RESUMO
Andrographolide (AGP) and 14-deoxy-11,12-didehydroandrographolide (DDAG), two main diterpenoid constituents of Andrographis paniculata were previously shown to ameliorate asthmatic symptoms in a mouse model. However, due to inadequacies of both compounds in terms of drug-likeness, DDAG analogues were semisynthesised for assessment of their anti-asthma activity. A selected analogue, 3,19-diacetyl-14-deoxy-11,12-didehydroandrographolide (SRS27), was tested for inhibitory activity of NF-κB activation in TNF-α-induced A549 cells and was subsequently evaluated in a mouse model of ovalbumin (OVA)-induced asthma. Female BALB/c mice, 6-8weeks old were sensitized on days 0 and 14, and challenged on days 22, 23 and 24 with OVA. Compound or vehicle (3% dimethyl sulfoxide) was administered intraperitoneally 1h before and 11h after each OVA aerosol challenge. On day 25, pulmonary eosinophilia, airway hyperresponsiveness, mucus hypersecretion, inflammatory cytokines such as IL-4, -5 and -13 in BAL fluid, gene expression of inflammatory mediators such as 5-LOX, E-selectin, VCAM-1, CCL5, TNF-α, AMCase, Ym2, YKL-40, Muc5ac, CCL2 and iNOS in animal lung tissues, and serum IgE were determined. SRS27 at 30µM was found to suppress NF-κB nuclear translocation in A549 cells. In the ovalbumin-induced mouse asthma model, SRS27 at 3mg/kg displayed a substantial decrease in pulmonary eosinophilia, BAL fluid inflammatory cytokines level, serum IgE production, mucus hypersecretion and gene expression of inflammatory mediators in lung tissues. SRS27 is the first known DDAG analogue effective in ameliorating inflammation and airway hyperresponsiveness in the ovalbumin-induced mouse asthma model.
Assuntos
Antiasmáticos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Diterpenos/uso terapêutico , Lactonas/uso terapêutico , NF-kappa B/antagonistas & inibidores , Células A549 , Animais , Antiasmáticos/farmacologia , Anti-Inflamatórios/farmacologia , Asma/sangue , Asma/imunologia , Asma/fisiopatologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Citocinas/imunologia , Modelos Animais de Doenças , Diterpenos/farmacologia , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Lactonas/farmacologia , Camundongos Endogâmicos BALB C , NF-kappa B/genética , NF-kappa B/imunologia , Ovalbumina , Transdução de SinaisRESUMO
BACKGROUND: Allergy is an acquired hypersensitivity reaction of the immune system mediated by cross-linking of allergen-specific IgE-bound high-affinity IgE receptors, leading to immediate mast cell degranulation. Artesunate is a semi-synthetic derivative of artemisinin, an active component of the medicinal plant Artemisia annua. Artesunate is a clinically effective anti-malarial drug and has recently been shown to attenuate allergic asthma in mouse models. This study investigated potential anti-allergic effects of artesunate in animal models of IgE-dependent anaphylaxis. METHODS: Anti-allergic actions of artesunate were evaluated in passive cutaneous anaphylaxis and passive systemic anaphylaxis mouse models, and in ovalbumin-induced contraction of bronchial rings isolated from sensitized guinea pigs. Direct mast cell-stabilizing effect of artesunate was examined in RBL-2H3 mast cell line and in mature human cultured mast cells. Anti-allergic signaling mechanisms of action of artesunate in mast cells were also investigated. RESULTS: Artesunate prevented IgE-mediated cutaneous vascular hyperpermeability, hypothermia, elevation in plasma histamine level, and tracheal tissue mast cell degranulation in mice in a dose-dependent manner. In addition, artesunate suppressed ovalbumin-mediated guinea pig bronchial smooth muscle contraction. Furthermore, artesunate concentration-dependently blocked IgE-mediated degranulation of RBL-2H3 mast cells and human culture mast cells. Artesunate was found to inhibit IgE-induced Syk and PLCγ1 phosphorylation, production of IP(3) , and rise in cytosolic Ca(+2) level in mast cells. CONCLUSIONS: We report here for the first time that artesunate possesses anti-allergic activity by blocking IgE-induced mast cell degranulation, providing a foundation for developing artesunate for the treatment of allergic asthma and other mast cell-mediated allergic disorders.
Assuntos
Anafilaxia/tratamento farmacológico , Antimaláricos/farmacologia , Artemisininas/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Anafilaxia/imunologia , Animais , Antialérgicos/farmacologia , Artesunato , Asma/tratamento farmacológico , Asma/imunologia , Asma/fisiopatologia , Degranulação Celular/imunologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Cobaias , Humanos , Imunidade Celular/fisiologia , Immunoblotting , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Técnicas In Vitro , Mastócitos/fisiologia , Camundongos , Distribuição AleatóriaRESUMO
BACKGROUND: We previously reported that prior nasal administration of highly attenuated Bordetella pertussis BPZE1 provides effective and sustained protection against lethal challenge with influenza A viruses. The protective effect was mediated by suppressing the production of major pro-inflammatory mediators. To further explore the anti-inflammatory properties of BPZE1, we investigated the effect of BPZE1 nasal pretreatment on two mouse models of allergic disease, allergic airway inflammation, and contact hypersensitivity (CHS). METHODS: Allergic reactions were induced in mice nasally pretreated with live attenuated BPZE1 bacteria using the ovalbumin (OVA)-induced allergic airway inflammation and dinitrochlorobenzene (DNCB)-induced CHS models. RESULTS: Prior BPZE1 nasal treatment suppressed OVA-induced lung inflammation and inflammatory cell recruitment and significantly reduced IgE levels and cytokine production. Similarly, BPZE1 nasal pretreatment markedly inhibited ear swelling, skin inflammation, and production of pro-inflammatory cytokines in the DNCB-induced CHS model. For both models, we showed that BPZE1 pretreatment does not affect the sensitization phase. Upon challenge, BPZE1 pretreatment selectively reduced the level of cytokines whose production is increased and did not affect the basal level of other cytokines. Together, our observations suggest that BPZE1 pretreatment specifically targets those cytokine-producing effector cells that are recruited and involved in the inflammatory reaction. CONCLUSION: Our study demonstrates the broad anti-inflammatory properties of the attenuated B. pertussis BPZE1 vaccine candidate and supports its development as a promising agent to prevent and/or treat allergic diseases.
Assuntos
Bordetella pertussis/imunologia , Dermatite de Contato/prevenção & controle , Modelos Animais de Doenças , Vacina contra Coqueluche/imunologia , Pneumonia/prevenção & controle , Vacinas Atenuadas/imunologia , Administração Intranasal , Animais , Citocinas/metabolismo , Dermatite de Contato/imunologia , Dinitroclorobenzeno/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Vacina contra Coqueluche/administração & dosagem , Pneumonia/imunologia , Vacinas Atenuadas/administração & dosagem , Coqueluche/imunologia , Coqueluche/prevenção & controleRESUMO
BACKGROUND: Glucocorticoids are the mainstream drugs used in the treatment and control of inflammatory diseases such as asthma. Annexin-1 (ANXA1) is an anti-inflammatory protein which has been described as an endogenous protein responsible for some anti-inflammatory glucocorticoid effects. Previous studies have identified its importance in other immune diseases such as rheumatoid arthritis and cystic fibrosis. ANXA1-deficient ((-/-)) mice are Th2 biased, and ANXA1 N-terminus peptide exhibits anti-inflammatory activity in a rat model of pulmonary inflammation. OBJECTIVE: ANXA1 protein is found in bronchoalveolar lavage fluid from asthmatics. However, the function of ANXA1 in the pathological development of allergy or asthma is unclear. Thus, in this study we intended to examine the effect of ANXA1 deficiency on allergen-specific antibody responses and airway responses to methacholine (Mch). METHODS: ANXA1(-/-) mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA. Airway resistance, lung compliance and enhanced pause (PenH) were measured in naïve, sensitized and saline or allergen-challenged wild-type (WT) and ANXA1(-/-) mice. Total and allergen-specific antibodies were measured in the serum. RESULTS: We show that allergen-specific and total IgE, IgG2a and IgG2b levels were significantly higher in ANXA1(-/-) mice. Furthermore, naïve ANXA1(-/-) mice displayed higher airway hypersensitivity to inhaled Mch, and significant differences were also observed in allergen-sensitized and allergen-challenged ANXA1(-/-) mice compared with WT mice. CONCLUSIONS: In conclusion, ANXA1(-/-) mice possess multiple features characteristic to allergic asthma, such as airway hyperresponsiveness and enhanced antibody responses, suggesting that ANXA1 plays a critical regulatory role in the development of asthma. CLINICAL RELEVANCE: We postulate that ANXA1 is an important regulatory factor in the development of allergic disease and dysregulation of its expression can lead to pathological changes which may affect disease progression.
Assuntos
Alérgenos/imunologia , Anexina A1/genética , Anexinas/genética , Asma/genética , Asma/imunologia , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/imunologia , Imunidade Humoral , Animais , Anexina A1/deficiência , Anexinas/deficiência , Especificidade de Anticorpos/imunologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/fisiopatologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Feminino , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ovalbumina/imunologiaRESUMO
SRS27, an andrographolide analogue, had been proven to have therapeutic properties at a dose of 3 mg/kg in both in vitro and in vivo asthma models of our previous study. The present study focuses on the pharmacokinetic and toxicity profile of this compound to provide further evidence for the development of this compound as an anti-asthma agent. A simple pharmacokinetic study was performed in female BALB/c mice to measure blood plasma concentration of the compound at therapeutic dose. At a single dose of 3 mg/kg, SRS27 had a relatively short half-life but was able to achieve a concentration range of 13-19 µM that is related to its in vitro bioactivities. With regard to toxicity profile, SRS27 appears to be safe, as no histopathological changes were observed in the liver, kidneys and ovaries of SRS27-treated female BALB/c mice. In addition, there was no significant change in the mean body weight and organ weight of the animals in the SRS27-treated groups compared with the vehicle-treated control group at the end of the treatment. This fully supports the absence of any significant changes in peripheral blood leukocyte counts of SRS27-treated mice. Rewardingly, this acute toxicity study also revealed that SRS27 has a wide therapeutic window as no toxicity symptoms were detected with a dose up to 60 mg/kg daily when tested for 14 days. These results provide strong justification for further investigation of SRS27 as a potential new anti-asthma agent.
Assuntos
Antiasmáticos/farmacocinética , Antiasmáticos/toxicidade , Diterpenos/farmacocinética , Diterpenos/toxicidade , Lactonas/farmacocinética , Lactonas/toxicidade , Animais , Antiasmáticos/sangue , Disponibilidade Biológica , Diterpenos/sangue , Feminino , Rim/anatomia & histologia , Rim/efeitos dos fármacos , Lactonas/sangue , Fígado/anatomia & histologia , Fígado/efeitos dos fármacos , Pulmão/anatomia & histologia , Pulmão/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Ovário/anatomia & histologia , Ovário/efeitos dos fármacosRESUMO
In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18-22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3-46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin alpha II chain (rat fragment, also known as alpha-fodrin or non-erythroid alpha chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24h after the induction of cerebral ischemia.
Assuntos
Isquemia Encefálica/genética , Encéfalo/fisiopatologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Proteômica/métodos , Espectrina/genética , Animais , Encéfalo/enzimologia , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Masculino , Pseudogenes , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND AND PURPOSE: Cigarette smoke is a major cause for chronic obstructive pulmonary disease (COPD). Andrographolide is an active biomolecule isolated from the plant Andrographis paniculata. Andrographolide has been shown to activate nuclear factor erythroid-2-related factor 2 (Nrf2), a redox-sensitive antioxidant transcription factor. As Nrf2 activity is reduced in COPD, we hypothesize that andrographolide may have therapeutic value for COPD. EXPERIMENTAL APPROACH: Andrographolide was given i.p. to BALB/c mice daily 2h before 4% cigarette smoke exposure for 1h over five consecutive days. Bronchoalveolar lavage fluid and lungs were collected for analyses of cytokines, oxidative damage markers and antioxidant activities. BEAS-2B bronchial epithelial cells were exposed to cigarette smoke extract (CSE) and used to study the antioxidant mechanism of action of andrographolide. KEY RESULTS: Andrographolide suppressed cigarette smoke-induced increases in lavage fluid cell counts; levels of IL-1ß, MCP-1, IP-10 and KC; and levels of oxidative biomarkers 8-isoprostane, 8-OHdG and 3-nitrotyrosine in a dose-dependent manner. Andrographolide promoted inductions of glutathione peroxidase (GPx) and glutathione reductase (GR) activities in lungs from cigarette smoke-exposed mice. In BEAS-2B cells, andrographolide markedly increased nuclear Nrf2 accumulation, promoted binding to antioxidant response element (ARE) and total cellular glutathione level in response to CSE. Andrographolide up-regulated ARE-regulated gene targets including glutamate-cysteine ligase catalytic (GCLC) subunit, GCL modifier (GCLM) subunit, GPx, GR and heme oxygenase-1 in BEAS-2B cells in response to CSE. CONCLUSIONS: Andrographolide possesses antioxidative properties against cigarette smoke-induced lung injury probably via augmentation of Nrf2 activity and may have therapeutic potential for treating COPD.
Assuntos
Antioxidantes/farmacologia , Diterpenos/farmacologia , Lesão Pulmonar/prevenção & controle , Fator 2 Relacionado a NF-E2/metabolismo , Fumar/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Citocinas/metabolismo , Feminino , Glutationa/genética , Glutationa/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/etiologia , Lesão Pulmonar/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/etiologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Regulação para Cima/efeitos dos fármacosAssuntos
Asma/fisiopatologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Asma/tratamento farmacológico , Butadienos/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Inibidores Enzimáticos/farmacologia , Cobaias , Imidazóis/farmacologia , Nitrilas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologiaRESUMO
Aeromonas hydrophila is a Gram-negative bacterium that is pathogenic in fish, causing motile aeromonad septicaemia. It can enter (invade) fish cells, and survive as an intracellular parasite. The host-pathogen interaction and signal transduction pathway were studied by screening signal transduction inhibitors using carp epithelial cells and a virulent strain of the bacterium, PPD134/91. Genistein, a tyrosine kinase inhibitor, postponed internalization of A. hydrophila into host cells, suggesting that tyrosine phosphorylation plays a role in internalization. In contrast, staurosporine, a protein kinase C inhibitor, and sodium orthovanadate, a protein tyrosine phosphatase inhibitor, accelerated internalization of PPD134/91. Other virulent strains of A. hydrophila were also examined and it is likely that all strains, irrespective of serogroup, use the same signalling pathway to facilitate bacterial uptake.
Assuntos
Aeromonas hydrophila , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Infecções por Bactérias Gram-Negativas/microbiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Actinas/metabolismo , Animais , Células Cultivadas , Células Epiteliais/microbiologia , Peixes , Interações Hospedeiro-Parasita , Microscopia Confocal , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Estaurosporina/farmacologia , Vanadatos/farmacologiaRESUMO
Vibrio anguillarum and Vibrio damselae are Gram-negative bacteria that cause systemic infections called vibriosis in fish. They can enter fish cells and survive as intracellular parasites. The host-pathogen interactions between these Vibrio species and the fish epithelial cell lines epithelioma papillosum of carp (EPC) and grunt-fin tissue (GF) cells, were examined using phase-contrast, scanning electron and confocal microscopy. In addition, potential signal transduction pathways that precede bacterial internalization were studied by using signal transduction inhibitors. Some Vibrio species induced morphological changes in fish cells and this allowed classification into a cytopathic group and a noncytopathic group. The cytopathic group could be subdivided into two invasive groups (I and II) and a cytotoxic group. Of the invasive strains V. anguillarum 811218-5W (group I) and G/Virus/5(3) (group II), genistein, a tyrosine kinase inhibitor, only inhibited internalization of V. anguillarum G/Virus/5(3) into EPC cells, whereas staurosporine, a protein kinase C inhibitor, accelerated internalization of both strains. Cytochalasin D, an inhibitor of microfilament polymerization, prevented internalization of both strains, whilst vincristin, a microtubule inhibitor, only inhibited internalization of V. anguillarum G/Virus/5(3). For the cytotoxic strain V. damselae ATCC 33539, extracellular products (ECP) alone caused morphological changes in EPC and GF. Bacterial internalization may not be important in the pathogenesis of this group. The non-cytopathic strain V. anguillarum S2/5/93(2) did not enter cells or induce any changes in EPC and GF monolayers. This study has identified some major differences between Vibrio species in their interactions with fish cells in vitro and will thus facilitate future studies of the molecular basis of pathogenesis of vibriosis.