ABSTRACT
BACKGROUND: Various health benefits have been attributed to Er-Miao-San (EMS), a traditional Chinese herbal formulation that contains equal amounts of cortex phellodendri (Phellodendron amurense Ruprecht) and rhizoma atractylodis (Atractylodes lancea D.C). However, its effect on the anti-inflammatory activity in human dermal fibroblasts (HDFs) and the mechanism underlying this effect are unknown. RESULTS: This study investigated the effects of EMS on TNF-α-induced MMP-1 expression in HDFs. Our data show that EMS inhibited TNF-α-induced MMP-1 expression in a concentration-dependent manner. Interestingly, EMS maintained IkB content without inhibiting the phosphorylation of MAPKs, which are well-established upstream kinases of NF-kB. Moreover, EMS reduced the level of nuclear p65 protein in HDFs. Luciferase assay revealed that EMS inhibits the transcriptional activity of NF-kBbystabilizing IkB. Our results show that EMS exerts its anti-inflammatory effect by inhibiting NF-kB-regulated genes such as IL-1ß and IL-8. Moreover, EMS effectively inhibited TNF-α-induced expression of MMP-1 via the NF-kBpathway. CONCLUSIONS: Taken together, our data suggest that EMS could potentially be used as an anti-inflammatory and anti-aging treatment.
Subject(s)
Humans , Aging/drug effects , Drugs, Chinese Herbal/pharmacology , Plant Extracts/pharmacology , Dermis/cytology , Matrix Metalloproteinase 1/biosynthesis , Fibroblasts/drug effects , Signal Transduction/drug effects , Cell Line , Cell Survival/drug effects , Enzyme Induction/drug effects , Gene Expression Regulation/drug effects , Interleukin-8/drug effects , Interleukin-8/metabolism , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , Mitogen-Activated Protein Kinases/drug effects , Interleukin-1beta/drug effects , Interleukin-1beta/metabolism , Real-Time Polymerase Chain Reaction , Fibroblasts/enzymology , Anti-Inflammatory Agents/administration & dosageABSTRACT
Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.
Subject(s)
Humans , Antibodies, Neutralizing/immunology , Cathepsin L/genetics , Cell Movement , Cells, Cultured , Comet Assay , Dependovirus/genetics , Endothelial Cells/cytology , Fibroblast Growth Factor 2/genetics , Gene Transfer Techniques , Immunoblotting , JNK Mitogen-Activated Protein Kinases , Lac Operon/genetics , Mass Spectrometry , Matrix Metalloproteinase 1/biosynthesis , Muscle, Skeletal/metabolism , Neovascularization, Physiologic , Plasminogen Activator Inhibitor 1/biosynthesis , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Skin exposure to low-dose ultraviolet B (UVB) light up-regulates the expression of matrix metalloproteinase-1 (MMP-1), thus contributing to premature skin aging (photo-aging). Although cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2), have been associated with UVB-induced signaling to MMP expression, very little are known about the roles of lipoxygenases and their products, especially leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), in MMP-1 expression in skin keratinocytes. In the present study, we demonstrate that BLT2, a cell surface receptor for LTB4 and 12(S)-HETE, plays a critical role in UVB-mediated MMP-1 upregulation in human HaCaT keratinocytes. Moreover, our results demonstrated that BLT2-mediated MMP-1 upregulation occurs through a signaling pathway dependent on reactive oxygen species (ROS) production and the subsequent stimulation of ERK. Blockage of BLT2 via siRNA knockdown or with the BLT2-antagonist LY255283 completely abolished the up-regulated expression of MMP-1 induced by low-dose UVB irradiation. Finally, when HaCaT cells were transiently transfected with a BLT2 expression plasmid, MMP-1 expression was significantly enhanced, along with ERK phosphorylation, suggesting that BLT2 overexpression alone is sufficient for MMP-1 up-regulation. Together, our results suggest that the BLT2-ROS-ERK-linked cascade is a novel signaling mechanism for MMP-1 upregulation in low-dose UVB-irradiated keratinocytes and thus potentially contributes to photo-aging.
Subject(s)
Humans , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/biosynthesis , Cell Line , Extracellular Signal-Regulated MAP Kinases/metabolism , Keratinocytes/metabolism , Leukotriene B4/biosynthesis , Matrix Metalloproteinase 1/biosynthesis , Phosphorylation , Reactive Oxygen Species/metabolism , Receptors, Leukotriene B4/physiology , Signal Transduction , Ultraviolet Rays/adverse effectsABSTRACT
El cartílago articular es un tejido paucicelular, con colágeno y proteoglicanos en la matriz extracelular. Su degradación es función de los sinoviocitos, que segregan metaloproteasas que catabolizan a los proteoglicanos. Se distinguen los sinoviocitos A o macrofágicos y los sinoviocitos B o fibroblásticos. La destrucción de proteoglicanos puede ser LT- dependiente o independiente. Nuestro objetivo fue estudiar ex vívo el rol de los sinoviocitos, sin la influencia del sistema inmune. Líquido sinovial de pacientes de ambos sexos, 70ñ2años, con OA(6) y AR(6), vírgenes de tratamiento, se centrifugó 30 minutos a 1500 g, para aislar sinoviocitos. El sedimento se incubó 6 hs en medio Dulbecco-Eagles, con 26 mM de HEPES Gibco, NaHCO3 ( 0.5g/l), glutamina (2 mM), estreptomicina (100mg/l), penicilina (1 U/ml) y anfotericina B (2.5mg/l). Identidad y viabilidad celulares se determinaron con técnicas citopatológicas. Las muestras control provinieron de artritis traumática o patología osteoarticular no-inflamatoria. Con anticuerpos monoclonales anti-MMPs(10mg/ml), previo bloqueo de producción de proteínas inespecíficas con albúmina sérica bovina, se midió actividad colagenasa (MMP-1) antes y después de incubar con ELISA-doble-sandwich. Con streptavidin-peroxidasa se desarrolló color y por absorbancia a 410 nm, se leyó la complejación de los anticuerpos marcados. La secreción de MMP-1 por sinovio-citos AR fue 1373ñ115 ng/ml. Con 6 hs de incubado aumentó hasta 2143ñ132ng/l (-56 por ciento)(p<0.0001), probablemente por la hipercelularidad. Los sinoviocitos OA secretaron 276 ñ 23 ng/ml , y 542 ñ 47 ng/ml tras la incubación (96 por ciente)(p<0.001). Hay paralelismo entre la producción de MMP-1 y la observación microscópica. Sinoviocitos con abundante citoplasma corresponden a altos niveles de enzima. La baja secreción de MMPs responde a escasa población celular y núcleos picnóticos. Aunque en AR la producción de MMPs fue 4.6 veces mayor que en OA, en cambio el incremento porcentual tras la incubación fue casi el doble en OA que en AR. Esos resultados confirman que la producción enzimática varía con la inflamación, que es mayor en los procesos agudos, y que la incubación de sinoviocitos permite detectar cambios patológicos locales