RESUMO
Alpha-1 antitrypsin (AAT) has protective functions in animal islet transplantation models. While the therapeutic effect of AAT therapy is currently being tested in clinical trials, we investigated the mechanism of AAT protection in a clinically relevant marginal intrahepatic human islet transplantation model. In recipients receiving islets and AAT, 68.9% (20/29) reached normoglycemia, compared to 35.7% (10/28) in those receiving islets only, at 60 days posttransplant (PT). AAT-treated mice had lower serum levels of inflammatory cytokines immediately PT. Reduced M1 macrophages were observed in livers of AAT-treated recipients compared to controls as evidenced by flow cytometry and RNA-seq transcriptional profiling analysis. In vitro AAT suppressed IFN-γ-induced M1 macrophage activation/polarization via suppression of STAT1 phosphorylation and iNOS production. AAT inhibits macrophage activation induced by cytokines or dying islets, and consequently leads to islet cell survival. In a macrophage depletion mouse model, the presence of M1 macrophages in the liver contributed to graft death. AAT, through suppressing macrophage activation, protected transplanted islets from death and dysfunction in the human islet and NOD-SCID mouse model. The protective effect of AAT was confirmed in a major mismatch allogeneic islet transplantation model. Taken together, AAT suppresses liver macrophage activation that contributes to graft survival after transplantation.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Sobrevivência de Enxerto , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , alfa 1-AntitripsinaRESUMO
Macrophage polarization contributes to obesity-induced insulin resistance. Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone specialized for folding and quality control of secreted and membrane proteins. To determine the role of GRP94 in macrophage polarization and insulin resistance, macrophage-specific GRP94 conditional knockout (KO) mice were challenged with a high-fat diet (HFD). Glucose tolerance, insulin sensitivity, and macrophage composition were compared with control mice. KO mice showed better glucose tolerance and increased insulin sensitivity. Adipose tissues from HFD-KO mice contained lower numbers of M1 macrophages, with lower expression of M1 macrophage markers, than wild-type (WT) mice. In vitro, WT adipocytes cocultured with KO macrophages retained insulin sensitivity, whereas those cultured with WT macrophages did not. In addition, compared with WT bone marrow-derived macrophages (BMDMs), BMDMs from GRP94 KO mice exhibited lower expression of M1 macrophage marker genes following stimulation with LPS or IFN-γ, and exhibited partially increased expression of M2 macrophage marker genes following stimulation with interleukin-4. These findings identify GRP94 as a novel regulator of M1 macrophage polarization and insulin resistance and inflammation.
Assuntos
Dieta Hiperlipídica , Resistência à Insulina/genética , Ativação de Macrófagos/genética , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Obesidade/imunologia , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Técnicas de Cocultura , Citocinas/genética , Citocinas/imunologia , Teste de Tolerância a Glucose , Inflamação/genética , Inflamação/imunologia , Resistência à Insulina/imunologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , RNA Mensageiro/metabolismoRESUMO
Browning of white adipose tissue (WAT) has been shown to reduce obesity and obesity-related complications, suggesting that factors that promote WAT browning may have applications in the development of therapeutic strategies for treating obesity. Here, we show that ablation of spinophilin (SPL), a ubiquitously expressed, multidomain scaffolding protein, increases metabolism and improves energy balance. Male and female SPL knockout (KO) and wild-type (WT) littermate controls were fed a chow diet or a high-fat diet (HFD). Body weight, hepatic steatosis, glucose and insulin tolerance, physical activity, and expression of browning genes in adipose tissues were measured and compared. Male SPL knockout (KO) mice fed a chow diet were significantly leaner, had lower body weights, and exhibited better glucose tolerance and insulin sensitivity than wild-type (WT) littermate controls. When fed an HFD, SPL KO mice were protected from increased body fat, weight gain, hepatic steatosis, hyperinsulinemia, and insulin resistance. Physical activity of SPL KO mice was markedly increased compared with WT controls. Furthermore, expression of the brown adipocyte marker, uncoupling protein-1 (UCP-1), and the mitochondrial activity markers, cd137 and c-idea, were significantly increased in visceral WAT (vWAT) of SPL KO mice, suggesting that SPL knockout protected the mice from HFD-induced obesity and its metabolic complications, at least in part, by promoting the browning of white adipocytes in vWAT. Our data identify a critical role of SPL in regulating glucose homeostasis, obesity, and adipocyte browning. These results suggest SPL may serve as a drug target for obesity and diabetes.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina/fisiologia , Proteínas dos Microfilamentos/deficiência , Proteínas do Tecido Nervoso/deficiência , Obesidade/prevenção & controle , Adiponectina/sangue , Tecido Adiposo Marrom/fisiopatologia , Tecido Adiposo Branco/fisiopatologia , Animais , Metabolismo Energético , Fígado Gorduroso/fisiopatologia , Fígado Gorduroso/prevenção & controle , Feminino , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Obesidade/etiologia , Obesidade/fisiopatologia , Esforço Físico/fisiologiaRESUMO
The objective of this study was to assess the capacity of adipose-derived mesenchymal stem cells (ASCs) to mitigate disease progression in an experimental chronic pancreatitis mouse model. Chronic pancreatitis (CP) was induced in C57BL/6 mice by repeated ethanol and cerulein injection, and mice were then infused with 4 × 105 or 1 × 106 GFP+ ASCs. Pancreas morphology, fibrosis, inflammation, and presence of GFP+ ASCs in pancreases were assessed 2 weeks after treatment. We found that ASC infusion attenuated pancreatic damage, preserved pancreas morphology, and reduced pancreatic fibrosis and cell death. GFP+ ASCs migrated to pancreas and differentiated into amylase+ cells. In further confirmation of the plasticity of ASCs, ASCs co-cultured with acinar cells in a Transwell system differentiated into amylase+ cells with increased expression of acinar cell-specific genes including amylase and chymoB1. Furthermore, culture of acinar or pancreatic stellate cell lines in ASC-conditioned medium attenuated ethanol and cerulein-induced pro-inflammatory cytokine production in vitro. Our data show that a single intravenous injection of ASCs ameliorated CP progression, likely by directly differentiating into acinar-like cells and by suppressing inflammation, fibrosis, and pancreatic tissue damage. These results suggest that ASC cell therapy has the potential to be a valuable treatment for patients with pancreatitis.
Assuntos
Tecido Adiposo/citologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Pancreatite Crônica/terapia , Células Acinares/citologia , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Tecido Adiposo/metabolismo , Amilases/genética , Amilases/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Ceruletídeo/administração & dosagem , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Etanol/administração & dosagem , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/genética , Pancreatite Crônica/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
High workload-induced cellular stress can cause pancreatic islet ß cell death and dysfunction, or ß cell failure, a hallmark of type 2 diabetes mellitus. Thus, activation of molecular chaperones and other stress-response genes prevents ß cell failure. To this end, we have shown that deletion of the glucose-regulated protein 94 (GRP94) in Pdx1+ pancreatic progenitor cells led to pancreas hypoplasia and reduced ß cell mass during pancreas development in mice. Here, we show that GRP94 was involved in ß cell adaption and compensation (or failure) in islets from leptin receptor-deficient (db/db) mice in an age-dependent manner. GRP94-deficient cells were more susceptible to cell death induced by various diabetogenic stress conditions. We also identified a new client of GRP94, insulin-like growth factor-1 receptor (IGF-1R), a critical factor for ß cell survival and function that may mediate the effect of GRP94 in the pathogenesis of diabetes. This study has identified essential functions of GRP94 in ß cell failure related to diabetes.
Assuntos
Células Secretoras de Insulina , Receptor IGF Tipo 1 , Animais , Camundongos , Morte Celular , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 1/genética , Receptores para Leptina/metabolismo , Receptores para Leptina/genéticaRESUMO
Onset of the mitochondrial permeability transition (MPT) plays a causative role in ischemia/reperfusion (I/R) injury. Current therapeutic strategies for reducing reperfusion injury remain disappointing. Autophagy is a lysosome-mediated, catabolic process that timely eliminates abnormal or damaged cellular constituents and organelles such as dysfunctional mitochondria. I/R induces calcium overloading and calpain activation, leading to degradation of key autophagy-related proteins (Atg). Carbamazepine (CBZ), an FDA-approved anticonvulsant drug, has recently been reported to increase autophagy. We investigated the effects of CBZ on hepatic I/R injury. Hepatocytes and livers from male C57BL/6 mice were subjected to simulated in vitro, as well as in vivo I/R, respectively. Cell death, intracellular calcium, calpain activity, changes in autophagy-related proteins (Atg), autophagic flux, MPT and mitochondrial membrane potential after I/R were analyzed in the presence and absence of 20 µM CBZ. CBZ significantly increased hepatocyte viability after reperfusion. Confocal microscopy revealed that CBZ prevented calcium overloading, the onset of the MPT and mitochondrial depolarization. Immunoblotting and fluorometric analysis showed that CBZ blocked calpain activation, depletion of Atg7 and Beclin-1 and loss of autophagic flux after reperfusion. Intravital multiphoton imaging of anesthetized mice demonstrated that CBZ substantially reversed autophagic defects and mitochondrial dysfunction after I/R in vivo. In conclusion, CBZ prevents calcium overloading and calpain activation, which, in turn, suppresses Atg7 and Beclin-1 depletion, defective autophagy, onset of the MPT and cell death after I/R.
Assuntos
Autofagia/efeitos dos fármacos , Calpaína/efeitos adversos , Carbamazepina/farmacologia , Fígado/efeitos dos fármacos , Animais , Anticonvulsivantes/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Cálcio/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/metabolismo , Lisossomos/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Traumatismo por Reperfusão/tratamento farmacológicoRESUMO
Activating brown adipose tissue (BAT) and stimulating white adipose tissue (WAT) browning is a prospective obesity treatment method. Dietary components derived from plants are the most effective approach to activate BAT and promote WAT browning in rodents. This study investigated the synergistic effects of Panax ginseng (PG) and Diospyros kaki leaf (DKL) extract on adipocyte differentiation and browning, as well as the molecular mechanism underlying their beneficial effects. The administration of PG and DKL to HFD-induced obese mice significantly decreased body weight and epididymal and abdominal adipose tissue mass. In in vitro, PG inhibited the adipogenesis of 3T3-L1 adipocytes by regulating the expression of key adipogenic regulators, such as peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein (C/EBP)-α. In contrast, DKL negligibly influenced the adipogenesis of 3T3-L1 adipocytes but greatly increased the protein expression of UCP-1, PGC-1α, and PPARα in BAT and/or WAT. Moreover, PG and DKL inhibited adipogenesis synergistically and activated white adipocyte browning via AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) pathways. These results suggest that a combination of PG and DKL regulates adipogenesis in white adipocytes and browning in brown adipocytes by activating AMPK/SIRT1 axis. The potential use of PG and DKL may represent an important strategy in obesity management that will be safer and more effective.
Assuntos
Diospyros , Panax , Camundongos , Animais , Adipócitos Brancos , Proteínas Quinases Ativadas por AMP/metabolismo , Panax/química , Sirtuína 1/metabolismo , Estudos Prospectivos , Adipogenia , PPAR gama/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Folhas de Planta/metabolismo , Células 3T3-L1RESUMO
Mori Folium (Morus alba leaf, MF) and Mori Cortex Radicis (Morus alba root cortex, MR) have been studied for their anti-obesity effects by enhancing the browning process and inhibiting adipogenesis. However, important aspects of their protective mechanisms have not been thoroughly investigated, which could aid in developing functional food. Thus, this study aims to determine the synergistic effects of MF and MR against obesity and its associated mechanisms. In an in vitro cell culture model of brown adipocytes, a 1:1 mixture of MF and MR showed a synergistic effect on the expression of brown adipocyte-specific genes, including Ucp-1, Ppargc1a, Cbp/p300-interacting transactivator (Cited), Prdm16, Tbx1, and Fgf21 compared with either MF- or MR-treated conditions. Moreover, they demonstrated the involvement of cAMP and Ca2+ in induction of brown adipocyte-specific genes. In an in vivo model using HFD-fed mice, MF/MR significantly inhibited weight gain, plasma cholesterol, LDL, TG content, fat mass, and adipocyte size. Furthermore, MF/MR inhibited morphological alteration and the expressions of fatty acid synthesis genes such as Srebp1 and Fasn in the white adipose tissue. Thermogenesis genes were recovered in the brown adipose tissue with MF/MR supplementation, indicating that MF/MR regulated adipocytic dysmetabolism where AMPK signaling is involved. In conclusion, these results suggested that MF/MR regulates brown and beige adipocyte processes, providing one of the preventive functional food/herbal medicines against obesity and its associated metabolic diseases.
Assuntos
Adipócitos Marrons , Obesidade , Animais , Camundongos , Obesidade/genética , Aumento de Peso , Tecido Adiposo MarromRESUMO
Stress that impairs endoplasmic reticulum (ER) function leads to an accumulation of unfolded or misfolded proteins in the ER (ER stress) and triggers the unfolded protein response (UPR). Recent studies suggest that ER stress is involved in idiopathic pulmonary fibrosis (IPF). The present study was undertaken to determine the role of ER stress on myofibroblastic differentiation of fibroblasts. Fibroblasts in fibroblastic foci of IPF showed immunoreactivity for GRP78. To determine the role of ER stress on α-smooth muscle actin (α-SMA) and collagen type I expression in fibroblasts, mouse and human lung fibroblasts were treated with TGF-ß1, and expression of ER stress-related proteins, α-SMA, and collagen type I was analyzed by Western blotting. TGF-ß1 significantly increased expression of GRP78, XBP-1, and ATF6α, which was accompanied by increases in α-SMA and collagen type I expression in mouse and human fibroblasts. A chemical chaperone, 4-PBA, suppressed TGF-ß1-induced UPR and α-SMA and collagen type I induction. We also showed that TGF-ß1-induced UPR was mediated through the reactive oxygen species generation. Our study provides the first evidence implicating the UPR in myofibroblastic differentiation during fibrosis. These findings of the role of ER stress and chemical chaperones in pulmonary fibrosis may improve our understanding of the pathogenesis of IPF.
Assuntos
Diferenciação Celular , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Pulmão/metabolismo , Animais , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse OxidativoRESUMO
Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H(+)-ATPase(V-ATPase), a proton pump, was activated, suggesting high H(+) uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Morte Celular/fisiologia , Linhagem Celular Tumoral , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
OBJECTIVES: Gastrodia elata (GE) Blume (Orchidaceae) has been previously known for its therapeutic benefits against neurodegenerative diseases. Microglial activation and death have been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease. In this study, GE and its pure components, gastrodin and 4-hydroxybenzyl alcohol (4HBA), were applied to ß-amyloid-induced BV2 mouse microglial cells. MATERIALS AND METHODS: Cell viability was assessed by the MTT assay and Western blotting was also performed. RESULTS: ß-amyloid-induced cell death was shown to be induced time- and dose-dependently. To examine the cell death mechanism, we confirmed the involvement of ER stress signaling. C/EBP homologous protein (CHOP), a pro-apoptotic ER stress protein, was expressed at high levels but glucose-regulated protein 78 (GRP78), an anti-apoptotic ER stress protein with chaperone activity, was only slightly affected by treatment with ß-amyloid. However, pretreatment with GE and its components inhibited the expression of CHOP but increased that of GRP78 in ß-amyloid-treated cells. This study also showed that a single treatment with GE extracts, gastrodin, or 4HBA induced the expression of GRP78, a marker for enhanced protein folding machinery, suggesting a protective mechanism for GE against ß-amyloid. CONCLUSIONS: This study reveals the protective effects of GE against ß-amyloid-induced cell death, possibly through the enhancement of protein folding machinery of a representative protein, GRP78, and the regulation of CHOP in BV2 mouse microglial cells.
Assuntos
Amiloide/farmacologia , Álcoois Benzílicos/farmacologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Gastrodia/química , Glucosídeos/farmacologia , Microglia/efeitos dos fármacos , Animais , Álcoois Benzílicos/isolamento & purificação , Chaperona BiP do Retículo Endoplasmático , Glucosídeos/isolamento & purificação , CamundongosRESUMO
Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER-mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory-like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. SIGNIFICANCE: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.
Assuntos
Monóxido de Carbono , eIF-2 Quinase , Apoptose , Autofagia , Monóxido de Carbono/farmacologia , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Linfócitos T/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
BACKGROUND: Salvia miltiorrhiza (SM) has long been used as a traditional oriental medicine for cardiovascular disease. Accumulating evidence also indicates that SM has anti-osteoporotic effects. This study was conducted to examine the SM-induced anti-osteoporotic effect and its possible mechanisms with various doses of SM. METHODS: We studied Sprague-Dawley female rats aged 12 weeks, divided into six groups: sham-operated control (SHAM), OVX rats supplemented with SM (1, 3, 10 and 30 mg/kg) orally for 8 weeks. At the end of the experiment, blood samples were collected and biochemistry analysis was performed. Specimens from both tibia and liver were processed for light microscopic examination. DEXA and µ-CT analyses of the tibia were also performed. RESULTS: SM treatment significantly ameliorated the decrease in BMD and trabecular bone mass according to DEXA and trabecular bone architecture analysis of trabecular bone structural parameters by µ-CT scanning. In serum biochemical analysis, SM decreased the released TRAP-5b, an osteoclast activation marker and oxidative stress parameters including MDA and NO induced by OVX. CONCLUSIONS: The preventive effect of SM was presumably due to its anti-oxidative stress partly via modulation of osteoclast maturation and number. In current study, SM appears to be a promising osteoporosis therapeutic natural product.
Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Densidade Óssea/efeitos dos fármacos , Osteoporose Pós-Menopausa/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/uso terapêutico , Salvia miltiorrhiza , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Conservadores da Densidade Óssea/farmacologia , Feminino , Humanos , Malondialdeído/sangue , Óxido Nítrico/sangue , Osteoclastos/efeitos dos fármacos , Osteoporose Pós-Menopausa/diagnóstico por imagem , Osteoporose Pós-Menopausa/prevenção & controle , Ovariectomia , Extratos Vegetais/farmacologia , Radiografia , Ratos , Ratos Sprague-Dawley , Tíbia/anatomia & histologia , Tíbia/efeitos dos fármacos , Tíbia/metabolismoRESUMO
Reactive oxygen species are important mediators that exert a toxic effect during ischemia-reperfusion (I/R) injury of various organs. Sulforaphane is known to be an indirect antioxidant that acts by inducing Nrf2-dependent phase 2 enzymes. In this study, we investigated whether sulforaphane protects heart against I/R injury. Sprague-Dawley rats received sulforaphane (500microg/kg/day) or vehicle intraperitoneally for 3 days and global ischemia was performed using isolated perfused Langendorff hearts. Hearts were perfused with Krebs-bicarbonate buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Treatment with sulforaphane inhibited an increase in the post-ischemic left ventricular end-diastolic pressure (LVEDP) and improved the post-ischemic left ventricular developed pressure (LVDP), +/-dP/dt, and coronary flow as compared with the untreated control hearts. Pretreatment with 5-hydroxydecanoic acid (5-HD), a mitochondrial K(ATP) channel blocker, for 10min before ischemia attenuated the improvement of LVEDP, LVDP, +/-dP/dt, and coronary flow induced by sulforaphane. Sulforaphane markedly decreased the infarcted size and attenuated the increased lactate dehydrogenase level in effluent during reperfusion. Pretreatment with 5-HD also blocked these protective effects of sulforaphane. Post-ischemia increased the concentration of atrial natriuretic peptide in coronary effluent, which attenuated by sulforaphane treatment. Decreases on Mn-superoxide dismutase (SOD), catalase, and heme oxygenase-1 levels by I/R were increased by sulforaphane treatment and pretreatment of 5-HD blocked the sulforaphane effects. Increases in Bax and caspase-3 levels, and decrease in Bcl-2 level by I/R were attenuated by sulforaphane treatment. These results suggest that the protective effects of sulforaphane against I/R injury may be partly mediated through mitochondrial K(ATP) channels and antioxidant pathway.
Assuntos
Antioxidantes/uso terapêutico , Canais KATP/metabolismo , Isquemia Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle , Tiocianatos/uso terapêutico , Animais , Antioxidantes/farmacologia , Fator Natriurético Atrial/metabolismo , Caspase 3/metabolismo , Ácidos Decanoicos/farmacologia , Modelos Animais de Doenças , Interações Medicamentosas , Sequestradores de Radicais Livres/metabolismo , Hidroxiácidos/farmacologia , Técnicas In Vitro , Isotiocianatos , L-Lactato Desidrogenase/metabolismo , Masculino , Mitocôndrias/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Isquemia Miocárdica/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Sulfóxidos , Tiocianatos/antagonistas & inibidores , Tiocianatos/farmacologia , Proteína X Associada a bcl-2/metabolismoRESUMO
Inflammation or hypoxia in gingival tissue can induce endoplasmic reticulum (ER) stress, which is related with autophagy. The autophagy is a catabolic process involving the degradation of a cell's own components. Although autophagy resulting in the total destruction of the cell is one of cell death types, no conclusive evidence exists for such a process. In order to examine the association of ER stress and autophagy in gingival system, ER stress agents brefeldin A, thapsigargin, and tunicamycin were exposed to human gingival cells. The ER stress agents induced cell death and the expression of ER stress proteins, glucose-regulated protein of 78 kDa (GRP78) and CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP). ER stress also increased the formation of autophagic vesicles with the expression of beclin and LC-3 (microtubule-associated protein1 light chain 3) II, two autophagic markers. ER stress induced the phosphorylation of p38MAPK (mitogen-activated protein kinase), and the p38MPAK inhibitor, SB203580, inhibited the resulting cell death and autophagy. In summary, ER stress induces cell death and autophagy through p38MAPK in human gingival cells.
Assuntos
Autofagia , Morte Celular , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Gengiva/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Autofagia/efeitos dos fármacos , Brefeldina A/farmacologia , Morte Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Inibidores Enzimáticos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Humanos , Imidazóis , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Inibidores da Síntese de Proteínas/farmacologia , Piridinas , Tapsigargina/farmacologia , Fator de Transcrição CHOP/metabolismo , Tunicamicina/farmacologiaRESUMO
OBJECTIVE: To investigate the differences between the effects of complex decongestive physiotherapy with and without active resistive exercise for the treatment of patients with breast cancer-related lymphedema (BCRL). DESIGN: Randomized control-group study. SETTING: An outpatient rehabilitation clinic. PARTICIPANTS: Patients (N=40) with diagnosed BCRL. INTERVENTIONS: Patients were randomly assigned to either the active resistive exercise group or the nonactive resistive exercise group. In the active resistive exercise group, after complex decongestive physiotherapy, active resistive exercise was performed for 15min/d, 5 days a week for 8 weeks. The nonactive resistive exercise group performed only complex decongestive physiotherapy. MAIN OUTCOME MEASURES: The circumferences of the upper limbs (proximal, distal, and total) for the volume changes, and the Short Form-36 version 2 questionnaire for the quality of life (QOL) at pretreatment and 8 weeks posttreatment for each patient. RESULTS: The volume of the proximal part of the arm was significantly more reduced in the active resistive exercise group than that of the nonactive resistive exercise group (P<.05). In the active resistive exercise group, there was significantly more improvement in physical health and general health, as compared with that of the nonactive resistive exercise group (P<.05). CONCLUSIONS: For the treatment of patients with BCRL, active resistive exercise with complex decongestive physiotherapy did not cause additional swelling, and it significantly reduced proximal arm volume and helped improve QOL.
Assuntos
Neoplasias da Mama/complicações , Terapia por Exercício/métodos , Linfedema/etiologia , Linfedema/terapia , Modalidades de Fisioterapia , Adulto , Idoso , Análise de Variância , Neoplasias da Mama/terapia , Distribuição de Qui-Quadrado , Feminino , Humanos , Pessoa de Meia-Idade , Qualidade de Vida , Resultado do TratamentoRESUMO
The excessive supply of fatty acids to the liver contributes to hepatic insulin resistance and endoplasmic reticulum (ER) stress associated with obesity or type 2 diabetes mellitus. Furthermore, excess and/or prolonged ER stress contributes to hepatic cell death deteriorating nonalcoholic fatty liver disease to steatohepatitis. The aim of this study was to investigate the effects of metformin on palmitate-induced ER stress and hepatic insulin resistance in HepG2 cells. Metformin significantly inhibited palmitate-induced cell death and apoptosis via caspase-3 activation. Metformin also blocked the induction of ER stress proteins (GRP78, Chop, Cleaved ATF-6, p-eIF2 alpha and XBP-1) and regulated serine phosphorylation of IRS-1. Metformin may therefore protect hepatocytes from death induced by saturated fatty acids. These data may also provide a further rationale for exploring the use of metformin in the treatment of non-alcoholic fatty liver disease, revealing its blocking effect for hepatic insulin resistance evoked by saturated fatty acids.
Assuntos
Apoptose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Palmítico/efeitos adversos , Western Blotting , Caspase 3/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/biossíntese , Células Hep G2 , Humanos , Imunoprecipitação , Insulina/biossíntese , Resistência à Insulina , Fígado/efeitos dos fármacos , Fígado/metabolismo , Microscopia de Fluorescência , FosforilaçãoRESUMO
Adriamycin is a potent antitumor drug that causes severe cardiotoxicity. However, the toxic mechanisms are not clear. We used a proteomics approach to analyze changes in protein profiles after adriamycin-induced changes in hemodynamic factors. Although adriamycin itself did not affect left ventricular developed pressure (LVDP) or left ventricular end diastolic pressure (LVEDP), the drug did enhance susceptibility to ischemia-reperfusion-induced changes in LVDP, LVEDP and heart rate. Adriamycin altered the expression of 52 proteins, primarily energy metabolism and cytoskeleton proteins. Adriamycin decreased the expression of the metabolism-related proteins, ATP synthase, Sdha protein, Triose phosphate isomerase 1 (TPI-1), pyruvate dehydrogenase E1 alpha1, 6-phosphofructokinase, and fructose-1,6-bisphosphatase, as did cytoskeletal proteins, such as actin. Alterations in energy metabolism and subsequent free radical production may affect cytoskeletal protein expression, producing adriamycin-induced changes in cardiac hemodynamics.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Doxorrubicina/efeitos adversos , Coração/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/induzido quimicamente , Proteínas/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Citoesqueleto/efeitos dos fármacos , Doxorrubicina/farmacologia , Metabolismo Energético/efeitos dos fármacos , Coração/fisiopatologia , Masculino , Camundongos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Proteínas/análise , Proteômica , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
The clinical utility of anthracycline anticancer agents, especially doxorubicin (DOX), is limited by progressive toxic cardiomyopathy linked to cardiomyocyte apoptosis. This study examined the protective effects of CO and bilirubin on DOX-induced cardiomyocyte toxicity. In vitro, DOX significantly decreased the viability of H9c2 cells and increased apoptotic features, such as changes in nuclear morphology and caspase protease activation. CO and bilirubin significantly inhibited DOX-induced cell death and caspase-3 activation, which may be explained by increased Bcl-2 expression and inhibition of Bax expression. CO and bilirubin up-regulated the heme oxygenase-1 (HO-1), which was required for the protective effect of CO, and a single bilirubin treatment increased DOX-induced apoptosis in H9c2 cells. The inhibition of HO-1 with ZnPP resulted in a striking increase in apoptosis in the CO, bilirubin, and DOX-treated cells. Furthermore, HO-1 overexpression increased resistance against DOX-induced cytotoxicity in H9c2 cells. In conclusion, CO and bilirubin can inhibit DOX-induced apoptosis in H9c2 cardiomyocytes. These findings imply that the therapeutic index of anthracycline cancer chemotherapeutics can be improved by protecting against cardiomyocyte death.
Assuntos
Apoptose/efeitos dos fármacos , Bilirrubina/farmacologia , Monóxido de Carbono/farmacologia , Doxorrubicina/antagonistas & inibidores , Doxorrubicina/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Protoporfirinas/farmacologia , RNA Interferente Pequeno/genética , Ratos , Transfecção , Proteína X Associada a bcl-2/metabolismoRESUMO
Cytokine-induced pancreatic ß cell death plays a pivotal role in both type 1 and type 2 diabetes. Our previous study showed that alpha-1 antitrypsin (AAT) inhibits ß cell death through the suppression of cytokine-induced c-Jun N-terminal kinase (JNK) activation in an islet transplantation model. The aim of this study was to further understand how AAT impacts ß cells by studying AAT endocytosis in human islets and a ßTC3 murine insulinoma cell line. Methods: In vitro, human islets and ßTC3 cells were stimulated with cytokines in the presence or absence of chlorpromazine (CPZ), a drug that disrupts clathrin-mediated endocytosis. Western blot, real-time PCR and cell death ELISA were performed to investigate ß cell death. The oxygen consumption rate (OCR) was measured on human islets. In vivo, islets were harvested from C57BL/6 donor mice treated with saline or human AAT and transplanted into the livers of syngeneic mice that had been rendered diabetic by streptozotocin (STZ). Islet graft survival and function were analyzed. Results: AAT was internalized by ß cells in a time- and dose-dependent manner. AAT internalization was mediated by clathrin as treatment with CPZ, profoundly decreased AAT internalization, cytokine-induced JNK activation and the downstream upregulation of c-Jun mRNA expression. Similarly, addition of CPZ attenuated cytokine-induced caspase 9 cleavage (c-casp 9) and DNA fragmentation, which was suppressed by AAT. Treatment of donor mice with AAT produced AAT internalization in islets, and resulted in a higher percentage of recipients reaching normoglycemia after syngeneic intraportal islet transplantation. Conclusion: Our results suggest that AAT is internalized by ß cells through clathrin-mediated endocytosis that leads to the suppression of caspase 9 activation. This process is required for the protective function of AAT in islets when challenged with proinflammatory cytokines or after islet transplantation.