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1.
Sheng Li Xue Bao ; 71(6): 846-854, 2019 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-31879740

RESUMO

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb (1, 2, 4 and 6 g/L) and control albumin (C-alb, 4 g/L) for 24 h, or preincubated with MCC950 (1 µmol/L) for 1 h and then treated with AGE-alb (4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase (LDH) activity and the levels of interleukin-1ß (IL-1ß) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL- or PI-positive cells in a concentration-dependent manner. Additionally, AGE-alb promoted IL-1ß and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950 (an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL- or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1ß and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.


Assuntos
Regulação da Expressão Gênica , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Albumina Sérica , Caspase 1 , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/genética , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Piroptose/efeitos dos fármacos , Albumina Sérica/farmacologia
2.
Molecules ; 24(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970533

RESUMO

Hepatocellular carcinoma (HCC) ranks fifth in occurrence and second in mortality of all cancers. The development of effective therapies for HCC is urgently needed. Anticancer drugs targeted to the liver-specific asialoglycoprotein receptors (ASGPRs) are viewed as a promising potential treatment for HCC. ASGPRs facilitate the recognition and endocytosis of molecules, and possibly vehicles with galactose end groups, by the liver. In this study, bovine serum albumin (BSA) was conjugated with lactose using a thermal treatment. The formation of lactosylated BSA (BSA-Lac) was confirmed by a change of the chemical structure, increased molecular mass, and Ricinus communis lectin recognition. Subsequently, the low-crosslinking BSA-Lac nanoparticles (LC BSA-Lac NPs) and high-crosslinking BSA-Lac nanoparticles (HC BSA-Lac NPs) were synthesized. These nanoparticles presented spherical shapes with a size distribution of 560 ± 18.0 nm and 539 ± 9.0 nm, as well as an estimated surface charge of -26 ± 0.15 mV and -24 ± 0.45 mV, respectively. Both BSA-Lac NPs were selectively recognized by ASGPRs as shown by biorecognition, competition, and inhibition assays using an in vitro model of HCC. This justifies pursuing the strategy of using BSA-Lac NPs as potential drug nanovehicles with selective direction toward hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/metabolismo , Portadores de Fármacos , Neoplasias Hepáticas/metabolismo , Nanopartículas , Soroalbumina Bovina , Albumina Sérica , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Nanopartículas/química , Nanopartículas/uso terapêutico , Albumina Sérica/química , Albumina Sérica/farmacocinética , Albumina Sérica/farmacologia , Soroalbumina Bovina/química , Soroalbumina Bovina/farmacocinética , Soroalbumina Bovina/farmacologia
3.
Blood ; 133(22): 2445-2451, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-30992271

RESUMO

Factor IX (FIX) binds to collagen IV (Col4) in the subendothelial basement membrane. In hemophilia B, this FIX-Col4 interaction reduces the plasma recovery of infused FIX and plays a role in hemostasis. Studies examining the recovery of infused BeneFix (FIXWT) in null (cross-reactive material negative, CRM-) hemophilia B mice suggest the concentration of Col4 readily available for binding FIX is ∼405 nM with a 95% confidence interval of 374 to 436 nM. Thus, the vascular cache of FIX bound to Col4 is several-fold the FIX level measured in plasma. In a mouse model of prophylactic therapy (testing hemostasis by saphenous vein bleeding 7 days after infusion of 150 IU/kg FIX), FIXWT and the increased half-life FIXs Alprolix (FIXFC) and Idelvion (FIXAlb) produce comparable hemostatic results in CRM- mice. In bleeding CRM- hemophilia B mice, the times to first clot at a saphenous vein injury site after the infusions of the FIX agents are significantly different, at FIXWT < FIXFC < FIXAlb Dysfunctional forms of FIX, however, circulate in the majority of patients with hemophilia B (CRM+). In the mouse prophylactic therapy model, none of the FIX products improves hemostasis in CRM+ mice expressing a dysfunctional FIX, FIXR333Q, that nevertheless competes with infused FIX for Col4 binding and potentially other processes involving FIX. The results in this mouse model of CRM+ hemophilia B demonstrate that the endogenous expression of a dysfunctional FIX can deleteriously affect the hemostatic response to prophylactic therapy.


Assuntos
Fator IX/farmacologia , Hemofilia B , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica/farmacologia , Animais , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Hemofilia B/sangue , Hemofilia B/tratamento farmacológico , Hemofilia B/genética , Camundongos , Camundongos Transgênicos
4.
Biomed Pharmacother ; 115: 108871, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31026729

RESUMO

This study sought to clarify the role and underlying mechanisms of human serum albumin (HSA) therapy in global cerebral ischemia/reperfusion (GCI/R)-induced brain damage in rats. Five groups of adult male Wistar rats (n = 12 per group) were created as follows: sham operation (Sham), global cerebral ischemia/reperfusion (GCI/R), HSA treatment (GCI/R + HSA), Dickkopf-1 (DDK1) treatment (GCI/R + DDK1), and DDK1 plus HSA treatment (GCI/R + DKK1 + HSA). The GCI/R injury model was created using the modified Pusinelli four-vessel occlusion method. After 24 h, rats were evaluated using neurological scoring, Nissl staining, and brain tissue water content. The mRNA expression of Wnt, GSK3ß, and ß-Catenin in the brain were detected by quantitative real time polymerase chain reaction. The protein expression of ß-Catenin and GSK-3ß were investigated by western blot and immunohistochemical analysis in the presence and absence of the Wnt/ß-Catenin antagonist, DKK-1. Complex I activity and ROS content were also measured. After 24 h of reperfusion, the behavior score and brain tissue water content in the GCI/R + HSA group were lower than that in the GCI/R group. In addition, the degree of neuronal injury was significantly reduced in the GCI/R + HSA group (P < 0.05). The ROS content was significantly decreased and Complex I activity was markedly raised in the GCI/R + HSA group compared to the GCI/R group (P < 0.05). Further, GSK-3ß expression in the GCI/R + HSA group was lower than that in the GCI/R group, while the Wnt and ß-catenin expression were increased. These effects were reversed by DKK1. Taken together, we showed that HSA attenuates GCI/R-induced brain damage and may be neuroprotective via regulation of the Wnt/ß-catenin/ROS signaling pathway.


Assuntos
Isquemia Encefálica/patologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Albumina Sérica/farmacologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo , Distribuição Aleatória , Ratos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Proteínas Wnt/genética , beta Catenina/genética
5.
Front Immunol ; 10: 12, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30733715

RESUMO

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5-10%), heat-inactivated (hiFCS, 0.1-10%) or human serum albumin (HSA, 0.05-2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.


Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Albumina Sérica/farmacologia , Soro , Animais , Biomarcadores , Ionóforos de Cálcio/farmacologia , Bovinos , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Humanos , Imuno-Histoquímica , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Ligação Proteica , Soro/metabolismo , Soroalbumina Bovina/metabolismo , Soroalbumina Bovina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia
6.
Sci Rep ; 8(1): 18012, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30573754

RESUMO

Podocyte injury is closely related to proteinuria and the progression of chronic kidney disease (CKD). Currently, there is no conclusive understanding about the mechanisms involved in albumin overload and podocyte apoptosis response. In this study, we sought to explore the ways by which intracellular albumin can mediate podocyte apoptosis. Here, immortalized mouse podocytes were treated with bovine serum albumin (BSA) at different times and concentrations, in the presence or absence of SB203580 (0.1 µM, inhibitor of mitogen-activated-protein kinase - p38MAPK). Using immunofluorescence images, flow cytometry and immunoblotting, we observed a time-dependent intracellular accumulation of fluorescent albumin-FITC-BSA, followed by concentration-and time-dependent effect of intracellular albumin overload on podocyte apoptosis, which was mediated by increased expression of the chaperone glucose-regulated-protein 78 (GRP 78) and phosphorylated inositol-requiring enzyme 1 alpha (pIRE1-α), as well as protein kinase C delta (PKC-δ), p38MAPK and cleaved caspase 12 expression. SB203580 prevented the cleavage of caspase 12 and the albumin-mediated podocyte apoptosis. These results suggest that intracellular albumin overload is associated with endoplasmic reticulum (ER) stress and upregulation of PKC-δ/p38MAPK/caspase 12 pathway, which may be a target for future therapeutic of albumin-induced podocyte apoptosis.


Assuntos
Albuminas/farmacologia , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Podócitos/fisiologia , Proteína Quinase C-delta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Albuminas/metabolismo , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Células Cultivadas , Citoplasma/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Podócitos/metabolismo , Albumina Sérica/metabolismo , Albumina Sérica/farmacologia , Soroalbumina Bovina/metabolismo , Soroalbumina Bovina/farmacologia
7.
Nanomedicine (Lond) ; 13(11): 1255-1265, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29949465

RESUMO

AIM: To explore the potential of albumin nanoparticles for oral drug delivery. METHODS: Sub-150 nm human serum albumin nanoparticles were fabricated via a desolvation technique. Nanoparticle cell uptake and epithelial translocation were tested in Caco-2 monolayers, while comparing with albumin solution. RESULTS: Data suggest epithelial transcytosis of albumin, applied in solution form, via neonatal Fc receptor. Cell uptake of albumin nanoparticles demonstrated behaviors indicating a different cell uptake pathway compared with albumin solution. Importantly, application of equivalent concentrations of albumin solution or nanoparticles resulted in higher epithelial transport capacity of the latter, suggesting improvement of intestinal delivery via nanoformulation. CONCLUSION: This study highlights for the first time that simply fabricated, nontoxic human serum albumin nanoparticles may find application in oral drug delivery.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Portadores de Fármacos/farmacologia , Nanopartículas/química , Albumina Sérica/farmacologia , Administração Oral , Transporte Biológico , Células CACO-2 , Neoplasias do Colo/patologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Humanos , Intestinos/química , Intestinos/efeitos dos fármacos , Tamanho da Partícula , Albumina Sérica/química , Albumina Sérica/farmacocinética , Propriedades de Superfície
8.
Theranostics ; 8(9): 2459-2476, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29721092

RESUMO

The inefficiency of recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based clinical regimens has been dominantly attributed to the short half-life of TRAIL. Affinity-controlled release using endogenous long-acting proteins, such as IgG and albumin, as carriers is extremely attractive for improving the pharmacokinetics of TRAIL. Up to now, it is unclear whether IgG-binding is efficient for affinity-controlled release of TRAIL. Methods: An IgG-binding affibody, IgBD, was genetically fused to the N-terminus of TRAIL to produce IgBD-TRAIL.The IgG-binding ability, cytotoxicity, serum half-life, and in vivo antitumor effect of IgBD-TRAIL were compared with that of TRAIL. In addition, an albumin-binding affibody, ABD, was fused to TRAIL to produce ABD-TRAIL. The cytototoxicity, serum half-life, and antitumor effect of IgBD-TRAIL and ABD-TRAIL were compared. Results: IgBD fusion endowed TRAIL with high affinity (nM) for IgG without interference with its cytotoxicity. The serum half-life of IgBD-TRAIL is 50-60 times longer than that of TRAIL and the tumor uptake of IgBD-TRAIL at 8-24 h post-injection was 4-7-fold that of TRAIL. In vivo antitumor effect of IgBD-TRAIL was at least 10 times greater than that of TRAIL. Owing to the high affinity (nM) for albumin, the serum half-life of ABD-TRAIL was 80-90 times greater than that of TRAIL. However, after binding to albumin, the cytotoxicity of ABD-TRAIL was reduced more than 10 times. In contrast, binding to IgG had little impact on the cytotoxicity of IgBD-TRAIL. Consequently, intravenously injected IgBD-TRAIL showed antitumor effects superior to those of ABD-TRAIL. Conclusions: Endogenous long-acting proteins, particularly IgG-based affinity-controlled release, prolonged the serum half-life as well as significantly enhanced the antitumor effect of TRAIL. IgBD-mediated endogenous IgG binding might be a novel approach for the affinity-controlled release of other protein drugs.


Assuntos
Antineoplásicos/farmacologia , Preparações de Ação Retardada/farmacologia , Imunoglobulina G/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Meia-Vida , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica/farmacologia
9.
Eur J Clin Invest ; 48 Suppl 2: e12949, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29734477

RESUMO

BACKGROUND: Chemokines play a critical role in orchestrating the distribution and trafficking of neutrophils in homeostasis and disease. RESULTS: The CXCR4/CXCL12 chemokine axis has been identified as a central regulator of these processes. CONCLUSION: In this review, we focus on the role of CXCR4/CXCL12 chemokine axis in regulating neutrophil release from the bone marrow and the trafficking of senescent neutrophils back to the bone marrow for clearance under homeostasis and disease. We also discuss the role of CXCR4 in fine-tuning neutrophil responses in the context of inflammation.


Assuntos
Homeostase/fisiologia , Neutrófilos/fisiologia , Receptores CXCR4/fisiologia , Animais , Benzilaminas/farmacologia , Medula Óssea/fisiologia , Sobrevivência Celular/fisiologia , Quimiocina CXCL12/genética , Quimiocina CXCL12/fisiologia , Proteína HMGB1/fisiologia , Fármacos Hematológicos/farmacologia , Compostos Heterocíclicos/farmacologia , Humanos , Imidazóis/farmacologia , Síndromes de Imunodeficiência/genética , Inflamação/fisiopatologia , Camundongos , Mutação/fisiologia , Neutrófilos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Albumina Sérica/farmacologia , Baço/fisiologia , Verrugas/genética
10.
PLoS One ; 13(5): e0197634, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29772022

RESUMO

Sulphonylurea drugs stimulate insulin secretion from pancreatic ß-cells primarily by inhibiting ATP sensitive potassium (KATP) channels in the ß-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare in vitro and in vivo data. We therefore measured the ability of gliclazide and glibenclamide to inhibit KATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of KATP channels at therapeutic concentrations in vivo. KATP currents from mouse pancreatic ß-cells and Xenopus oocytes were measured using the patch-clamp technique. Gliclazide and glibenclamide binding to human plasma were determined in spiked plasma samples using an ultrafiltration-mass spectrometry approach. Bovine serum albumin (60g/l) produced a mild, non-significant reduction of gliclazide block of KATP currents in pancreatic ß-cells and Xenopus oocytes. In contrast, glibenclamide inhibition of recombinant KATP channels was dramatically suppressed by albumin (predicted free drug concentration <0.1%). Insulin secretion was also reduced. Free concentrations of gliclazide and glibenclamide in the presence of human plasma measured in binding experiments were 15% and 0.05%, respectively. Our data suggest the free concentration of glibenclamide in plasma is too low to account for the drug's therapeutic effect. In contrast, the free gliclazide concentration in plasma is high enough to close KATP channels and stimulate insulin secretion.


Assuntos
Gliclazida/farmacologia , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Canais KATP/antagonistas & inibidores , Albumina Sérica/farmacologia , Animais , Bovinos , Células Cultivadas , Gliclazida/sangue , Gliclazida/metabolismo , Gliclazida/farmacocinética , Glibureto/sangue , Glibureto/metabolismo , Glibureto/farmacocinética , Humanos , Hipoglicemiantes/sangue , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacocinética , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Proteínas Recombinantes/metabolismo , Albumina Sérica/metabolismo , Soroalbumina Bovina/metabolismo , Soroalbumina Bovina/farmacologia , Xenopus laevis
11.
Shock ; 50(3): 366-372, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29049137

RESUMO

BACKGROUND: Dextran-70 is a more potent plasma volume expander than albumin but use has been hampered because of its antithrombotic properties. However, also albumin has antithrombotic properties and little is known about relative effects of these two colloids on coagulation in vivo when controlling for differences in efficacy as plasma volume expanders. AIM: Compare effects of dextran-70 and albumin on coagulation at a dose resulting in equal plasma volume expansion. METHODS: Guinea pigs were subjected to a 25 mL/kg hemorrhage during 20 min and randomized to resuscitation with either 6% dextran-70 at a dose of 15 mL/kg or 5% albumin at a dose of 25 mL/kg (n = 14 in each group) during 30 min starting 1 h of shock. Blood samples were collected at the completion of resuscitation and at 4 h. Plasma volume was measured using I-albumin and the effect on coagulation was evaluated using whole blood thrombelastography (TEG), measurement of plasma fibrinogen and von Willebrand factor (vWF) concentrations and vWF glycoprotein 1b (GP1b) A activity. RESULTS: Plasma volumes after resuscitation were similar in the groups at both time points. Dextran-70 resulted in a transient prolongation of TEG clot amplification time (K) at the completion of resuscitation compared with albumin. TEG clot initiation (R) and strength (MA) did not differ between the treatments at any of the time points. Albumin reduced vWF concentrations to a larger extent than dextran at both time points, whereas no difference in vWF GP1bA activity or in plasma fibrinogen and could be detected. CONCLUSION: In equipotent doses with regard to plasma volume expansion, dextran-70 transiently prolongs clot amplification time more than albumin whereas dextran-70 reduces plasma vWF concentrations less than albumin.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Dextranos/farmacologia , Hemorragia , Albumina Sérica/farmacologia , Animais , Modelos Animais de Doenças , Cobaias , Hemorragia/sangue , Hemorragia/tratamento farmacológico , Hemorragia/patologia
12.
Sheng Li Xue Bao ; 69(6): 767-774, 2017 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-29270592

RESUMO

The purpose of this study was to investigate whether activating transcription factor 6 (ATF6), a sensor to endoplasmic reticulum stress (ERS), would mediate advanced glycated albumin (AGE-alb)-induced macrophage apoptosis and to elucidate the possible molecular mechanisms. RAW264.7 macrophages were cultured in vitro and treated with AGE-alb (2, 4 and 6 g/L), normal control albumin or tunicamycin (TM, 4 mg/L) for 24 h. ATF6 small interfering RNA (siRNA) was transfected to RAW264.7 cells by Lipofectamine 2000. Cell viability and apoptosis were determined by MTT method and Annexin V-FITC/propidium iodide apoptosis detection kit, respectively. The activities of lactate dehydrogenase (LDH) in medium and caspase-3 in cells were measured by corresponding detection kits. ATF6 nuclear translocation was detected by Western blot and immunofluorescence cytochemistry. Protein and mRNA levels of C/EBP homologous protein (CHOP, a key-signaling component of ERS-induced apoptosis) were detected by Western blot and real-time fluorescence quantitative PCR, respectively. The results showed that similar to TM, AGE-alb increased the expression of CHOP at both the protein and mRNA levels in a concentration dependent manner. ATF6, as a factor that positively regulates CHOP expression, was activated by AGE-alb in a concentration dependent manner. siRNA-mediated knockdown of ATF6 significantly inhibited AGE-alb-induced macrophage injury, as indicated by the increased cell viability and the decreased LDH release, apoptosis and caspase-3 activation. Additionally, ATF6 siRNA attenuated AGE-alb-induced CHOP upregulation at both the protein and mRNA levels. These results suggest that ATF6 and its downstream molecule CHOP are involved in AGE-alb-induced macrophage apoptosis.


Assuntos
Fator 6 Ativador da Transcrição/fisiologia , Apoptose/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Albumina Sérica/farmacologia , Fator de Transcrição CHOP/fisiologia , Animais , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Transdução de Sinais/fisiologia
13.
Mutat Res ; 824: 42-51, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29150049

RESUMO

Glucose, in the presence of reactive oxygen species (ROS), acts as an as an oxidative agent and drives deleterious processes in Diabetes Mellitus. We have studied the mechanism and the toxicological effects of glucose-dependent glycoxidation reactions driven by copper and ROS, using a model peptide based on the exposed sequence of Human Serum Albumin (HSA) and containing a lysine residue susceptible to copper complexation. The main products of these reactions are Advanced Glycation End-products (AGEs). Carboxymethyl lysine and pyrraline condensed on the model peptide, generating a Modified Peptide (MP). These products were isolated, purified, and tested on cultured motor neuron cells. We observed DNA damage, enhancement of membrane roughness, and formation of domes. We evaluated nuclear abnormalities by the cytokinesis-blocked micronucleus assay and we measured cytostatic and cytotoxic effects, chromosomal breakage, nuclear abnormalities, and cell death. AGEs formed by glycoxidation caused large micronucleus aberrations, apoptosis, and large-scale nuclear abnormalities, even at low concentrations.


Assuntos
Cobre/química , Citotoxinas , Produtos Finais de Glicação Avançada , Neurônios Motores/metabolismo , Peptídeos , Espécies Reativas de Oxigênio/química , Albumina Sérica , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Quebra Cromossômica/efeitos dos fármacos , Citotoxinas/síntese química , Citotoxinas/química , Citotoxinas/farmacologia , Glucose/química , Produtos Finais de Glicação Avançada/síntese química , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/farmacologia , Humanos , Neurônios Motores/patologia , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Albumina Sérica/química , Albumina Sérica/farmacologia
14.
PLoS One ; 12(8): e0182997, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28800610

RESUMO

Albumin is the most abundant plasma protein. Critical illness is often associated with altered, predominately decreased, serum albumin levels. This hypoalbuminaemia is usually corrected by administration of exogenous albumin. This study aimed to track the concentration-dependent influence of albumin on blood coagulation in vitro. Whole blood (WB) samples from 25 volunteers were prepared to contain low (19.3 ± 7.7 g/L), physiological (45.2 ± 7.8 g/L), and high (67.5 ± 18.1 g/L) levels of albumin. Haemostatic profiling was performed using a platelet function analyzer (PFA) 200, impedance aggregometry, a Cone and Platelet analyzer (CPA), calibrated automated thrombogram, and thrombelastometry (TEM). Platelet aggregation-associated ATP release was assessed via HPLC analysis. In the low albumin group, when compared to the physiological albumin group, we found: i) shortened PFA 200-derived closure times indicating increased primary haemostasis; ii) increased impedance aggregometry-derived amplitudes, slopes, ATP release, as well as CPA-derived average size indicating improved platelet aggregation; iii) increased TEM-derived maximum clot firmness and alpha angles indicating enhanced clot formation. TEM measurements indicated impaired clot formation in the high albumin group compared with the physiological albumin group. Thus, albumin exerted significant anticoagulant action. Therefore, low albumin levels, often present in cancer or critically ill patients, might contribute to the frequently occurring venous thromboembolism.


Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Albumina Sérica/farmacologia , Adulto , Testes de Coagulação Sanguínea , Plaquetas/citologia , Plaquetas/fisiologia , Células Cultivadas , Feminino , Humanos , Masculino , Testes de Função Plaquetária
15.
Biofactors ; 43(4): 577-592, 2017 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-28543688

RESUMO

Diabetes and obesity are strongly associated with increased levels of circulating advanced glycation end products (AGEs) and reactive oxygen species (ROS). These two molecular phenomena affect the physiology of adipose tissue, a biological driver of the metabolic syndrome, leading to an inflammatory profile and insulin resistance, which could contribute to obesity/diabetes-associated complications, such as cardiovascular diseases. Herein, we investigated the impact of AGEs on mitochondrial bioenergetics in murine preadipocyte cells (3T3-L1) and cellular redox homeostasis. We show that incubation of preadipocytes with AGEs stimulates mitochondrial activity and respiration while inducing oxidative stress. This AGE-induced intracellular ROS production was blocked by diphenylene iodonium, an NAD(P)H oxidase inhibitor. In parallel, antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) were found to be activated upon AGE treatment. Our results suggest that AGE-induced oxidative stress is generated by NAD(P)H oxidase and leads to a cellular proliferation arrest associated with enhanced mitochondrial metabolism and biogenesis, and with increased levels of ROS-detoxifying enzymes, as well. These new data show how AGEs may be involved in hyperglycemia-induced oxidative damage in preadipocytes and their potential links to diabetes progression. © 2017 BioFactors, 43(4):577-592, 2017.


Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Albumina Sérica/farmacologia , Células 3T3-L1 , Animais , Antioxidantes/farmacologia , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
J Inorg Biochem ; 171: 67-75, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28371680

RESUMO

Aggregation of amyloid ß-proteins (Aß) induced by Cu2+ is a crucial element in the pathogenesis of Alzheimer's disease (AD), and cerebral acidosis is a common complication of AD. Under mildly acidic conditions, Cu2+-Aß species have higher tendency to generate neurotoxic aggregates. Hence it is of significance to develop potent agents that inhibit Cu2+-mediated Aß aggregation under a mildly acidic condition. Herein we synthesized acidulated human serum albumin (A-HSA) to mitigate Cu2+-mediated Aß42 aggregation and cytotoxicity at pH6.6. Extensive experiments showed that A-HSA altered the pathway of Cu2+-mediated Aß42 aggregation and protected SH-SY5Y cells from cytotoxicity and oxidative damage induced by Cu2+-Aß42 species. Equimolar A-HSA increased cell viability from 52% to 91% as compared to Cu2+-Aß42-treated group. Stopped-flow fluorescence analysis revealed that A-HSA changed the Cu2+-Aß42 coordination mode from component I to II on the second timescale at pH6.6, which avoided the formation of aggregation-prone Cu2+-Aß42 aggregates. The findings revealed that the more negative charges on A-HSA surface could stabilize the protonated form of the adjacent histidine residues of Aß42. Hence, component I, which is necessary to form toxic aggregates, became unstable in the presence of A-HSA. On the other hand, hydrophobic binding and electrostatic repulsion could work simultaneously on the bound Aß42 on A-HSA surface. The two opposite forces stretched Aß42 conformations, which inhibited the formation of toxic Cu2+-Aß42 aggregates. Thus, A-HSA worked as a bifunctional inhibitor against Cu2+-mediated Aß42 aggregation and cytotoxicity under a mildly acidic condition.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Cobre/metabolismo , Agregação Patológica de Proteínas/fisiopatologia , Albumina Sérica/química , Sobrevivência Celular/efeitos dos fármacos , Cobre/toxicidade , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Ligação Proteica , Albumina Sérica/farmacologia
17.
Eur J Pharmacol ; 804: 57-67, 2017 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-28322835

RESUMO

Oxidative stress aggravates renal fibrosis, a pathway involved in almost all forms of chronic kidney disease (CKD). However, the underlying mechanism involved in the pathogenesis of renal oxidative stress has not been completely elucidated. In this study, we explored the role and mechanism of hypochlorite-modified albumin (HOCl-alb) in mediating oxidative stress and fibrotic response in a remnant-kidney rat model. Five-sixths nephrectomy (5/6 NX) was performed on the rats and then the animals were randomly assigned to intravenous treatment with either vehicle alone, or HOCl-rat serum albumin (RSA) in the presence or absence of SS-31 (administered intraperitoneally). A sham-operation control group was set up concurrently. Compared with the control group, 5/6 NX animals displayed marked mitochondrial (mt) dysfunction, as evidenced by decrease of mitochondrial membrane potential (MMP), ATP production, mtDNA copy number alterations and manganese superoxide dismutase (MnSOD) activity, release of cytochrome C (Cyto C) from mitochondria to the cytoplasm, and increase of mitochondrial reactive oxygen species in renal tissues. They also displayed increased levels of HOCl-alb in both plasma and renal tissues. These changes were accompanied by accumulation of extracellular matrix, worsened proteinuria, deteriorated renal function, and a marked increase of macrophage infiltration along with up-regulation of monocyte chemoattractant protein (MCP)-1 and transforming growth factor (TGF)-ß1 expression. HOCl-alb challenge further exacerbated the above biological effects in 5/6 NX animals, but these adverse effects were prevented by administration of SS-31, a mitochondrial targeted antioxidant peptide. These data suggest that accumulation of HOCl-alb may promote renal inflammation and fibrosis, probably related to mitochondrial oxidative stress and dysfunction and that the mitochondrial targeted peptide SS-31 might be a novel therapy for renal fibrosis and chronic renal failure (CRF).


Assuntos
Antioxidantes/farmacologia , Ácido Hipocloroso/química , Rim/efeitos dos fármacos , Rim/patologia , Mitocôndrias/efeitos dos fármacos , Oligopeptídeos/farmacologia , Albumina Sérica/farmacologia , Animais , Biomarcadores/metabolismo , Citoproteção/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibrose , Rim/metabolismo , Rim/fisiologia , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Albumina Sérica/química , Regulação para Cima/efeitos dos fármacos
18.
J Pharm Biomed Anal ; 139: 238-246, 2017 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-28314215

RESUMO

Tobacco smoke exposure is the principal cause of lung tissue destruction, which in turn results in emphysema that leads into shortness of breath. Liver growth factor (LGF, a cell and tissue regenerating factor with therapeutic activity in several organs) has antifibrotic and antioxidant properties that could be useful to promote lung tissue regenerating capacity in damaged lungs. The current study has examined differences in metabolite profiles (fingerprints) of plasma from mice (strain C57BL/6J, susceptible to develop emphysema) exposed to tobacco smoke during six months. One group of mice received a treatment with Liver Growth Factor (LGF) after emphysema was established, whereas the other group did not receive the treatment. Age and sex-matched mice not exposed to smoke were also maintained with or without treatment as controls. Metabolic fingerprints (untargeted analysis) of plasma after protein precipitation were obtained by LC-QTOF-MS. The signals were processed and a large number of possible metabolites were found (23944). Multivariate data analysis provided models that highlighted the differences between control and smoke exposed mice in both conditions. Accurate masses of features (possible compounds) representing significant differences were searched using online public databases. Lipid mediators, related to intracellular signaling in inflammation, were found among the metabolites putatively identified as markers of the different conditions and among them, sphingosine, sphingosine 1-phosphate and lysophospholipids point at the relevance of such metabolites in the regulation of the processes related to tissue regeneration mediated by LGF. These results also suggest that metabolomic fingerprinting could potentially guide the characterization of relevant metabolites leading the regeneration of lungs in emphysema disease.


Assuntos
Bilirrubina/uso terapêutico , Lisofosfolipídeos/metabolismo , Metabolômica/métodos , Enfisema Pulmonar/metabolismo , Albumina Sérica/uso terapêutico , Fumar/efeitos adversos , Esfingosina/análogos & derivados , Animais , Bilirrubina/farmacologia , Exposição por Inalação/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Enfisema Pulmonar/tratamento farmacológico , Albumina Sérica/farmacologia , Albumina Sérica Humana , Fumar/tratamento farmacológico , Esfingosina/metabolismo
19.
Lipids Health Dis ; 16(1): 36, 2017 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-28179022

RESUMO

BACKGROUND: High-density Lipoprotein (HDL) attenuates endothelial cell apoptosis induced by different cell-death stimuli such as oxidation or growth factor deprivation. HDL is the main plasma carrier of the bioactive lipid sphingosine 1-phosphate (S1P), which it is a signaling molecule that promotes cell survival in response to several apoptotic stimuli. In HDL, S1P is bound to Apolipoprotein M (ApoM), a Lipocalin that is only present in around 5% of the HDL particles. The goal of this study is to characterize ApoM-bound S1P role in endothelial apoptosis protection and the signaling pathways involved. METHODS: Human umbilical vein endothelial cells (HUVEC) cultures were switched to serum/grow factor deprivation medium to induce apoptosis and the effect caused by the addition of ApoM and S1P analyzed. RESULTS: The addition of HDL+ApoM or recombinant ApoM-bound S1P promoted cell viability and blocked apoptosis, whereas HDL-ApoM had no protective effect. Remarkably, S1P exerted a more potent anti-apoptotic effect when carried by ApoM as compared to albumin, or when added as free molecule. Mechanistically, cooperation between S1P1 and S1P3 was required for the HDL/ApoM/S1P-mediated anti-apoptotic ability. Furthermore, AKT and ERK phosphorylation was also necessary to achieve the anti-apoptotic effect of the HDL/ApoM/S1P complex. CONCLUSIONS: Altogether, our results indicate that ApoM and S1P are key elements of the anti-apoptotic activity of HDL and promote optimal endothelial function.


Assuntos
Apolipoproteínas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Lipocalinas/metabolismo , Lipoproteínas HDL/metabolismo , Lisofosfolipídeos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Apolipoproteínas/genética , Apolipoproteínas/farmacologia , Apolipoproteínas M , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/química , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Lipocalinas/genética , Lipocalinas/farmacologia , Lipoproteínas HDL/genética , Lipoproteínas HDL/farmacologia , Lisofosfolipídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisoesfingolipídeo/genética , Albumina Sérica/farmacologia , Esfingosina/metabolismo , Esfingosina/farmacologia
20.
Protein Eng Des Sel ; 30(5): 359-372, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28180900

RESUMO

Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and pharmacokinetics (PK) of the DKK2 polypeptide. Specifically, human serum albumin (HSA) was identified as a highly effective fusion partner. Substitution of selected amino acid residues in DKK2 designed to decrease heparan sulfate binding by HSA-DKK2 variants, further improved the PK properties of the molecule in rodents. The HSA-DKK2 variants were monomeric, as thermally stable as wild type, and active as measured by their ability to bind to and prevent phosphorylation of the Wnt coreceptor LRP6. Our engineering efforts resulted in potent long-lived variants of the canonical Wnt inhibitor DKK2, applicable for Wnt pathway manipulation either by systematic delivery or focused administration at sites of tissue injury.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Engenharia de Proteínas , Proteínas Recombinantes de Fusão , Albumina Sérica , Proteínas Wnt/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica/biossíntese , Albumina Sérica/química , Albumina Sérica/isolamento & purificação , Albumina Sérica/farmacologia
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