RESUMO
We synthesized phenylboronic acid pinacol ester (PBPE)-conjugated hyaluronic acid (HA) via thiobis(ethylamine) (TbEA) linkage (abbreviated as HAsPBPE conjugates) to fabricate the radiosensitive delivery of caffeic acid phenetyl ester (CAPE) and for application in radioprotection. PBPE was primarily conjugated with TbEA and then PBPE-TbEA conjugates were conjugated again with hyaluronic acid using carbodiimide chemistry. CAPE-incorporated nanoparticles of HAsPBPE were fabricated by the nanoprecipitation method and then the organic solvent was removed by dialysis. CAPE-incorporated HAsPBPE nanoparticles have a small particle size of about 80 or 100 nm and they have a spherical shape. When CAPE-incorporated HAsPBPE nanoparticles were irradiated, nanoparticles became swelled or disintegrated and their morphologies were changed. Furthermore, the CAPE release rate from HAsPBPE nanoparticles were increased according to the radiation dose, indicating that CAPE-incorporated HAsPBPE nanoparticles have radio-sensitivity. CAPE and CAPE-incorporated HAsPBPE nanoparticles appropriately prevented radiation-induced cell death and suppressed intracellular accumulation of reactive oxygen species (ROS). CAPE and CAPE-incorporated HAsPBPE nanoparticles efficiently improved survivability of mice from radiation-induced death and reduced apoptotic cell death. We suggest that HAsPBPE nanoparticles are promising candidates for the radio-sensitive delivery of CAPE.
Assuntos
Ácidos Borônicos/química , Ácidos Cafeicos/farmacologia , Glicóis/química , Ácido Hialurônico/química , Nanopartículas/química , Álcool Feniletílico/análogos & derivados , Proteção Radiológica , Animais , Ácidos Borônicos/síntese química , Ácidos Cafeicos/síntese química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Peróxido de Hidrogênio/toxicidade , Fígado/metabolismo , Camundongos Endogâmicos BALB C , Nanopartículas/ultraestrutura , Tamanho da Partícula , Álcool Feniletílico/síntese química , Álcool Feniletílico/farmacologia , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
INTRODUCTION: Extracellular nucleotides are released and detectable in a high concentration within the tumor microenvironment. G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is activated equipotently by adenosine triphosphate (ATP) and uridine 5'-triphosphate (UTP), which mediate proinflammatory responses such as cell migration and proliferation. However, the role of P2Y2R in the process of cancer metastasis remains unclear. This study aimed to determine the role of P2Y2R in the proliferation, migration and invasion of highly metastatic MDA-MB-231 breast cancer cells through crosstalk with endothelial cells (ECs). METHODS: ATP release and P2Y2R activity between high metastatic breast cancer cell MDA-MB-231 and low metastatic breast cancer cell MCF-7 were compared. Then, the role of P2Y2R on tumor growth and invasion via crosstalk with ECs was examined in vitro, using MDA-MB-231 cells and ECs transfected with control- or P2Y2R-siRNA, and in vivo, using an animal model injected with control-shRNA- or P2Y2R-shRNA-transfected MDA-MB-231 cells. RESULTS: We found that this highly metastatic breast cancer cell line released higher levels of ATP and showed a higher P2Y2R activity in comparison to a low metastatic breast cancer cell line, MCF-7. In MDA-MB-231 cells, P2Y2R activation by ATP or UTP increased proliferation at 24 or 72 hours, which was abolished by P2Y2R knock-down. In addition, the adhesion of MDA-MB-231 cells to ECs and cell migration were both significantly increased by ATP or UTP through the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in MDA-MB-231 or ECs but not in cells where P2Y2R was knocked down. Furthermore, ATP- or UTP-mediated activation of P2Y2R induced MDA-MB-231 invasion through ECs, increased matrix metalloproteinase-9 (MMP-9) activity and vascular endothelial growth factor (VEGF) production in MDA-MB-231 and induced the phosphorylation of vascular endothelial (VE)-cadherin in ECs. Tumor growth and metastasis to other tissues were dramatically reduced, and body weight was increased in mice injected with P2Y2R-shRNA-transfected MDA-MB-231 cells compared to mice injected with control shRNA-transfected MDA-MB-231 cells. CONCLUSION: This study suggests that P2Y2R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.
Assuntos
Trifosfato de Adenosina/fisiologia , Neoplasias da Mama/patologia , Células Endoteliais/metabolismo , Neoplasias Pulmonares/secundário , Receptores Purinérgicos P2Y2/metabolismo , Animais , Antígenos CD/metabolismo , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Comunicação Celular , Movimento Celular , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Processamento de Proteína Pós-Traducional , Uridina Trifosfato/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
P2Y2 R has been shown to be upregulated in a variety of tissues in response to stress or injury and to mediate tissue regeneration through its ability to activate multiple signalling pathways. This study aimed to investigate the role of P2Y2 R in the wound-healing process and the mechanisms by which P2Y2 R activation promotes wound healing in fibroblasts. The role of P2Y2 R in skin wound healing was examined using a full-thickness skin wound model in wildtype (WT) and P2Y2 R(-/-) mice and an in vitro scratch wound model in control or P2Y2 R siRNA-transfected fibroblasts. WT mice showed significantly decreased wound size compared with P2Y2 R(-/-) mice at day 14 post-wounding, and immunohistochemical analysis showed that a proliferation marker Ki67 and extracellular matrix (ECM)-related proteins VEGF, collagen I, fibronectin and α-SMA were overexpressed in WT mice, which were reduced in P2Y2 R(-/-) mice. Scratch-wounded fibroblasts increased ATP release, which peaked at 5 min. In addition, scratch wounding increased the level of P2Y2 R mRNA. Activation of P2Y2 R by ATP or UTP enhanced proliferation and migration of fibroblasts in in vitro scratch wound assays and were blocked by P2Y2 R siRNA. Finally, ATP or UTP also increased the levels of ECM-related proteins through the activation of P2Y2 R in fibroblasts. This study suggests that P2Y2 R may be a potential therapeutic target to promote wound healing in chronic wound diseases.
Assuntos
Trifosfato de Adenosina/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Uridina Trifosfato/metabolismo , Cicatrização , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Inativação Gênica , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Interferente Pequeno/metabolismo , Receptores Purinérgicos P2Y2/genética , Pele/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Lipoprotein oxidation, inflammation, and immune responses involving the vascular endothelium and immune cells contribute to the pathogenesis of atherosclerosis. In an atherosclerotic animal model, P2Y2 receptor (P2Y2R) upregulation and stimulation were previously shown to induce intimal hyperplasia and increased intimal monocyte infiltration. Thus, we investigated the role of P2Y2R in oxidized low-density lipoprotein (oxLDL)-mediated oxidative stress and the subsequent interaction between endothelial cells (ECs) and immune cells. The treatment of human ECs with oxLDL caused the rapid release of ATP (maximum after 5 min). ECs treated with oxLDL or the P2Y2R agonists ATP/UTP for 1h exhibited significant reactive oxygen species (ROS) production, but this effect was not observed in P2Y2R siRNA-transfected ECs. In addition, oxLDL and ATP/UTP both induced RAGE expression, which was P2Y2R dependent. Oxidized LDL- and ATP/UTP-mediated ROS production was diminished in RAGE siRNA-transfected ECs, suggesting that RAGE is an important mediator in P2Y2R-mediated ROS production. Treatment with oxLDL for 24h induced P2Y2R expression in the human monocyte cell line THP-1 and increased THP-1 cell migration toward ECs. The addition of apyrase, an enzyme that hydrolyzes nucleotides, or diphenyleneiodonium (DPI), a well-known inhibitor of NADPH oxidase, significantly inhibited the increase in cell migration caused by oxLDL. P2Y2R siRNA-transfected THP-1 cells did not migrate in response to oxLDL or ATP/UTP treatment, indicating a critical role for P2Y2R and nucleotide release in oxLDL-induced monocyte migration. Last, oxLDL and ATP/UTP effectively increased ICAM-1 and VCAM-1 expression and the subsequent binding of THP-1 cells to ECs, which was inhibited by pretreatment with DPI or by siRNA against P2Y2R or RAGE, suggesting that P2Y2R is an important mediator in oxLDL-mediated monocyte adhesion to ECs through the regulation of ROS-dependent adhesion molecule expression in ECs. Taken together, our findings suggest that P2Y2R could be a therapeutic target for the prevention of vascular disorders, including atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Receptor para Produtos Finais de Glicação Avançada/biossíntese , Receptores Purinérgicos P2Y/biossíntese , Trifosfato de Adenosina/administração & dosagem , Aterosclerose/patologia , Aterosclerose/terapia , Linhagem Celular , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Lipoproteínas LDL/administração & dosagem , Lipoproteínas LDL/metabolismo , Terapia de Alvo Molecular , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Uridina Trifosfato/administração & dosagem , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
Glioblastoma is one of the most lethal and prevalent malignant human brain tumors, with aggressive proliferation and highly invasive properties. There is still no effective cure for patients with glioblastoma. Honokiol, derived from Magnolia officinalis, can cross the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), making it a strong candidate for an effective drug for the treatment of brain tumors, including glioblastoma. In our previous study, we demonstrated that honokiol effectively induced apoptotic cell death in glioblastoma. Metastasis poses the largest problem to cancer treatment and is the primary cause of death in cancer patients. Thus, in this study, we investigated the effect of honokiol on the cell invasion process of U87MG human glioblastoma cells through brain microvascular endothelial cells (BMECs) and its possible mechanisms. Honokiol dose-dependently inhibited TNF-α-induced VCAM-1 expression in BMECs and adhesion of U87MG to BMECs. Moreover, honokiol effectively blocked U87MG invasion through BMEC-Matrigel-coated transwell membranes. Increased phosphorylation of VE-cadherin and membrane permeability by TNF-α were suppressed by honokiol in BMECs. Furthermore, we investigated the effect of honokiol on the epithelial-mesenchymal transition (EMT) in U87MG cells. Honokiol reduced the expression levels of Snail, N-cadherin and ß-catenin, which are mesenchymal markers, but increased E-cadherin, an epithelial marker. In conclusion, these results suggest that honokiol inhibits metastasis by targeting the interaction between U87MG and BMECs, regulating the adhesion of U87MG to BMECs by inhibiting VCAM-1, and regulating the invasion of U87MG through BMECs by reducing membrane permeability and EMT processes of U87MG cells.
Assuntos
Compostos de Bifenilo/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Permeabilidade da Membrana Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Lignanas/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Células Endoteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
Sepsis is a severe systemic inflammatory response that is associated with high morbidity and mortality. A previous study using an animal model of sepsis showed that survival was significantly lower in WT mice than in P2Y(2) receptor (P2Y(2)R)-deficient mice, suggesting that P2Y(2)R plays a role in septic death. We therefore investigated the role of P2Y(2)R in the inflammatory responses of RAW264.7 murine macrophages to LPS. LPS time-dependently upregulated P2Y(2)R mRNA levels, with a prominent increase observed at 4 h. In addition, LPS increased ATP release in a time dependent manner (5-120 min post LPS treatment). Accordingly, we pretreated cells with LPS for 4 h to induce P2Y(2)R expression and then stimulated the cells with UTP or ATP for 16 h. Interestingly, ATP- or UTP-dependent P2Y(2)R activation in LPS-pretreated cells resulted in dramatically enhanced HMGB1 secretion, COX-2 and iNOS expression, and furthermore PGE2 and NO production compared to LPS treatment alone (4 h) or ATP or UTP treatment alone (16 h), an effect that was inhibited by P2Y(2)R silencing. In addition, these increases in HMGB1 secretion, COX-2 and iNOS expression and PGE(2) and NO production commonly involved the JNK, PKC and PDK pathways. Taken together, these data demonstrate that LPS-dependent upregulation of P2Y(2)R plays a critical role in facilitating the inflammatory responses induced by LPS.
Assuntos
Trifosfato de Adenosina/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Receptores Purinérgicos P2Y2/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Expressão Gênica , Proteína HMGB1/genética , Inflamação/genética , Inflamação/metabolismo , MAP Quinase Quinase 4/metabolismo , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Proteína Quinase C/metabolismo , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Regulação para Cima , Uridina Trifosfato/farmacologiaRESUMO
The nuclear DNA binding protein high mobility group box 1 (HMGB1) has recently been suggested to act as a late mediator of septic shock. The effect of ((S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid, also known as THI-56, in an experimental model of sepsis was investigated. THI-56 exhibited potent anti-inflammatory properties in response to LPS in RAW 264.7 cells. In particular, THI-56 significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and the release of HMGB1 in activated macrophages. THI-56 activated NE-F2-regulated factor 2 (Nrf-2)/heme oxygenase 1 (HO-1). The specific knockdown of the HO-1 gene by HO-1 siRNA significantly reversed the inhibitory effects of THI-56 on iNOS expression and HMGB1 release in LPS-stimulated macrophages. Importantly, THI-56 administration protected animals from death induced by either a lethal dose of LPS or cecal ligation and puncture (CLP). Furthermore, the ALT, AST, BUN, creatinine, and HMGB1 levels in the blood were significantly increased in CLP-induced septic mice, and the administration of THI-56 reduced these levels in a concentration-dependent and zinc protoporphyrin IX (ZnPPIX)-sensitive manner. In addition, the administration of THI-56 significantly ameliorated not only lung damage but also macrophage infiltration in the livers of CLP-induced septic mice, and these effects were also abrogated in the presence of ZnPPIX. Thus, we conclude that THI-56 significantly attenuates the proinflammatory response induced by LPS and reduces organ damage in a CLP-induced sepsis model through the upregulation of Nrf-2/HO-1.
Assuntos
Proteína HMGB1/metabolismo , Heme Oxigenase-1/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Sepse/metabolismo , Tetra-Hidroisoquinolinas/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/genética , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Interferência de RNA , Sepse/tratamento farmacológico , Sepse/etiologia , Sepse/genética , Sepse/mortalidade , Tetra-Hidroisoquinolinas/administração & dosagemRESUMO
High mobility group box 1 (HMGB1) plays a crucial mediator in the pathogenesis of many inflammatory diseases. We recently proposed that heme oxygenase-1 (HO-1) negatively regulates HMGB1 in inflammatory conditions. We investigated whether ethanol extract of Inula helenium L. (EIH) activates p38 MAPK/Nrf2/HO-1 pathways in RAW264.7 cells and reduces inflammation in CLP-induced septic mice. EIH induced expression of HO-1 protein in a time- and concentration-dependent manner. EIH significantly diminished HO-1 expression in siNrf2 RNA-transfected cells. As expected, the inhibited expression of iNOS/NO, COX-2/PGE2, HMGB1 release by EIH in LPS-activated RAW264.7 cells was significantly reversed by siHO-1RNA transfection. Furthermore, EIH not only inhibited NF-κB luciferase activity, phosphorylation of IκBα in LPS-activated cells but also significantly suppressed expression of adhesion molecules (ICAM-1 and VCAM-1) in TNF-α activated human umbilical vein endothelial cells. The induction of HO-1 by EIH was inhibited by SB203580 but not by SP600125, PD98059, nor LY294002. Most importantly, administration of EIH significantly reduced not only increase in blood HMGB1, ALT, AST, BUN, creatinine levels but also decrease macrophage infiltrate in the liver of septic mice, which were reversed by ZnPPIX, a HO-1 inhibitor. We concluded that EIH has anti-inflammatory effect via the induction of p38 MAPK-dependent HO-1 signaling pathway.
Assuntos
Heme Oxigenase-1/biossíntese , Inflamação/prevenção & controle , Inula/química , Lipopolissacarídeos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Extratos Vegetais/farmacologia , Sepse/fisiopatologia , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Indução Enzimática , Etanol/química , Inflamação/enzimologia , Inflamação/metabolismo , Camundongos , Sepse/enzimologia , Sepse/metabolismoRESUMO
BACKGROUND AND PURPOSE: Given the importance of VEGF and haem oxygenase (HO)-1 in wound healing, the present study tested the hypothesis that CKD712, a synthetic tetrahydroisoquinoline alkaloid, activated VEGF production through the induction of HO-1 in human dermal fibroblasts (HDFs) and in mouse skin to stimulate wound healing. EXPERIMENTAL APPROACH: Using HDFs, the effects of CKD712 on the production of VEGF and migration were evaluated. The mechanisms responsible were investigated using various signal inhibitors and small interfering RNA techniques. The ability of CKD712 to promote wound healing was also investigated in full-thickness skin-wounded mice. KEY RESULTS: CKD712 treatment of HDFs increased VEGF production and accelerated migration, which was antagonized by anti-VEGF antibodies. Both an AMPK inhibitor (compound C) and a HO-1 activity inhibitor (SnPPIX) but not inhibitors of MAPKs, PI3K and PKC reduced the production of VEGF by CKD712. Interestingly, SnPPIX inhibited HO-1 expression but not p-AMPK, whereas compound C inhibited both p-AMPK and HO-1 induction by CKD712. Moreover, CKD712 decreased HO-1 expression without affecting the expression of p-AMPK by siHO-1 transfection, but it failed to induce HO-1 in siAMPKα1-transfected cells, suggesting that AMPK is involved in HO-1 induction by CKD712 in HDFs. Also, CKD712 shortened the time of wound closure in an SnPPIX-sensitive manner in a full-thickness skin-wounded mouse model. CONCLUSION AND IMPLICATIONS: CKD712 accelerated cutaneous wound healing, at least in part, by the production of VEGF through HO-1 induction in HDFs and mouse skin.
Assuntos
Fármacos Dermatológicos/uso terapêutico , Heme Oxigenase-1/metabolismo , Pele/efeitos dos fármacos , Tetra-Hidroisoquinolinas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/biossíntese , Cicatrização/efeitos dos fármacos , Ferimentos Penetrantes/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Fármacos Dermatológicos/farmacologia , Inibidores Enzimáticos/farmacologia , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Interferência de RNA , RNA Interferente Pequeno , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Pele/citologia , Pele/lesões , Pele/metabolismo , Tetra-Hidroisoquinolinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ferimentos Penetrantes/metabolismoRESUMO
The goal of this study was to investigate the differential effect of tanshinone IIA on the induction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by TNF-α and the possible molecular mechanisms by which it regulates ICAM-1 and VCAM-1 expression differentially. Stimulation of human umbilical vein endothelial cells (HUVEC) with TNF-α increased ICAM-1 and VCAM-1 expressions, and the pretreatment with tanshinone IIA concentration dependently inhibited VCAM-1 expression but not ICAM-1 expression. In previous study, PI3K/Akt, PKC and Jak/STAT-3 pathways were involved in the TNF-α-mediated induction of VCAM-1 but not ICAM-1. Thus, we examined the effect of tanshinone IIA on TNF-α-mediated activations of PI3K/Akt, PKC and Jak/STAT-3 pathways. Tanshinone IIA efficiently inhibited the phosphorylations of Akt, PKC and STAT-3 by TNF-α. Moreover, we determined the effect of tanshinone IIA on IRF-1 or GATAs induction and binding activity to VCAM-1 promoter since the upstream promoter region of VCAM-1 but not ICAM-1 contains IRF-1 and GATA binding motifs. Western blot analysis and ChIP assay showed that tanshinone IIA efficiently inhibited TNF-α-increased nuclear level of IRF-1 and GATA-6 and their binding affinity to VCAM-1 promoter region. Taken together, tanshinone IIA selectively inhibits TNF-α-mediated expression of VCAM-1 but not ICAM-1 through modulation of PI3/Akt, PKC and Jak/STAT-3 pathway as well as IRF-1 and GATA-6 binding activity.
Assuntos
Abietanos/farmacologia , Fator de Transcrição GATA6/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Abietanos/química , Anti-Inflamatórios não Esteroides/farmacologia , Adesão Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Fator Regulador 1 de Interferon/genética , Estrutura Molecular , Monócitos/fisiologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Glioblastoma is one of the most lethal and common malignant human brain tumors, with aggressive proliferation and highly invasive properties. Honokiol derived from Magnolia officinalis is able to cross the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), suggesting a strong possibility that it could be an effective drug for the treatment of brain tumors, including glioblastoma. Thus, we investigated the effects of honokiol on the expression of adhesion molecules in TNF-α-stimulated endothelial cells, and cancer growth and invasion were determined in T98G human glioblastoma cells. Honokiol dose-dependently inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) stimulated with TNF-α for 6 h. Pretreatment with honokiol significantly reduced the adhesion of T98G cells to HUVECs. Moreover, honokiol inhibited the invasion of T98G cells, suggesting that honokiol has an anti-metastatic effect. In addition, honokiol increased the cytotoxicity of T98G cells in a dose- and time-dependent manner as assayed by MTT. TUNEL assay showed that honokiol significantly induced apoptosis in T98G cells at doses of 10 µM or more. The induction of apoptotic cell death was mediated by the downregulation of the anti-apoptotic protein Bcl-2 and the upregulation of the pro-apoptotic protein Bax. Taken together, the results of this study suggest that honokiol exerts an anticancer effect by preventing metastasis and inducing apoptotic cell death of brain tumor cells.
Assuntos
Compostos de Bifenilo/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Molécula 1 de Adesão Intercelular/metabolismo , Lignanas/administração & dosagem , Extratos Vegetais/administração & dosagem , Molécula 1 de Adesão de Célula Vascular/metabolismo , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/genética , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Magnolia/química , Extratos Vegetais/química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
AIMS: We investigated the molecular mechanism by which ethyl pyruvate (EP) induces heme oxygenase-1 (HO-1) in RAW 264.7 cells and its effect on survival rate in cecal ligation and puncture (CLP)-induced wild-type (WT) and HO-1 knockout (HO-1(-/-)) septic mice. RESULTS: EP induced HO-1 in a dose- and time-dependent manner, which was mediated through p38 mitogen-activated protein kinase (MAPK) and NF-E2-related factor 2 (Nrf2) signaling cascade in RAW 264.7 cells. EP significantly inhibited the lipopolysaccharide (LPS)-stimulated inducible nitric oxide synthase (iNOS) expression and high-mobility group box 1 (HMGB1) release in RAW 264.7 cells. The inhibitory effect of EP on LPS-stimulated iNOS expression and HMGB1 release was reversed by transfection with siHO-1RNA in RAW 264.7 cells, but EP failed to reduce them in HO-1(-/-) peritoneal macrophages treated with LPS. Moreover, treatment of cells with glutathione ethyl ester (GSH-Et), SB203580 (p38 MAPK inhibitor), siHO-1, or p38-siRNA transfection inhibited anti-inflammatory effect of EP. Interestingly, both HO-1 induction and phosphorylation of p38 by EP were reversed by GSH-Et, and antioxidant redox element-luciferase activity by EP was reversed by SB203580 in LPS-activated cells. EP increased survival and decreased serum HMGB1 in CLP-WT mice, whereas it did not increase survival or decrease circulating HMGB1 in HO-1(-/-) CLP-mice. INNOVATION AND CONCLUSION: Our work provides new insights into the understanding the molecular mechanism by showing that EP induces HO-1 through a p38 MAPK- and NRF2-dependent pathway by decreasing GSH cellular levels. We conclude that EP inhibits proinflammatory response to LPS in macrophages and increases survival in CLP-induced septic mice by upregulation of HO-1 level, in which p38 MAPK and Nrf2 play an important role.
Assuntos
Glutationa/metabolismo , Heme Oxigenase-1/metabolismo , Sepse/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Heme Oxigenase-1/genética , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Cell adhesion molecules (CAMs) are involved in a variety of pathologies including cancer, inflammation, pathogenic infections and autoimmune disease. In particular, VCAM-1, rather than ICAM-1, plays a major role in the initiation of atherosclerosis and tumor progression. Therefore, we attempted to elucidate differential mechanisms that regulate VCAM-1 and ICAM-1 expressions. Down-regulation of JNK by a specific inhibitor (SP600125) or dominant negative (DN) JNK1 plasmid enhanced TNF-α-induced VCAM-1 but not ICAM-1 expression. Moreover, transfection with a JNK1-overexpressing vector resulted in the inhibition of VCAM-1 expression stimulated by TNF-α in HUVECs, suggesting that JNK negatively regulates TNF-α-induced VCAM-1 expression in endothelial cells (ECs). Next, we investigated whether JNK signaling affects IRF-1 and/or GATA6, which are transcription factors that mediate TNF-α induction of VCAM-1 but not ICAM-1. The DN-JNK1 plasmid-transfected cells enhanced TNF-α up-regulation of IRF-1 whereas JNK1-overexpressing cells displayed down-regulation; however, neither DN-JNK1 transfection nor JNK1 overexpression affected GATA6 protein levels in the nuclear fraction. Chromatin immunoprecipitation (ChIP) assay confirmed that the inhibition of JNK by DN-JNK1 transfection increases the binding of IRF-1 to the VCAM-1 promoter whereas the overexpression of JNK1 inhibits IRF-1 binding to the VCAM-1 promoter. However, neither DN-JNK1 nor JNK1 overexpression altered GATA6 affinity for the VCAM-1 promoter region. We also examined whether MKP-7 affects ICAM-1 or VCAM-1 by regulating JNK. TNF-α-induced phosphor-JNK levels increased after 5min, peaked at 10 min, and decreased after 30 min. Interestingly, MKP-7 protein levels increased after 30 min, when phosphor-JNK induction by TNF-α was decreased. In addition, silencing MKP-7 with specific siRNA resulted in an increase in phosphor-JNK and inhibited the expression of VCAM-1 but not ICAM-1. Moreover, silencing MKP-7 caused the down-regulation of IRF-1 protein levels and binding to the VCAM-1 promoter. Thus, we suggest that MKP-7, a negative regulator of JNK, regulates VCAM-1 expression in activated endothelial cells through IRF-1 but not GATA6.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Regulação da Expressão Gênica , MAP Quinase Quinase 4/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Transdução de Sinais/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Imunoprecipitação da Cromatina , Fosfatases de Especificidade Dupla/genética , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Genes Reporter , Células Endoteliais da Veia Umbilical Humana , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Luciferases , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Plasmídeos , Inibidores de Proteínas Quinases/farmacologia , Transfecção , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia Blume has been used as a traditional Chinese herbal medicine for alleviation of fever, inflammation, chronic bronchitis, and to improve blood circulation. AIM OF THE STUDY: We addressed whether 2-methoxycinnamaldehyde (2-MCA), one of active ingredients of Cinnamomum cassia, reduces vascular cell adhesion molecule-1 (VCAM-1) expression in tumor necrosis factor-alpha (TNF-α)-activated endothelial cells and protects ischemia/reperfusion (I/R)-injury due to heme oxygenase (HO)-1 induction. MATERIALS AND METHODS: Adult male rats were subjected to 30 min of ischemia by occlusion of the left anterior descending coronary artery followed by 24h of reperfusion. Rats were randomized to receive vehicle or 2-MCA (i.v.) 10 min before reperfusion. RESULTS: Administration of 2-MCA significantly improved I/R-induced myocardial dysfunction by increasing the values of the first derivative (±dp/dt) of left ventricular pressure and decreased infarct size. In addition, 2-MCA reduced the expression of high mobility group box 1 (HMGB1), an activator of the inflammatory cascade when released into the extracellular space, and VCAM-1 in I/R myocardium along with increase of HO-1 induction. The reduced injury was accompanied by significantly reduction of neutrophils infiltration and increased SOD activity in ischemic tissues and reduced serum level of cardiac troponin I (cTnI). Furthermore, 2-MCA significantly increased HO-1 induction by translocation of Nrf-2 from cytosol to nucleus in endothelial cells. Inhibition of VCAM-1 expression by 2-MCA was reversed both by SnPPIX, a HO-1 inhibitor and siHO-1 RNA trasfection in TNF-α-activated cells. In addition, 2-MCA significantly inhibited NF-κB luciferase activity in TNF-α-activated endothelial cells. As expected, 2-MCA significantly inhibited monocyte (U937) adhesion to endothelial cells. CONCLUSION: We concluded that 2-MCA protects of myocardial I/R-injury due to antioxidant and anti-inflammatory action possibly by HO-1 induction which can be explained why Cinnamomum cassia has been used in inflammatory disorders.
Assuntos
Acroleína/análogos & derivados , Cardiotônicos/farmacologia , Cinnamomum aromaticum , Heme Oxigenase (Desciclizante)/biossíntese , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Extratos Vegetais/farmacologia , Acroleína/isolamento & purificação , Acroleína/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Cardiotônicos/isolamento & purificação , Cinnamomum aromaticum/química , Técnicas de Cocultura , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Proteína HMGB1/metabolismo , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1/biossíntese , Hemodinâmica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Masculino , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Fatores de Tempo , Transfecção , Troponina I/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células U937 , Molécula 1 de Adesão de Célula Vascular/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacosRESUMO
The ethanol extract of the flower of P. vulgaris var. lilacina (EEPV) has been used traditionally as an antiinflammatory agent in many countries. Inducers of heme oxygenase-1 (HO-1) reduce high mobility group box 1 (HMGB1), a late phase cytokine, in sepsis. Although EEPV has been used as an antiinflammatory agent, no report is available as to whether it modifies HMGB1 in sepsis due to HO-1 induction. It was found that EEPV increased HO-1 protein expression in RAW 264.7 cells, which was significantly inhibited by LY294002, but not PD98059, SB203580 or SP600125. In addition, EEPV activated NF-E2-related factor (Nrf2) to move from the cytosol to the nucleus, and EEPV-induced HO-1 and activation of ARE-luciferase activity were significantly reduced by siNrf2 transfection and LY294002 but not SB203508. EEPV also significantly inhibited NF-κB luciferase activity, and decreased both iNOS/NO and COX-2/PGE(2) production in lipopolysaccharide (LPS)-stimulated macrophages which was reversed by siHO-1 RNA transfection. Importantly, EEPV inhibited HMGB1 release in LPS-activated macrophages in a PI3K-sensitive manner and reduced serum HMGB1 level and lung HMGB1 expression in cecal ligation and puncture (CLP)-induced septic mice. It is concluded that EEPV induces HO-1 expression through PI3K/Nrf2 signal pathways, which may be beneficial for the treatment of sepsis due to a reduction of HMGB1 release.
Assuntos
Ceco/lesões , Proteína HMGB1/antagonistas & inibidores , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Extratos Vegetais/farmacologia , Prunella/química , Animais , Antracenos/farmacologia , Ceco/patologia , Linhagem Celular , Sobrevivência Celular , Cromonas/farmacologia , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Indução Enzimática , Etanol , Flavonoides/farmacologia , Flores/química , Vetores Genéticos , Proteína HMGB1/sangue , Heme Oxigenase-1/antagonistas & inibidores , Imidazóis/farmacologia , Lipopolissacarídeos/efeitos adversos , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Morfolinas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Transporte Proteico , Piridinas/farmacologia , Sepse/patologia , Transdução de Sinais , TransfecçãoRESUMO
Growing lines of evidence suggests that high mobility group box-1 (HMGB1) plays an important role for promoting inflammation and apoptosis in brain ischemia. Previously, we demonstrated that inducers of heme oxygenase-1 (HO-1) significantly reduce HMGB1 release in inflammatory conditions in vitro and in vivo. Thus, we tested our hypothesis that higenamine protects brain injury by inhibition of middle cerebral artery occlusion (MCAO)-mediated HMGB1 release in vivo, and glucose/glucose oxidase (GOX)-induced apoptosis in C6 cells in vitro due to HO-1 induction. Higenamine increased HO-1 expression in C6 cells in both hypoxia and normoxia, in which the former was much more significant than the latter. Higenamine increased Nrf-2 luciferase activity, translocated Nrf-2 to nucleus, and increased phosphorylation of Akt in C6 cells. Consistent with this, LY 294002, a PI3K inhibitor, inhibited HO-1 induction by higenamine and apoptosis induced by glucose/GOX in C6 cells was prevented by higenamine, which effect was reversed by LY 294002. Importantly, administration of higenamine (i.p) significantly reduced brain infarct size, mortality rate, MPO activity and tissue expression of HMGB1 in MCAO rats. In addition, recombinant high mobility group box 1 induced apoptosis in C6 cells by increasing ratio of Bax/bcl-2 and cleaved caspase c, which was inhibited by higenamine, and all of these effects were reversed by co-treatment with ZnPPIX. Therefore, we conclude that higenamine, at least in part, protects brain cells against hypoxic damages by up-regulation of HO-1. Thus, higenamine may be beneficial for the use of ischemic injuries such as stroke.
Assuntos
Alcaloides/farmacologia , Indução Enzimática/efeitos dos fármacos , Proteína HMGB1/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Transdução de Sinais , Tetra-Hidroisoquinolinas/farmacologia , Alcaloides/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Proteína HMGB1/farmacologia , Hipóxia-Isquemia Encefálica/enzimologia , Hipóxia-Isquemia Encefálica/patologia , Infarto da Artéria Cerebral Média , Masculino , Morfolinas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Tetra-Hidroisoquinolinas/uso terapêuticoRESUMO
Vascular inflammation process has been suggested to be an important risk factor in the development of atherosclerosis. Recently we reported that induction of peroxisome proliferator-activated receptor-γ (PPAR-γ) selectively inhibits vascular cell adhesion molecule-1 (VCAM-1) but not intercellular cell adhesion molecule-1 (ICAM-1) in tumor necrosis factor (TNF)-α-activated human umbilical vein endothelial cells (HUVEC). In this study, we investigated whether genipin inhibits expression of cellular adhesion molecules, which is relevant to inflammation. Pretreatment with genipin reduced reactive oxygen species (ROS) production and expression of VCAM-1, but not ICAM-1 in TNF-α-activated HUVEC. Genipin dose- and time-dependently increased PPAR-γ expression and inhibited TNF-α-induced phosphorylation of Akt and PKC with different degrees. Finally, genipin prevented TNF-α-induced adhesion of U937 monocytic cells to HUVEC. Taken together, these results indicate that upregualtion of PPAR-γ by genipin selectively inhibits TNF-α-induced expression of VCAM-1, in which regulation of Akt and/or PKC play a key role. We concluded that genipin can be used for the treatment of cardiovascular disorders such as atherosclerosis.
RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The rhizomes of Cyperus rotundus have been used as traditional folk medicine for the treatment of inflammatory diseases. However, the mechanism by which extract of rhizomes of Cyperus rotundus (ECR) elicits anti-inflammation has not been extensively investigated so far. The aim of the present study was to test whether heme oxygenase (HO)-1 induction is involved in the anti-inflammatory action of ECR. MATERIALS AND METHODS: Induction of HO-1 and inhibition of inducible nitric oxide synthase (iNOS)/NO production by ECR and its 12 constituents (3 monoterpenes, 5 sesquiterpenes, and 4 aromatic compounds) were investigated using RAW264.7 cells in vitro. In addition, anti-inflammatory action of ECR and its two active ingredients (nookkatone, valencene) were confirmed in sepsis animal model in vivo. RESULTS: ECR increased HO-1 expression in a concentration-dependent manner, which was correlated with significant inhibition of iNOS/NO production in LPS-activated RAW264.7 cells. Among 12 compounds isolated from ECR, mostly sesquiterpenes induced stronger HO-1 expression than monoterpenes in macrophage cells. Nootkatone and valencene (sesquiterpenes) significantly inhibited iNOS expression and NO production in LPS-simulated RAW264.7 cells. Inhibition of iNOS expression by nootkatone, valencene, and ECR were significantly reduced in siHO-1 RNA transfected cells. Furthermore, all three showed marked inhibition of high mobility group box-1 (HMGB1) in LPS-activated macrophages and increased survival rates in cecal ligation and puncture (CLP)-induced sepsis in mice. CONCLUSIONS: Taken together, we concluded that possible anti-inflammatory mechanism of ECR is, at least, due to HO-1 induction, in which sesquiterpenes such as nootkatone and valencene play a crucial role.
Assuntos
Anti-Inflamatórios/farmacologia , Cyperus , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Extratos Vegetais/farmacologia , Sepse/tratamento farmacológico , Sesquiterpenos/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Linhagem Celular , Cyperus/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Proteína HMGB1/metabolismo , Heme Oxigenase-1/genética , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Sesquiterpenos Policíclicos , Interferência de RNA , Rizoma , Sepse/enzimologia , Sesquiterpenos/isolamento & purificação , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
Apoptosis is a highly conserved genetic process leading to death in mammalian cells. A critical step in apoptosis is mitochondrial membrane permeabilization, which results in the release of proteins critical to downstream events. Transmembrane protein 14A (TMEM14A) was identified as a novel suppressor of Bax using yeast-based functional screening. TMEM14A is a novel mitochondria-associated membrane protein containing a putative transmembrane domain. Over-expression of TMEM14A in U87MG cells inhibited N-(4-hydroxyphenyl)retinamide (4-HPR)-induced apoptosis. TMEM14A prevented 4-HPR-induced loss of mitochondrial membrane potential (MMP), the release of cytochrome c, and the activation of caspase-3, but not the generation of reactive oxygen species, suggesting that TMEM14A regulates mitochondrial membrane potential in a ROS-independent manner. As expected, cyclosporin A, an inhibitor of membrane potential transition, inhibited 4-HPR-induced loss of MMP and apoptosis in U87MG cells, indicating that loss of MMP plays a pivotal role in 4-HPR-induced apoptosis. Suppression of TMEM14A expression using shRNA significantly increased apoptosis and MMP loss in untreated and 4-HPR-treated cells. These findings show for the first time that TMEM14A inhibits apoptosis by blocking the mitochondrial permeability transition and stabilizing mitochondrial membrane potential.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Fenretinida/farmacologia , Potencial da Membrana Mitocondrial , Proteínas de Membrana/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Ciclosporina/metabolismo , Citocromos c/biossíntese , Inibidores Enzimáticos , Fenretinida/metabolismo , Citometria de Fluxo , Glioblastoma , Humanos , Immunoblotting , Mitocôndrias/fisiologia , Interferência de RNA , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/metabolismoRESUMO
High mobility group box (HMGB)-1 plays an important role in sepsis-associated death in experimental studies. Heme oxygenase-1 (HO-1) inducers were reported to reduce HMGB1 release in experimental sepsis. Previously, we reported on the importance of the ß1-adrenergic receptor and protein kinase A pathway in the regulation of HO-1 expression by isoproterenol (ISO) in RAW 264.7 cells. We investigated whether ISO reduces HMGB1 release in LPS-activated RAW 264.7 cells and improves survival rate in septic mice due to HO-1 induction. ISO concentration-dependently increased HO-1 via Nrf-2 translocation and inhibited release of HMGB1 through the ß1-adrenergic receptor (ß1-AR) in LPS-activated RAW 264.7 cells. This conclusion was supported by the finding that dobutamine but not salbutamol increased HO-1 expression in both RAW 264.7 cells. ISO failed to inhibit HMGB1 release when HO-1 expression was suppressed by ZnPPIX, an HO-1 inhibitor in RAW 264.7 cells. ISO significantly inhibited phosphorylation of IκB-α and NF-κB-driven luciferase activity in LPS-activated RAW 264.7 cells. In addition, LY294002, a PI3K inhibitor, and SB203580, a p38 MAPK inhibitor, significantly inhibited not only HO-1 induction but also HMGB1 release by ISO. Importantly, ISO increased HO-1 protein expression in heart and lung tissues, reduced HMGB1 in plasma and increased survival rate in CLP-treated septic mice, which was significantly reversed by co-treatment with ZnPPIX. Taken together, we conclude that inhibition of HMGB1 release during sepsis via ß1-AR-mediated HO-1 induction is a novel mechanism for the beneficial effects of ISO in the treatment of sepsis.