Sergio M. Borghi

Vitexin Inhibits Inflammatory Pain in Mice by Targeting TRPV1, Oxidative Stress, and Cytokines

The flavonoid vitexin (1) is a flavone C-glycoside (apigenin-8-C-β-D-glucopyranoside) present in several medicinal and other plants. Plant extracts containing 1 are reported to possess antinociceptive, anti-inflammatory, and antioxidant activities. However, the only evidence that 1 exhibits antinociceptive activity was demonstrated in the acetic acid-induced writhing model. Therefore, the analgesic effects and mechanisms of 1 were evaluated. In the present investigation, intraperitoneal treatment with 1 dose-dependently inhibited acetic acid-induced writhing. Furthermore, treatment with 1 also inhibited pain-like behavior induced by phenyl-p-benzoquinone, complete Freunds adjuvant (CFA), capsaicin (an agonist of transient receptor potential vanilloid 1, TRPV1), and both phases of the formalin test. It was also observed that inhibition of carrageenan-, capsaicin-, and chronic CFA-induced mechanical and thermal hyperalgesia occurred. Regarding the antinociceptive mechanisms of 1, it prevented the decrease of reduced glutathione levels, ferric-reducing ability potential, and free-radical scavenger ability, inhibited the production of hyperalgesic cytokines such as TNF-α, IL-1β, IL-6, and IL-33, and up-regulated the levels of the anti-hyperalgesic cytokine IL-10. These results demonstrate that 1 exhibits an analgesic effect in a variety of inflammatory pain models by targeting TRPV1 and oxidative stress and by modulating cytokine production.

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 citrus flavonone naringenin reduces lipopolysaccharide-induced inflammatory pain and leukocyte recruitment by inhibiting NF-κB activation

Lipopolysaccharide (LPS) is the major structural component of Gram-negative bacteria cell wall and a highly pro-inflammatory toxin. Naringenin is found in Citrus fruits and exhibits antioxidant and anti-inflammatory properties through inhibition of NF-κB activation but its effects in LPS-induced inflammatory pain and leukocyte recruitment were not investigated yet. We investigated the effects of naringenin in mechanical hyperalgesia, thermal hyperalgesia and leukocyte recruitment induced by intraplantar injection of LPS in mice. We found that naringenin reduced hyperalgesia to mechanical and thermal stimuli, myeloperoxidase (MPO, a neutrophil and macrophage marker) and N-acetyl-β-D-glucosaminidase (NAG, a macrophage marker) activities, oxidative stress and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production in the paw skin. In the peritoneal cavity, naringenin reduced neutrophil and mononuclear cell recruitment, and abrogated MPO and NAG activity, cytokine and superoxide anion production, and lipid peroxidation. In vitro, pre-treatment with naringenin inhibited superoxide anion and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production by LPS-stimulated RAW 264.7 macrophages. Finally, we demonstrated that naringenin inhibited NF-κB activation in vitro and in vivo. Therefore, naringenin is a promising compound to treat LPS-induced inflammatory pain and leukocyte recruitment.

Role of TNF-α/TNFR1 in intense acute swimming-induced delayed onset muscle soreness in mice

The injection of cytokines such as TNF-α induces muscle pain. Herein, it was addressed the role of endogenous TNF-α/TNFR1 signaling in intense acute swimming-induced muscle mechanical hyperalgesia in mice. Mice were exposed to water during 30 s (sham) or to a single session of 30-120 min of swimming. Intense acute swimming induced a dose-dependent (time of exercise-dependent) muscle mechanical hyperalgesia, which peaked after 24 h presenting characteristics of delayed onset muscle soreness (DOMS). The intense acute swimming (120 min)-induced muscle mechanical hyperalgesia was reduced in etanercept (soluble TNF receptor) treated and TNFR1 deficient ((-/-)) mice. TNF-α levels increased 2 and 4 h after intense acute swimming in soleus muscle (but not in gastrocnemius), and spinal cord, respectively. Exercise induced an increase of myeloperoxidase activity and decrease in reduced glutathione levels in an etanercept-sensitive and TNFR1-dependent manners in the soleus muscle, but not in the gastrocnemius muscle. Concluding, TNF-α/TNFR1 signaling mediates intense acute swimming-induced DOMS by an initial role in the soleus muscle followed by spinal cord, inducing muscle inflammatory hyperalgesia and oxidative stress. The knowledge of these mechanisms might contribute to improve the training of athletes, individuals with physical impairment and intense training such as military settings.

Vinpocetine reduces diclofenac-induced acute kidney injury through inhibition of oxidative stress, apoptosis, cytokine production, and NF-κB activation in mice

&NA; Acute kidney injury (AKI) represents a complex clinical condition associated with significant morbidity and mortality. Approximately, 19–33% AKI episodes in hospitalized patients are related to drug‐induced nephrotoxicity. Although, considered safe, non‐steroidal anti‐inflammatory drugs such as diclofenac have received special attention in the past years due to the potential risk of renal damage. Vinpocetine is a nootropic drug known to have anti‐inflammatory properties. In this study, we investigated the effect and mechanisms of vinpocetine in a model of diclofenac‐induced AKI. We observed that diclofenac increased proteinuria and blood urea, creatinine, and oxidative stress levels 24 h after its administration. In renal tissue, diclofenac also increased oxidative stress and induced morphological changes consistent with renal damage. Moreover, diclofenac induced kidney cells apoptosis, up‐regulated proinflammatory cytokines, and induced the activation of NF‐&kgr;B in renal tissue. On the other hand, vinpocetine reduced diclofenac‐induced blood urea and creatinine. In the kidneys, vinpocetine inhibited diclofenac‐induced oxidative stress, morphological changes, apoptosis, cytokine production, and NF‐&kgr;B activation. To our knowledge, this is the first study demonstrating that diclofenac‐induced AKI increases NF‐&kgr;B activation, and that vinpocetine reduces the nephrotoxic effects of diclofenac. Therefore, vinpocetine is a promising molecule for the treatment of diclofenac‐induced AKI. Graphical abstract Figure. No caption available.

Granulocyte-colony stimulating factor (G-CSF)-induced mechanical hyperalgesia in mice: Role for peripheral TNFα, IL-1β and IL-10.

Granulocyte-colony stimulating factor (G-CSF) is a therapeutic approach to increase peripheral neutrophil counts after anti-tumor therapies. Pain is the major side effect of G-CSF. Intraplantar administration of G-CSF in mice induces mechanical hyperalgesia. However, the peripheral mechanisms involved in this effect were not elucidated. Therefore, the participation of pronociceptive cytokines tumor necrosis factor (TNF) alpha (TNFα), interleukin (IL)-1 beta (IL-1β) and antinociceptive cytokine IL-10 in G-CSF-induced mechanical hyperalgesia in mice was investigated. G-CSF-induced mechanical hyperalgesia was inhibited by systemic and local treatment with etanercept and IL-1 receptor antagonist (IL-1ra) or TNF receptor 1 (TNFR1) deficiency and increased in IL-10 deficient mice. In agreement, G-CSF injection induced significant TNFα, IL-1β and IL-10 production in paw tissue. G-CSF-induced hyperalgesia was dose-dependently inhibited by thalidomide (5-45mg/kg) and pentoxifylline (0.5-13.5mg/kg), and treatment with these drugs inhibited G-CSF-induced TNFα, IL-1β and IL-10 production. The combined treatment with pentoxifylline or thalidomide with morphine, at doses that are ineffective as single treatment, diminished G-CSF-induced hyperalgesia through inhibiting cytokine production. Indomethacin also reduces G-CSF hyperalgesia alone or combined with pentoxifylline or thalidomide. Thus, G-CSF-induced hyperalgesia might be mediate by peripheral production of pronociceptive cytokines TNFα and IL-1β and down-regulated by IL-10. Systemic IL-1ra reduced G-CSF-induced increase of peripheral neutrophil counts. However, local treatment with morphine, IL-1ra or etanercept, and systemic treatment with indomethacin, etanercept, thalidomide and pentoxifylline did not alter G-CSF-induced mobilization of neutrophils. Therefore, this study advances in the understanding of G-CSF-induced hyperalgesia and suggests therapeutic approaches for its control.

Targeting interleukin-1β reduces intense acute swimming-induced muscle mechanical hyperalgesia in mice

The role of interleukin (IL)‐1β in intense acute swimming‐induced muscle mechanical hyperalgesia was investigated in mice.

Pyrrolidine dithiocarbamate inhibits superoxide anion-induced pain and inflammation in the paw skin and spinal cord by targeting NF-κB and oxidative stress

We evaluated the effect of pyrrolidine dithiocarbamate (PDTC) in superoxide anion-induced inflammatory pain. Male Swiss mice were treated with PDTC and stimulated with an intraplantar or intraperitoneal injection of potassium superoxide, a superoxide anion donor. Subcutaneous PDTC treatment attenuated mechanical hyperalgesia, thermal hyperalgesia, paw oedema and leukocyte recruitment (neutrophils and macrophages). Intraplantar injection of superoxide anion activated NF-κB and increased cytokine production (IL-1β, TNF-α and IL-10) and oxidative stress (nitrite and lipid peroxidation levels) at the primary inflammatory foci and in the spinal cord (L4–L6). PDTC treatment inhibited superoxide anion-induced NF-κB activation, cytokine production and oxidative stress in the paw and spinal cord. Furthermore, intrathecal administration of PDTC successfully inhibited superoxide anion-induced mechanical hyperalgesia, thermal hyperalgesia and inflammatory response in peripheral foci (paw). These results suggest that peripheral stimulus with superoxide anion activates the local and spinal cord oxidative- and NF-κB-dependent inflammatory nociceptive mechanisms. PDTC targets these events, therefore, inhibiting superoxide anion-induced inflammatory pain in mice.

Quercetin Inhibits Peripheral and Spinal Cord Nociceptive Mechanisms to Reduce Intense Acute Swimming-Induced Muscle Pain in Mice.

The present study aimed to evaluate the effects of the flavonoid quercetin (3,3´,4´,5,7-pentahydroxyflavone) in a mice model of intense acute swimming-induced muscle pain, which resembles delayed onset muscle soreness. Quercetin intraperitoneal (i.p.) treatment dose-dependently reduced muscle mechanical hyperalgesia. Quercetin inhibited myeloperoxidase (MPO) and N-acetyl-β-D- glucosaminidase (NAG) activities, cytokine production, oxidative stress, cyclooxygenase-2 (COX-2) and gp91phox mRNA expression and muscle injury (creatinine kinase [CK] blood levels and myoblast determination protein [MyoD] mRNA expression) as well as inhibited NFκB activation and induced Nrf2 and HO-1 mRNA expression in the soleus muscle. Beyond inhibiting those peripheral effects, quercetin also inhibited spinal cord cytokine production, oxidative stress and glial cells activation (glial fibrillary acidic protein [GFAP] and ionized calcium-binding adapter molecule 1 [Iba-1] mRNA expression). Concluding, the present data demonstrate that quercetin is a potential molecule for the treatment of muscle pain conditions related to unaccustomed exercise.

Bosentan, a mixed endothelin receptor antagonist, induces antidepressant-like activity in mice

Endothelins are peptides described initially as potent vasoactive mediators. Recently, studies reported that endothelins can modulate the production and release of cytokines by immune cells. In turn, cytokines are involved in depression disorders and also in the effectiveness of some antidepressants. Therefore, we investigated the effects of treating mice with bosentan, a mixed endothelin receptor antagonist, in widely used models for assessing antidepressant activity of compounds, the forced swimming (FST) and the tail suspension tests (TST). Moreover, the influence of bosentan treatment on circulating IL-6 levels was also addressed after FST. The results show that bosentan treatment induced a bell shaped dose-dependent antidepressant-like effect with increase in circulating IL-6 levels in animals exposed to FST. Bosentan also presented antidepressant-like effect in TST. Similar results were obtained with nortriptyline treatment in the FST and TST. Possible anxiogenic effect of bosentan was excluded using the elevated plus maze test. Therefore, this is the first study to demonstrate the antidepressant-like activity of bosentan in mice, unveiling a previous unrecognized role of endothelin in depression and its possible relation with increased circulating IL-6 levels.