Kenji W. Ruiz-Miyazawa

Vinpocetine reduces lipopolysaccharide-induced inflammatory pain and neutrophil recruitment in mice by targeting oxidative stress, cytokines and NF-κB.

In response to lipopolysaccharide (LPS), tissue resident macrophages and recruited neutrophils produce inflammatory mediators through activation of Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway. These mediators include inflammatory cytokines and reactive oxygen species that, in turn, sensitize nociceptors and lead to inflammatory pain. Vinpocetine is a nootropic drug widely used to treat cognitive and neurovascular disorders, and more recently its anti-inflammatory properties through inhibition of NF-κB activation have been described. In the present study, we used the intraplantar and intraperitoneal LPS stimulus in mice to investigate the effects of vinpocetine pre-treatment (3, 10, or 30mg/kg by gavage) in hyperalgesia, leukocyte recruitment, oxidative stress, and pro-inflammatory cytokine production (TNF-α, IL-1β, and IL-33). LPS-induced NF-κB activation and cytokine production were investigated using RAW 264.7 macrophage cell in vitro. Vinpocetine (30mg/kg) significantly reduces hyperalgesia to mechanical and thermal stimuli, and myeloperoxidase (MPO) activity (a neutrophil marker) in the plantar paw skin, and also inhibits neutrophil and mononuclear cell recruitment, superoxide anion and nitric oxide production, oxidative stress, and cytokine production (TNF-α, IL-1β and IL-33) in the peritoneal cavity. At least in part, these effects seem to be mediated by direct effects of vinpocetine on macrophages, since it inhibited the production of the same cytokines (TNF-α, IL-1β and IL-33) and the NF-κB activation in LPS-stimulated RAW 264.7 macrophages. Our results suggest that vinpocetine represents an important therapeutic approach to treat inflammation and pain induced by a gram-negative bacterial component by targeting NF-κB activation and NF-κB-related cytokine production in macrophages.

Quercetin inhibits gout arthritis in mice: induction of an opioid-dependent regulation of inflammasome

We investigated the anti-inflammatory and analgesic effects of quercetin in monosodium urate crystals (MSU)-induced gout arthritis, and the sensitivity of quercetin effects to naloxone, an opioid receptor antagonist. Mice were treated with quercetin, and mechanical hyperalgesia was assessed at 1–24xa0h after MSU injection. In vivo, leukocyte recruitment, cytokine levels, oxidative stress, NFκB activation, and gp91phox and inflammasome components (NLRP3, ASC, Pro-caspase-1, and Pro-IL-1β) mRNA expression by qPCR were determined in the knee joints at 24xa0h after MSU injection. Inflammasome activation was determined, in vitro, in lipopolysaccharide-primed macrophages challenged with MSU. Quercetin inhibited MSU-induced mechanical hyperalgesia, leukocyte recruitment, TNFα and IL-1β production, superoxide anion production, inflammasome activation, decrease of antioxidants levels, NFκB activation, and inflammasome components mRNA expression. Naloxone pre-treatment prevented all the inhibitory effects of quercetin over MSU-induced gout arthritis. These results demonstrate that quercetin exerts analgesic and anti-inflammatory effect in the MSU-induced arthritis in a naloxone-sensitive manner.

Vinpocetine reduces carrageenan-induced inflammatory hyperalgesia in mice by inhibiting oxidative stress, cytokine production and NF-κB activation in the paw and spinal cord.

Vinpocetine is a safe nootropic agent used for neurological and cerebrovascular diseases. The anti-inflammatory activity of vinpocetine has been shown in cell based assays and animal models, leading to suggestions as to its utility in analgesia. However, the mechanisms regarding its efficacy in inflammatory pain treatment are still not completely understood. Herein, the analgesic effect of vinpocetine and its anti-inflammatory and antioxidant mechanisms were addressed in murine inflammatory pain models. Firstly, we investigated the protective effects of vinpocetine in overt pain-like behavior induced by acetic acid, phenyl-p-benzoquinone (PBQ) and formalin. The intraplantar injection of carrageenan was then used to induce inflammatory hyperalgesia. Mechanical and thermal hyperalgesia were evaluated using the electronic von Frey and the hot plate tests, respectively, with neutrophil recruitment to the paw assessed by a myeloperoxidase activity assay. A number of factors were assessed, both peripherally and in the spinal cord, including: antioxidant capacity, reduced glutathione (GSH) levels, superoxide anion, tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) levels, as well as nuclear factor kappa B (NF-κB) activation. Vinpocetine inhibited the overt pain-like behavior induced by acetic acid, PBQ and formalin (at both phases), as well as the carrageenan-induced mechanical and thermal hyperalgesia and associated neutrophil recruitment. Both peripherally and in the spinal cord, vinpocetine also inhibited: antioxidant capacity and GSH depletion; increased superoxide anion; IL-1β and TNF-α levels; and NF-κB activation. As such, vinpocetine significantly reduces inflammatory pain by targeting oxidative stress, cytokine production and NF-κB activation at both peripheral and spinal cord levels.

The nitroxyl donor Angeli's salt ameliorates Staphylococcus aureus-induced septic arthritis in mice

Abstract Septic arthritis is a severe and rapidly debilitating disease associated with severe joint pain, inflammation and oxidative stress. Nitroxyl (HNO) has become a nitrogen oxide of significant interest due to its pharmacological endpoints that are potentially favorable for treating varied diseases. However, whether HNO also serves as a treatment to septic arthritis is currently unknown. The aim of this study was to investigate the effect of the HNO donor, Angelis salt (AS), in the outcome of chronic Staphylococcus aureus (S. aureus)‐induced septic arthritis in mice. Daily treatment with AS inhibited mechanical hyperalgesia and inflammation (edema, leukocyte migration, cytokines release and NF‐&kgr;B activation, and oxidative stress) resulting in reduced disease severity (clinical course, histopathological changes, proteoglycan levels in the joints, and osteoclastogenesis). In addition, AS decreased the number of S. aureus colony forming unities in synovial tissue, enhanced the bactericidal effect of macrophages and inhibited the worsening of systemic inflammatory response (leukocyte counts in the lung and systemic proinflammatory cytokine concentration). Our results suggest for the first time the therapeutic potential of AS in a model of septic arthritis by mechanisms involving microbicidal effects, anti‐inflammatory actions and reduction of disease severity. Graphical abstract Figure. No Caption available. HighlightsAngelis salt (AS) reduces S. aureus‐induced articular pain and inflammation.AS reduced S. aureus‐induced cartilage damage and osteoclastogenesis.AS reduced S. aureus‐induced cytokines production and NF‐&kgr;B activation.AS reduced S. aureus‐induced oxidative stress.AS is microbicide and prevents systemic inflammatory response.

Erratum to: The Sesquiterpene Lactone, Budlein A, Inhibits Antigen-Induced Arthritis in Mice: Role of NF-κB and Cytokines

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by debilitating pain, cartilage destruction, and loss of joint function. Management of RA includes drugs that target NF-κB and downstream cytokine production. Therefore, molecules that act by inhibiting this signaling pathway without the severe side effects of, for instance, corticoids would be suitable therapeutic strategies. Budlein A is a sesquiterpene lactone with antinociceptive and anti-inflammatory properties related to the inhibition of pro-inflammatory cytokines and neutrophil recruitment. In this study, the effect of budlein A was evaluated in antigen-induced arthritis (AIA) in mice. At the 26th day, leukocyte recruitment to the knee joint, knee contents of proteoglycans, blood levels of ALT and AST, stomach tissue myeloperoxidase activity, and RT-qPCR for pro-inflammatory gene mRNA expression in knee joint samples was performed. NF-κB luciferase activity was evaluated in RAW 264.7 macrophages. Budlein A treatment dose-dependently inhibited AIA-induced mechanical hyperalgesia, edema, total leukocytes and neutrophil recruitment, and proteoglycan degradation. Budlein A did not induce gastric or liver damage. Budlein also inhibited AIA-induced Il-33, Tnf, Il-1β, preproET-1, and Cox-2 mRNA expression. In vitro, budlein reduced TNF- and IL-1β-induced NF-κB activity in RAW 264.7 macrophages. Altogether, we demonstrate that budlein A ameliorates AIA-induced inflammation and pain by targeting NF-κB. Importantly, budlein A does not induce in vivo side effects, suggesting that it possesses a favorable pre-clinical profile as analgesic and it is a prosperous molecule to be further investigated for the treatment of RA.

Quercetin attenuates zymosan-induced arthritis in mice

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by articular lesions, recruitment of inflammatory cells and increased levels of pro-inflammatory cytokine. The intra-articular administration of zymosan is an experimental model that promotes inflammatory parameters resembling RA. Therefore, this model was used to investigate the efficacy of quercetin as a treatment of articular inflammation. Treatment with quercetin dose-dependently reduced zymosan-induced hyperalgesia, articular edema and the recruitment of neutrophils to the knee joint cavity. Histological analysis confirmed that quercetin inhibited zymosan-induced arthritis. The treatment with quercetin also inhibited zymosan-induced depletion of reduced glutathione (GSH) levels, TNFα and IL-1β production, and gp91phox, prepro-endothelin-1 (preproET-1), and cyclooxygenase-2 mRNA expression. These molecular effects of quercetin were related to the inhibition of the nuclear factor kappa-B and induction of Nuclear factor erythroid 2- related factor (Nrf2)/home oxygenase (HO-1) pathway. Thus, quercetin exerted anti-inflammatory, analgesic and antioxidant effects in experimental arthritis, suggesting quercetin is a possible candidate for arthritis treatment.

Leishmania (L). amazonensis induces hyperalgesia in balb/c mice: Contribution of endogenous spinal cord TNFα and NFκB activation

Cutaneous leishmaniasis (CL) is the most common form of the leishmaniasis in humans. Ulcerative painless skin lesions are predominant clinical features of CL. Wider data indicate pain accompanies human leishmaniasis, out with areas of painless ulcerative lesions per se. In rodents, Leishmania (L.) major infection induces nociceptive behaviors that correlate with peripheral cytokine levels. However, the role of the spinal cord in pain processing after Leishmania infection has not been investigated. Balb/c mice received intraplantar (i.pl.) injection of Leishmania (L). amazonensis and hyperalgesia, edema, parasitism, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression, and NFκB activation were evaluated. The effects of intrathecal (i.t.) injection of morphine, TNFα, TNFα inhibitors (etanercept and adalimumab) and NFκB inhibitor (PDTC) were investigated. The present study demonstrates that Leishmania (L.) amazonensis infection in balb/c mice induces chronic mechanical and thermal hyperalgesia in an opioid-sensitive manner. Spinal cord TNFα mRNA expression increased in a time-dependent manner, peaking between 30 and 40 days after infection. At the peak of TNFα mRNA expression (day 30), there was a concomitant increase in TNFR1 and TNFR2 mRNA expression. TNFα i.t. injection enhanced L.xa0(L.) amazonensis-induced hyperalgesia. Corroborating a role for TNFα in L.xa0(L.) amazonensis-induced hyperalgesia, i.t. treatment with the TNFα inhibitors, etanercept and adalimumab inhibited the hyperalgesia. L.xa0(L.) amazonensis also induced spinal cord activation of NFκB, and PDTC (given i.t.), also inhibited L.xa0(L.) amazonensis-induced hyperalgesia, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression. Moreover, L.xa0(L.) amazonensis-induced spinal cord activation of NFκB was also inhibited by etanercept and adalimumab as well as PDTC i.t.nnnTREATMENTnThese results demonstrate that endogenous spinal cord TNFα and NFκB activation contribute to L.xa0(L.) amazonensis-induced hyperalgesia in mice. Thus, spinal cord TNFα and NFκB are potential therapeutic targets for Leishmania infection-induced pain.

Pyrrolidine dithiocarbamate inhibits mouse acute kidney injury induced by diclofenac by targeting oxidative damage, cytokines and NF-κB activity

Aims: Non‐steroidal anti‐inflammatory drugs (NSAIDs) are widely used and effective anti‐inflammatories despite the well‐known side effects such as gastrointestinal damage, acute kidney injury (AKI), and cardiovascular dysfunctions. Diclofenac is among the most prescribed NSAIDs due to its efficient analgesic and anti‐inflammatory properties. Patients using diclofenac possess 77% risk increase to develop AKI. Activation of NF‐&kgr;B contributes to diclofenac‐induced AKI, which is in line with the use of glucocorticoids as one of the management choices to treat AKI patients. Main methods: In this work, we investigate the efficacy of pyrrolidine dithiocarbamate (PDTC) in diclofenac‐induced AKI in mice given it is a known NF‐&kgr;B inhibitor. Key findings: We observed that diclofenac increased proteinuria and urine neutrophil gelatinase‐associated lipocalin (NGAL), blood levels of urea, creatinine, oxidative stress, C‐reactive protein (CRP), and pro‐inflammatory cytokine after 24 h of a bolus administration. In renal tissue, diclofenac also induced morphological changes consistent with kidney damage, modulated cytokine production, increased oxidative stress and reduced antioxidant defenses. These alterations induced by diclofenac were accompanied by activation of NF‐&kgr;B in the kidney. Treatment with PDTC dose‐dependently reduced diclofenac‐induced blood urea, creatinine, and oxidative stress. In addition, PDTC reduced proteinuria and urine NGAL levels and blood CRP and pro‐inflammatory cytokines. In the kidney, PDTC inhibited diclofenac‐induced morphological changes, pro‐inflammatory cytokine production, oxidative stress, and NF‐&kgr;B activation, and increased antioxidant defenses and anti‐inflammatory cytokine IL‐10. Significance: Our data demonstrate that PDTC ameliorates diclofenac‐induced AKI and that targeting NF‐&kgr;B signaling pathway is a promising therapeutic approach for the treatment of diclofenac‐induced AKI.

Hesperidin Methylchalcone Suppresses Experimental Gout Arthritis in Mice by Inhibiting NF-κB Activation

Gout arthritis is a painful inflammatory disease induced by monosodium urate (MSU) crystals. We evaluate the therapeutic potential of the flavonoid hesperidin methylchalcone (HMC) in a mouse model of gout arthritis induced by intra-articular injection of MSU (100 μg/10 μL). Orally given HMC (3-30 mg/kg, 100 μL) reduced in a dose-dependent manner the MSU-induced hyperalgesia (44%, p < 0.05), edema (54%, p < 0.05), and leukocyte infiltration (70%, p < 0.05). HMC (30 mg/kg) inhibited MSU-induced infiltration of LysM-eGFP+ cells (81%, p < 0.05), synovitis (76%, p < 0.05), and oxidative stress (increased GSH, FRAP, and ABTS by 62, 78, and 73%, respectively; reduced O2- and NO by 89 and 48%, p < 0.05) and modulated cytokine production (reduced IL-1β, TNF-α, IL-6, and IL-10 by 35, 72, 37, and 46%, respectively, and increased TGF-β by 90%, p < 0.05). HMC also inhibited MSU-induced NF-κB activation (41%, p < 0.05), gp91phox (66%, p < 0.05) and NLRP3 inflammasome components mRNA expression in vivo (72, 77, 71, and 73% for NLRP3, ASC, pro-caspase-1, and pro-IL-1 β, respectively, p < 0.05), and induced Nrf2/HO-1 mRNA expression (3.9- and 5.1-fold increase, respectively, p < 0.05). HMC (30, 100, and 300 μM) did not inhibit IL-1β secretion by macrophages primed by LPS and challenged with MSU (450 μg/mL), demonstrating that the anti-inflammatory effect of HMC in gout arthritis depends on inhibiting NF-κB but not on direct inhibition of inflammasome. The pharmacological effects of HMC indicate its therapeutic potential for the treatment of gout.

15d-PGJ2-loaded nanocapsules ameliorate experimental gout arthritis by reducing pain and inflammation in a PPAR-gamma-sensitive manner in mice

Gout arthritis (GA) is a painful inflammatory disease in response to monosodium urate (MSU) crystals in the joints. 15deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a natural activator of PPAR-γ with analgesic, anti-inflammatory, and pro-resolution properties. Thus, we aimed to evaluate the effect and mechanisms of action of 15d-PGJ2 nanocapsules (NC) in the model of GA in mice, since a reduction of 33-fold in the dose of 15d-PGJ2 has been reported. Mice were treated with 15d-PGJ2-loaded NC, inert NC, free 15d-PGJ2 (without NC), or 15d-PGJ2-loaded NC+ GW9662, a PPAR-γ inhibitor. We show that 15d-PGJ2-loaded NC provided analgesic effect in a dose that the free 15d-PGJ2 failed to inhibiting pain and inflammation. Hence, 15d-PGJ2-loaded NC reduced MSU-induced IL-1β, TNF-α, IL-6, IL-17, and IL-33 release and oxidative stress. Also, 15d-PGJ2-loaded NC decreased the maturation of IL-1β in LPS-primed BMDM triggered by MSU. Further, 15d-PGJ2-loaded NC decreased the expression of the components of the inflammasome Nlrp3, Asc, and Pro-caspase-1, as consequence of inhibiting NF-κB activation. All effects were PPAR-γ-sensitive. Therefore, we demonstrated that 15d-PGJ2-loaded NC present analgesic and anti-inflammatory properties in a PPAR-γ-dependent manner inhibiting IL-1β release and NF-κB activation in GA. Concluding, 15d-PGJ2-loaded NC ameliorates MSU-induced GA in a PPAR-γ-sensitive manner.