Waldiceu A. Verri

Role of cytokines in mediating mechanical hypernociception in a model of delayed-type hypersensitivity in mice

In the present study, we used the electronic version of the von Frey test to investigate the role of cytokines (TNF‐α and IL‐1β) and chemokines (KC/CXCL‐1) in the genesis of mechanical hypernociception during antigen‐induced inflammation in mice. The nociceptive test consisted of evoking a hindpaw flexion reflex with a hand‐held force transducer (electronic anesthesiometer) adapted with a 0.5mm2 polypropylene tip. The intraplantar administration of methylated bovine serum albumin (mBSA) in previously immunized (IM), but not in sham‐immunized (SI) mice, induced mechanical hypernociception in a dose‐dependent manner. Hypernociception induced by antigen was reduced in animals pretreated with IL‐1ra and reparixin (a non‐competitive allosteric inhibitor of CXCR2), and in TNF receptor type 1 deficient (TNFR1−/−) mice. Consistently, antigen challenge induced a time‐dependent release of TNF‐α, IL‐1β and KC/CXCL‐1 in IM, but not in SI, mice. The increase in TNF‐α levels preceded the increase in IL‐1β and KC/CXCL1. Antigen‐induced release of IL‐1β and KC/CXCL1 was reduced in TNFR1−/− mice, and TNF‐α‐induced hypernociception was inhibited by IL‐1ra and reparixin. Hypernociception induced by IL‐1β in immunized mice was inhibited by indomethacin, whereas KC/CXCL1‐induced hypernociception was inhibited by indomethacin and guanethidine. Antigen‐induced hypernociception was reduced by indomethacin and guanethidine and abolished by the two drugs combined. Together, these results suggest that inflammation associated with an adaptive immune response induces hypernociception that is mediated by an initial release of TNF‐α, which triggers the subsequent release of IL‐1β and KC/CXCL1. The latter cytokines in turn stimulate the release of the direct‐acting final mediators, prostanoids and sympathetic amines.

Hypernociception elicited by tibio-tarsal joint flexion in mice: A novel experimental arthritis model for pharmacological screening

Mice have been used as animal model to study mechanisms underlying inflammatory and immune diseases. The present study describes a model of joint inflammation-induced hypernociception to discriminate pharmacological tests in mice. A polypropylene tip probe with a large area (4.15 mm2) applied on the plantar surface of the hind paw was used to produce a dorsal flexion of tibio-tarsal joint. Experiments were performed to demonstrate that the probe application did not provoke cutaneous nociception. The decrease in the withdrawal threshold of inflamed joint was used as nociceptive parameter. Administration of zymosan in the tibio-tarsal joint induced a dose and time-dependent hypernociception elicited by articular dorsal flexion movement. Maximal joint hypernociception was detected between 7 and 24 h after zymosan injection, and matched maximal inflammation score as determined by histopathology and neutrophil migration assay. In agreement with the inflammatory hypernociceptive paradigm, flexion-elicited hypernociception induced by zymosan was dose-dependently inhibited by morphine (2-8 mg/kg) and by an effective dose of indomethacin (5 mg/kg). The present study demonstrated that the tibio-tarsal flexion reflex is a behavioral nociceptive model that allows a quantitative evaluation of inflammatory joint hypernociception in mice and its pharmacological modulation.

Role of IL-18 in overt pain-like behaviour in mice

There are evidences that targeting IL-18 might be beneficial to inhibit inflammatory symptoms, including hypernociception (decrease in nociceptive threshold). The mechanism of IL-18 mechanical hypernociception depends on endothelin in rats and mice. However, the role of IL-18 in overt pain-like behaviour remains undetermined. Therefore, we addressed the role of IL-18 in writhing response induced by intraperitoneal (i.p.) injection of phenyl-p-benzoquinone (PBQ) and acetic acid in mice. Firstly, it was detected that PBQ and acetic acid i.p. injection induced a dose-dependent number of writhes in Balb/c mice. Subsequently, it was observed that the PBQ - but not the acetic acid-induced writhes were diminished in IL-18 deficient ((-/-)) mice. Therefore, considering that IFN-gamma, endothelin and prostanoids mediate IL-18-induced mechanical hypernociception, we also investigated the role of these mediators in the same model of writhing response in which IL-18 participates. It was noticed that PBQ-induced writhes were diminished in IFN-gamma(-/-) mice and by the treatment with bosentan (mixed endothelin ETA/ETB receptor antagonist), BQ 123 (cyclo[DTrp-DAsp-Pro-DVal-Leu], selective endothelin ETA receptor antagonist), BQ 788 (N-cys-2,6 dimethylpiperidinocarbonyl-l-methylleucyl-d-1-methoxycarboyl-d-norleucine, selective endothelin ETB receptor antagonist) or indomethacin (cycloxigenase inhibitor). Thus, IL-18, IFN-gamma, endothelin acting on endothelin ETA and ETB receptors, and prostanoids mediate PBQ-induced writhing response in mice. To conclude, these results further advance the understanding of the physiopathology of overt pain-like behaviour, and suggest for the first time a role for IL-18 in writhing response in mice.

Quercetin in Lyotropic Liquid Crystalline Formulations: Physical, Chemical and Functional Stability

The purpose of this study was to develop a lyotropic liquid crystalline formulation using the emulsifier vitamin E TPGS and evaluate its behavior after incorporation of a flavonoid, quercetin. The physical (macro and microscopic), chemical (determination of quercetin content by the HPLC method) and functional (determination of quercetin antioxidant activity by DPPH• assay) stability of the lamellar liquid crystalline formulation containing flavonoid was evaluated when stored at 4u2009±u20092xa0°C; 30u2009±u20092xa0°C/70u2009±u20095% RH (relative humidity) and 40u2009±u20092xa0°C/70u2009±u20095% RH during 12xa0months. The lamellar liquid crystalline structure of the formulation was maintained during the experiment, however chemical and functional stability results showed a great influence of the storage period in all conditions tested. A significant decrease in quercetin content (approximately 40%) was detected during the first month of storage and a similar significant loss in antioxidant activity was detected after 6xa0months. The remaining flavonoid content was unchanged during the final 6xa0months of the experimental period. The results suggest possible interactions between quercetin and the liquid crystalline formulation, which could inhibit or reduce the quercetin activity incorporated in the system. In conclusion, the present study demonstrated that incorporation of quercetin (1%) did not affect the liquid crystalline structure composed of vitamin E TPGS/IPM/PG–H2O (1:1) at 63.75/21.25/15 (w/w/w). Nevertheless, of the total quercetin incorporated in the system only 60% was free to act as an antioxidant.

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.

The protein LJM 111 from Lutzomyia longipalpis Salivary Gland Extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model

Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis. We aimed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF-α and IFN-γ released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry, TNF-α and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF-α and IFN-γ. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF-α whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils.

Effect of gedunin on acute articular inflammation and hypernociception in mice.

Gedunin, a natural limonoid from Meliaceae species, has been previously described as an antiinflammatory compound in experimental models of allergic inflammation. Here, we report the antiinflammatory and antinociceptive effects of gedunin in an acute model of articular inflammation induced by zymosan (500 μg/cavity; intra-articular) in C57BL/6 mice. Intraperitoneal (i.p.) pretreatment with gedunin (0.005–5 mg/kg) impaired zymosan-induced edema formation, neutrophil accumulation and hypernociception in mouse knee joints, due to decreased expression of preproET-1 mRNA and production of LTB4, PGE2, TNF-α and IL-6. Mouse post-treatment with gedunin (0.05 mg/kg; i.p.) 1 and 6 h after stimulation also impaired articular inflammation, by reverting edema formation, neutrophil accumulation and the production of lipid mediators, cytokines and endothelin. In addition, gedunin directly modulated the functions of neutrophils and macrophages in vitro. The pre-incubation of neutrophil with gedunin (100 µM) impaired shape change, adhesion to endothelial cells, chemotaxis and lipid body formation triggered by different stimuli. Macrophage pretreatment with gedunin impaired intracellular calcium mobilization, nitric oxide production, inducible nitric oxide synthase expression and induced the expression of the antiinflammatory chaperone heat shock protein 70. Our results demonstrate that gedunin presents remarkable antiinflammatory and anti-nociceptive effects on zymosan-induced inflamed knee joints, modulating different cell populations.

Diosmin reduces chronic constriction injury-induced neuropathic pain in mice

Injury or dysfunction of somatosensory system induces a complex syndrome called neuropathic pain, which still needs adequate pharmacological control. The current pharmacological treatments were in part developed from natural compounds. Flavonoids are natural polyphenolic molecules presenting varied biological activities and low toxicity. The flavonoid diosmin is a safe compound with good tolerability and low toxicity. This study evaluated the antinociceptive effect of diosmin in the sciatic nerve chronic constriction injury (CCI)-induced neuropathic pain model. Male Swiss mice were submitted to CCI and 7 days after, diosmin at 1 or 10xa0mg/kg was administrated intraperitoneally. Mechanical (electronic analgesimeter) and thermal (hot plate) hyperalgesia were evaluated 1-24xa0h after treatment. The role of the NO/cGMP/PKG/KATP channel signaling pathway in the analgesic effect of diosmin was evaluated using the pretreatment with L-NAME (an inhibitor of NOS), ODQ (an inhibitor of soluble guanylate cyclase), KT5823 (an inhibitor of PKG), or glibenclamide (an ATP-sensitive K+ channels blocker). Single treatment with diosmin inhibited in a dose-dependent manner CCI-induced mechanical and thermal hyperalgesia by activating the NO/cGMP/PKG/KATP channel signaling pathway and inhibiting spinal cord cytokine (Il-1β and Il-33/St2) and glial cells activation (microglia - Iba-1, oligodendrocytes - Olig2) mRNA expression markers. Daily treatment during 7 days with diosmin inhibited CCI-induced mechanical and thermal hyperalgesia by inhibiting spinal cord cytokine (Il-1β, Tnfα, and Il-33/St2) and glial cells activation (astrocytes - Gfap, Iba-1, and Olig2) markers mRNA expression. In conclusion, diosmin inhibits neuropathic spinal cord nociceptive mechanisms suggesting this flavonoid as a potential therapeutic molecule to reduce nerve lesion-induced neuropathic pain.

Budlein A, a Sesquiterpene Lactone From Viguiera robusta, Alleviates Pain and Inflammation in a Model of Acute Gout Arthritis in Mice

Background: Gout is the most common inflammatory arthritis worldwide. It is a painful inflammatory disease induced by the deposition of monosodium urate (MSU) crystals in the joints and peri-articular tissues. Sesquiterpene lactones (SLs) are secondary metabolite biosynthesized mainly by species from the family Asteraceae. It has been demonstrated that SLs present anti-inflammatory, analgesic, antitumoral, antiparasitic, and antimicrobial activities. In this study, we aimed at evaluating the efficacy of the SL budlein A in a model of acute gout arthritis in mice. Methods: Experiments were conducted in male Swiss or male LysM-eGFP mice. Animals were treated with budlein A (1 or 10 mg/kg) or vehicle 30 min before stimulus with MSU (100 μg/10 μL, intra-articular). Knee joint withdrawal threshold and edema were evaluated using electronic von Frey and caliper, respectively, 1–15 h after MSU injection. Leukocyte recruitment was determined by counting cells (Neubauer chamber), H&E staining, and using LysM-eGFP mice by confocal microscopy. Inflammasome components, Il-1β, and Tnf-α mRNA expression were determined by RT-qPCR. IL-1β and TNF-α production (in vitro) and NF-κB activation (in vitro and in vivo) were evaluated by ELISA. In vitro analysis using LPS-primed bone marrow-derived macrophages (BMDMs) was performed 5 h after stimulation with MSU crystals. For these experiments, BMDMs were either treated or pre-treated with budlein A at concentrations of 1, 3, or 10 μg/mL. Results: We demonstrated that budlein A reduced mechanical hypersensitivity and knee joint edema. Moreover, it reduced neutrophil recruitment, phagocytosis of MSU crystals by neutrophils, and Il-1β and Tnf-α mRNA expression in the knee joint. In vitro, budlein A decreased TNF-α production, which might be related to the inhibition of NF-κB activation. Furthermore, budlein A also reduced the IL-1β maturation, possibly by targeting inflammasome assembly in macrophages. Conclusion: Budlein A reduced pain and inflammation in a model of acute gout arthritis in mice. Therefore, it is likely that molecules with the ability of targeting NF-κB activation and inflammasome assembly, such as budlein A, are interesting approaches to treat gout flares.