperalgesic mechanisms of jararhagin were investigated focusing on the role of proinflammatory cytokines (TNF-α and IL-1β) and the transcription factor NFκB. Intraplantar administration of jararhagin (1, 10, 100 and 1000 ng/paw) induced mechanical hyperalgesia, and increased TNF-α levels at 1, 3 and 5 h, and IL-1β levels at 0.5, 1 and 3 h after its injection in the paw tissue. Pre-treatment with morphine (2, 6, 12 μg/paw) inhibited jararhagin-induced mechanical hyperagesia. The systemic or local pre-treatment with etanercept (10 mg/kg and 100 μg/paw) and IL-1ra (30 mg/kg and 100 pg/paw) inhibited jararhagin-induced mechanical hyperalgesia. Co-administration of jararhagin (0.1 ng/paw) and TNF-α (0.1 pg/paw) or jararhagin (0.1 ng/paw) and IL-1β (1 pg/paw) enhanced the mechanical hyperalgesia. The systemic or local pre-treatment with PDTC (NFκB inhibitor; 100 mg/kg and 100 μg/paw) inhibited jararhagin-induced mechanical hyperalgesia as well as PDTC decreased the jararhagin-induced production of TNF-α and IL-1β. Thus, these data demonstrate the involvement of pro-inflammatory cytokines TNF-α and IL-1β and nuclear transcription factor NFκB in jararhagin-induced mechanical hyperalgesia indicating that targeting these mechanisms might contribute to reduce the pain induced by B. jararaca snake venom.

Targeting IL-33/ST2 signaling: regulation of immune function and analgesia

ABSTRACT Introduction: IL-33 signals through ST2 receptor and promotes inflammation by activating downstream pathways culminating in the production of pro-inflammatory mediators such as IL-1β, TNF-α, and IL-6 in an NF-κB-dependent manner. In fact, compelling evidence has demonstrated the importance of IL-33/ST2 in both innate and adaptive immune responses in diseases presenting pain as an important clinical symptom. Areas covered: IL-33 is a pleiotropic cytokine with varied immune functions. Dysregulation of this pathway has been described as a key step in varied immune responses. Further, IL-33 contributes to peripheral and spinal cord nociceptor neuron sensitization in innate and adaptive inflammatory immune responses as well as in neuropathic and cancer pain. In this sense, targeting IL-33/ST2 signaling is a promising therapeutic approach. Expert opinion: The modulation of IL-33/ST2 signaling represents a possible approach in regulating immune functions. In addition to immune function, strategies targeting IL-33/ST2 signaling pathway display a favorable preclinical analgesic profile in both acute and chronic models of pain. Therefore, IL-33-targeting therapies represent a potential target for the development of novel analgesic drugs given that IL-33 activates, for instance, neutrophils, mast cells, macrophages, astrocytes, and microglia that are important cells in the induction and maintenance of chronic pain states.

Probucol attenuates lipopolysaccharide-induced leukocyte recruitment and inflammatory hyperalgesia: effect on NF-кB activation and cytokine production

&NA; Probucol 4,4′‐ (Isopropylidenedithio)bis(2,6‐di‐tert‐butylphenol) is a synthetic molecule clinically used for prevention and treatment of hypercholesterolemia and atherosclerosis. Recent studies have shown that the beneficial effects of probucol mainly derive from its anti‐inflammatory and antioxidant properties. Gram‐negative bacteria are common infectious agents and their wall components, e.g. lipopolysaccharide (LPS), are important elicitors of inflammation. LPS is sensed by tissue resident cells and it triggers a Toll‐like receptor 4/MyD88‐dependent signaling cascade resulting in endothelial activation, leukocyte recruitment and nociception. Therefore the present study aimed to investigate the anti‐inflammatory and analgesic effects of probucol in models of LPS‐induced acute inflammation. Probucol at 0.3–30 mg/kg was administrated to male Swiss mice per oral 1 h before intraplantar or intraperitoneal lipopolysaccharide stimulus. Probucol at 3 mg/kg reduced lipopolysaccharide‐induced mechanical and thermal hyperalgesia. These effects were accompanied by reduced leukocyte influx and cytokine production in both paw skin and peritoneum exudate. Unexpectedly, probucol did not alter lipopolysaccharide‐induced tissue oxidative stress at anti‐inflammatory /analgesic dose. On the other hand, probucol inhibited lipopolysaccharide‐induced nuclear factor kappa B (NF‐&kgr;B) activation in paw tissue as well as NF‐&kgr;B activity in cultured macrophages in vitro, reinforcing the inhibitory effect of probucol over the NF‐&kgr;B signaling pathway. In this sense, we propose that probucol acts on resident immune cells, such as macrophages, targeting the NF‐&kgr;B pathway. As a result, it prevents the amplification and persistence of the inflammatory response by attenuating NF‐&kgr;B‐dependent cytokine production and leukocyte recruitment explaining its analgesic effects as well.