RESUMO
MCS-18, a natural product isolated from Helleborus purpurascens has been shown to have several beneficial effects in inflammatory and autoimmune disorders. However, very little is known regarding the immuno-modulatory capacity of MCS-18 in respect to murine bone marrow-derived dendritic cells (BM-DC) and B-cells. Thus, in the present study we examined the effect of MCS-18 on murine BM-DC and B-cells. Interestingly MCS-18 inhibited the expression of important DC-specific molecules and lead to an impaired T-cell stimulation capacity. In addition, MCS-18 also reduced B-cell proliferation and immunoglobulin production.
Assuntos
Linfócitos B/efeitos dos fármacos , Produtos Biológicos/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Linfócitos T/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Produtos Biológicos/isolamento & purificação , Células da Medula Óssea/imunologia , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Helleborus/química , Imunoglobulina G/biossíntese , Imunoglobulina G/efeitos dos fármacos , Imunoglobulina M/biossíntese , Imunoglobulina M/efeitos dos fármacos , Fatores Imunológicos/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Here we report for the first time that MCS-18, a novel natural product isolated from Helleborus purpurascens, is able to inhibit the expression of typical molecules of mature dendritic cells (DC) such as CD80, CD86, and especially of CD83 subsequently leading to a clear and dose-dependent inhibition of the DC-mediated T-cell stimulation. Furthermore, MCS-18 impeded the formation of the typical DC/T-cell clusters, which are essential to induce potent immune responses. Interestingly, MCS-18 also inhibited CCR7 expression on DC which subsequently lead to a dose-dependent block of the CCL19-mediated DC migration. MCS-18 not only inhibited the DC-mediated T-cell stimulation but also the anti-CD3/anti-CD28-mediated T-cell stimulation. Strikingly, MCS-18 also strongly reduced the paralysis associated with the experimental autoimmune encephalomyelitis (EAE), which is a murine model for human multiple sclerosis, in a prophylactic as well as in a "real" therapeutic setting. Even when the EAE was induced for a second time, the MCS-18-treated animals were still protected, suggesting that MCS-18 induces a long-lasting suppressive effect. In addition, and very important for the potential practical application in humans, MCS-18 was also active when administered orally. MCS-18 treatment almost completely reduced leukocyte infiltration in the brain and in the spinal cord. In conclusion, using in vitro as well in vivo assays we were able to show that MCS-18 exerts a strong immunosuppressive activity with remarkable potential for the therapy of diseases characterized by a pathologically over-activated immune system.
Assuntos
Autoimunidade/efeitos dos fármacos , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Helleborus/química , Imunossupressores/farmacologia , Extratos Vegetais/farmacologia , Animais , Autoimunidade/imunologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/metabolismo , Imunossupressores/uso terapêutico , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Fitoterapia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
OBJECTIVE: Atherosclerosis is associated with chronic inflammatory responses of the arterial blood vessels. The previously observed protective effect of the MCS-18 substance against the initiation of atherosclerosis in a murine model was explained by its pronounced anti-inflammatory activity. Here, we investigated its impact on murine plaque progression in advanced atherosclerosis and on proatherogenic processes. APPROACH & RESULTS: ApoE-deficient mice were fed a high-fat diet for 12 weeks to induce atherosclerosis, followed by normal chow and intraperitoneal injections of either MCS-18 (500 µg, n = 10) or saline (n = 10) twice a week for another 12 weeks. Plaque size was reduced in MCS-18 treated mice compared to controls (p = 0.001), which was associated with a reduced size of the lipid core (p = 0.01). There was a decrease in apoptotic cells (p = 0.02), endothelial ICAM-1 expression (p < 0.001), and macrophage density (p = 0.01) in the MCS-18 group. In addition, human and murine dendritic cells (DCs) and human umbilical vein endothelial cells (HUVECs) were treated with MCS-18 (50-200 µg/ml) to analyze cell migration and adhesion under flow conditions. MCS-18 reduced human (p = 0.01) and murine (p = 0.006) DC migration. Furthermore, adhesion of MCS-18-treated DCs to a HUVEC monolayer was decreased (p < 0.001). Compared to controls, CD209 (p < 0.001) and CCR7 (p = 0.003) expression was decreased in MCS-18-treated DCs, while in HUVECs lower levels of ICAM-1 (p < 0.001) and of phosphorylated NF-κB-p65 (p = 0.002) were observed. Blocking of ICAM-1 reduced DC adhesion (p < 0.001). CONCLUSIONS: MCS-18 exhibits interesting therapeutic effects when applied in advanced murine atherosclerosis. Its antiatherogenic impact might be associated with a suppressed adhesion to the endothelium due to down-regulation of endothelial ICAM-1 expression.
Assuntos
Aterosclerose/genética , Produtos Biológicos/farmacologia , Molécula 1 de Adesão Intercelular/genética , Leucócitos/metabolismo , Animais , Apolipoproteínas E/deficiência , Aterosclerose/tratamento farmacológico , Aterosclerose/patologia , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Camundongos , Camundongos Knockout , NF-kappa B/biossíntese , NF-kappa B/efeitos dos fármacos , NF-kappa B/genéticaRESUMO
Recently discovered macrocyclic carbon suboxide (MCS) factors with the general formula (C(3)O(2))(n) were found to strongly inhibit rabbit and rat Na,K-ATPase as well as SR Ca-ATPase. Highly active MCS factors were obtained by a base/acid treatment of their lipophilic precursor isolated from plants. In the ESI-MS spectra, the dominant molar mass ion of 431 Da corresponds to a 1:1 complex of the carbon suboxide hexamer (n=6; M(r)=408 Da) with a Na(+) ion. Additional mass ions identified in positive and negative ion mode were assigned as complexes of the MCS hexamer (n=6) and octamer (n=8) with Na(+) or with TFA(-) in various ratios. The dominant mass ion values of these active MCS factors from plants are also found in mass spectra of previously described endogenous digitalis-like factors (EDLF) from animals. This would suggest that ubiquitously distributed MCS factors may function as putative endogenous regulatory substances of Na,K-ATPase and possibly of other ATPases. With the symmetric display of several equivalent carbonyl or hydroxy groups, the structure of MCS factors is particularly suited for interactions with proteins and other bio-molecules. This could explain the high biological activity and the unusual properties of the MCS factors.
Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , Compostos Inorgânicos de Carbono/farmacologia , Inibidores Enzimáticos/farmacologia , Óxidos/farmacologia , Retículo Sarcoplasmático/enzimologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The Na(+)/K(+)-ATPase is a membrane ion-transporter protein, specifically inhibited by digitalis glycosides used in cardiac therapy. The existence in mammals of some endogenous digitalis-like factors (EDLFs) as presumed ATPase ligands is generally accepted. But the chemical structure of these factors remained elusive because no weighable amounts of pure EDLFs have been isolated. Recent high-resolution crystal structure data of Na(+)/K(+)-ATPase have located the hydrophobic binding pocket of the steroid glycoside ouabain. It remained uncertain if the EDLF are targeting this steroid-receptor or another specific binding site(s). Our recently disclosed spherical oligo-silicic acids (SOSA) fulfill the main criteria to be identified with the presumed EDL factors. SOSA was found as a very potent inhibitor of the Na(+)/K(+)-ATPase, Ca(2+)-ATPase, H(+)/K(+)-ATPase, and of K-dp-ATPase, with IC50 values between 0.2 and 0.5 µg/mL. These findings are even more astonishing while so far, neither monosilicic acid nor its poly-condensed forms have been remarked biologically active. With the diameter Ï between 1 and 3 nm, SOSA still belong to molecular species definitely smaller than silica nano-particles with Ï > 5 nm. In SOSA molecules, almost all Si-OH bonds are displayed on the external shell, which facilitates the binding to hydrophilic ATPase domains. SOSA is stable for long term in solution but is sensitive to freeze-drying, which could explain the failure of countless attempts to isolate pure EDLF. There is a strong resemblance between SOSA and vanadates, the previously known general inhibitors of P-type ATPases. SOSA may be generated endogenously by spherical oligomerization of the ubiquitously present monosilicic acid in animal fluids. The structure of SOSA is sensitive to the concentration of Na(+), K(+), Ca(2+), Mg(2+), and other ions suggesting a presumably archaic mechanism for the regulation of the ATPase pumps.
RESUMO
INTRODUCTION: Inflammation accelerates both plaque progression and instability in the pathogenesis of atherosclerosis. The inhibition of dendritic cell (DC) maturation is a promising approach to suppress excessive inflammatory immune responses and has been shown to be protective in several autoimmune models. The aim of this study was to investigate the immune modulatory effects of the natural substance MCS-18, an inhibitor of DC maturation, regarding the progression of atherosclerosis in ApoE-deficient mice. MATERIALS AND METHODS: ApoE-deficient mice were fed for twelve weeks with a Western-type diet (n = 32) or normal chow (control group; n = 16). Animals receiving high-fat diet were treated with MCS-18 (500 µg/kg body weight, n = 16) or saline (n = 16) twice a week. After 12 weeks, animals were transcardially perfused and sacrificed. The percentage of mature DCs (CD3(-)/CD19(-)/CD14(-)/NK1.1(-)/CD11c(+)/MHCII(+)/CD83(+)/CD86(+)) and T cell subpopulations (CD4(+)/CD25(+)/Foxp3(+), CD3/CD4/CD8) was analyzed in peripheral blood and in the spleen using flow cytometry. Plaque size was determined in the aortic root and the thoracoabdominal aorta using en-face staining. Immunohistochemical stainings served to detect inflammatory cells in the aortic root. Several cytokines and chemokines were determined in serum using multiplex assays. RESULTS: In splenic cells derived from saline-treated atherosclerotic mice an increased DC maturation, reflected by the upregulation of CD83 and CD86 expression, was observed. The enhanced expression of both maturation markers was absent in MCS-18 treated atherosclerotic mice. While the percentage of splenic Foxp3 expressing Treg was increased in animals receiving MCS-18 compared to saline-treated atherosclerotic mice, cytotoxic T cells were reduced in the spleen and in atherosclerotic lesions of the aortic root. Furthermore, proatherogenic cytokines (e.g. IL-6 and IFN-γ) and chemokines (e.g. MIP-1ß) were decreased in serum of MCS-18-treated animals when compared to saline-treated atherosclerotic mice. Also plaque size in the aortic root and the thoracoabdominal aorta was significantly lower following administration of MCS-18. CONCLUSION: This study provides for the first time evidence that MCS-18 is able to prevent the onset of atherosclerosis in ApoE-deficient mice. The observed anti-atherogenic effect is associated with the suppression of DC maturation and an inhibited migration and proliferation of cytotoxic T cells.
Assuntos
Aterosclerose/prevenção & controle , Produtos Biológicos/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Placa Aterosclerótica/prevenção & controle , Animais , Anti-Inflamatórios , Apolipoproteínas E/deficiência , Aterosclerose/patologia , Células Dendríticas/imunologia , Dieta Hiperlipídica , Progressão da Doença , Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
There is still a vital need for new therapies in order to prevent or treat type I diabetes. In this respect, we report that MCS-18 a novel natural product isolated from the plant Helleborus purpurascens (i.e. Christmas rose) is able to increase diabetes free survival using the NOD-mouse model, which is accompanied with a diminished IFN-γ secretion of splenocytes. In the animal group which has been treated with MCS-18 during week 8 and week 12 of age 70% of the animals showed a diabetes free survival at week 30, whereas in contrast in the untreated animals less than 10% were free of diabetes. MCS-18 treatment significantly reduced islet T-cell infiltrates as well as the rate of T-cell proliferation. Periinsular infiltrates in the MCS-18 treated animals showed a significantly enhanced number of Foxp3(+) CD25(+) T cells, indicating the increased presence of regulatory T cells. These studies show that MCS-18 exerts an efficient immunosuppressive activity with remarkable potential for the therapy of diseases characterized by pathological over-activation of the immune system.
Assuntos
Produtos Biológicos/uso terapêutico , Diabetes Mellitus Tipo 1/prevenção & controle , Imunossupressores/uso terapêutico , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Progressão da Doença , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/metabolismo , Secreção de Insulina , Interferon gama/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Estimativa de Kaplan-Meier , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , RNA Mensageiro/genética , Baço/imunologia , Baço/metabolismo , Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoAssuntos
Compostos Inorgânicos de Carbono/farmacologia , Inibidores Enzimáticos/farmacologia , Óxidos/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Compostos Inorgânicos de Carbono/química , Inibidores Enzimáticos/química , Corantes Fluorescentes , Cinética , Ouabaína/farmacologia , Óxidos/química , Compostos de Piridínio , Espectrometria de Fluorescência/métodos , EstirenosRESUMO
Thionins are relatively small-sized multiple-cystine peptides that are probably involved in the plant defense against pathogens. As such, these peptides constitute promising candidates for engineered plant resistance in the agricultural industry. More recently, thionins have been proposed as potential immunotoxins in tumor therapy. In the search for pharmacologically active natural products, a new family of thionins was recently discovered in the roots of Helleborus purpurascens that accordingly were termed hellethionins. The structural characterization by NMR of one representative member of this family, i.e., of hellethionin D, clearly reveals that thionins from different sources share a highly conserved overall fold. In fact, the well-defined 3D structure of hellethionin D is very similar to those reported so far for viscotoxins, purothionins, or crambin, although distinct differences could be detected in the C-terminal portion, especially for loop 36-39. These differences may derive from the unusual distribution of charged residues in the C-terminal half of the peptide sequence compared to other thionins and from the uncommon occurrence of four contiguous threonine residues in loop 36-39. As expected, reduction of the disulfide bonds in hellethionin D leads to complete unfolding, but upon oxidative refolding by air oxygen in the presence of glutathione the correct isomer is recovered in high yields, confirming the very robust fold of this class of bioactive cystine peptides.