RESUMO

The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.

Assuntos

Pró-Colágeno-Prolina Dioxigenase , Isomerases de Dissulfetos de Proteínas , Humanos , Oxirredução , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/metabolismo , Zinco/química , Zinco/metabolismo

RESUMO

Chronic Chagas disease (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis compared to other cardiomyopathies. We show the expression and activity of Matrix Metalloproteinases (MMP) and of their inhibitors TIMP (tissue inhibitor of metalloproteinases) in myocardial samples of end stage CCC, idiopathic dilated cardiomyopathy (DCM) patients, and from organ donors. Our results showed significantly increased mRNA expression of several MMPs, several TIMPs and EMMPRIN in CCC and DCM samples. MMP-2 and TIMP-2 protein levels were significantly elevated in both sample groups, while MMP-9 protein level was exclusively increased in CCC. MMPs 2 and 9 activities were also exclusively increased in CCC. Results suggest that the balance between proteins that inhibit the MMP-2 and 9 is shifted toward their activation. Inflammation-induced increases in MMP-2 and 9 activity and expression associated with imbalanced TIMP regulation could be related to a more extensive heart remodeling and poorer prognosis in CCC patients.

Assuntos

Cardiomiopatia Dilatada , Cardiomiopatia Chagásica , Cardiomiopatia Dilatada/metabolismo , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Miocárdio

RESUMO

BACKGROUND: Protein Disulfide Isomerases are thiol oxidoreductase chaperones from thioredoxin superfamily with crucial roles in endoplasmic reticulum proteostasis, implicated in many diseases. The family prototype PDIA1 is also involved in vascular redox cell signaling. PDIA1 is coded by the P4HB gene. While forced changes in P4HB gene expression promote physiological effects, little is known about endogenous P4HB gene regulation and, in particular, gene modulation by alternative splicing. This study addressed the P4HB splice variant landscape. RESULTS: Ten protein coding sequences (Ensembl) of the P4HB gene originating from alternative splicing were characterized. Structural features suggest that except for P4HB-021, other splice variants are unlikely to exert thiol isomerase activity at the endoplasmic reticulum. Extensive analyses using FANTOM5, ENCODE Consortium and GTEx project databases as RNA-seq data sources were performed. These indicated widespread expression but significant variability in the degree of isoform expression among distinct tissues and even among distinct locations of the same cell, e.g., vascular smooth muscle cells from different origins. P4HB-02, P4HB-027 and P4HB-021 were relatively more expressed across each database, the latter particularly in vascular smooth muscle. Expression of such variants was validated by qRT-PCR in some cell types. The most consistently expressed splice variant was P4HB-021 in human mammary artery vascular smooth muscle which, together with canonical P4HB gene, had its expression enhanced by serum starvation. CONCLUSIONS: Our study details the splice variant landscape of the P4HB gene, indicating their potential role to diversify the functional reach of this crucial gene. P4HB-021 splice variant deserves further investigation in vascular smooth muscle cells.

Assuntos

Pró-Colágeno-Prolina Dioxigenase , Isomerases de Dissulfetos de Proteínas , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Mutação , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Transdução de Sinais

RESUMO

BACKGROUND: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. METHODS: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. RESULTS: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M-1 s-1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. CONCLUSIONS: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. GENERAL SIGNIFICANCE: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.

Assuntos

Peróxidos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Ácido Úrico/análogos & derivados , Domínio Catalítico , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Sobrevivência Celular/fisiologia , Cromatografia Líquida/métodos , Cisteína/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Oxirredução , Peroxidases/metabolismo , Agregação Plaquetária , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Isomerases de Dissulfetos de Proteínas/fisiologia , Compostos de Sulfidrila/metabolismo , Espectrometria de Massas em Tandem/métodos , Trombose/metabolismo , Ácido Úrico/metabolismo

RESUMO

Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (>median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses.

Assuntos

Células Endoteliais/metabolismo , Fenótipo , Isomerases de Dissulfetos de Proteínas/sangue , Proteoma , Proteômica , Adulto , Biomarcadores , Sobrevivência Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Voluntários Saudáveis , Humanos , Masculino , Oxirredução , Agregação Plaquetária , Proteômica/métodos , Reprodutibilidade dos Testes

RESUMO

Protein disulfide isomerases including PDIA1 are implicated in cancer progression, but underlying mechanisms are unclear. PDIA1 is known to support vascular Nox1 NADPH oxidase expression/activation. Since deregulated reactive oxygen species (ROS) production underlies tumor growth, we proposed that PDIA1 is an upstream regulator of tumor-associated ROS. We focused on colorectal cancer (CRC) with distinct KRas activation levels. Analysis of RNAseq databanks and direct validation indicated enhanced PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) and basal (Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we first reported a dual effect correlated with Ras-level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production, while in HCT116 cells PDIA1 restricted superoxide production, a behavior associated with increased Rac1 expression/activity. Transfection of Rac1G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide, while in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide. PDIA1 silencing promoted diminished cell proliferation and migration in HKE3, not detectable in HCT116 cells. Screening of cell signaling routes affected by PDIA1 silencing highlighted GSK3ß and Stat3. Also, E-cadherin expression after PDIA1 silencing was decreased in HCT116, consistent with PDIA1 support of epithelial-mesenchymal transition. Thus, Ras overactivation switches the pattern of PDIA1-dependent Rac1/Nox1 regulation, so that Ras-induced PDIA1 bypass can directly activate Rac1. PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression.

Assuntos

Neoplasias do Colo/metabolismo , NADPH Oxidase 1/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células CACO-2 , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HCT116 , Humanos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Transfecção , Proteínas rac1 de Ligação ao GTP/metabolismo

RESUMO

Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial struc-ture/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here,we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. Wevalidated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual(median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably,plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (> median)plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping func-tions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, in-flammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs.PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phe-notype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene ex-pression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such sig-natures translated into functional responses, with PDI-poor plasma promoting impairment of endothelialadhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients withcardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels asreporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes andfunctional responses.

RESUMO

Protein disulfide isomerases are thiol oxidoreductase chaperones from thioredoxin superfamily. As redox folding catalysts from the endoplasmic reticulum (ER), their roles in ER-related redox homeostasis and signaling are well-studied. PDIA1 exerts thiol oxidation/reduction and isomerization, plus chaperone effects. Also, substantial evidence indicates that PDIs regulate thiol-disulfide switches in other cell locations such as cell surface and possibly cytosol. Subcellular PDI translocation routes remain unclear and seem Golgi-independent. The list of signaling and structural proteins reportedly regulated by PDIs keeps growing, via thiol switches involving oxidation, reduction and isomerization, S-(de)nytrosylation, (de)glutathyonylation and protein oligomerization. PDIA1 is required for agonist-triggered Nox NADPH oxidase activation and cell migration in vascular cells and macrophages, while PDIA1-dependent cytoskeletal regulation appears a converging pathway. Extracellularly, PDIs crucially regulate thiol redox signaling of thrombosis/platelet activation, e.g., integrins, and PDIA1 supports expansive caliber remodeling during injury repair via matrix/cytoskeletal organization. Some proteins display regulatory PDI-like motifs. PDI effects are orchestrated by expression levels or post-translational modifications. PDI is redox-sensitive, although probably not a mass-effect redox sensor due to kinetic constraints. Rather, the "all-in-one" organization of its peculiar redox/chaperone properties likely provide PDIs with precision and versatility in redox signaling, making them promising therapeutic targets.

Assuntos

Retículo Endoplasmático/metabolismo , Oxirredução , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Citoesqueleto/metabolismo , Citosol/metabolismo , Dissulfetos/química , Homeostase , Humanos , Cinética , Camundongos , Chaperonas Moleculares/metabolismo , NADPH Oxidases/metabolismo , Peróxidos/química , Processamento de Proteína Pós-Traducional , Transdução de Sinais

RESUMO

OBJECTIVE: We investigated the effect of two different saline solutions on the mechanisms of injury after intestinal ischemia: oxidative stress and inflammatory responses. METHODS: Wistar rats underwent transient superior mesenteric artery occlusion and were studied for 6 hours after reperfusion. After randomization, the animals were divided into four groups: Sham; Hypertonic Saline, in which they received infusion of 4 mL/kg body weight of 7.5% hypertonic saline; Saline, in which they received infusion of 33 mL/kg body weight of 0.9% saline; and Non Treatment. The infusion was performed immediately prior to the reperfusion. The plasma concentrations of interleukin 6 and interleukin 10 were measured. Tissue samples (lung, liver, and intestine) were collected for malondialdehyde, myeloperoxidase, and interleukin measurements. RESULTS: The animals that received infusions (Hypertonic Saline and Saline) showed lower levels of tissue malondialdehyde, myeloperoxidase, interleukin 6, and interleukin 10 compared with the Non Treatment group. The plasma concentrations of interleukin 6 and interleukin 10 were higher in the animals treated with 7.5% hypertonic saline compared with Saline and Non Treatment groups. CONCLUSION: In this model of transient intestinal ischemia, the adequate maintenance of intravascular volume decreased oxidative stress and the synthesis of inflammatory markers. Both 7.5% Hypertonic Saline and Saline attenuated the deleterious effects observed after intestinal ischemia.

Assuntos

Isquemia/tratamento farmacológico , Traumatismo por Reperfusão/tratamento farmacológico , Solução Salina Hipertônica/farmacologia , Cloreto de Sódio/farmacologia , Animais , Modelos Animais de Doenças , Inflamação/etiologia , Inflamação/prevenção & controle , Interleucinas/metabolismo , Intestinos/irrigação sanguínea , Intestinos/efeitos dos fármacos , Intestinos/patologia , Isquemia/patologia , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia

RESUMO

Objetivo: Investigar o papel de duas diferentes soluções salinas nos mecanismos de lesão após isquemia intestinal: estresse oxidativo e respostas inflamatórias. Métodos: Ratos Wistar foram submetidos a oclusão transitória da artéria mesentérica superior e estudados durante as 6 horas seguintes à reperfusão. Após randomização, os animais foram divididos em quatro grupos: Falso; Solução Hipertônica, os quais receberam infusão de solução salina hipertônica a 7,5% (4mL/kg de peso corpóreo); Solução Fisiológica, os quais receberam infusão de solução salina a 0,9% (33mL/kg); e Sem Tratamento. A infusão foi realizada imediatamente antes da reperfusão. Foram realizadas dosagens sequenciais de interleucina 6 e interleucina 10 no plasma. Foram coletadas amostras de tecidos (pulmão, fígado e intestino) para medir malondialdeído, mieloperoxidase e interleucina. Resultados: Em comparação ao Grupo Sem Tratamento, os animais que receberam volume (Grupos Solução Hipertônica e Solução Fisiológica) mostraram níveis tissulares mais baixos de malondialdeído, mieloperoxidase, interleucina 6 e interleucina 10. As concentrações plasmáticas de interleucina 6 e interleucina 10 foram mais altas nos animais tratados com solução hipertônica do que nos tratados com solução fisiológica e nos sem tratamento. Conclusão: Neste modelo de isquemia intestinal transitória, a manutenção adequada de volume intravascular diminuiu o estresse oxidativo e a síntese de marcadores de inflamação. Tanto a solução hipertônica quanto a fisiológica atenuaram os efeitos deletérios observados após isquemia intestinal. .

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Objective: We investigated the effect of two different saline solutions on the mechanisms of injury after intestinal ischemia: oxidative stress and inflammatory responses. Methods: Wistar rats underwent transient superior mesenteric artery occlusion and were studied for 6 hours after reperfusion. After randomization, the animals were divided into four groups: Sham; Hypertonic Saline, in which they received infusion of 4mL/kg body weight of 7.5% hypertonic saline; Saline, in which they received infusion of 33mL/kg body weight of 0.9% saline; and Non Treatment. The infusion was performed immediately prior to the reperfusion. The plasma concentrations of interleukin 6 and interleukin 10 were measured. Tissue samples (lung, liver, and intestine) were collected for malondialdehyde, myeloperoxidase, and interleukin measurements. Results: The animals that received infusions (Hypertonic Saline and Saline) showed lower levels of tissue malondialdehyde, myeloperoxidase, interleukin 6, and interleukin 10 compared with the Non Treatment group. The plasma concentrations of interleukin 6 and interleukin 10 were higher in the animals treated with 7.5% hypertonic saline compared with Saline and Non Treatment groups. Conclusion: In this model of transient intestinal ischemia, the adequate maintenance of intravascular volume decreased oxidative stress and the synthesis of inflammatory markers. Both 7.5% Hypertonic Saline and Saline attenuated the deleterious effects observed after intestinal ischemia. .

Assuntos

Animais , Masculino , Ratos , Isquemia/tratamento farmacológico , Traumatismo por Reperfusão/tratamento farmacológico , Solução Salina Hipertônica/farmacologia , Cloreto de Sódio/farmacologia , Modelos Animais de Doenças , Inflamação/etiologia , Inflamação/prevenção & controle , Interleucinas/metabolismo , Intestinos/irrigação sanguínea , Intestinos/efeitos dos fármacos , Intestinos/patologia , Isquemia/patologia , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Ratos Wistar , Traumatismo por Reperfusão/patologia

RESUMO

Protein disulfide isomerase (PDI) is a dithiol-disulfide oxidoreductase that has essential roles in redox protein folding. PDI has been associated with protective roles against protein aggregation, a hallmark of neurodegenerative diseases. Intriguingly, PDI has been detected in the protein inclusions found in the central nervous system of patients of neurodegenerative diseases. Oxidized proteins are also consistently detected in such patients, but the agents that promote these oxidations remain undefined. A potential trigger of protein oxidation is the bicarbonate-dependent peroxidase activity of the human enzyme superoxide dismutase 1 (hSOD1). Therefore, we examined the effects of this activity on PDI structure and activity. The results showed that PDI was oxidized to radicals that lead to PDI inactivation and aggregation. The aggregates are huge and apparently produced by covalent cross-links. Spin trapping experiments coupled with MS analysis indicated that at least 3 residues of PDI are oxidized to tyrosyl radicals (Y(63), Y(116) and Y(327)). Parallel experiments showed that PDI is also oxidized to radicals, inactivated and aggregated by the action of photolytically generated carbonate radical and by UV light. PDI is prone to inactivation and aggregation by one-electron oxidants and UV light probably because of its high content of aromatic amino acids.

Assuntos

Bicarbonatos/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Superóxido Dismutase/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas/métodos , Oxirredução , Raios Ultravioleta

RESUMO

Severe acute pancreatitis (AP) induces a systemic inflammatory disease that is responsible for high mortality rates, particularly when it is complicated by infection. Therefore, differentiating sepsis from the systemic inflammation caused by AP is a serious clinical challenge. Considering the high metabolic rates of leukocytes in response to stress induced by infection, we hypothesized that the transcription coactivator peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1α), a master regulator of mitochondrial biogenesis and function, would be distinctly expressed during inflammation or infection and, therefore, could constitute a useful marker to differentiate between these two conditions. Rats were subjected to injection of taurocholate into the main pancreatic duct, which caused a severe AP with high amylase levels and white blood cell counts. In these animals, a marked increase in PGC-1α mRNA levels in circulating leukocytes was observed 48 h after the surgical procedure, a time when bacteremia is present. Antibiotic treatment abolished PGC-1α up-regulation. Moreover, PGC-1α expression was higher in peritoneal macrophages from animals subjected to a bacterial insult (cecal ligation and puncture) than in animals with AP. In isolated macrophages, we also observed that PGC-1α expression is more prominent in the presence of a phagocytic stimulus (zymosan) when compared to lipopolysaccharide-induced aseptic inflammation. Moreover, abolishing PGC-1α expression with antisense oligos impaired zymosan phagocytosis. Together, these findings suggest that PGC-1α is differentially expressed during aseptic inflammation and infection and that it is necessary for adequate phagocytosis. These results could be useful in developing new tests for differentiating infection from inflammation for clinical purposes in patients with AP.

Assuntos

Regulação da Expressão Gênica , Leucócitos/metabolismo , Pancreatite/metabolismo , Fatores de Transcrição/metabolismo , Doença Aguda , Animais , Ceco/cirurgia , Inflamação/metabolismo , Macrófagos/citologia , Masculino , Mitocôndrias/metabolismo , Oligonucleotídeos Antissenso/química , Pancreatite/induzido quimicamente , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fagocitose , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Sepse/metabolismo , Regulação para Cima

RESUMO

PURPOSE: To investigate if the ethyl-pyruvate solution could reduce mortality in AP and/or diminish the acute lung injury. METHODS: Forty male rats, weighing between 270 to 330 grams were operated. An experimental model of severe AP by injection of 0.1ml/100g of 2.5% sodium taurocholate into the bilio-pancreatic duct was utilized. The rats were divided into two groups of ten animals each: CT - control (treatment with 50ml/kg of Ringer's solution, intraperitoneal) and EP (treatment with 50ml/kg of Ringer ethyl- pyruvate solution, intra-peritoneal), three hours following AP induction. After six hours, a new infusion of the treatment solution was performed in each group. Two hours later, the animals were killed and the pulmonary parenchyma was resected for biomolecular analysis, consisting of: interleukin, myeloperoxidase, MDA, nitric oxide, metalloproteinases and heat shock protein. In the second part of the experiment, another, 20 rats were randomly divided into EP and CT groups, in order to evaluate a survival comparison between the two groups. RESULTS: There were no significant differences in IL-1B,IL-10, MMP-9, HSP70, nitric oxide, MPO, MDA (lipidic peroxidation) concerning both groups. The levels of IL-6 were significantly diminished in the EP group. Furthermore, the MMP-2 levels were also reduced in the EP group (p<0.05). The animals from the EP treatment groups had improved survival, when compared to control group (p<0.05). CONCLUSION: The ethyl-pyruvate diminishes acute lung injury inflammatory response in acute pancreatitis and ameliorates survival when compared to control group, in the experimental model of necrotizing acute pancreatitis.

Assuntos

Animais , Masculino , Ratos , Lesão Pulmonar Aguda/tratamento farmacológico , Citocinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Pancreatite Necrosante Aguda/tratamento farmacológico , Piruvatos/farmacologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Modelos Animais de Doenças , Immunoblotting , Soluções Isotônicas/farmacologia , Estimativa de Kaplan-Meier , Pancreatite Necrosante Aguda/mortalidade , Distribuição Aleatória , Ratos Wistar , Valores de Referência , Reprodutibilidade dos Testes , Fatores de Tempo , Resultado do Tratamento

RESUMO

PURPOSE: To investigate if the ethyl-pyruvate solution could reduce mortality in AP and/or diminish the acute lung injury. METHODS: Forty male rats, weighing between 270 to 330 grams were operated. An experimental model of severe AP by injection of 0.1ml/100g of 2.5% sodium taurocholate into the bilio-pancreatic duct was utilized. The rats were divided into two groups of ten animals each: CT - control (treatment with 50ml/kg of Ringer's solution, intraperitoneal) and EP (treatment with 50ml/kg of Ringer ethyl- pyruvate solution, intra-peritoneal), three hours following AP induction. After six hours, a new infusion of the treatment solution was performed in each group. Two hours later, the animals were killed and the pulmonary parenchyma was resected for biomolecular analysis, consisting of: interleukin, myeloperoxidase, MDA, nitric oxide, metalloproteinases and heat shock protein. In the second part of the experiment, another, 20 rats were randomly divided into EP and CT groups, in order to evaluate a survival comparison between the two groups. RESULTS: There were no significant differences in IL-1B,IL-10, MMP-9, HSP70, nitric oxide, MPO, MDA (lipidic peroxidation) concerning both groups. The levels of IL-6 were significantly diminished in the EP group. Furthermore, the MMP-2 levels were also reduced in the EP group (p<0.05). The animals from the EP treatment groups had improved survival, when compared to control group (p<0.05). CONCLUSION: The ethyl-pyruvate diminishes acute lung injury inflammatory response in acute pancreatitis and ameliorates survival when compared to control group, in the experimental model of necrotizing acute pancreatitis.(AU)

Assuntos

Animais , Ratos , Lesão Pulmonar/patologia , Citocinas/análise , Inflamação/patologia , Pancreatite/patologia , Ratos/classificação , Necrose

RESUMO

It is known that low level laser therapy is able to improve skin flap viability by increasing angiogenesis. However, the mechanism for new blood vessel formation is not completely understood. Here, we investigated the effects of 660 nm and 780 nm lasers at fluences of 30 and 40 J/cm(2) on three important mediators activated during angiogenesis. Sixty male Wistar rats were used and randomly divided into five groups with twelve animals each. Groups were distributed as follows: skin flap surgery non-irradiated group as a control; skin flap surgery irradiated with 660 nm laser at a fluence of 30 or 40 J/cm(2) and skin flap surgery irradiated with 780 nm laser at a fluence of 30 or 40 J/cm(2). The random skin flap was performed measuring 10×4 cm, with a plastic sheet interposed between the flap and the donor site. Laser irradiation was performed on 24 points covering the flap and surrounding skin immediately after the surgery and for 7 consecutive days thereafter. Tissues were collected, and the number of vessels, angiogenesis markers (vascular endothelial growth factor, VEGF and hypoxia inducible factor, HIF-1α) and a tissue remodeling marker (matrix metalloproteinase, MMP-2) were analyzed. LLLT increased an angiogenesis, HIF-1α and VEGF expression and decrease MMP-2 activity. These phenomena were dependent on the fluences, and wavelengths used. In this study we showed that LLLT may improve the healing of skin flaps by enhancing the amount of new vessels formed in the tissue. Both 660 nm and 780 nm lasers were able to modulate VEGF secretion, MMP-2 activity and HIF-1α expression in a dose dependent manner.

Assuntos

Isquemia/radioterapia , Terapia com Luz de Baixa Intensidade , Neovascularização Fisiológica/efeitos da radiação , Retalhos Cirúrgicos/irrigação sanguínea , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Isquemia/fisiopatologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Neovascularização Fisiológica/fisiologia , Ratos , Ratos Wistar , Pele/irrigação sanguínea , Pele/metabolismo , Pele/efeitos da radiação , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo

RESUMO

PURPOSE: To investigate if the ethyl-pyruvate solution could reduce mortality in AP and/or diminish the acute lung injury. METHODS: Forty male rats, weighing between 270 to 330 grams were operated. An experimental model of severe AP by injection of 0.1 ml/100g of 2.5% sodium taurocholate into the bilio-pancreatic duct was utilized. The rats were divided into two groups of ten animals each: CT - control (treatment with 50 ml/kg of Ringer's solution, intraperitoneal) and EP (treatment with 50 ml/kg of Ringer ethyl-pyruvate solution, intra-peritoneal), three hours following AP induction. After six hours, a new infusion of the treatment solution was performed in each group. Two hours later, the animals were killed and the pulmonary parenchyma was resected for biomolecular analysis, consisting of: interleukin, myeloperoxidase, MDA, nitric oxide, metalloproteinases and heat shock protein. In the second part of the experiment, another, 20 rats were randomly divided into EP and CT groups, in order to evaluate a survival comparison between the two groups. RESULTS: There were no significant differences in IL-1B,IL-10, MMP-9, HSP70, nitric oxide, MPO, MDA (lipidic peroxidation) concerning both groups. The levels of IL-6 were significantly diminished in the EP group. Furthermore, the MMP-2 levels were also reduced in the EP group (p<0.05). The animals from the EP treatment groups had improved survival, when compared to control group (p<0.05). CONCLUSION: The ethyl-pyruvate diminishes acute lung injury inflammatory response in acute pancreatitis and ameliorates survival when compared to control group, in the experimental model of necrotizing acute pancreatitis.

Assuntos

Lesão Pulmonar Aguda/tratamento farmacológico , Citocinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Pancreatite Necrosante Aguda/tratamento farmacológico , Piruvatos/farmacologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Animais , Modelos Animais de Doenças , Immunoblotting , Soluções Isotônicas/farmacologia , Estimativa de Kaplan-Meier , Masculino , Pancreatite Necrosante Aguda/mortalidade , Distribuição Aleatória , Ratos , Ratos Wistar , Valores de Referência , Reprodutibilidade dos Testes , Fatores de Tempo , Resultado do Tratamento

RESUMO

The experimental model of aortocaval fistula is a useful model of cardiac hypertrophy in response to volume overload. In the present study it has been used to investigate the pathologic subendocardial remodeling associated with the development of heart failure during the early phases (day 1, 3, and 7) following volume overload. Compared with sham treated rats, aortocaval fistula rats showed lower systemic blood pressure and higher left ventricular end-diastolic pressure This resulted in lower coronary driving pressure and left ventricular systolic and diastolic dysfunction. Signs of myocyte necrosis, leukocyte cell infiltration, fibroplasia and collagen deposition appeared sequentially in the subendocardium where remodeling was more prominent than in the non-subendocardium. Accordingly, increased levels of TNF-alpha, IL-1 beta, and IL-6, and enhanced MMP-2 activity were all found in the subendocardium of rats with coronary driving pressure ≤ 60 mmHg. The coronary driving pressure was inversely correlated with MMP-2 activity in subendocardium in all time-points studied, and blood flow in this region showed positive correlation with systolic and diastolic function at day 7. Thus the predominant subendocardial remodeling that occurs in response to low myocardial perfusion pressure during the acute phases of aortocaval fistula contributes to early left ventricular dysfunction.

Assuntos

Aorta Abdominal/fisiopatologia , Fístula Arteriovenosa/fisiopatologia , Pressão Sanguínea/fisiologia , Veia Cava Inferior/fisiopatologia , Função Ventricular Esquerda/fisiologia , Remodelação Ventricular/fisiologia , Doença Aguda , Animais , Aorta Abdominal/patologia , Fístula Arteriovenosa/metabolismo , Fístula Arteriovenosa/patologia , Circulação Coronária/fisiologia , Modelos Animais de Doenças , Hemodinâmica/fisiologia , Interleucinas/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Peroxidase/metabolismo , Fluxo Pulsátil/fisiologia , Ratos , Ratos Wistar , Veia Cava Inferior/patologia

RESUMO

Muscle regeneration is a complex phenomenon, involving replacement of damaged fibers by new muscle fibers. During this process, there is a tendency to form scar tissue or fibrosis by deposition of collagen that could be detrimental to muscle function. New therapies that could regulate fibrosis and favor muscle regeneration would be important for physical therapy. Low-level laser therapy (LLLT) has been studied for clinical treatment of skeletal muscle injuries and disorders, even though the molecular and cellular mechanisms have not yet been clarified. The aim of this study was to evaluate the effects of LLLT on molecular markers involved in muscle fibrosis and regeneration after cryolesion of the tibialis anterior (TA) muscle in rats. Sixty Wistar rats were randomly divided into three groups: control, injured TA muscle without LLLT, injured TA muscle treated with LLLT. The injured region was irradiated daily for four consecutive days, starting immediately after the lesion using an AlGaAs laser (808 nm, 30 mW, 180 J/cm(2); 3.8 W/cm(2), 1.4 J). The animals were sacrificed on the fourth day after injury. LLLT significantly reduced the lesion percentage area in the injured muscle (p<0.05), increased mRNA levels of the transcription factors MyoD and myogenin (p<0.01) and the pro-angiogenic vascular endothelial growth factor (p<0.01). Moreover, LLLT decreased the expression of the profibrotic transforming growth factor TGF-ß mRNA (p<0.01) and reduced type I collagen deposition (p<0.01). These results suggest that LLLT could be an effective therapeutic approach for promoting skeletal muscle regeneration while preventing tissue fibrosis after muscle injury.

Assuntos

Terapia com Luz de Baixa Intensidade , Músculo Esquelético/lesões , Regeneração/efeitos da radiação , Animais , Colágeno Tipo I/metabolismo , Fibrose , Lasers Semicondutores/uso terapêutico , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos da radiação , Proteína MyoD/genética , Miogenina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Regeneração/genética , Regeneração/fisiologia , Fator de Crescimento Transformador beta1/genética , Fator A de Crescimento do Endotélio Vascular/genética

RESUMO

OBJETIVO: A falência hepática é uma consequência da inflamação sistêmica após pancreatite aguda. Avaliou-se o efeito da reposição volêmica com soluções salinas fisiológicas ou hipertônica na produção hepática de citocinas e na expressão de proteínas ativadas por choque térmico e proteínas ligadas à apoptose durante a pancreatite aguda. MÉTODOS: Ratos Wistar foram divididos em quatro grupos: C - animais controles não submetidos à lesão e nem ao tratamento; NT - animais submetidos à indução de pancreatite aguda e não tratados; SN - animais submetidos à indução de pancreatite aguda e tratados com solução salina normal (NaCl 0,9%); SH - animais submetidos à pancreatite aguda e tratados com solução salina hipertônica (NaCl 7,5%). A pancreatite aguda foi induzida por infusão retrógrada transduodenal de taurocolato de sódio 2,5% no ducto pancreático. Após 4, 12 e 24 horas da indução da pancreatite aguda, analisaram-se, no fígado, TNF-α, IL-1β, IL-6 e IL-10, caspase-2, caspase-7, APAF-1, AIF, HSP60 e HSP90. RESULTADOS: A caspase-2 diminuiu nos grupos SN e SH (p<0,05 versus C) após 12 horas. APAF-1, AIF e HSP90 permaneceram inalterados. Após 4 horas da indução, a capsase-7 aumentou no grupo NT (p<0,01 versus C), embora se mantendo em níveis basais nos grupos reperfundidos. A HSP60 aumentou em todos os grupos após 4 horas (p<0,001 versus C). No entanto, o grupo SH mostrou menor expressão de HSP60 que o grupo SN (p<0,05). A solução salina hipertônica manteve a produção de citocinas em níveis normais. A reperfusão com volume com solução salina normal ou hipertônica, modulou significativamente a expressão de caspase-7. CONCLUSÃO: A reposição volêmica com solução salina normal ou hipertônica foi efetiva em reduzir a caspase-7. Entretanto, somente a solução salina hipertônica foi capaz de regular a produção de citocinas e a expressão de HSP60 em todos os momentos analisados.

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OBJECTIVE: Liver failure can occur as a consequence of the systemic inflammation after acute pancreatitis. We assessed the effect of volume repositioning with hypertonic saline solution or normal saline on hepatic cytokine production and the expression of heat-shock proteins and apoptotic proteins after acute pancreatitis. METHODS: Wistar rats were divided in four groups: C - control animals that were not subjected to insult or treatment; NT - animals that were subjected to acute pancreatitis and received no treatment; normal saline - animals that were subjected to acute pancreatitis and received normal saline (NaCl 0.9%); and HS - animals that were subjected to acute pancreatitis and received hypertonic saline solution (NaCl 7.5%). Acute pancreatitis was induced by retrograde transduodenal infusion of 2.5% sodium taurocholate into the pancreatic duct. At 4, 12 and 24 h following acute pancreatitis induction, TNF-alpha, IL-1-beta, IL-6 and IL-10, caspase-2 and -7, Apaf-1, AIF and HSP60 and 90 were analyzed in the liver. RESULTS: Casp2 decreased in the normal saline and hypertonic saline groups (p<0.05 versus. C) at 12 h. Apaf-1, AIF and HSP90 remained unchanged. At 4 h, Casp7 increased in the NT group (p<0.01 versus C), although it remained at the baseline levels in the reperfused groups. HSP60 increased in all of the groups at 4 h (p< 0.001 vs. C). However, the hypertonic saline group showed lower expression of HSP60 than the normal saline group (p<0.05). Hypertonic saline solution maintained the production of cytokines at normal levels. Volume reperfusion with normal or hypertonic saline significantly modulated the expression of Casp7. CONCLUSION: Volume replacement with hypertonic or normal saline was effective in reducing caspase 7. However, only hypertonic solution was capable of regulating cytokine production and HSP60 expression at all time points.