RESUMO
Psoriasis, a chronic inflammatory skin disease, is characterized by the excessive proliferation and impaired differentiation of epidermal keratinocytes and is accompanied by the increased infiltration of inflammatory cells. The condition requires longterm treatment and has no definitive cure. Hence, supplements and therapeutic agents have been intensely investigated. Gomisin M2 (GM2), a lignan extracted from Schisandra chinensis (Turcz). Baill. (Schisandraceae; S. chinensis), has demonstrated diverse pharmacological properties, including anticancer, antiinflammatory and antiallergic effects. Based on these findings, the present study examined the effects of GM2 on an imiquimod (IMQ)induced psoriasis mouse model and on keratinocytes stimulated by tumor necrosis factor (TNF)α and interferonγ. IMQ was topically applied to the back skin of mice for 7 consecutive days, and the mice were orally administered CD. These results showed that the oral administration of GM2 suppressed the symptoms of psoriasis, as evidenced by reductions in skin thickness, psoriasis area severity index scores for psoriasis lesions, transepidermal water loss and myeloperoxidase (MPO)associated cell infiltration. Furthermore, GM2 reduced the pathologically increased levels of immunoglobulin G2a, MPO and TNFα in the serum and T helper (Th)1 and Th17 cell populations in the spleen. GM2 decreased the gene expression of inflammatoryrelated cytokines and chemokines and inhibited the expression of signal transducer and activator of transcription 1 and nuclear factorκB in the activated keratinocytes. These results suggested that GM2 from S. chinensis is a potential therapeutic candidate to alleviate psoriasislike skin inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lignanas/farmacologia , Psoríase/tratamento farmacológico , Psoríase/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Humanos , Imiquimode/toxicidade , Inflamação/induzido quimicamente , Inflamação/genética , Interferon gama/toxicidade , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Lignanas/uso terapêutico , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Psoríase/induzido quimicamente , Psoríase/patologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.
Assuntos
Caquexia/tratamento farmacológico , Caproatos/farmacologia , Interferon gama/toxicidade , Interleucina-6/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Caquexia/induzido quimicamente , Caquexia/metabolismo , Caquexia/patologia , Linhagem Celular , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Metilistidinas/metabolismo , Camundongos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Fosforilação , Biossíntese de Proteínas , ProteóliseRESUMO
Host defenses in the brain are modulated by the activation of several factors such as oxygen free radical species (ROS), Ca2+ influx, and TRPM2 activation, and they are well-known adverse factors in neurotoxicity and neurodegenerative diseases. Importantly, recent data indicated a protective action of curcumin (CRC) via inhibition of TRPM2 on the inflammation factors, ROS, and apoptosis in hypoxia-induced SH-SY5Y neuronal cells. However, the relationship between interferon gamma (IFNg) exposure and TRPM2 activation in the SH-SY5Y cells are not fully identified. The SH-SY5Y cells as a neuronal cell line model were used in several neuroinflammation studies. Hence, we used the SH-SY5Y cells in the current study, and they were divided into four main groups as control, CRC, IFNg, and IFNg+CRC. The data presented here indicate that IFNg induced excessive Ca2+ influx via activation of TRPM2. The IFNg treatment further increased cell death, cell debris amount, apoptosis, and cytokine generations (IL-1ß, IL-6, and TNF-α) which were due to increased cytosolic and mitochondrial ROS generations as well as increased activations of caspase-3 and caspase-9. The expression levels of TRPM2, PARP-1, Bax, caspase-3, and caspase-9 were increased in the cells by the IFNg treatment. However, CRC treatment reduced the increase of expression levels, cytokine generations, caspase activations, ROS release, Ca2+ influx, cell death, and apoptosis levels via inhibition of TRPM2 in the SH-SY5Y cells that were treated with IFNg. Moreover, the treatment of TRPM2 blockers (ACA and 2-APB) potentiated the modulator effects of CRC. In conclusion, these results suggest that neuroinflammation via IFNg lead to the TRPM2 activation in the SH-SY5Y cells, whereas CRC prevents IFNg-mediated TRPM2 activation, cell death, and cytokine generations.
Assuntos
Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Mediadores da Inflamação/metabolismo , Interferon gama/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Zinco/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Scurrula ferruginea (Jack) Danser (locally known as 'Dedalu' or 'dian nan ji sheng' in Malaysia and China) is a hemi-parasitic shrub that is widely used as herbal medicine to treat inflammation, rheumatism, and stroke. However, the scientific basis of its anti-inflammatory function and mechanism remain to be proven. AIM OF THE STUDY: To evaluate the anti-inflammatory activity as well as the preliminary mechanism of S. ferruginea parasitizing on Tecoma stans. MATERIALS AND METHODS: The anti-inflammatory capability of freeze-dried stem aqueous extract was assessed via inhibition of inflammatory cytokines interleukin- (IL-) 1ß, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) production in lipopolysaccharide (LPS) and interferon-γ (IFN-γ) stimulated RAW 264.7 macrophages. The underlying anti-inflammatory mechanism was deciphered through reverse transcriptase and real time quantitative polymerase chain reactions (RT-PCR and qPCR) for inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1ß, and TNF-α mRNA expression. RESULTS: The results exhibited that aqueous extract of freeze-dried S. ferruginea stem sample concentration-dependently inhibited IL-1ß protein production along with the down regulation of iNOS and IL-1ß mRNA expression. Moreover, it significantly suppressed the protein release of IL-6 and IL-10 in a concentration-dependent manner. However, it slightly reduced TNF-α at higher sample concentration (250 µg/mL) without affecting the mRNA expression levels of COX-2 and TNF-α. CONCLUSIONS: This study suggests that S. ferruginea parasitizing on Tecoma stans exerted anti-inflammatory capability attributed to inhibition of iNOS and IL-1ß mRNA expression, NO creation, IL-1ß, IL-6, IL-10, and TNF-α protein production, indicating this plant might be a useful plant-derived candidate against inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Bignoniaceae , Interferon gama/toxicidade , Lipopolissacarídeos/toxicidade , Loranthaceae , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Relação Dose-Resposta a Droga , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Extratos Vegetais/isolamento & purificação , Células RAW 264.7RESUMO
Cordycepin, a natural derivative of adenosine, has been shown to exert pharmacological properties including anti-oxidation, antitumor, and immune regulation. It is reported that cordycepin is involved in the regulation of macrophage function. However, the effect of cordycepin on inflammatory cell infiltration in inflammation remains ambiguous. In this study, we investigated the potential role of cordycepin playing in macrophage function in CFA-induced inflammation mice model. In this model, we found that cordycepin prevented against macrophage infiltration in paw tissue and reduced interferon-γ (IFN-γ) production in both serum and paw tissue. Using luciferase reporter assay, we found that cordycepin suppressed IFN-γ-induced activators of transcription-1 (STAT1) transcriptional activity in a dose-dependent manner. Moreover, western blotting data demonstrated that cordycepin inhibited IFN-γ-induced STAT1 activation through attenuating STAT1 phosphorylation. Further investigations revealed that cordycepin inhibited the expressions of IFN-γ-inducible protein 10 (IP-10) and monokine induced by IFN-γ (Mig), which were the effector genes in IFN-γ-induced STAT1 signaling. Meanwhile, the excessive inflammatory cell infiltration in paw tissue was reduced by cordycepin. These findings demonstrate that cordycepin alleviates excessive inflammatory cell infiltration through down-regulation of macrophage IP-10 and Mig expressions via suppressing STAT1 phosphorylation. Thus, cordycepin may be a potential therapeutic approach to prevent and treat inflammation-associated diseases.
Assuntos
Quimiocina CXCL10/antagonistas & inibidores , Quimiocina CXCL9/antagonistas & inibidores , Desoxiadenosinas/uso terapêutico , Interferon gama/toxicidade , Macrófagos/efeitos dos fármacos , Fator de Transcrição STAT1/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Quimiocina CXCL10/biossíntese , Quimiocina CXCL9/biossíntese , Desoxiadenosinas/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Adjuvante de Freund/toxicidade , Expressão Gênica , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Distribuição Aleatória , Fator de Transcrição STAT1/metabolismoRESUMO
BACKGROUND: Alterations to epithelial tight junctions can compromise the ability of the epithelium to act as a barrier between luminal contents and the underlying tissues, thereby increasing intestinal permeability, an early critical event in inflammatory bowel disease (IBD). Tofacitinib (Xeljanz), an orally administered pan-Janus kinase (JAK) inhibitor, was recently approved for the treatment of moderate to severe ulcerative colitis. Nevertheless, the effects of tofacitinib on intestinal epithelial cell functions are largely unknown. The aim of this study was to determine if JAK inhibition by tofacitinib can rescue cytokine-induced barrier dysfunction in intestinal epithelial cells (IECs). METHODS: T84 IECs were used to evaluate the effects of tofacitinib on JAK-signal transducer and activator of transcription (STAT) activation, barrier permeability, and expression and localization of tight junction proteins. The impact of tofacitinib on claudin-2 promoter activity was assessed in HT-29 IECs. Tofacitinib rescue of barrier function was also tested in human colonic stem cell-derived organoids. RESULTS: Pretreatment with tofacitinib prevented IFN-γ-induced decreases in transepithelial electrical resistance (TER) and increases in 4 kDa FITC-dextran permeability (FD4), partly due to claudin-2 transcriptional regulation and restriction of ZO-1 rearrangement at tight junctions. Although tofacitinib administered after IFN-γ challenge only partially normalized TER and claudin-2 levels, FD4 permeability and ZO-1 localization were fully recovered. The IFN-γ-induced FD4 permeability in primary human colonoids was fully rescued by tofacitinib. CONCLUSIONS: These data suggest differential therapeutic efficacy of tofacitinib in the rescue of pore vs leak-tight junction barrier defects and indicate a potential contribution of improved epithelial barrier function to the beneficial effects of tofacitinib in IBD patients.
Assuntos
Células Epiteliais/efeitos dos fármacos , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Inibidores de Janus Quinases/farmacologia , Piperidinas/farmacologia , Pirimidinas/farmacologia , Claudinas/metabolismo , Colo/citologia , Células Epiteliais/metabolismo , Células HT29 , Humanos , Interferon gama/toxicidade , Mucosa Intestinal/fisiopatologia , Intestinos/citologia , Permeabilidade/efeitos dos fármacos , Proteínas de Junções Íntimas/genética , Junções Íntimas/metabolismoRESUMO
Host inflammatory responses must be tightly regulated to ensure effective immunity while limiting tissue injury. IFN gamma (IFNγ) primes macrophages to mount robust inflammatory responses. However, IFNγ also induces cell death, and the pathways that regulate IFNγ-induced cell death are incompletely understood. Using genome-wide CRISPR/Cas9 screening, we identified autophagy genes as central mediators of myeloid cell survival during the IFNγ response. Hypersensitivity of autophagy gene-deficient cells to IFNγ was mediated by tumor necrosis factor (TNF) signaling via receptor interacting protein kinase 1 (RIPK1)- and caspase 8-mediated cell death. Mice with myeloid cell-specific autophagy gene deficiency exhibited marked hypersensitivity to fatal systemic TNF administration. This increased mortality in myeloid autophagy gene-deficient mice required the IFNγ receptor, and mortality was completely reversed by pharmacologic inhibition of RIPK1 kinase activity. These findings provide insight into the mechanism of IFNγ-induced cell death via TNF, demonstrate a critical function of autophagy genes in promoting cell viability in the presence of inflammatory cytokines, and implicate this cell survival function in protection against mortality during the systemic inflammatory response.
Assuntos
Autofagia/genética , Interferon gama/toxicidade , Células Mieloides/patologia , Fator de Necrose Tumoral alfa/toxicidade , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citoproteção/efeitos dos fármacos , Genoma , Camundongos Knockout , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Mieloides/ultraestrutura , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
The anti-oxidant lipoic acid (LA) is beneficial in murine models of multiple sclerosis (MS) and has recently been shown to slow brain atrophy in secondary progressive MS. The mechanism of these effects by LA is incompletely understood but may involve effects on microglia. The objective of this study is to understand how LA affects microglial cells. We cultured primary microglial cells from C57BL/6 adult mice brains and stimulated the cells with lipopolysaccharide (LPS) and interferon gamma (IFN-γ) in the presence or absence of LA. We demonstrate the inhibition of phagocytosis, rearrangement of actin, and formation of membrane blebs in stimulated microglia in the presence of LA. These experiments suggest that LA causes changes in microglial actin, which may lead to alterations in phagocytosis, mobility, and migration.
Assuntos
Antioxidantes/farmacologia , Movimento Celular/fisiologia , Microglia/fisiologia , Fagocitose/fisiologia , Ácido Tióctico/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Interferon gama/toxicidade , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Fagocitose/efeitos dos fármacosRESUMO
BACKGROUND: Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood. METHODS: Counter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction. RESULTS: Both iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. CONCLUSION: Large scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.
Assuntos
Interferon alfa-2/farmacologia , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Monócitos/metabolismo , Polietilenoglicóis/farmacologia , Animais , Contagem de Células , Morte Celular/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Criopreservação , Feminino , Humanos , Interferon alfa-2/toxicidade , Interferon-alfa/toxicidade , Interferon gama/toxicidade , Camundongos , Monócitos/efeitos dos fármacos , Polietilenoglicóis/toxicidade , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/toxicidadeRESUMO
Disruption of intestinal barrier homeostasis is an important pathogenic factor in conditions such as irritable bowel syndrome (IBS). Lactobacillus rhamnosus GG (LGG) improves IBS symptoms through unclear mechanisms. Previous studies utilizing colorectal adenocarcinoma cell lines showed that LGG metabolites prevented interferon gamma (IFN-gamma) induced barrier damage but the model employed limited these findings. We aimed to interrogate the protective effects of LGG on epithelial barrier function using human intestinal epithelial cultures (enteroids and colonoids) as a more physiologic model. To investigate how LGG affects epithelial barrier function, we measured FITC-Dextran (FD4) flux across the epithelium as well as tight junction zonula occludens 1 (ZO-1) and occludin (OCLN) expression. Colonoids were incubated with fecal supernatants from IBS patients (IBS-FSN) and healthy controls in the presence or absence of LGG to examine changes in gut permeability. Enteroids incubated with IFN-gamma demonstrated a downregulation of OCLN and ZO-1 expression by 67% and 50%, respectively (p<0.05). This was accompanied by increased paracellular permeability as shown by leakage of FD4. Pretreatment of enteroids with LGG prevented these changes and normalized OCLN and ZO-1 to control levels. These actions were independent of its action against apoptosis. However, these protective effects were not seen with LGG cell wall extracts, LGG DNA, or denatured (boiled) LGG. Intriguingly, IBS-FSN injected into colonoids increased paracellular permeability, which was prevented by LGG. LGG, likely due to secreted proteins, protects against epithelial barrier dysfunction. Bacterial-derived factors to modulate gut barrier function may be a treatment option in disorders such as IBS.
Assuntos
Fezes/química , Interferon gama/toxicidade , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiopatologia , Síndrome do Intestino Irritável/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Junções Íntimas/metabolismo , Técnicas de Cultura de Células , Colo/citologia , Meios de Cultivo Condicionados/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Permeabilidade/efeitos dos fármacos , Proteínas de Junções Íntimas/genéticaRESUMO
Melaleuca styphelioides, known as the prickly-leaf tea tree, contains a variety of bioactive compounds. The purposes of this study were to characterize the polyphenols extracted from Melaleuca styphelioides leaves and assess their potential antioxidant and anti-inflammatory effects. The polyphenol extracts were prepared by maceration with solvents of increasing polarity. The LC/MS-MS technique was used to identify and quantify the phenolic compounds. An assessment of the radical scavenging activity of all extracts was performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTSâº), and ferric reducing antioxidant power (FRAP) assays. The anti-inflammatory activity was determined on interferon gamma (IFN-γ)/histamine (H)-stimulated human NCTC 2544 keratinocytes by Western blot and RT-PCR. Compared to other solvents, methanolic extract presented the highest level of phenolic contents. The most frequent phenolic compounds were quercetin, followed by gallic acid and ellagic acid. DPPH, ABTSâº, and FRAP assays showed that methanolic extract exhibits strong concentration-dependent antioxidant activity. IFN-γ/H treatment of human NCTC 2544 keratinocytes induced the secretion of high levels of the pro-inflammatory mediator inter-cellular adhesion molecule-1 (ICAM-1), nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB), which were inhibited by extract. In conclusion, the extract of Melaleuca styphelioides leaves is rich in flavonoids, and presents antioxidant and anti-inflammatory proprieties. It can be proposed as a useful compound to treat inflammatory skin diseases.
Assuntos
Inflamação/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Melaleuca/química , Polifenóis/química , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular , Flavonoides/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Histamina/toxicidade , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Interferon gama/toxicidade , Queratinócitos/patologia , Extratos Vegetais/química , Folhas de Planta/química , Polifenóis/isolamento & purificação , Polifenóis/farmacologiaRESUMO
BACKGROUND: Overactivated microglial cells exhibit chronic inflammatory response and can lead to the continuous production of pro-inflammatory cytokines, perpetuating inflammation, and ultimately resulting in neuronal injury. 1,2,3,4,6-Penta-O-Galloyl-ß-d-Glucose (PGG), which is a naturally occurring polyphenolic compound, has exhibited anti-inflammatory effect through the inhibition of many cytokines in different experimental models, but its effect on activated microglia cells was never described. In the present study, we investigated PGG effect in proteins involved in the NFÆB and MAPK signaling pathways, which play a central role in inflammation through their ability to induce transcription of pro-inflammatory genes. METHODS: PCR arrays and RT-PCR with individual primers were used to determine the effect of PGG on mRNA expression of genes involved in NFÆB and MAPK signaling pathways. Western blots were performed to confirm PCR results. RESULTS: The data obtained showed that PGG modulated the expression of 5 genes from the NFÆB (BIRC3, CHUK, IRAK1, NFÆB1, NOD1) and 2 genes from MAPK signaling pathway (CDK2 and MYC) when tested in RT-PCR assays. Western blots confirmed the PCR results at the protein level, showing that PGG attenuated the expression of total and phosphorylated proteins (CDK2, CHUK, IRAK1, and NFÆB1) involved in NFÆB and MAPK signaling. CONCLUSION: These findings show that PGG could modulate the expression of genes and proteins involved in the production of pro-inflammatory cytokines in microglia cells.
Assuntos
Taninos Hidrolisáveis/farmacologia , Mediadores da Inflamação/metabolismo , Interferon gama/toxicidade , Lipopolissacarídeos/toxicidade , Sistema de Sinalização das MAP Quinases/fisiologia , Microglia/metabolismo , NF-kappa B/metabolismo , Animais , Linhagem Celular , Mediadores da Inflamação/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacosRESUMO
Several studies have identified the aryl hydrocarbon receptor (AhR) as a negative regulator of the innate and adaptive immune responses. However, the molecular mechanisms by which this transcription factor exerts such modulatory effects are not well understood. Interaction between AhR and RelA/p65 has previously been reported. RelA/p65 is the major NFκB subunit that plays a critical role in immune responses to infection. The aim of the present study was to determine whether the activation of AhR disrupted RelA/p65 signaling in mouse peritoneal macrophages by decreasing its half-life. The data demonstrate that the activation of AhR by TCDD and ß-naphthoflavone (ß-NF) decreased protein levels of the pro-inflammatory cytokines TNF-α, IL-6 and IL-12 after macrophage activation with LPS/IFNγ. In an AhR-dependent manner, TCDD treatment induces RelA/p65 ubiquitination and proteosomal degradation, an effect dependent on AhR transcriptional activity. Activation of AhR also induced lysosome-like membrane structure formation in mouse peritoneal macrophages and RelA/p65 lysosome-dependent degradation. In conclusion, these results demonstrate that AhR activation promotes RelA/p65 protein degradation through the ubiquitin proteasome system, as well as through the lysosomes, resulting in decreased pro-inflammatory cytokine levels in mouse peritoneal macrophages.
Assuntos
Mediadores da Inflamação/metabolismo , Lisossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Fator de Transcrição RelA/metabolismo , Ubiquitina/metabolismo , Animais , Células Cultivadas , Feminino , Interferon gama/toxicidade , Lipopolissacarídeos/toxicidade , Lisossomos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Bone marrow (BM) mesenchymal stromal cells (MSCs) provide microenvironmental support to hematopoietic stem and progenitor cells (HSPCs). Culture-expanded MSCs are interesting candidates for cellular therapies due to their immunosuppressive and regenerative potential which can be further enhanced by pretreatment with interferon-gamma (IFN-γ). However, it remains unknown whether IFN-γ can also influence hematopoietic support by BM-MSCs. In this study, we elucidate the impact of IFN-γ on the hematopoietic support of BM-MSCs. We found that IFN-γ increases expression of interleukin (IL)-6 and stem cell factor by human BM-MSCs. IFN-γ-treated BM-MSCs drive HSPCs toward myeloid commitment in vitro, but impair subsequent differentiation of HSPC. Moreover, IFN-γ-ARE-Del mice with increased IFN-γ production specifically lose their BM-MSCs, which correlates with a loss of hematopoietic stem cells' quiescence. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, we conclude that IFN-γ negatively affects maintenance of BM-MSCs and their hematopoietic support in vitro and in vivo.
Assuntos
Hematopoese/efeitos dos fármacos , Interferon gama/toxicidade , Células-Tronco Mesenquimais/patologia , Adolescente , Adulto , Idoso , Animais , Citocinas/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Adulto JovemRESUMO
Until recently, fermentation was the only processing used to improve the functionality of wheat germ. The release of 2,6-dimethoxy-1,4-benzoquinone (DMBQ) from hydroquinone glycosides during the fermentation process is considered a marker of quality control. Here, we treated wheat germ extract with citric acid (CWG) to release DMBQ and examined the anti-inflammatory activity of this extract using a lipopolysaccharide-activated macrophage model. Treatment of wheat germ with citric acid resulted in detectable release of DMBQ but reduced total phenolic and total flavonoid contents compared with untreated wheat germ extract (UWG). CWG inhibited secretion of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6, and IL-12 and the synthesis of cyclooxygenase-2, while UWG only decreased IL-12 production. CWG and UWG induced high levels of anti-inflammatory IL-10 and heme oxygenase-1. CWG specifically inhibited phosphorylation of NF-κB p65 and p38 kinase at 15 min after LPS stimulation. Our study showed that citric acid treatment enhanced the anti-inflammatory activity of wheat germ extract.
Assuntos
Anti-Inflamatórios/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ácido Cítrico/química , Macrófagos Peritoneais/efeitos dos fármacos , Extratos Vegetais/farmacologia , Triticum/química , Animais , Anti-Inflamatórios/química , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Interferon gama/toxicidade , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Extratos Vegetais/químicaRESUMO
Chronic and acute central nervous system (CNS) inflammation are contributors toward neurological injury associated with head trauma, stroke, infection, Parkinsons or Alzheimers disease. CNS inflammatory illnesses can also contribute toward risk of developing glioblastoma multiforme (GBM). With growing public interest in complementary and alternative medicines (CAMs), we conduct a high throughput (HTP) screening of >1400 natural herbs, plants and over the counter (OTC) products for anti-inflammatory effects on lipopolysaccharide (LPS)/interferon gamma (IFNγ) activated C6 glioma cells. Validation studies were performed showing a pro-inflammatory profile of [LPS 3 µg/ml/ IFNγ 3 ng/ml] consistent with greater release [>8.5 fold] of MCP-1, NO2-, cytokine-induced neutrophil chemo-attractants (CINC) 1, CINC 2a and CINC3. The data show no changes to the following, IL-13, TNF-a, fracktaline, leptin, LIX, GM-CSF, ICAM1, L-Selectin, activin A, agrin, IL-1α, MIP-3a, B72/CD86, NGF, IL-1b, MMP-8, IL-1 R6, PDGF-AA, IL-2, IL-4, prolactin R, RAGE, IL-6, Thymus Chemokine-1, CNTF,IL-10 or TIMP-1. A HTP screening was conducted, where we employ an in vitro efficacy index (iEI) defined as the ratio of toxicity (LC50)/anti-inflammatory potency (IC50). The iEI was precautionary to ensure biological effects were occurring in fully viable cells (ratio > 3.8) independent of toxicity. Using NO2- as a guideline molecule, the data show that 1.77% (25 of 1410 tested) had anti-inflammatory effects with iEI ratios >3.8 and IC50s <250µg/ml. These include reference drugs (hydrocortisone, dexamethasone N6-(1-iminoethyl)-l-lysine and NSAIDS: diclofenac, tolfenamic acid), a histone deacetylase inhibitor (apicidin) and the following natural products; Ashwaganda (Withania somnifera), Elecampagne Root (Inula helenium), Feverfew (Tanacetum parthenium), Green Tea (Camellia sinensis), Turmeric Root (Curcuma longa) Ganthoda (Valeriana wallichii), Tansy (Tanacetum vulgare), Maddar Root (Rubia tinctoria), Red Sandle wood (Pterocarpus santalinus), Bay Leaf (Laurus nobilis, Lauraceae), quercetin, cardamonin, fisetin, EGCG, biochanin A, galangin, apigenin and curcumin. The herb with the largest iEI was Ashwaganda where the IC50/LC50 was 11.1/>1750.0µg/ml, and the compound with the greatest iEI was quercetin where the IC50/LC50 was 10.0/>363.6µg/ml. These substances also downregulate the production of iNOS expression and attenuate CINC-3 release. In summary, this HTP screening provides guideline information about the efficacy of natural products that could prevent inflammatory processes associated with neurodegenerative disease and aggressive glioma tumor growth.
Assuntos
Produtos Biológicos/farmacologia , Quimiocina CXCL2/metabolismo , Citocinas/metabolismo , Glioma/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Dióxido de Nitrogênio/metabolismo , Animais , Produtos Biológicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Quimiocina CXCL2/antagonistas & inibidores , Citocinas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Glioma/tratamento farmacológico , Interferon gama/toxicidade , Lipopolissacarídeos/toxicidade , Neutrófilos , Dióxido de Nitrogênio/antagonistas & inibidores , RatosRESUMO
Components of Pelagia noctiluca (P. noctiluca) venom were evaluated for their anticancer and nitric Oxide (NO) inhibition activities. Three fractions, out of four, obtained by gel filtration on Sephadex G75 of P. noctiluca venom revealed an important selective anti-proliferative activity on several cell lines such as human bladder carcinoma (RT112), human glioblastoma (U87), and human myelogenous leukemia (K562) but not on mitogen-stimulated peripheral blood mononuclear cells. Interestingly, P. noctiluca components showed an important dose-dependent anti-inflammatory activity, through inhibition of NO production via transcriptional regulation of Inducible NO Synthase (iNOS), in IFN-γ/LPS stimulated RAW 264.7 macrophages. These data strongly suggest that P. noctiluca venom could be used as a natural inhibitor of cancer cell lines and a potent anti-inflammatory agent for the treatment of anti-inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Venenos de Cnidários/farmacologia , Interferon gama/toxicidade , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Animais , Anti-Inflamatórios/isolamento & purificação , Linhagem Celular , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Venenos de Cnidários/isolamento & purificação , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Células K562 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
Epidemiologic studies indicate that coffee consumption reduces the risk of Parkinson's disease and Alzheimer's disease. To determine the factors involved, we examined the protective effects of coffee components. The test involved prevention of neurotoxicity to SH-SY5Y cells that was induced by lipopolysaccharide plus interferon-γ or interferon-γ released from activated microglia and astrocytes. We found that quercetin, flavones, chlorogenic acid, and caffeine protected SH-SY5Y cells from these toxins. They also reduced the release of tumor necrosis factor-α and interleukin-6 from the activated microglia and astrocytes and attenuated the activation of proteins from P38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFκB). After exposure to toxin containing glial-stimulated conditioned medium, we also found that quercetin reduced oxidative/nitrative damage to DNA, as well as to the lipids and proteins of SH-SY5Y cells. There was a resultant increase in [GSH]i in SH-SY5Y cells. The data indicate that quercetin is the major neuroprotective component in coffee against Parkinson's disease and Alzheimer's disease.
Assuntos
Doença de Alzheimer/prevenção & controle , Café/química , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/prevenção & controle , Fitoterapia , Quercetina/farmacologia , Astrócitos/metabolismo , Cafeína/isolamento & purificação , Cafeína/farmacologia , Células Cultivadas , Ácido Clorogênico/isolamento & purificação , Ácido Clorogênico/farmacologia , Dano ao DNA/efeitos dos fármacos , Flavonas/isolamento & purificação , Flavonas/farmacologia , Humanos , Interferon gama/toxicidade , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , Microglia/metabolismo , NF-kappa B/metabolismo , Fármacos Neuroprotetores/isolamento & purificação , Quercetina/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND AND AIM: Studies have verified the protective effect of Hydrogen Sulfide (H2S) on gastric ulcer and ulcerative colitis, but the mechanisms are not fully illustrated. In this study, the possible protective effect of H2S on TNF-α/IFN-γ induced barrier dysfunction was investigated in Caco-2 cell monolayers. METHOD: The barrier function of Caco-2 monolayers was evaluated by measuring trans-epithelial electrical resistance (TEER) and FITC-Dextran 4 kDa (FD-4) trans-membrane flux. ZO-1 and Occludin were chosen as markers of the localization of tight junction (TJ) proteins for immunofluorescence. The expression of MLCK and phosphorylation level of myosin light chain (MLC) were measured by immunoblotting. The activation of NF-kB p65 was analyzed by EMSA and immunofluorescence. RESULTS: NaHS at 500 uM significantly attenuated TNF-α/IFN-γ-indueced Caco-2 monolayer barrier injury. The increased expression of MLCK and increased phosphorylation level of MLC induced by TNF-α/IFN-γ was also inhibited significantly by NaHS. Additionally, NaHS inhibited TNF-α/IFN-γ induced activation and nuclear translocation of NF-kB p65. CONCLUSION: The present study reveals the protective effect of H2S on TNF-α and IFN-γ-induced injury of intestinal epithelial barrier function in Caco-2 monolayers and suggests that the suppression of MLCK-P-MLC signaling mediated by NF-kB P65 might be one of the mechanisms underlying the protective effect of H2S.
Assuntos
Epitélio/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Interferon gama/antagonistas & inibidores , Interferon gama/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/toxicidade , Biomarcadores/metabolismo , Células CACO-2 , Humanos , Mucosa Intestinal/citologia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Ocludina/metabolismo , Fosforilação , Úlcera Gástrica/patologia , Úlcera Gástrica/prevenção & controle , Proteínas de Junções Íntimas/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Microglia-mediated neuroinflammation has been reported as a common feature of familial and sporadic forms of Parkinson's disease (PD), and a growing body of evidence indicates that onset and progression of PD correlates with the extent of neuroinflammatory responses involving Interferon γ (IFNγ). Transforming growth factor ß1 (TGFß1) has been shown to be a major player in the regulation of microglia activation states and functions and, thus, might be a potential therapeutic agent by shaping microglial activation phenotypes during the course of neurodegenerative diseases such as PD. In this study, we demonstrate that TGFß1 is able to block IFNγ-induced microglia activation by attenuating STAT1 phosphorylation and IFNγRα expression. Moreover, we identified a set of genes involved in microglial IFNγ signaling transduction that were significantly down-regulated upon TGFß1 treatment, resulting in decreased sensitivity of microglia toward IFNγ stimuli. Interestingly, genes mediating negative regulation of IFNγ signaling, such as SOCS2 and SOCS6, were up-regulated after TGFß1 treatment. Finally, we demonstrate that TGFß1 is capable of protecting midbrain dopaminergic (mDA) neurons from IFNγ-driven neurotoxicity in mixed neuron-glia cultures derived from embryonic day 14 (E14) midbrain tissue. Together, these data underline the importance of TGFß1 as a key immunoregulatory factor for microglia by silencing IFNγ-mediated microglia activation and, thereby, rescuing mDA neurons from IFNγ-induced neurotoxicity. Interferon γ (IFNγ) is a potent pro-inflammatory factor that triggers the activation of microglia and the subsequent release of neurotoxic factors. Transforming growth factor ß1 (TGFß1) is able to inhibit the IFNγ-mediated activation of microglia, which is characterized by the release of nitric oxide (NO) and tumor necrosis factor α (TNFα). By decreasing the expression of IFNγ-induced genes as well as the signaling receptor IFNγR1, TGFß1 reduces the responsiveness of microglia towards IFNγ. In mixed neuron-glia cultures, TGFß1 protects midbrain dopaminergic (mDA) neurons from IFNγ-induced neurotoxicity.