Preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral able to attenuate virus-induced inflammation.

Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary because of the continuous circulation of new variants that challenge immunized individuals. Because severe COVID-19 is a virus-triggered immune and inflammatory dysfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that kinetin (MB-905) inhibits the in vitro replication of SARS-CoV-2 in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce error-prone virus replication. Coinhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously incorporated nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. MB-905 shows good oral absorption, its metabolites are stable, achieving long-lasting plasma and lung concentrations, and this drug is not mutagenic nor cardiotoxic in acute and chronic treatments. SARS-CoV-2-infected hACE-mice and hamsters treated with MB-905 show decreased viral replication, lung necrosis, hemorrhage and inflammation. Because kinetin is clinically investigated for a rare genetic disease at regimens beyond the predicted concentrations of antiviral/anti-inflammatory inhibition, our investigation suggests the opportunity for the rapid clinical development of a new antiviral substance for the treatment of COVID-19.

Radical Anions and Dianions of Naphthalenediimides Generated within Layer-by-Layer Zirconium Phosphonate Thin Films.

Chemical reduction of N,N'-bis(2-phosphonoethyl)-1,4,5,8-naphthalenediimide (PNDI) with the reducing agent sodium dithionite gave stable colored reduced species, both in homogeneous solutions and in self-assembled thin films. When colorless PNDI aqueous solutions were titrated with the reducing agent, stepwise reduction was observed, giving first the radical anion (PNDI-•) and then the dianion (PNDI2-) species, which were detected by UV-visible-NIR spectroscopy, allowing the unambiguous determination of absorption maxima and molar absorptivities for each species. The radical anion PNDI-• was found to form π-dimers in water, but monomeric PNDI-• was formed in the presence of the cationic surfactant cetyltrimethylammonium bromide, indicating association with the micelles. Thin films of PNDI with 25 layers were grown by the zirconium phosphonate method on quartz substrates. Reduction of the films with sodium dithionite also produced radical anions and dianions of PNDI. However, reduction in the films was much slower than in solution, evidencing the compactness of the films. Moreover, reduction in the films did not proceed to completion, even with excess of the reducing agent, which can be attributed to the repulsion of negative charges within the film.

Amiloidose pulmonar com apresentação traqueobrônquica: relato de caso clínico / Pulmonary amyloidosis with tracheobronchial presentation: clinical case report

Introdução: Amiloidose é o termo utilizado para designar doenças que fazem deposição extracelular de proteínas fibrilares patológicas em órgãos e tecidos, podendo ser sistêmica ou restrita a um único órgão. As manifestações clínicas são diversas, como cardiomiopatia, falência renal, esplenomegalia, problemas intestinais, neuropatias, problemas pulmonares, entre outros. Objetivo: relatar um caso clínico de paciente com amiloidose traqueobrônquica. Metodologia: revisão de bibliografias em comparação ao relato de caso, o qual foi descrito a partir de dados retirados do prontuário e de exames complementares da paciente. Caso clínico: paciente do sexo feminino, 70 anos, procurou assistência médica por dorsalgia, apresentando também chiado, tosse seca, dispneia paroxística noturna e ortopneia. Realizou-se investigação diagnóstica durante a internação, na qual biópsia da mucosa traqueobrônquica e coleta de lavado alveolar foram positivos para o teste Vermelho Congo, o que confirmou o diagnóstico de amiloidose. A paciente, então, foi encaminhada para terapia de ablação a laser. Conclusão: portanto, diante de um paciente com quadro clínico inespecífico e suspeita diagnóstica principal de amiloidose pulmonar, é imprescindível investigar e descartar diagnósticos diferenciais como neoplasia ou discrasia de células plasmáticas. Para isso, é necessário que haja alta precisão na análise dos exames de imagem, de modo a sugerir esse diagnóstico, o qual deve ser confirmado através da fibrobroncoscopia com biópsia de tecido brônquico, que através da coloração Vermelho do Congo, evidenciará presença de substância amorfa e birrefringente, compatível com substância amiloide [au]

Introduction: Amyloidosis is the term used to describe diseases that cause extracellular deposition of pathological fibrillar proteins in organs and tissues, which can be systemic or restricted to a single organ. The clinical manifestations are diverse, such as cardiomyopathy, renal failure, splenomegaly, intestinal problems, neuropathies, lung problems, among others. Objective: to report a clinical case of a patient with pulmonary amioloidosis. Methodology: review of bibliographies in comparison to the case report, which was described based on data taken from the patient's record and complementary exams. Clinical case: a seventy-year-old female patient sought medical assistance because of back pain, also presenting wheezing, dry cough, paroxysmal nocturnal dyspnea and orthopnea. Diagnostic investigation was carried out during hospitalization, in which biopsy of the tracheobronchial mucosa and collection of alveolar lavage were positive for the Congo Red test,wich confirmed the amyloidosis diagnosis. The patient was then referred for laser ablation therapy.Conclusion: hence, in a patient with a nonspecific clinical presentation and main diagnostic suspicion of pulmonary amyloidosis, it is essential to investigate and rule out differential diagnoses such as malignancy or plasma cell dyscrasia. Therefore, it is necessary to use high precision in the analysis of image exams in order to suggest this diagnosis, which should be confirmed through fibrobronchoscopy with bronchial tissue biopsy, that through the Congo Red dye, will show the presence of amorphous and birefringent substance, compatible with amyloid substance [au]

A new series of acetohydroxamates shows in vitro and in vivo anticancer activity against melanoma.

Cancer treatment is challenging, mainly due to high levels of drug toxicity and the resistance of tumours to chemotherapy. Hydroxamic acid derivatives have recently aroused attention due to their potential to treat malignancies. In the present study, we sought to investigate the anticancer effects of a new series of synthetic acetohydroxamates. The in vitro cytotoxic and antiproliferative effects of 11 synthetic acetohydroxamates were evaluated against the melanoma cell line A375. Apoptosis, cell cycle, and autophagy assays were employed to elucidate the cell death pathways induced by the compounds. The in vivo pharmacokinetic profiles of the most promising compounds were determined in CD-1 mice, while the in vivo antitumour efficacies were evaluated using the A375 melanoma xenograft model in nude mice. MTT assays revealed that all compounds presented concentration-dependent cytotoxicity against the A375 cell line. AKS 61 produced the most favourable antiproliferative activity according to the sulphorhodamine B and clonogenic assays. AKS 61 treatment resulted in decreased mitochondrial membrane potential and increased apoptosis and autophagy in the A375 cell line. However, AKS 61 failed to prevent in vivo tumour growth in a melanoma xenograft, whereas compound AKS 7 was able to inhibit tumour growth when administered orally. These in vivo findings may be explained by a more favourable pharmacokinetic profile presented by AKS 7 when compared to AKS 61. Taken together, these results suggest that acetohydroxamates have potential anticancer effects and will guide future optimisation of these molecules to allow for further non-clinical development.

Physical Exercise Attenuates Experimental Autoimmune Encephalomyelitis by Inhibiting Peripheral Immune Response and Blood-Brain Barrier Disruption.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) caused by demyelination, immune cell infiltration, and axonal damage. Herein, we sought to investigate the influence of physical exercise on mice experimental autoimmune encephalomyelitis (EAE), a reported MS model. Data show that both strength and endurance training protocols consistently prevented clinical signs of EAE and decreased oxidative stress, an effect which was likely due to improving genomic antioxidant defense-nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response elements (ARE) pathway-in the CNS. In addition, physical exercise inhibited the production of pro-inflammatory cytokines interferon (IFN)-γ, interleukin (IL)-17, and IL-1ß in the spinal cord of mice with EAE. Of note, spleen cells obtained from strength training group incubated with MOG35-55 showed a significant upregulation of CD25 and IL-10 levels, with a decrease of IL-6, MCP-1, and tumor necrosis factor (TNF)-α production, mainly, during acute and chronic phase of EAE. Moreover, these immunomodulatory effects of exercise were associated with reduced expression of adhesion molecules, especially of platelet and endothelial cell adhesion molecule 1 (PECAM-1). Finally, physical exercise also restored the expression of tight junctions in spinal cord. Together, these results demonstrate that mild/moderate physical exercise, when performed regularly in mice, consistently attenuates the progression and pathological hallmarks of EAE, thereby representing an important non-pharmacological intervention for the improvement of immune-mediated diseases such as MS. Graphical Abstract Schematic diagram illustrating the beneficial effects of physical exercise during experimental model of MS. Physical exercise, especially strength (ST) and endurance (ET) training protocols, inhibits the development and progression of disease, measured by the mean maximal clinical score (1.5 and 1.0, respectively), with inhibition of 30 % and 50 %, respectively, based on the AUC, compared with EAEuntreated group. In addition, ST and ET decreased oxidative stress, possibly, through genomic antioxidant defense, Nrf2-Keap1 signaling pathway, in the CNS. Physical exercise inhibited the production of inflammatory cytokines, such as IFN-γ, IL-17 and IL-1ß in the spinal cord after EAE induction, as well as spleen cells obtained from ST group showed a significant upregulation of regulatory T cell markers, such as CD25 and IL-10 levels, and blocked IL-6, MCP-1 and TNF-α production, mainly, during acute and chronic phase of EAE. Finally, these immunomodulatory effects of exercise were associated with inhibition of adhesion molecules and reestablishment of tight junctions expression in spinal cord tissue, thereby limiting BBB permeability and transmigration of autoreactive T cells to the CNS. NO, nitric oxide; GPx, glutathione peroxidase, GSH, glutathione; Nrf2, nuclear factor (erythroid-derived 2)-like 2; CNS, central nervous system; BBB, blood-brain barrier; IFN-g, interferon-gamma; IL-17, interleukin 17; IL-1b, interleukin-1beta.

EPA- and DHA-derived resolvins' actions in inflammatory bowel disease.

Inflammatory bowel diseases are chronic diseases divided into two major forms, ulcerative colitis and Crohn's disease, which are both associated with a chronic inflammatory condition of the gastrointestinal tract. Recent studies have shown that the resolution of inflammatory conditions is a biosynthetically active process where new pro-resolution lipid mediators derived from omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), such as E- and D-series resolvins, protectins, and macrophage mediator in resolving inflammation (maresins), have potent anti-inflammatory activity and serve as specialised mediators that play an important role in the resolution of inflammation. Recent studies have also shown the role of resolvins in referred hyperalgesia associated with different inflammatory processes, such as the visceral pain caused by inflammatory bowel disease. There are many reports describing the principal effects of EPA- and DHA-derived mediators in experimental models of inflammatory bowel diseases. This review focuses on the recent studies on the important role played by pro-resolution lipid mediators in controlling the inflammatory process associated with inflammatory bowel diseases.

Essential oil from Pterodon emarginatus seeds ameliorates experimental autoimmune encephalomyelitis by modulating Th1/Treg cell balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Pterodon emarginatus Vogel is a medicinal plant commonly used in Brazilian traditional medicine as a folk therapy due to its immunosuppressive, anti-inflammatory, anti-rheumatic, healing, tonic and depurative activities. The essential oil (EO) of Pterodon emarginatus is composed of volatile aromatic terpenes and phenyl propanoids, mainly, ß-elemene and ß-caryophyllene sesquiterpenes. Here we reported the effects and some underlying mechanisms of action of EO during murine model of MS, the experimental autoimmune encephalomyelitis (EAE). MATERIALS AND METHODS: EO (50 and 100 mg/kg) was orally administered during the entire period of development of EAE (preventive treatment, day 0-25). In vitro and in vivo immunological responses were evaluated by ELISA, immunohistochemistry, immunofluorescence and flow cytometry. RESULTS: We provide evidence that EO of Pterodon emarginatus (100 mg/kg, p.o.) significantly attenuates neurological signs and also the development of EAE. Furthermore, at the same dose EO consistently inhibited Th1 cell-mediated immune response and upregulated Treg response in vitro. Moreover, the EO inhibited both microglial activation and expression of iNOS, associated with inhibition of axonal demyelization and neuronal death during the development of the disease. CONCLUSION: This is the first experimental evidence showing that oral administration of EO consistently reduces and limits the severity and development of EAE, mainly, through the modulation of Th1/Treg immune balance, and might represent a helpful new tool for control immunoinflammatory conditions, such as MS.

Maresin 1, a proresolving lipid mediator derived from omega-3 polyunsaturated fatty acids, exerts protective actions in murine models of colitis.

It has been previously reported that dietary fish oils, which are rich in the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid, can exert beneficial effects in inflammatory bowel disease. In this study, we investigated the effects of docosahexaenoic acid-derived lipid mediator maresin 1 (MaR1) in dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice. Systemic treatment with MaR1 significantly attenuated both DSS- and 2,4,6-trinitrobenzene sulfonic acid-induced colonic inflammation by improving the disease activity index and reducing body weight loss and colonic tissue damage. MaR1 treatment also induced a significant decrease in levels of inflammatory mediators, such as IL-1ß, TNF-α, IL-6, and IFN-γ, in the acute protocol, as well as IL-1ß and IL-6, but not TNF-α and INF-γ, in the chronic DSS colitis protocol. Additionally, MaR1 decreased ICAM-1 mRNA expression in both the acute and chronic protocols of DSS-induced colitis. Furthermore, the beneficial effects of MaR1 seem to be associated with inhibition of the NF-κB pathway. Moreover, incubation of LPS-stimulated bone marrow-derived macrophage cultures with MaR1 reduced neutrophil migration and reactive oxygen species production, besides decreasing IL-1ß, TNF-α, IL-6, and INF-γ production. Interestingly, macrophages incubated only with MaR1 showed a significant upregulation of mannose receptor C, type 1 mRNA expression, an M2 macrophage phenotype marker. These results indicate that MaR1 consistently protects mice against different models of experimental colitis, possibly by inhibiting the NF-κB pathway and consequently multiple inflammatory mediators, as well as by enhancing the macrophage M2 phenotype.

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.

Spatial reference memory deficits precede motor dysfunction in an experimental autoimmune encephalomyelitis model: the role of kallikrein-kinin system.

Multiple sclerosis (MS) is a progressive T cell-mediated autoimmune demyelinating inflammatory disease of the central nervous system (CNS). Although it is recognized that cognitive deficits represent a manifestation of the disease, the underlying pathogenic mechanisms remain unknown. Here we provide evidence of spatial reference memory impairments during the pre-motor phase of experimental autoimmune encephalomyelitis (EAE) in mice. Specifically, these cognitive deficits were accompanied by down-regulation of choline acetyltransferase (ChAT) mRNA expression on day 5 and 11 post-immunization, and up-regulation of inflammatory cytokines in the hippocampus and prefrontal cortex. Moreover, a marked increase in B1R mRNA expression occurred selectively in the hippocampus, whereas protein level was up-regulated in both brain areas. Genetic deletion of kinin B1R attenuated cognitive deficits and cholinergic dysfunction, and blocked mRNA expression of both IL-17 and IFN-γ in the prefrontal cortex, lymph node and spleen of mice subjected to EAE. The discovery of kinin receptors, mainly B1R, as a target for controlling neuroinflammatory response, as well as the cognitive deficits induced by EAE may foster the therapeutic exploitation of the kallikrein-kinin system (KKS), in particular for the treatment of autoimmune disorders, such as MS, mainly during pre-symptomatic phase.

The role of kinin B1 and B2 receptors in the persistent pain induced by experimental autoimmune encephalomyelitis (EAE) in mice: evidence for the involvement of astrocytes.

Multiple sclerosis (MS) is a progressive, demyelinating inflammatory disease of the human central nervous system (CNS). While the primary symptoms of MS affect motor function, it is now recognized that chronic pain is a relevant symptom that affects both animals and MS patients. There is evidence that glial cells, such as astrocytes, play an important role in the development and maintenance of chronic pain. Kinins, notably bradykinin (BK) acting through B1 (B1R) and B2 (B2R) receptors, play a central role in pain and inflammatory processes. However, it remains unclear whether kinin receptors are involved in neuropathic pain in MS. Here we investigated by genetic and pharmacological approaches the role of kinin receptors in neuropathic pain behaviors induced in the experimental autoimmune encephalomyelitis (EAE) mouse model. Our results showed that gene deletion or antagonism of kinin receptors, especially B1R, significantly inhibited both tactile and thermal hypersensitivity in EAE animals. By contrast, animals with EAE and treated with a B1R selective agonist displayed a significant increase in tactile hypersensitivity. We also observed a marked increase in B1R mRNA and protein level in the mouse spinal cord 14days after EAE immunization. Blockade of B1R significantly suppressed the levels of mRNAs for IL-17, IFN-γ, IL-6, CXCL-1/KC, COX-2 and NOS2, as well as glial activation in the spinal cord. Of note, the selective B1 antagonist DALBK consistently prevented IFN-induced up-regulation of TNF-α and IL-6 release in astrocyte culture. Finally, both B1R and B2R antagonists significantly inhibited COX-2 and NOS2 expression in primary astrocyte culture. The B1R was co-localized with immunomarker of astrocytes in the spinal cord of EAE-treated animals. The above data constitute convincing experimental evidence indicating that both kinin receptors, especially the B1 subtype, exert a critical role in the establishment of persistent hypersensitivity observed in the EAE model, an action that seems to involve a central inflammatory process, possibly acting on astrocytes. Thus, B1 selective antagonists or drugs that reduce kinin release may have the potential to treat neuropathic pain in patients suffering from MS.

Preventive and therapeutic oral administration of the pentacyclic triterpene α,ß-amyrin ameliorates dextran sulfate sodium-induced colitis in mice: the relevance of cannabinoid system.

The pentacyclic triterpene α,ß-amyrin has been previously reported as an effective compound in the treatment of several inflammatory conditions. Recent evidence indicates that α,ß-amyrin displayed its effects through interaction with the cannabinoid pathway. We assessed the anti-inflammatory effects of the α,ß-amyrin in the dextran sulfate sodium (DSS)-induced colitis in mice and investigated whether its effects were associated with the interaction with the cannabinoid system. Our results showed that the oral preventive or therapeutic treatment with α,ß-amyrin significantly reduced disease activity, body weight loss, colonic damage, as well as colonic myeloperoxidase and N-acetylglucosaminidase activities. Moreover, α,ß-amyrin decreases the colonic pro-inflammatory mediators tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and keratinocyte-derived chemokine (CXCL1/KC), while up-regulating the IL-4 levels. Additionally, we also observed that the α,ß-amyrin caused a significant reduction of the adhesion molecules mRNA expression for intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet cell adhesion molecule 1 (PCAM-1), ß(2)-integrin and protein expression for proliferation marker Ki67, the macrophage molecule CD68 and for adhesion molecule P-selectin. Interestingly, our results also showed that the cannabinoid receptor 1 (CB(1)), but not CB(2), pharmacological blockade significantly reversed the beneficial effects of α,ß-amyrin in DSS-induced colitis. Besides, our data demonstrated that mRNA expression for both the endocannabinoid hydrolase monoglyceride lipase 1 (MGL1) and fatty acid amide hydrolase (FAAH) were significantly reduced in the colon of α,ß-amyrin-treated mice. Altogether, these results suggest that the α,ß-amyrin might possess potential therapeutic interest for the treatment of IBD, and also provide new insights for the underlying mechanisms.

The role of PKC/ERK1/2 signaling in the anti-inflammatory effect of tetracyclic triterpene euphol on TPA-induced skin inflammation in mice.

Inflammation underlies the development and progression of a number of skin disorders including psoriasis, atopic dermatitis and cancer. Therefore, novel antiinflammatory agents are of great clinical interest for prevention and treatment of these conditions. Herein, we demonstrated the underlying molecular mechanisms of the antiinflammatory activity of euphol, a tetracyclic triterpene isolated from the sap of Euphorbia tirucalli, in skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice. Topical application of euphol (100 µg/ear) significantly inhibited TPA-induced ear edema and leukocyte influx through the reduction of keratinocyte-derived chemokine (CXCL1/KC) and macrophage inflammatory protein (MIP)-2 levels. At the intracellular level, euphol reduced TPA-induced extracellular signal-regulated protein kinase (ERK) activation and cyclooxygenase-2 (COX-2) upregulation. These effects were associated with euphol's ability to prevent TPA-induced protein kinase C (PKC) activation, namely PKCα and PKCδ isozymes. Our data indicate that topical application of euphol markedly inhibits the inflammatory response induced by TPA. Thus, euphol represents a promising agent for the management of skin diseases with an inflammatory component.

Chemical composition and cytotoxicity activity of the essential oil of Pterodon emarginatus

Pterodon emarginatus Vogel, Fabaceae, is a native aromatic tree distributed by central region of Brazil. Hydroalcoholic infusions of the seeds are used in folk medicine for their anti-rheumatic and anti-inflammatory properties. The objective of this work was identified the chemical components and verify the cytotoxic effect of the essential oil (EO) from P. emarginatus seeds. Thus, the EO of P. emarginatus seeds was analyzed by GC/MS analysis followed by brine shrimp lethality test and cytotoxic activity against tumor cell lines and human peripheral mononuclear blood cells (PBMC). The cancer cell lines tested were C6 (rat glioma), MeWo (human melanoma), CT26.WT (mouse colon carcinoma), MDA (human breast cancer), A549 (human lung carcinoma), B16-F1 (mouse melanoma), CHO-K1 (hamster ovary cell) and BHK-21 (hamster kidney fibroblast). Eleven compounds were identified by GC and CG/MS analyses. The main compounds with concentrations higher than 5% were β-elemene (15.3%), trans-caryophyllene (35.9%), α-humulene (6.8%), germacrene-D (9.8%), bicyclo germacrene (5.5%) and spathulenol (5.9%). The EO of P. emarginatus seeds showed toxicity to Artemia salina (LC50 1.63 µg/mL) and was active against all the cell lines tested. The potent cytotoxic activity had IC50 values ranging from 24.9 to 47 µg/mL. However, EO (1-100 µg/mL) had less cytotoxicity in PBMCs isolated from a healthy subject. In summary, the present study showed the potential antiproliferative of the EO of P. emarginatus seeds.

Evaluation of chemical mediators and cellular response during acute and chronic gut inflammatory response induced by dextran sodium sulfate in mice.

Inflammatory bowel disease (IBD) affects millions of people worldwide but its pathophysiology remains unclear. Therefore, experimental models of colitis have contributed crucially for the understanding of IBD, and also in the investigations for effective therapies. Herein we investigated the kinetics of inflammatory mediator production and cell infiltration during acute and chronic dextran sodium sulfate (DSS)-induced colitis. The induction phases with DSS were characterized by severe disease activity with massive colonic polymorphonuclear infiltration and increased levels of tumor necrosis factor-α (TNF-α), keratinocyte-derived chemokine (CXCL1/KC), interleukin (IL)-17 and vascular adhesion molecule-1 (VCAM-1). Interestingly, in the recovery periods, we found marked increase of anti-inflammatory mediators IL-10, IL-4, transforming growth factor-ß (TGF-ß) and cyclooxygenase 2 (COX-2) that seems be essential for the resolution of intestinal inflammation. Furthermore, nuclear factor κB (NFκB) and regulatory T cell marker forkhead box P3 (FoxP3) were increased gradually during experimental colitis, demonstrating a discrepant profile response and evident immune disbalance in the chronic phase of intestinal mucosal inflammation. Taken together, these results provide valuable information for studies on DSS-induced colitis and especially for the identification of biomarkers that predict disease course and possible therapeutic interventions.

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.

Euphol prevents experimental autoimmune encephalomyelitis in mice: evidence for the underlying mechanisms.

Multiple sclerosis (MS) is a severe chronic T cell-mediated autoimmune inflammatory disease of the central nervous system (CNS), the existing therapy of which is only partially effective and is associated with undesirable side effects. Euphol, an alcohol tetracyclic triterpene, has a wide range of pharmacological properties and is considered to have anti-inflammatory action. However there are no reports about the effects and mechanisms of euphol in experimental autoimmune encephalomyelitis (EAE), an established model of MS. Here we report the effects and the underlying mechanisms of action of euphol in EAE. Euphol (1-10mg/kg) was administered orally at different time-points of EAE. Immunological and inflammatory responses were evaluated by real-time PCR, Western blot and flow cytometry assays. We provide evidence that euphol significantly attenuates neurological signs of EAE. These beneficial effects of euphol seem to be associated with the down-regulation of mRNA and protein expression of some pro-inflammatory mediators such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the CNS. Furthermore, in vitro, euphol consistently inhibited the T cell-mediated immune response including the production of T(H)1 and T(H)17 cytokines in spleen cells of untreated EAE animals. Likewise, oral euphol treatment inhibited the infiltration of T(H)17 myelin-specific cells into the CNS through the adhesion molecule, lymphocyte function-associated antigen 1 (LFA-1). Our findings reveal that oral administration of euphol consistently reduces and limits the severity and development of EAE. Therefore, euphol might represent a potential molecule of interest for the treatment of MS and other T(H)17 cell-mediated inflammatory diseases.

Antinociception of ß-D-glucan from Pleurotus pulmonarius is possibly related to protein kinase C inhibition.

ß-D-Glucan, a polysaccharide isolated from an edible mushroom Pleurotus pulmonarius (Fr.) Quel., presented antinociceptive activity in mice. This study evaluated the involvement of transient receptor potential (TRP) channels and protein kinase C (PKC) on antinociceptive effect of a (1→3),(1→6)-linked ß-D-glucan (GL) in mice. Intraperitoneal administration of GL potently inhibited nociceptive responses induced by intraplantar injections of capsaicin, cinnamaldehyde, menthol, acidified saline and phorbol myristate acetate (PMA). Moreover, Western blot analysis revealed that GL treatment also prevented PMA-induced PKCɛ activation. Collectively, present results demonstrate that GL could constitute an attractive molecule of interest for the development of new analgesic drugs.

Preventive and therapeutic euphol treatment attenuates experimental colitis in mice.

BACKGROUND: The tetracyclic triterpene euphol is the main constituent found in the sap of Euphorbia tirucalli. This plant is widely known in Brazilian traditional medicine for its use in the treatment of several kinds of cancer, including leukaemia, prostate and breast cancers. Here, we investigated the effect of euphol on experimental models of colitis and the underlying mechanisms involved in its action. METHODOLOGY/PRINCIPAL FINDINGS: Colitis was induced in mice either with dextran sulfate sodium (DSS) or with 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the effect of euphol (3, 10 and 30 mg/kg) on colonic injury was assessed. Pro-inflammatory mediators and cytokines were measured by immunohistochemistry, enzyme-Linked immunoabsorbent assay (ELISA), real time-polymerase chain reaction (RT-PCR) and flow cytometry. Preventive and therapeutic oral administration of euphol attenuated both DSS- and TNBS-induced acute colitis as observed by a significant reduction of the disease activity index (DAI), histological/microscopic damage score and myeloperoxidase (MPO) activity in colonic tissue. Likewise, euphol treatment also inhibited colon tissue levels and expression of IL-1ß, CXCL1/KC, MCP-1, MIP-2, TNF-α and IL-6, while reducing NOS2, VEGF and Ki67 expression in colonic tissue. This action seems to be likely associated with inhibition of activation of nuclear factor-κB (NF-κB). In addition, euphol decreased LPS-induced MCP-1, TNF-α, IL-6 and IFN-γ, but increased IL-10 secretion from bone marrow-derived macrophages in vitro. Of note, euphol, at the same schedule of treatment, markedly inhibited both selectin (P- and E-selectin) and integrin (ICAM-1, VCAM-1 and LFA-1) expression in colonic tissue. CONCLUSIONS/SIGNIFICANCE: Together, these results clearly demonstrated that orally-administered euphol, both preventive or therapeutic treatment were effective in reducing the severity of colitis in two models of chemically-induced mouse colitis and suggest this plant-derived compound might be a potential molecule in the management of inflammatory bowel diseases.

Omega-3 fatty acid-derived mediators 17(R)-hydroxy docosahexaenoic acid, aspirin-triggered resolvin D1 and resolvin D2 prevent experimental colitis in mice.

Resolvins of the D series are generated from docosahexaenoic acid, which are enriched in fish oils and are believed to exert beneficial roles on diverse inflammatory disorders, including inflammatory bowel disease (IBD). In this study, we investigated the anti-inflammatory effects of the aspirin-triggered resolvin D1 (AT-RvD1), its precursor (17(R)-hydroxy docosahexaenoic acid [17R-HDHA]) and resolvin D2 (RvD2) in dextran sulfate sodium (DSS)- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Our results showed that the systemic treatment with AT-RvD1, RvD2, or 17R-HDHA in a nanogram range greatly improved disease activity index, body weight loss, colonic damage, and polymorphonuclear infiltration in both colitis experimental models. Moreover, these treatments reduced colonic cytokine levels for TNF-α, IL-1ß, MIP-2, and CXCL1/KC, as well as mRNA expression of NF-κB and the adhesion molecules VCAM-1, ICAM-1, and LFA-1. Furthermore, AT-RvD1, but not RvD2 or 17R-HDHA, depended on lipoxin A4 receptor (ALX) activation to inhibit IL-6, MCP-1, IFN-γ, and TNF-α levels in bone marrow-derived macrophages stimulated with LPS. Similarly, ALX blockade reversed the beneficial effects of AT-RvD1 in DSS-induced colitis. To our knowledge, our findings showed for the first time the anti-inflammatory effects of resolvins of the D series and precursor 17R-HDHA in preventing experimental colitis. We also demonstrated the relevant role exerted by ALX activation on proresolving action of AT-RvD1. Moreover, AT-RvD1 showed a higher potency than 17R-HDHA and RvD2 in preventing DSS-induced colitis. The results suggest that these lipid mediators possess a greater efficacy when compared with other currently used IBD therapies, such as monoclonal anti-TNF, and have the potential to be used for treating IBD.