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 Freund's 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.

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.

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.

The Ehrlich tumor is a mammary adenocarcinoma of mice that can be developed in solid and ascitic forms depending on its administration in tissues or cavities, respectively. The present study investigates whether the subcutaneous plantar administration of the Ehrlich tumor cells induces pain-like behavior and initial pharmacological susceptibility characteristics. The Ehrlich tumor cells (1 × 104–107 cells) induced dose-dependent mechanical hyperalgesia (electronic version of the von Frey filaments), paw edema/tumor growth (caliper), and flinches compared with the saline group between days 2 and 12. There was no difference between doses of cells regarding thermal hyperalgesia in the hot-plate test. Indomethacin (a cyclooxygenase inhibitor) and amitriptyline hydrochloride (a tricyclic antidepressant) treatments did not affect flinches or thermal and mechanical hyperalgesia. On the other hand, morphine (an opioid) inhibited the flinch behavior and the thermal and mechanical hyperalgesia. These effects of morphine on pain-like behavior were prevented by naloxone (an opioid receptor antagonist) treatment. None of the treatments affected paw edema/tumor growth. The results showed that, in addition to tumor growth, administration of the Ehrlich tumor cells may represent a novel model for the study of cancer pain, specially the pain that is susceptible to treatment with opioids, but not to cyclooxygenase inhibitor or to tricyclic antidepressant.

The present study evaluated the anti-inflammatory and analgesic effects of the superoxide dismutase mimetic agent tempol in superoxide anion-induced pain and inflammation. Mice were treated intraperitoneally with tempol (10–100 mg/kg) 40 min before the intraplantar injection of a superoxide anion donor, potassium superoxide (KO2, 30 μg). Mechanical hyperalgesia and thermal hyperalgesia, paw edema, and mRNA expression of peripheral and spinal cord mediators involved in inflammatory pain, TNFα, IL-1β, IL-10, COX-2, preproET-1, gp91phox, Nrf2, GFAP, and Iba-1, were evaluated. Peripheral and spinal cord reductions of antioxidant defenses and superoxide anion were also assessed. Tempol reduced KO2-induced mechanical hyperalgesia and thermal hyperalgesia and paw edema. The increased mRNA expression of the evaluated mediators and oxidative stress in the paw skin and spinal cord and increased mRNA expression of glial markers in the spinal cord induced by KO2 were successfully inhibited by tempol. KO2-induced reduction in Nrf2 mRNA expression in paw skin and spinal cord was also reverted by tempol. Corroborating the effect of tempol in the KO2 model, tempol also inhibited carrageenan and CFA inflammatory hyperalgesia. The present study demonstrates that tempol inhibits superoxide anion-induced molecular and behavioral alterations, indicating that tempol deserves further preclinical studies as a promising analgesic and anti-inflammatory molecule for the treatment of inflammatory pain.

Cancer pain directly affects the patient's quality of life. We have previously demonstrated that the subcutaneous administration of the mammary adenocarcinoma known as Ehrlich tumor induces pain in mice. Several studies have shown that the flavonoid quercetin presents important biological effects, including anti-inflammatory, antioxidant, analgesic, and antitumor activity. Therefore, the analgesic effect and mechanisms of quercetin were evaluated in Ehrlich tumor-induced cancer pain in mice. Intraperitoneal (i.p.) treatments with quercetin reduced Ehrlich tumor-induced mechanical and thermal hyperalgesia, but not paw thickness or histological alterations, indicating an analgesic effect without affecting tumor growth. Regarding the analgesic mechanisms of quercetin, it inhibited the production of hyperalgesic cytokines IL-1β and TNFα and decreased neutrophil recruitment (myeloperoxidase activity) and oxidative stress. Naloxone (opioid receptor antagonist) inhibited quercetin analgesia without interfering with neutrophil recruitment, cytokine production, and oxidative stress. Importantly, cotreatment with morphine and quercetin at doses that were ineffective as single treatment reduced the nociceptive responses. Concluding, quercetin reduces the Ehrlich tumor-induced cancer pain by reducing the production of hyperalgesic cytokines, neutrophil recruitment, and oxidative stress as well as by activating an opioid-dependent analgesic pathway and potentiation of morphine analgesia. Thus, quercetin treatment seems a suitable therapeutic approach for cancer pain that merits further investigation.

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.

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