A novel diselenide attenuates the carrageenan-induced inflammation by reducing neutrophil infiltration and the resulting tissue damage in mice.

Selenium-containing compounds have emerged as promising treatment for redox-based and inflammatory diseases. This study aimed to investigate the in vitro and in vivo anti-inflammatory activity of a novel diselenide named as dibenzyl[diselanediyIbis(propane-3-1diyl)] dicarbamate (DD). DD reacted with HOCl (k = 9.2 x 107 M-1s-1), like glutathione (k = 1.2 x 108 M-1s-1), yielding seleninic and selenonic acid derivatives, and it also decreased HOCl formation by activated human neutrophils (IC50=4.6 µM) and purified myeloperoxidase (MPO) (IC50=3.8 µM). However, tyrosine, MPO-I and MPO-II substrates, did not restore HOCl formation in presence of DD. DD inhibited the oxidative burst in dHL-60 cells with no toxicity up to 25 µM for 48h. Next, an intraperitoneal administration of 25, 50, and 75 mg/kg DD decreased total leukocyte, neutrophil chemotaxis, and inflammation markers (MPO activity, lipid peroxidation, albumin exudation, nitrite, TNF-α, IL-1ß, CXCL1/KC, and CXCL2/MIP-2) on a murine model of carrageenan-induced peritonitis. Likewise, 50 mg/kg DD (i.p.) decreased carrageenan-induced paw edema over 5h. Histological and immunohistochemistry analyses of the paw tissue showed decreased neutrophil count, edema area, and MPO, carbonylated, and nitrated protein staining. Furthermore, DD treatment decreased the fMLP-induced chemotaxis of human neutrophils (IC50=3.7 µM) in vitro with no toxicity. Lastly, DD presented no toxicity in a single-dose model using mice (50 mg/kg, i.p.) over 15 days and in Artemia salina bioassay (50 to 2000 µM), corroborating findings from in silico toxicological study. Altogether, these results demonstrate that DD attenuates carrageenan-induced inflammation mainly by reducing neutrophil migration and the resulting damage from MPO-mediated oxidative burst.

Inflammatory stimulus worsens the effects of UV-A exposure on J774 cells.

UV-A radiation affects skin homeostasis by promoting oxidative distress. Endogenous photosensitizers in the dermis and epidermis of human skin absorb UV-A radiation forming excited states (singlet and triplet) and reactive oxygen species (ROS) producing oxidized compounds that trigger biological responses. The activation of NF-kB induces the expression of pro-inflammatory cytokines and can intensify the generation of ROS. However, there is no studies evaluating the cross talks between inflammatory stimulus and UV-A exposure on the levels of redox misbalance and inflammation. In here, we evaluated the effects of UV-A exposure on J774 macrophage cells previously challenged with LPS in terms of oxidative distress, release of pro-inflammatory cytokines, and activation of regulated cell death pathways. Our results showed that LPS potentiates the dose-dependent UV-A-induced oxidative distress and cytokine release, in addition to amplifying the regulated (autophagy and apoptosis) and non-regulated (necrosis) mechanisms of cell death, indicating that a previous inflammatory stimulus potentiates UV-A-induced cell damage. We discuss these results in terms of the current-available skin care strategies.

A Purine Derivative Containing an Organoselenium Group Protects Against Memory Impairment, Sensitivity to Nociception, Oxidative Damage, and Neuroinflammation in a Mouse Model of Alzheimer's Disease.

In the present study, the effect of 6-((4-fluorophenyl) selanyl)-9H-purine (FSP) was tested against memory impairment and sensitivity to nociception induced by intracerebroventricular injection of amyloid-beta peptide (Aß) (25-35 fragment), 3 nmol/3 µl/per site in mice. Memory impairment was determined by the object recognition task (ORT) and nociception by the Von-Frey test (VFT). Aß caused neuroinflammation with upregulation of glial fibrillary acidic protein (GFAP) (in hippocampus), nuclear factor-κB (NF-κB), and the proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in cerebral cortex and hippocampus. Additionally, Aß increased oxidant levels and lipid peroxidation in cerebral cortex and hippocampus, but decreased heme oxygenase-1 (HO-1) and peroxiredoxin-1 (Prdx1) expression in the hippocampus. Anti-neuroinflammatory effects of FSP were demonstrated by a decrease in the expression of GFAP and NF-κB in the hippocampus, as well as a decrease in proinflammatory cytokines in both the hippocampus and cerebral cortex FSP protected against oxidative stress by decreasing oxidant levels and lipid peroxidation and by increasing HO-1 and Prdx1 expressions in the hippocampus of mice. Moreover, FSP prevented the activation of nuclear factor erythroid 2-related factor 2 (Nrf-2) in the hippocampus of mice induced by Aß. In conclusion, treatment with FSP attenuated memory impairment, nociception sensitivity by decreasing oxidative stress, and neuroinflammation in a mouse model of Alzheimer's disease.

Uric Acid Reacts with Peroxidasin, Decreases Collagen IV Crosslink, Impairs Human Endothelial Cell Migration and Adhesion.

Uric acid is considered the main substrate for peroxidases in plasma. The oxidation of uric acid by human peroxidases generates urate free radical and urate hydroperoxide, which might affect endothelial function and explain, at least in part, the harmful effects of uric acid on the vascular system. Peroxidasin (PXDN), the most recent heme-peroxidase described in humans, catalyzes the formation of hypobromous acid, which mediates collagen IV crosslinks in the extracellular matrix. This enzyme has gained increasing scientific interest since it is associated with cardiovascular disease, cancer, and renal fibrosis. The main objective here was to investigate whether uric acid would react with PXDN and compromise the function of the enzyme in human endothelial cells. Urate decreased Amplex Red oxidation and brominating activity in the extracellular matrix (ECM) from HEK293/PXDN overexpressing cells and in the secretome of HUVECs. Parallelly, urate was oxidized to 5-hydroxyisourate. It also decreased collagen IV crosslink in isolated ECM from PFHR9 cells. Urate, the PXDN inhibitor phloroglucinol, and the PXDN knockdown impaired migration and adhesion of HUVECs. These results demonstrated that uric acid can affect extracellular matrix formation by competing for PXDN. The oxidation of uric acid by PXDN is likely a relevant mechanism in the endothelial dysfunction related to this metabolite.

Loss of mTORC2 Activity in Neutrophils Impairs Fusion of Granules and Affects Cellular Metabolism Favoring Increased Bacterial Burden in Sepsis.

Sepsis is a complex infectious syndrome in which neutrophil participation is crucial for patient survival. Neutrophils quickly sense and eliminate the pathogen by using different effector mechanisms controlled by metabolic processes. The mammalian target of rapamycin (mTOR) pathway is an important route for metabolic regulation, and its role in neutrophil metabolism has not been fully understood yet, especially the importance of mTOR complex 2 (mTORC2) in the neutrophil effector functions. In this study, we observed that the loss of Rictor (mTORC2 scaffold protein) in primary mouse-derived neutrophils affects their chemotaxis by fMLF and their microbial killing capacity, but not the phagocytic capacity. We found that the microbicidal capacity was impaired in Rictor-deleted neutrophils because of an improper fusion of granules, reducing the hypochlorous acid production. The loss of Rictor also led to metabolic alterations in isolated neutrophils, increasing aerobic glycolysis. Finally, myeloid-Rictor-deleted mice (LysMRic Δ/Δ) also showed an impairment of the microbicidal capacity, increasing the bacterial burden in the Escherichia coli sepsis model. Overall, our results highlight the importance of proper mTORC2 activation for neutrophil effector functions and metabolism during sepsis.

Peroxiredoxin AhpC1 protects Pseudomonas aeruginosa against the inflammatory oxidative burst and confers virulence.

Pseudomonas aeruginosa is an opportunistic bacterium in patients with cystic fibrosis and hospital acquired infections. It presents a plethora of virulence factors and antioxidant enzymes that help to subvert the immune system. In this study, we identified the 2-Cys peroxiredoxin, alkyl-hydroperoxide reductase C1 (AhpC1), as a relevant scavenger of oxidants generated during inflammatory oxidative burst and a mechanism of P. aeruginosa (PA14) escaping from killing. Deletion of AhpC1 led to a higher sensitivity to hypochlorous acid (HOCl, IC50 3.2 ± 0.3 versus 19.1 ± 0.2 µM), hydrogen peroxide (IC50 91.2 ± 0.3 versus 496.5 ± 6.4 µM) and the organic peroxide urate hydroperoxide. ΔahpC1 strain was more sensitive to the killing by isolated neutrophils and less virulent in a mice model of infection. All mice intranasally instilled with ΔahpC1 survived as long as they were monitored (15 days), whereas 100% wild-type and ΔahpC1 complemented with ahpC1 gene (ΔahpC1 attB:ahpC1) died within 3 days. A significantly lower number of colonies was detected in the lung and spleen of ΔahpC1-infected mice. Total leucocytes, neutrophils, myeloperoxidase activity, pro-inflammatory cytokines, nitrite production and lipid peroxidation were much lower in lungs or bronchoalveolar liquid of mice infected with ΔahpC1. Purified AhpC neutralized the inflammatory organic peroxide, urate hydroperoxide, at a rate constant of 2.3 ± 0.1 × 106 M-1s-1, and only the ΔahpC1 strain was sensitive to this oxidant. Incubation of neutrophils with uric acid, the urate hydroperoxide precursor, impaired neutrophil killing of wild-type but improved the killing of ΔahpC1. Hyperuricemic mice presented higher levels of serum cytokines and succumbed much faster to PA14 infection when compared to normouricemic mice. In summary, ΔahpC1 PA14 presented a lower virulence, which was attributed to a poorer ability to neutralize the oxidants generated by inflammatory oxidative burst, leading to a more efficient killing by the host. The enzyme is particularly relevant in detoxifying the newly reported inflammatory organic peroxide, urate hydroperoxide.

Integration of an Inhibitor-like Rule and Structure-based Virtual Screening for the Discovery of Novel Myeloperoxidase Inhibitors.

Myeloperoxidase (MPO) is an attractive therapeutic target against inflammation. Herein, we developed an inhibitor-like rule, based on known MPO inhibitors, and generated a target database containing 6546 molecules with privileged inhibitory properties. Using a structure-based approach validated by decoys, robust statistical metrics, redocking, and cross-docking, we selected 10 putative MPO inhibitors with high chemical diversity. At 20 µM, six of these 10 compounds (i.e., 60% success rate) inhibited more than 20% of the chlorinating activity of the enzyme. Additionally, we found that compound ZINC9089086 forms hydrogen bonds with Arg233 and with the hemic carboxylate. It makes a π-stacking interaction with the heme group and displays a high affinity for the enzyme active site. When incubated with purified MPO, ZINC9089086 inhibited the chlorinating activity of the enzyme with an IC50 of 2.2 ± 0.1 µM in a reversible manner. Subsequent experiments revealed that ZINC9089086 inhibited hypochlorous acid production in dHL-60 cells and human neutrophils. Furthermore, the theoretical ADME/Tox profile indicated that this compound exhibits low toxicity risks and adequate pharmacokinetic parameters, thus making ZINC9089086 a very promising candidate for preclinical anti-inflammatory studies. Overall, our study shows that integrating an inhibitor-like rule with a validated structure-based methodology is an excellent approach for improving the success rate and molecular diversity of novel MPO inhibitors with good pharmacokinetics and toxicological profiles. By combining these tools, it was possible to increase the assurance rate, which ultimately diminishes the costs and time needed for the acquisition, synthesis, and evaluation of new compounds.

Urate hydroperoxide oxidizes endothelial cell surface protein disulfide isomerase-A1 and impairs adherence.

BACKGROUND: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. METHODS: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. RESULTS: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M-1 s-1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. CONCLUSIONS: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. GENERAL SIGNIFICANCE: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.

Methylene blue photodynamic therapy induces selective and massive cell death in human breast cancer cells.

BACKGROUND: Breast cancer is the main cause of mortality among women. The disease presents high recurrence mainly due to incomplete efficacy of primary treatment in killing all cancer cells. Photodynamic therapy (PDT), an approach that causes tissue destruction by visible light in the presence of a photosensitizer (Ps) and oxygen, appears as a promising alternative therapy that could be used adjunct to chemotherapy and surgery for curing cancer. However, the efficacy of PDT to treat breast tumours as well as the molecular mechanisms that lead to cell death remain unclear. METHODS: In this study, we assessed the cell-killing potential of PDT using methylene blue (MB-PDT) in three breast epithelial cell lines that represent non-malignant conditions and different molecular subtypes of breast tumours. Cells were incubated in the absence or presence of MB and irradiated or not at 640 nm with 4.5 J/cm2. We used a combination of imaging and biochemistry approaches to assess the involvement of classical autophagic and apoptotic pathways in mediating the cell-deletion induced by MB-PDT. The role of these pathways was investigated using specific inhibitors, activators and gene silencing. RESULTS: We observed that MB-PDT differentially induces massive cell death of tumour cells. Non-malignant cells were significantly more resistant to the therapy compared to malignant cells. Morphological and biochemical analysis of dying cells pointed to alternative mechanisms rather than classical apoptosis. MB-PDT-induced autophagy modulated cell viability depending on the cell model used. However, impairment of one of these pathways did not prevent the fatal destination of MB-PDT treated cells. Additionally, when using a physiological 3D culture model that recapitulates relevant features of normal and tumorous breast tissue morphology, we found that MB-PDT differential action in killing tumour cells was even higher than what was detected in 2D cultures. CONCLUSIONS: Finally, our observations underscore the potential of MB-PDT as a highly efficient strategy which could use as a powerful adjunct therapy to surgery of breast tumours, and possibly other types of tumours, to safely increase the eradication rate of microscopic residual disease and thus minimizing the chance of both local and metastatic recurrence.

The transient receptor potential ankyrin-1 mediates mechanical hyperalgesia induced by the activation of B1 receptor in mice.

The kinin receptor B1 and the transient receptor potential ankyrin 1 (TRPA1) work as initiators and gatekeepers of nociception and inflammation. This study reports that the nociceptive transmission induced by activation of B1 receptor is dependent on TRPA1 ion channel. The mechanical hyperalgesia was induced by intrathecal (i.t.) injection of B1 agonist des-Arginine9-bradykinin (DABK) or TRPA1 agonist cinnamaldehyde and was evaluated by the withdrawal response after von Frey Hair application in the hind paw. After behavioral experiments, lumbar spinal cord and dorsal root ganglia (DRG) were harvested to assess protein expression and mRNA by immunohistochemistry and real time-PCR, respectively. The pharmacological antagonism (HC030031) or the down-regulation of TRPA1 greatly inhibited the mechanical hyperalgesia induced by DABK. Intrathecal injection of DABK up regulated the ionized calcium binding adaptor molecule (Iba-1) in lumbar spinal cord (L5-L6); TRPA1 protein and mRNA in lumbar spinal cord; and B1 receptor mRNA in both lumbar spinal cord and DRG. The knockdown of TRPA1 prevented microglia activation induced by DABK. Furthermore, the mechanical hyperalgesia induced by either DABK or by cinnamaldehyde was significantly reduced by inhibition of cyclooxygenase (COX), protein kinase C (PKC) or phospholipase C (PLC). In summary, this study revealed that TRPA1 positively modulates the mechanical hyperalgesia induced by B1 receptor activation in the spinal cord and that the classical GPCR downstream molecules PLC, diacylglycerol (DAG), 3,4,5-inositide phosphate (IP3) and PKC are involved in the nociceptive transmission triggered by these two receptors.

TRP modulation by natural compounds.

The use of medicinal plants or other naturally derived products to relieve illness can be traced back over several millennia, and these natural products are still extensively used nowadays. Studies on natural products have, over the years, enormously contributed to the development of therapeutic drugs used in modern medicine. By means of the use of these substances as selective agonists, antagonists, enzyme inhibitors or activators, it has been possible to understand the complex function of many relevant targets. For instance, in an attempt to understand how pepper species evoke hot and painful actions, the pungent and active constituent capsaicin (from Capsicum sp.) was isolated in 1846 and the receptor for the biological actions of capsaicin was cloned in 1997, which is now known as TRPV1 (transient receptor potential vanilloid 1). Thus, TRPV1 agonists and antagonists have currently been tested in order to find new drug classes to treat different disorders. Indeed, the transient receptor potential (TRP) proteins are targets for several natural compounds, and antagonists of TRPs have been synthesised based on the knowledge of naturally derived products. In this context, this chapter focuses on naturally derived compounds (from plants and animals) that are reported to be able to modulate TRP channels. To clarify and make the understanding of the modulatory effects of natural compounds on TRPs easier, this chapter is divided into groups according to TRP subfamilies: TRPV (TRP vanilloid), TRPA (TRP ankyrin), TRPM (TRP melastatin), TRPC (TRP canonical) and TRPP (TRP polycystin). A general overview on the naturally derived compounds that modulate TRPs is depicted in Table 1.

Oral administration of the flavonoid myricitrin prevents dextran sulfate sodium-induced experimental colitis in mice through modulation of PI3K/Akt signaling pathway.

SCOPE: We investigated the protective effect of the flavonoid myricitrin in dextran sulfate sodium (DSS) induced colitis as promising candidate for the treatment of ulcerative colitis which is considered an important worldwide public health problem. METHODS AND RESULTS: Male CD1 mice were provided with a solution of filtered water containing 3% w/v DSS ad libitum over a 5-day period followed by 2 days with normal drinking water. Myricitrin was administered orally, once a day, at the doses 1, 3, and 10 mg/kg of body weight. At the end of day 7th, the animals were euthanized and the colonic tissue was collected to be analyzed by RT-PCR, immunohistochemistry and Western blot. Our results showed that oral treatment with myricitrin exerts consistent anti-inflammatory action in DSS-induced acute colitis in mice by the inhibition of the Akt/phosphatidylinositol-3 kinase-dependent phosphorylation. Consequently, the phosphorylation of mitogen-activated protein kinases (MAPK) p38, extracellular signal-regulated protein kinase (ERK1/2), and c-Jun N-terminal kinase and of the nuclear factor B (NF-κB) was reduced and prevented an increase in the cytokines/chemokines levels. CONCLUSION: Together, these data reveal that the anti-inflammatory effect of myricitrin in DSS-induced colitis in mice is likely associated with its ability to prevent the activation of upstream kinases, such as phosphatidylinositol-3 kinase-dependent Akt, NF-κB, and mitogen-activated protein kinase.

Euphol, a tetracyclic triterpene produces antinociceptive effects in inflammatory and neuropathic pain: the involvement of cannabinoid system.

Persistent pains associated with inflammatory and neuropathic states are prevalent and debilitating diseases, which still remain without a safe and adequate treatment. Euphol, an alcohol tetracyclic triterpene, has a wide range of pharmacological properties and is considered to have anti-inflammatory action. Here, we assessed the effects and the underlying mechanisms of action of euphol in preventing inflammatory and neuropathic pain. Oral treatment with euphol (30 and 100 mg/kg) reduced carrageenan-induced mechanical hyperalgesia. Likewise, euphol given through the spinal and intracerebroventricular routes prevented mechanical hyperalgesia induced by carrageenan. Euphol consistently blocked the mechanical hyperalgesia induced by complete Freund's adjuvant, keratinocyte-derived chemokine, interleukin-1ß, interleukin-6 and tumor necrosis factor-alpha associated with the suppression of myeloperoxidase activity in the mouse paw. Oral treatment with euphol was also effective in preventing the mechanical nociceptive response induced by ligation of the sciatic nerve and also significantly reduced the levels and mRNA of cytokines/chemokines in both paw and spinal cord tissues following i.pl. injection of complete Freund's adjuvant. In addition, the pre-treatment with either CB1R or CB2R antagonists, as well as the knockdown gene of the CB1R and CB2R, significantly reversed the antinociceptive effect of euphol. Interestingly, even in higher doses, euphol did not cause any relevant action in the central nervous system. Considering that few drugs are currently available for the treatment of chronic pain states, the present results provided evidence that euphol constitutes a promising molecule for the management of inflammatory and neuropathic pain states.

The nociception induced by glutamate in mice is potentiated by protons released into the solution.

UNLABELLED: In this study we compare the effect of a glutamate solution with pH adjusted to 7 (3-30 micromol/paw), a non-pH-adjusted glutamate solution (.3-30 micromol/paw, pH range 2.24-1.14), and an acid solution (2% acetic acid, pH 1.4-7) in terms of causing licking behavior in mice. The sum of licking seconds was recorded in the first 15 minutes following the intraplantar (i.pl.) injection of the solutions. Protons potentiated the nociception induced by glutamate. The ED(50) values were 2.5 (1.5-4.2) and 15.1 (11.5-19.9) micromol/paw for the non-pH-adjusted and pH-adjusted glutamate solutions, respectively. The acid solutions at pH 1.4, 2 and 4 induced a similar nociception. The blocking of the acid-sensitive ion channels (ASICs) by amiloride and the antagonism of the transient receptor potential vanilloid subtype-1 (TRPV1) by capsazepine, injected via i.pl., significantly decreased the nociception mediated by acid and by non-pH-adjusted glutamate solutions, but did not affect the nociception caused by the pH-adjusted glutamate solution. The pretreatment with the NMDA-receptor antagonist (MK-801, i.pl.), with the cyclooxygenase inhibitor (indomethacin, i.pl.) or the disruption of the sensorial C fibers by capsaicin, decreased the nociceptive effect of the 3 algogen tested. In summary, the protons present in aqueous solution of glutamate can cause nociception per se or can potentiate the nociception caused by glutamate. These effects are related to the activation of ASICs, TRPV1 and NMDA receptors, inhibition of the synthesis of prostanoids, and disruption of the C fibers. PERSPECTIVE: The nociception induced by glutamate is a useful method for investigation of the mechanisms of nociception and the effects of new analgesic drugs. Our findings showed that the protons released from glutamic acid have to be removed from the solution to avoid misinterpretation of results in the search for new analgesic drugs.

The involvement of the transient receptor potential A1 (TRPA1) in the maintenance of mechanical and cold hyperalgesia in persistent inflammation.

This study investigated the role of TRPA1 in the development and maintenance of mechanical and cold hyperalgesia in persistent inflammation induced by Complete Freund's Adjuvant (CFA) in mice. The intraplantar (i.pl.) injection of CFA induced a long lasting (28 days) hyperalgesia for both mechanical and thermal (cold) stimuli. The intraperitoneal (i.p., 30-300 mg/kg), intraplantar (i.pl., 100 microg/site) or intrathecal (i.t., 10 microg/site) injection of the TRPA1 selective antagonist HC-030031 significantly reduced the mechanical hyperalgesia evaluated by the von Frey hair test. The effect of HC-030031 was evidenced on the day after CFA injection and was kept throughout the test. However, the intracerebroventricular (i.c.v., 10 microg/site) injection of HC-030031 did not interfere with CFA-induced hyperalgesia. Treatment with HC-030031 (300 mg/kg, i.p.) completely inhibited the noxious cold hyperalgesia induced by tetrafluoroethane in mice that received CFA. The pre-treatment with the TRPA1 oligonucleotide antisense (AS-ODN, i.t.) consistently prevented both mechanical and cold hyperalgesia. Interestingly, both TRPA1 protein expression and mRNA were over-expressed in spinal cord and dorsal root ganglia (DRG) of mice treated with CFA, an effect that was fully prevented by the pre-treatment with the TRPA1 antagonist HC-030031. Collectively, the present results showed that TRPA1 present at either peripheral or spinal sites play a relevant role in the development and maintenance of both mechanical and cold hyperalgesia during CFA-induced inflammation. Thus, TRPA1 selective antagonists represent promising candidates to treat hyperalgesia in persistent inflammatory states.

Synthetic derivatives of the alpha- and beta-amyrin triterpenes and their antinociceptive properties.

Fifteen different derivatives of an alpha- and beta-amyrin mixture were synthesized by acylation with appropriate anhydride or acid chlorides and oxidation in the presence of tert-butyl chromate or PCC. The molecular structures of the obtained compounds were confirmed by means of IR and (1)H NMR spectra. The compounds were screened for antinociceptive activity using the acetic acid pain model. The 3-O-acyl derivatives alpha- and beta-amyrin propionate 4, alpha- and beta-amyrin hexanoate 6, and alpha- and beta-amyrin octanoate 7 were found to be the most active compounds of the series. In addition, we also have found that alpha- and beta-amyrin octanoate 7 was able to reduce acetic acid-induced abdominal constriction when administered by oral route. Furthermore, this compound reduced the nociceptive response induced by intraplantar injection of formalin.

Myricitrin as a substrate and inhibitor of myeloperoxidase: implications for the pharmacological effects of flavonoids.

Flavonoids are increasingly being ingested by the general population as chemotherapeutic and anti-inflammatory agents. They are potentially toxic because of their conversion to free radicals and reactive quinones by peroxidases. Little detailed information is available on how flavonoids interact with myeloperoxidase, which is the predominant peroxidase present at sites of inflammation. This enzyme uses hydrogen peroxide to oxidize chloride to hypochlorous acid, as well as to produce an array of reactive free radicals from organic substrates. We investigated how the flavonoid myricitrin is oxidized by myeloperoxidase and how it affects the activities of this enzyme. Myricitrin was readily oxidized by myeloperoxidase in the presence of hydrogen peroxide. Its main oxidation product was a dimer that underwent further oxidation. In the presence of glutathione, myricitrin was oxidized to a hydroquinone that was conjugated to glutathione. When myeloperoxidase oxidized myricitrin and related flavonoids it became irreversibly inactivated. The number of hydroxyl groups in the B ring of the flavonoids and the presence of a free hydroxyl m-phenol group in the A ring were important for the inhibitory effects. Less enzyme inactivation occurred in the presence of chloride. Neutrophils also oxidized myricitrin to dimers in a reaction that was partially dependent on myeloperoxidase. Myricitrin did not affect the production of hypochlorous acid by neutrophils. We conclude that myricitrin will be oxidized by neutrophils at sites of inflammation to produce reactive free radicals and quinones. It is unlikely to affect hypochlorous acid production by neutrophils.

Toxicological evaluation of subchronic exposure to diphenyl diselenide in rats.

The aim of this study was to investigate the effects caused by subchronic exposure to diphenyl diselenide in rats. Adult Wistar rats were exposed to diphenyl diselenide (5-300 micromol kg(-1), subcutaneously) once a day for 14 days. The subchronic administration of diphenyl diselenide at a dose of 300 micromol kg(-1) significantly increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in plasma. Conversely, this exposure did not alter lactate dehydrogenase (LDH) activity, urea and creatinine levels in plasma. The activity of delta-aminolevulinate dehydratase (delta-ALA-D) from liver and kidney was inhibited by high dosages of diphenyl diselenide. Diphenyl diselenide did not alter renal Na(+)/K(+)ATPase. A decline in body weight gain was associated with a decrease in food consumption in rats treated with 100 or 300 micromol kg(-1) diphenyl diselenide. At these dosages (100 and 300 micromol kg(-1)), diphenyl diselenide did not cause histological alterations in the liver of rats. Taken together, these results demonstrated that subchronic exposure to diphenyl diselenide at high doses induced minor toxicological effects.

Involvement of p38MAPK on the antinociceptive action of myricitrin in mice.

Previous studies from our group investigated the analgesic and anti-inflammatory properties of the flavonoid myricitrin. Here, we demonstrated the role of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and mitogen-activated protein kinases (MAPKs) on the antinociceptive action of myricitrin. The nociceptive response was evaluated by monitoring biting behaviour following intratecal (i.t.) administration of IL-1beta and TNF-alpha in mice. Western blot analyses of total and phosphorylated MAPKs: p38(MAPK), extracellular-signal regulated kinase (ERK1/2) and c-Jun amino-terminal kinases (JNK1/2) from the spinal cord of mice injected with cytokines were measured. Myricitrin (0.03-30mg/kg) or vehicle (control) was administered 30 min beforehand by intraperitoneal (i.p.) injection. Myricitrin pre-treatment prevented cytokine-induced biting behaviour. The calculated ID(50) of myricitrin were 6.8 (4.6-9.0) and 2.6 (0.3-4.9) mg/kg and maximal inhibition of 83+/-9 and 100+/-0% for IL-1beta and TNF-alpha, respectively. Intrathecal injection of IL-1beta and TNF-alpha significantly increased p38(MAPK) phosphorylation and this was inhibited by myricitrin treatment. Cytokines administration did not alter ERK1/2 and JNK1/2 phosphorylation. Myricitrin prevented cytokine-induced biting behaviour and inhibited p38(MAPK) phosphorylation in response to cytokines stimulation. Taken together, it suggests that the mechanism for antinociceptive action of myricitrin in response to cytokines may involve a blockage on p38(MAPK) pathway. This finding could explain, at least in part, the antinociceptive action of this flavonoid in process like neuropathic and inflammatory chronic pain.