RESUMO
Asthma is a chronic inflammatory disease of lung airways, and pharmacological inhibitors of cyclic adenosine monophosphate-specific phosphodiesterase 4 (PDE4) have been considered as therapeutics for the treatment of asthma. However, development of PDE4 inhibitors in clinical trials has been hampered because of the severe side effects of non-selective PDE4 inhibitors. Here, screening of a plant extract library in conjunction with dereplication technology led to identification of baicalin as a new type of PDE4-selective inhibitor. We demonstrated that while rolipram inhibited the enzyme activity of a range of PDE4 subtypes in in vitro enzyme assays, baicalin selectively inhibited the enzyme activity of PDE4A and 4B. In addition, baicalin suppressed lipopolysaccharide-induced TNF-α expression in macrophage where PDE4B plays a key role in lipopolysaccharide-induced signaling. Furthermore, baicalin treatment in an animal model of allergic asthma reduced inflammatory cell infiltration and TNF-α levels in bronchoalveolar lavage fluids, indicating that the antiinflammatory effects of baicalin in vivo are attributable, in part, to its ability to inhibit PDE4.
Assuntos
Anti-Inflamatórios/farmacologia , Asma/tratamento farmacológico , Flavonoides/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Extratos Vegetais/farmacologia , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Células RAW 264.7 , Rolipram/farmacologia , Rosaceae/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Despite the significance of Alzheimer's disease, the link between metal-associated amyloid-ß (metal-Aß) and disease etiology remains unclear. To elucidate this relationship, chemical tools capable of specifically targeting and modulating metal-Aß species are necessary, along with a fundamental understanding of their mechanism at the molecular level. Herein, we investigated and compared the interactions and reactivities of the green tea extract, (-)-epigallocatechin-3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate; EGCG], with metal [Cu(II) and Zn(II)]-Aß and metal-free Aß species. We found that EGCG interacted with metal-Aß species and formed small, unstructured Aß aggregates more noticeably than in metal-free conditions in vitro. In addition, upon incubation with EGCG, the toxicity presented by metal-free Aß and metal-Aß was mitigated in living cells. To understand this reactivity at the molecular level, structural insights were obtained by ion mobility-mass spectrometry (IM-MS), 2D NMR spectroscopy, and computational methods. These studies indicated that (i) EGCG was bound to Aß monomers and dimers, generating more compact peptide conformations than those from EGCG-untreated Aß species; and (ii) ternary EGCG-metal-Aß complexes were produced. Thus, we demonstrate the distinct antiamyloidogenic reactivity of EGCG toward metal-Aß species with a structure-based mechanism.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/efeitos dos fármacos , Catequina/análogos & derivados , Metais/química , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/toxicidade , Camellia sinensis/química , Catequina/química , Catequina/farmacologia , Cobre/química , Cobre/farmacologia , Cobre/toxicidade , Humanos , Metais/farmacologia , Metais/toxicidade , Modelos Moleculares , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Espectrometria de Massas em Tandem , Zinco/química , Zinco/farmacologia , Zinco/toxicidadeRESUMO
Ellagic acid, a polyphenol compound present in berries and pomegranate, has received attention as an agent that may have potential bioactivities preventing chronic diseases. This study examined photoprotective effects of ellagic acid on collagen breakdown and inflammatory responses in UV (ultraviolet)-B irradiated human skin cells and hairless mice. Ellagic acid attenuated the UV-B-induced toxicity of HaCaT keratinocytes and human dermal fibroblasts. Non-toxic ellagic acid markedly prevented collagen degradation by blocking matrix metalloproteinase production in UV-B-exposed fibroblasts. Anti-wrinkle activity of ellagic acid was further investigated in hairless mice exposed to UV-B, in which it attenuated UV-B-triggered skin wrinkle formation and epidermal thickening. Topical application of 10 micromol/l ellagic acid diminished production of pro-inflammatory cytokines IL-1beta and IL-6, and blocked infiltration of inflammatory macrophages in the integuments of SKH-1 hairless mice exposed to UV-B for 8 weeks. In addition, this compound mitigated inflammatory intracellular cell adhesion molecule-1 expression in UV-B-irradiated keratinocytes and photoaged mouse epidermis. These results demonstrate that ellagic acid prevented collagen destruction and inflammatory responses caused by UV-B. Therefore, dietary and pharmacological interventions with berries rich in ellagic acid may be promising treatment strategies interrupting skin wrinkle and inflammation associated with chronic UV exposure leading to photoageing.
Assuntos
Dermatite/tratamento farmacológico , Ácido Elágico/farmacologia , Ácido Elágico/uso terapêutico , Envelhecimento da Pele/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Colágeno/metabolismo , Dermatite/etiologia , Dermatite/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Pelados , Modelos Animais , Envelhecimento da Pele/patologia , Envelhecimento da Pele/efeitos da radiação , Resultado do TratamentoRESUMO
The matrix metalloproteinases (MMP) play an important role in tumor invasion, angiogenesis and inflammatory tissue destruction. Increased expression of MMP was observed in benign tissue hyperplasia and in atherosclerotic lesions. Invasive cancer cells utilize MMP to degrade the extracellular matrix and vascular basement membrane during metastasis, where MMP-2 has been implicated in the development and dissemination of malignancies. The present study attempted to examine the antiangiogenic activity of the medicinal herbs of Aspergillus usamii var. shirousamii-transformed Angelicae Gigantis Radix and Zizyphus jujube (tAgR and tZj) with respect to MMP-2 production and endothelial motility in phorbol 12-myristate 13-acetate (PMA)- or VEGF-exposed human umbilical vein endothelial cells (HUVEC). Nontoxic tAgR and tZj substantially suppressed PMA-induced MMP-2 secretion. In addition, 25 microg/mL tAgR and tZj prevented vascular endothelial growth factor-stimulated endothelial cell transmigration and tube formation. The results reveal that tAgR and tZj dampened endothelial MMP-2 production leading to endothelial transmigration and tube formation. tAgR and tZj-mediated inhibition of endothelial MMP may boost a therapeutic efficacy during vascular angiogenesis.
RESUMO
Fruits of bog blueberry (Vaccinium uliginosum L.) are rich in anthocyanins that contribute pigmentation. Anthocyanins have received much attention as agents with potentials preventing chronic diseases. This study investigated the capacity of anthocyanin-rich extract from bog blueberry (ATH-BBe) to inhibit photoaging in UV-B-irradiated human dermal fibroblasts. BBe anthocyanins were detected as cyanidin-3-glucoside, petunidin-3-glucoside, malvidin-3-glucoside, and delphinidin3-glucoside. ATH-BBe attenuated UV-B-induced toxicity accompanying reactive oxygen species (ROS) production and the resultant DNA damage responsible for activation of p53 and Bad. Preincubation of ATH-BBe markedly suppressed collagen degradation via blunting production of collagenolytic matrix metalloproteinases (MMP). Additionally, ATH-BBe enhanced UV-B-downregulated procollagen expression at transcriptional levels. We next attempted to explore whether ATH-BBe mitigated the MMP-promoted collagen degradation through blocking nuclear factor kappaB (NF-kappaB) activation and MAPK-signaling cascades. UV-B radiation enhanced nuclear translocation of NF-kappaB, which was reversed by treatment with ATH-BBe. The UV-B irradiation rapidly activated apoptosis signal-regulating kinase-1 (ASK-1)-signaling cascades of JNK and p38 mitogen-activated protein kinase (p38 MAPK), whereas ATH-BBe hampered phosphorylation of c-Jun, p53, and signal transducers and activators of transcription-1 (STAT-1) linked to these MAPK signaling pathways. ATH-BBe diminished UV-B augmented-release of inflammatory interleukin (IL)-6 and IL-8. These results demonstrate that ATH-BBe dampens UV-B-triggered collagen destruction and inflammatory responses through modulating NF-kappaB-responsive and MAPK-dependent pathways. Therefore, anthocyanins from edible bog blueberry may be protective against UV-induced skin photoaging.
Assuntos
Antocianinas/farmacologia , Mirtilos Azuis (Planta)/química , Extratos Vegetais/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Células Cultivadas , Colágeno/análise , Fibroblastos/efeitos da radiação , Humanos , MAP Quinase Quinase Quinase 5/fisiologia , Sistema de Sinalização das MAP Quinases , Metaloproteinase 1 da Matriz/genética , Raios UltravioletaRESUMO
Diabetes can cause a wide variety of vascular complications and endothelial dysfunction. In this study, human vascular endothelial cells were exposed to 5.5 mM and 33 mM glucose for 5 d in the absence and presence of 1 to 20 mug/mL roasted licorice (Glycyrrhiza inflata Bat.) ethanol extracts (rLE). Caspase-3 activation and Annexin V staining revealed that high glucose induced endothelial apoptotic toxicity with a generation of reactive oxygen species (ROS) and these effects were reversed by rLE at >/=1 mug/mL in a dose-dependent manner. Cytoprotective rLE substantially reduced high glucose-induced expression of endothelial nitric oxide synthase (eNOS), and hence attenuated the formation of peroxynitrite radicals derived from NO. In addition, rLE suppressed expression of PKCbeta2 and activation of NADPH oxidase subunit of p22phox promoted by high glucose. However, rLE =1 mug/mL did not modulate the high glucose-triggered activation of ASK-JNK signaling pathway. Our results suggest that PKCbeta2 expression and NADPH oxidase-dependent superoxide production and eNOS-mediated peroxynitrite generation may be essential mechanisms responsible for increased oxidative stress and endothelial apoptosis in chronic hyperglycemic conditions. Thus, rLE may be a beneficial agent most likely contributing to prevention of vascular NADPH oxidase induction and preservation of endothelial nitric oxide availability, resulting in blunting diabetes-associated endothelial dysfunction and vascular complications.
Assuntos
Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Glucose/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Etilaminas/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceínas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Glucose/metabolismo , Glycyrrhiza/metabolismo , Humanos , Imuno-Histoquímica , Óxido Nítrico Sintase Tipo III/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Veias Umbilicais/citologiaRESUMO
Hyperglycemia is a causal factor in the development of diabetic vascular complications including impaired vascular smooth muscle contractility and increased cell proliferation. The present study was designed to investigate the effects of Sasa borealis water-extract (SBwE) on chronic hyperglycemia-induced oxidative stress and apoptosis in human umbilical endothelial cells (HUVEC). HUVEC were cultured in 5.5 mM low glucose, 5.5 mM glucose plus 27.5 mM mannitol as an osmotic control, or 33 mM high glucose for 5 days in the absence and presence of 1-30 microg/ ml SBwE. Caspase-3 activation and Annexin V staining revealed chronic high glucose-induced endothelial apoptotic toxicity with a generation of oxidants detected by DCF-fluorescence, and these effects were reversed by SBwE at > or =1 microg/ml in a dose-dependent manner. Cytoprotective SBwE substantially reduced the sustained high glucose-induced expression of endothelial nitric oxide synthase and attenuated the formation of peroxynitrite radicals. The suppressive effects of SBwE were most likely mediated through blunting activation of PKC beta 2 and NADPH oxidase promoted by high glucose. In addition, this bamboo extract modulated the high glucose-triggered mitogen-activated protein kinase-dependent upregulation of heat-shock proteins. Our results suggest that SBwE suppressed these detrimental effects caused by PKC-dependent peroxynitrite formation via activation of NADPH oxidase and induction of nitric oxide synthase and heat-shock protein family that may be essential mechanisms responsible for increased apoptotic oxidative stress in diabetic vascular complications. Moreover, the blockade of high glucose-elicited heat-shock protein induction appeared to be responsible for SBwE-alleviated endothelial apoptosis. Therefore, SBwE may be a therapeutic agent for the prevention and treatment of diabetic endothelial dysfunction and related complications.
Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Glucose/farmacologia , Extratos Vegetais/farmacologia , Sasa/química , Células Cultivadas , Células Endoteliais/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Peroxinitroso/biossíntese , Fitoterapia , Extratos Vegetais/química , Proteína Quinase C/metabolismo , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
Numerous polyphenolic compounds have been found to inhibit adhesion and migration of leukocytes to sites of inflammation that are partly regulated by the expression of cell adhesion molecules (CAM) such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and platelet endothelial cell adhesion molecule-1 (PECAM-1). Licorice root extracts have been used in traditional Chinese, Tibetan, and Indian medicine for the treatment of pulmonary diseases and inflammatory processes. Expression of CAM proteins was examined in human umbilical vein endothelial cells (HUVEC) treated with a licorice component (isoliquiritigenin, 18beta-glycyrrhetinic acid, glycyrrhizin, formononetin, or ononin) and exposed to TNF-alpha. The involvement of NF-kappaB in the transcriptional control of CAM proteins was assessed by degradation of IkappaBalpha and nuclear translocation of NF-kappaB using Western blotting techniques and immunocytochemical staining. At nontoxic > or =10 microM, isoliquiritigenin blocked the induction of VCAM-1 and E-selectin on activated HUVEC and markedly interfered with THP-1 monocyte adhesion to TNF-alpha-activated endothelial cells. Isoliquiritigenin abolished TNF-alpha-induced mRNA accumulation of VCAM-1 and E-selectin. Additionally, immunocytochemical staining revealed that isoliquiritigenin attenuated PECAM-1 expression induced by TNF-alpha. In contrast, other components recognized in licorice, 18beta-glycyrrhetinic acid, glycyrrhizin, formononetin, and ononin did not down-regulate the expression of VCAM-1 and/or PECAM-1 activated by TNF-alpha, implying that these components are inactive in modulating adhesion of leukocytes to stimulated endothelial cells. Isoliquiritigenin downregulated CAM proteins in TNF-alpha-activated HUVEC at the transcriptional levels by blocking degradation of IkappaBalpha and nuclear translocation of NF-kappaB. These results demonstrate that the induction blockade of VCAM-1 and E-selectin by isoliquiritigenin was directly mediated by its interference with the CAM mRNA transcription through NF-kappaB-dependent mechanisms under inflammatory conditions.