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INTRODUCTION: Proteinuria in burn patients is common, and may be associated with acute kidney injury (AKI) and adverse outcomes. We evaluated the incidences, outcomes, characteristics and determinants of proteinuria and its influence on AKI and outcomes in burn patients. METHODS: This retrospective study was carried out in a hospital's burn department. The study population consisted of patients with burn injuries admitted during a five-year period. Positive urine dipstick readings were defined as mild (± or 1+) or heavy (≥ 2+) proteinuria, and AKI was diagnosed and staged according to the Risk, Injury, Failure, Loss, End Stage (RIFLE) classification system. Patient characteristics, management and outcomes were evaluated for associations with proteinuria using nonparametric tests, chi-square (χ(2)) tests and binary logistic regression. RESULTS: Of the patients admitted to the burn unit during the study period (n = 2,497), 865 (34.64%) were classified as having proteinuria. In the patients whose total burn surface areas (TBSA) were > 30% (n = 396), 271 patients (68.43%) had proteinuria and 152 of these patients (56.09%) met AKI criteria. No patients without proteinuria developed AKI. Intensive care unit (ICU) mortality rates were 0.8%, 16.67% and 30.77% (P < 0.001) in the groups with no, mild and heavy proteinuria, respectively. Logistic regression analysis identified proteinuria (OR 4.48; 95% CI, 2.824 to 7.108; P < 0.001) and sequential organ failure assessment (OR 1.383; 95% CI, 1.267 to 1.509; P < 0.001) as risk factors for AKI. CONCLUSIONS: We observed a high prevalence of proteinuria in patients with severe burns (> 30% TBSA). Severely burned patients with proteinuria had a high risk of developing AKI and a poor prognosis for survival. This suggests that proteinuria should be used for identifying burn patients at risk of developing AKI.
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Injúria Renal Aguda/epidemiologia , Queimaduras/epidemiologia , Proteinúria/epidemiologia , Índice de Gravidade de Doença , Injúria Renal Aguda/diagnóstico , Adulto , Queimaduras/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteinúria/diagnóstico , Estudos Retrospectivos , Adulto JovemRESUMO
The aim of this study is to investigate the effects of enalapril, an angiotensin-converting enzyme inhibitor, on multiple organ damage after scald injury. Healthy adult rats (half male and half female; 8-12 weeks old) were randomly assigned to the following treatments: sham operation, scald injury, and intraperitoneal enalapril (1, 2, and 4 mg/kg body weight) treatment after scalding. At 1, 12, and 24 H postscald, left ventricular and aortic hemodynamics were measured using a multichannel physiological recorder. Functional and pathological changes of the heart, liver, and kidney were examined by biochemical and histological methods. Compared with sham controls, untreated scalded animals showed decreased hemodynamic parameters and increased myocardial angiotensin II, serum creatine kinase heart isoenzyme, and serum cardiac troponin I and histopathological inflammation in the myocardium 12 H postscald. These hemodynamic, functional, and pathological changes were attenuated by 1 mg/kg enalapril. Enalapril reversed scald-induced elevations in aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and blood creatinine 12 H postscald, and ameliorated focal necrosis in the liver and erythrocyte cast formation in renal tubules. However, higher doses of enalapril yielded less or no improvement in organ dysfunction. Enalapril at 1 mg/kg attenuates scald-induced multiple organ damage in rats.
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Inibidores da Enzima Conversora de Angiotensina/farmacologia , Queimaduras/complicações , Enalapril/farmacologia , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/prevenção & controle , Peptidil Dipeptidase A/metabolismo , Animais , Biomarcadores/metabolismo , Feminino , Hemodinâmica/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Masculino , Insuficiência de Múltiplos Órgãos/patologia , Insuficiência de Múltiplos Órgãos/fisiopatologia , Especificidade de Órgãos , Ratos , Ratos Sprague-DawleyRESUMO
In both cardiomyocytes and HeLa cells, hypoxia (1% O(2)) quickly leads to microtubule disruption, but little is known about how microtubule dynamics change during the early stages of hypoxia. We demonstrate that microtubule associated protein 4 (MAP4) phosphorylation increases while oncoprotein 18/stathmin (Op18) phosphorylation decreases after hypoxia, but their protein levels do not change. p38/MAPK activity increases quickly after hypoxia concomitant with MAP4 phosphorylation, and the activated p38/MAPK signaling leads to MAP4 phosphorylation and to Op18 dephosphorylation, both of which induce microtubule disruption. We confirmed the interaction between phospho-p38 and MAP4 using immunoprecipitation and found that SB203580, a p38/MAPK inhibitor, increases and MKK6(Glu) overexpression decreases hypoxic cell viability. Our results demonstrate that hypoxia induces microtubule depolymerization and decreased cell viability via the activation of the p38/MAPK signaling pathway and changes the phosphorylation levels of its downstream effectors, MAP4 and Op18.
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Hipóxia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Estatmina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anaerobiose , Animais , Hipóxia Celular , Sobrevivência Celular , Células HeLa , Humanos , Microtúbulos/enzimologia , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
SITSH (syndrome of inappropriate secretion of thyrotropin) is a rare clinical state defined as uninhibited serum thyroid stimulating hormone in the presence of elevated thyroid hormone. This state is complicated and mainly caused by the abnormal feedback of hypothalamus-pituitary thyroid axis. The TSH adenoma (TSH-oma) and resistance to thyroid hormones (RTH) are the main etiologies of SITSH. As is well known that the treatment strategies of RTH and TSH-oma are apparently different, thus identifying the difference between RTH and TSH-oma is of great significance for the diagnosis and treatment of SITSH. CASE DESCRIPTION: A 62-year-old man with a state of elevated thyroid hormones and inappropriate elevated serum TSH level was hospitalized in 2016. Results of the pituitary enhanced magnetic resonance imaging and the somatostatin test respectively demonstrated a space-occupying lesion of pituitary and an elevated serum sex hormone binding globulin (SHBG) and inhibited TSH secretion, which indicated the occurrence of TSH-oma. In 2019, a 23-year-old girl with a state of elevated thyroid hormones and inappropriate normal serum TSH was hospitalized. Interestingly, whole exome sequencing detection suggested a pathogenic mutation in thyroid hormone receptor ß (THRB) gene, which has been shown to be associated with RTH. CONCLUSIONS: The difference between TSH-oma and RTH ought to be clarified for their accurate diagnose and treatment. The clinical experiences of the two cases reported here suggest that more detail information such as family medical history, serum SHBG level, and THRB gene test is helpful for the diagnose and treatment of TSH-oma and RTH. Additionally, we also summarized the identification points, diagnosis process, and treatment strategies for these two rare diseases.
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Adenoma , Neoplasias Hipofisárias , Síndrome da Resistência aos Hormônios Tireóideos , Adenoma/diagnóstico por imagem , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/diagnóstico por imagem , Síndrome da Resistência aos Hormônios Tireóideos/genética , Hormônios Tireóideos , Tireotropina , Adulto JovemRESUMO
Phosphorylation of MAP4 (p-MAP4) causes cardiac remodeling, with the cardiac microvascular endothelium being considered a vital mediator of this process. In the current study, we investigated the mechanism underlying p-MAP4 influences on cardiac microvascular density. We firstly confirmed elevated MAP4 phosphorylation in the myocardium of MAP4 knock-in (KI) mice. When compared with the corresponding control group, we detected the decreased expression of CD31, CD34, VEGFA, VEGFR2, ANG2, and TIE2 in the myocardium of MAP4 KI mice, accompanied by a reduced plasma concentration of VEGF. Moreover, we observed apoptosis and mitochondrial disruption in the cardiac microvascular endothelium of MAP4 KI animals. Consistently, we noted a decreased cardiac microvascular density, measured by CD31 and lectin staining, in MAP4 KI mice. To explore the underlying mechanism, we targeted the NLRP3-related pyroptosis and found increased expression of the corresponding proteins, including NLRP3, ASC, mature IL-1ß, IL-18, and GSDMD-N in the myocardium of MAP4 KI mice. Furthermore, we utilized a MAP4 (Glu) adenovirus to mimic cellular p-MAP4. After incubating HUVECs with MAP4 (Glu) adenovirus, the angiogenic ability was inhibited, and NLRP3-related pyroptosis were significantly activated. Moreover, both cytotoxicity and PI signal were upregulated by the MAP4 (Glu) adenovirus. Finally, NLRP3 inflammasome blockage alleviated the inhibited angiogenic ability induced by MAP4 (Glu) adenovirus. These results demonstrated that p-MAP4 reduced cardiac microvascular density by activating NLRP3-related pyroptosis in both young and aged mice. We thus managed to provide clues explaining MAP4 phosphorylation-induced cardiac remodeling and enriched current knowledge regarding the role of MAP4.
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1. Astragaloside IV (AST-IV) is purified from a natural plant product. Previous studies have shown that AST-IV has anti-oxidant activity. In the present study, we investigated the effect and mechanism of action AST-IV on rat cardiomyocytes subjected to hypoxic conditions (up to 12 h). 2. Cardiomyocytes were prepared from neonatal rats and cultured under normoxic or hypoxic conditions in the absence or presence of AST-IV (12.5, 25 or 50 microg/mL). Cell viability, malondialdehyde (MDA) levels, activity and expression of superoxide dismutase (SOD)-1 (mRNA and protein levels determined by reverse transcription-polymerase chain reaction and western blotting, respectively) and reactive oxygen species (ROS; determined by 2',7'-dichlorodihydrofluorescein diacetate) were investigated under these culture conditions. Intracellular localization of AST-IV was tested using fluorescein isothiocyanate-labelled AST-IV. 3. Hypoxic culture reduced the viability of cardiomyocytes, which was improved following treatment with 25 or 50 microg/mL AST-IV. Under hypoxic conditions, MDA levels were double those under control conditions. Astragaloside IV (25 and 50 microg/mL) dose-dependently reduced the increase in MDA seen in hypoxic cardiomyocytes. 4. Fluorescein isothiocyanate-labelled AST-IV entered cardiomyocytes and was localized mainly within the cytoplasm. 5. Under hypoxic conditions, SOD-1 activity was decreased, but mRNA and protein expression increased, compared with normoxia. Following treatment with 25 microg/mL AST-IV, SOD-1 activity and expression were increased under both normoxic and hypoxic conditions. The ROS scavenging effect of AST-IV was abolished in the presence of the SOD inhibitor sodium diethyl dithiocarbamate (25 micromol/L). 6. These in vitro results show that AST-IV protects cardiomyocytes from oxidative stress-mediated injury under hypoxic conditions. A major part of this action is achieved by upregulation of SOD-1 content and activity within the cell cytoplasm.
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Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Saponinas/farmacologia , Superóxido Dismutase/genética , Triterpenos/farmacologia , Animais , Animais Recém-Nascidos , Hipóxia Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoproteção/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Modelos Biológicos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Saponinas/farmacocinética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Triterpenos/farmacocinética , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the dose-effect relationship of enalaprilat (ENA) injection on the organ damage following early burn injury in rats. METHODS: A total of 54 SD rats were subjected to 30% total body surface area III scald injury, and were randomly divided into simple scald group (B group, with conventional fluid transfusion after scald), ENA treated group (E1, E2, E3 group, with intraperitoneal enalaprilat injection of 1, 2, 4 mg/kg after scald respectively). Other 6 rats were taken as normal control. Aortic systolic pressure (AOSP), aortic diastolic blood pressure (AODP), mean arterial pressure (MAP), angiotensin 1, blood urea nitrogen (Bun), creatinine (Cr), creatinine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST) of the simple scald group, E1 group, E2 group and E3 group were investigated at 6 h and 12 h post burn. RESULTS: Ang II, Bun, Cr, CK, ALT, AST levels in ENA treated group after 6 h and 12 hours were significantly lower than those of simple scald group (all P < 0.05). AOSP, AODP, MAP in ENA treated group after 6 and 12 hours were significantly higher than those of simple scald group (all P < 0.05). CONCLUSION: Low-dose enalaprilat, injection (1 mg/kg) could alleviate organ damage in post-burned rats, but has little effect on AOSP and AODP.
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Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Queimaduras/tratamento farmacológico , Enalaprilato/uso terapêutico , Vísceras/patologia , Animais , Queimaduras/sangue , Queimaduras/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Vísceras/efeitos dos fármacosRESUMO
(Macro)autophagy mediates the bulk degradation of defective organelles, long-lived proteins and protein aggregates in lysosomes and plays a critical role in cellular and tissue homeostasis. Defective autophagy processes have been found to contribute to a variety of metabolic diseases. However, the regulatory mechanisms of autophagy are not fully understood. Increasing data indicate that nicotinamide adenine nucleotide (NAD(+)) homeostasis correlates intimately with autophagy. NAD(+) is a ubiquitous coenzyme that functions primarily as an electron carrier of oxidoreductase in multiple redox reactions. Both NAD(+) homeostasis and its metabolism are thought to play critical roles in regulating autophagy. In this review, we discuss how the regulation of NAD(+) and its metabolism can influence autophagy. We focus on the regulation of NAD(+)/NADH homeostasis and the effects of NAD(+) consumption by poly(ADP-ribose) (PAR) polymerase-1 (PARP-1), NAD(+)-dependent deacetylation by sirtuins and NAD(+) metabolites on autophagy processes and the underlying mechanisms. Future studies should provide more direct evidence for the regulation of autophagy processes by NAD(+). A better understanding of the critical roles of NAD(+) and its metabolites on autophagy will shed light on the complexity of autophagy regulation, which is essential for the discovery of new therapeutic tools for autophagy-related diseases.
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Autofagia/fisiologia , NAD/metabolismo , NAD/fisiologia , Animais , Homeostase , Humanos , Poli(ADP-Ribose) PolimerasesRESUMO
Purpura fulminans (PF) is a life-threatening hemorrhagic condition. Because of the rarity and randomness of the disease, no improvement in treatment has been made for a long time. In this study, we assessed the serum proteome response to PF by comparing serum proteins between healthy controls and PF patient. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach was used after depleting 6 abundant proteins of serum. In total, 262 proteins were confidently identified with 2 unique peptides, and 38 proteins were identified significantly up- (≥ 2) or downregulated (≤ 0.5) based on spectral counting ratios (SpCPF/N). In the 38 proteins with significant abundance changes, 11 proteins were previously known to be associated with burn or sepsis response, but 27 potentially novel proteins may be specifically associated with PF process. Two differentially expressed proteins, alpha-1-antitrypsin (SERPINA1) and alpha-2 antiplasmin (SERPINF2), were validated by Western blot. This is the first study where PF patient and healthy controls are compared in a proteomic study to elucidate proteins involved in the response to PF. This study provides an initial basis for future studies of PF, and the differentially expressed proteins might provide new therapeutic targets to decrease the mortality of PF.
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Queimaduras/sangue , Púrpura Fulminante/sangue , Sepse/sangue , alfa 1-Antitripsina/sangue , alfa 2-Antiplasmina/metabolismo , Biomarcadores/sangue , Queimaduras/complicações , Estudos de Casos e Controles , Feminino , Expressão Gênica , Ontologia Genética , Humanos , Pessoa de Meia-Idade , Proteoma/genética , Proteoma/metabolismo , Púrpura Fulminante/microbiologia , Sepse/microbiologia , alfa 1-Antitripsina/genética , alfa 2-Antiplasmina/genéticaRESUMO
Hypoxia-induced microtubule disruption and mitochondrial permeability transition (mPT) are crucial events leading to fatal cell damage and recent studies showed that microtubules (MTs) are involved in the modulation of mitochondrial function. Dynein light chain Tctex-type 1 (DYNLT1) is thought to be associated with MTs and mitochondria. Previously we demonstrated that DYNLT1 knockdown aggravates hypoxia-induced mitochondrial permeabilization, which indicates a role of DYNLT1 in hypoxic cytoprotection. But the underlying regulatory mechanism of DYNLT1 remains illusive. Here we aimed to investigate the phosphorylation alteration of DYNLT1 at serine 82 (S82) in hypoxia (1% O2). We therefore constructed recombinant adenoviruses to generate S82E and S82A mutants, used to transfect H9c2 and HeLa cell lines. Development of hypoxia-induced mPT (MMP examining, Cyt c release and mPT pore opening assay), hypoxic energy metabolism (cellular viability and ATP quantification), and stability of MTs were examined. Our results showed that phosph-S82 (S82-P) expression was increased in early hypoxia; S82E mutation (phosphomimic) aggravated mitochondrial damage, elevated the free tubulin in cytoplasm and decreased the cellular viability; S82A mutation (dephosphomimic) seemed to diminish the hypoxia-induced injury. These data suggest that DYNLT1 phosphorylation at S82 is involved in MTs and mitochondria regulation, and their interaction and cooperation contribute to the cellular hypoxic tolerance. Thus, we provide new insights into a DYNLT1 mechanism in stabilizing MTs and mitochondria, and propose a potential therapeutic target for hypoxia cytoprotective studies.
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Hipóxia Celular , Dineínas/genética , Dineínas/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Serina/metabolismo , Animais , Hipóxia Celular/genética , Linhagem Celular , Sobrevivência Celular , Regulação da Expressão Gênica , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Mutagênese Sítio-Dirigida , Permeabilidade , Fosforilação , RatosRESUMO
BACKGROUND: Interactions between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXCR4 are crucial for the recruitment of mesenchymal stem cells (MSCs) from bone marrow (BM) reservoirs to damaged tissues for repair during alarm situations. MicroRNAs are differentially expressed in stem cell niches, suggesting a specialized role in stem cell regulation. Here, we gain insight into the molecular mechanisms involved in regulating SDF-1α. METHODS: MSCs from green fluorescent protein transgenic male mice were transfused to irradiated recipient female C57BL/6 mice, and skin burn model of bone marrow-chimeric mice were constructed. Six miRNAs with differential expression in burned murine skin tissue compared to normal skin tissue were identified using microarrays and bioinformatics. The expression of miR-27b and SDF-1α was examined in burned murine skin tissue using quantitative real-time PCR (qPCR) and immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA). The Correlation of miR-27b and SDF-1α expression was analyzed by Pearson analysis Correlation. miRNAs suppressed SDF-1α protein expression by binding directly to its 3'UTR using western blot and luciferase reporter assay. The importance of miRNAs in MSCs chemotaxis was further estimated by decreasing SDF-1α in vivo and in vitro. RESULTS: miR-23a, miR-27a and miR-27b expression was significantly lower in the burned skin than in the normal skin (p<0.05). We also found that several miRNAs suppressed SDF-1α protein expression, while just miR-27a and miR-27b directly bound to the SDF-1α 3'UTR. Moreover, the forced over-expression of miR-27a and miR-27b significantly reduced the directional migration of mMSCs in vitro. However, only miR-27b in burn wound margins significantly inhibited the mobilization of MSCs to the epidermis. CONCLUSION: miR-27b may be a unique signature of the stem cell niche in burned mouse skin and can suppress the directional migration of mMSCs by targeting SDF-1α by binding directly to its 3'UTR.
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Queimaduras/genética , Movimento Celular/genética , Quimiocina CXCL12/genética , Inativação Gênica , Células-Tronco Mesenquimais/patologia , MicroRNAs/genética , Cicatrização/genética , Animais , Queimaduras/patologia , Queimaduras/fisiopatologia , Quimiocina CXCL12/deficiência , Biologia Computacional , Regulação para Baixo , Feminino , Temperatura Alta , Masculino , Camundongos , Pele/patologiaRESUMO
The aim of this study was to address the effects of shaking stress (a.k.a. physical agitation) on burn-induced remote organ injury and to evaluate the application of delayed fluid resuscitation to treat severe burns under shaking conditions. Healthy adult male rabbits, weighing 2.50±0.40 kg, were randomly assigned to the following groups: control group, burn group, and burn+shaking group. One half of burned animals received a 6-h delayed fluid resuscitation and the other half remained untreated. Cardiovascular hemodynamics and functional and pathological changes of the heart and kidney were examined. Compared to normal controls, untreated burned animals showed decreased hemodynamic parameters, increased serum lactic acid, and severe myocardial inflammation. The burn-induced hemodynamic abnormalities and cardiac injury were aggravated by shaking stress. Burn injury led to reduced urine volume, elevated serum creatinine and blood urea nitrogen, and formation of erythrocyte casts in renal tubules. Shaking stimulation worsened the burn-associated functional and pathological changes of the kidney. Fluid resuscitation markedly mitigated cardiac and renal injury in burned animals, and, to a lesser extent, in the presence of shaking stimulation. Shaking stimulation aggravates burn-induced cardiovascular and renal disturbances. Delayed fluid resuscitation attenuates cardiac and renal damages in burn injury under shaking conditions.
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Queimaduras/complicações , Doenças Cardiovasculares/fisiopatologia , Nefropatias/fisiopatologia , Agitação Psicomotora/fisiopatologia , Estresse Fisiológico/fisiologia , Animais , Nitrogênio da Ureia Sanguínea , Queimaduras/terapia , Doenças Cardiovasculares/etiologia , Creatinina/sangue , Modelos Animais de Doenças , Hidratação , Hemodinâmica/fisiologia , Inflamação/terapia , Nefropatias/etiologia , Ácido Láctico/sangue , Masculino , CoelhosRESUMO
Mitochondrial damage plays an important role in mediating postburn cardiac injury. To elucidate the pivotal mitochondrial proteins and pathways underlying postburn cardiac injury, mitochondria were purified from control and postburn rat hearts. 2-dimensional gel electrophoresis (2-DE) and HPLC-chip-MS/MS analyses revealed 9 differentially expressed proteins, 3 of which were further validated by Western blotting. The differential expression of these mitochondrial proteins was accompanied by increased levels of oxidative cardiac damage and decreased levels of cardiac output. One of the differentially expressed proteins, mitochondria translation elongation factor Tu (EF-Tumt), was hypothesized to contribute crucially to postburn oxidative cardiac damage. The small interfering RNA (siRNA)-mediated downregulation of EF-Tumt in cultured rat cardiomyocytes increased reactive oxygen species (ROS) generation and protein carbonyl levels, and led to cell damage. The potential pathway of this process was associated with respiratory chain complex I deficiency. Together, these results demonstrate the mitochondrial responses to severe burn, and indicate a pathway by which decreased EF-Tumt expression mediates oxidative damage in postburn myocardium.
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Queimaduras/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Animais , Queimaduras/patologia , Complexo I de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica , Masculino , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondrial membrane permeability has received considerable attention recently because of its key role in apoptosis and necrosis induced by physiological events such as hypoxia. The manner in which mitochondria interact with other molecules to regulate mitochondrial permeability and cell destiny remains elusive. Previously we verified that hypoxia-induced phosphorylation of microtubule-associated protein 4 (MAP4) could lead to microtubules (MTs) disruption. In this study, we established the hypoxic (1% O(2)) cell models of rat cardiomyocytes, H9c2 and HeLa cells to further test MAP4 function. We demonstrated that increase in the pool of MAP4 could promote the stabilization of MT networks by increasing the synthesis and polymerization of tubulin in hypoxia. Results showed MAP4 overexpression could enhance cell viability and ATP content under hypoxic conditions. Subsequently we employed a yeast two-hybrid system to tag a protein interacting with mitochondria, dynein light chain Tctex-type 1 (DYNLT1), by hVDAC1 bait. We confirmed that DYNLT1 had protein-protein interactions with voltage-dependent anion channel 1 (VDAC1) using co-immunoprecipitation; and immunofluorescence technique showed that DYNLT1 was closely associated with MTs and VDAC1. Furthermore, DYNLT1 interactions with MAP4 were explored using a knockdown technique. We thus propose two possible mechanisms triggered by MAP4: (1) stabilization of MT networks, (2) DYNLT1 modulation, which is connected with VDAC1, and inhibition of hypoxia-induced mitochondrial permeabilization.
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Dineínas/metabolismo , Regulação da Expressão Gênica , Hipóxia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sobrevivência Celular , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia Confocal/métodos , Permeabilidade , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The aim of this study was to test the hypothesis that circulating factors released after a severe burn cause endothelial barrier dysfunction by triggering endothelial cell (EC) contraction through a p38 mitogen-activated protein (MAP) kinase-dependent mechanism. Human umbilical vein ECs (ECV304 cell line) were cultured to create a monolayer of cells that were then cultured with 20% human normal or burn serum. Monolayer permeability was measured by the influx of labeled albumin across the cells. Endothelial cells contraction was determined by alterations of cell surface area and formation of intracellular gaps. P38 MAP kinase activation, F-actin arrangement, and L-caldesmon phosphorylation were assessed by Western blots or immunofluorescence staining. These studies showed that exposure to burn serum resulted in a significant increase in endothelial permeability in a time-dependent manner, which was paralleled by a rapid and persistent activation of p38 MAP kinases. Morphologically, increased intercellular gaps, reduced cell surface area, and a unique rearrangement of F-actin cytoskeleton were observed in burn serum-treated ECs. Inhibition of p38 MAP kinase suppressed the rearrangement of F-actin cytoskeleton, reduced the occurrence of burn serum-induced formation of intercellular gaps, and ameliorated endothelial hyperpermeability. Further study showed that phosphorylation of L-caldesmon was enhanced in burn serum-treated cells via p38 MAP kinase; overexpression of L-caldesmon by adenovirus transfection, however, attenuated the increase in endothelial permeability by burn serum challenge. Collectively, these results have demonstrated for the first time that p38 MAP kinase is an important participant in mediating burn serum-induced endothelial barrier dysfunction through rearrangement of the F-actin cytoskeleton and phosphorylation of L-caldesmon. Inhibition of p38 MAP kinase in vivo, thus, would be a promising therapeutic strategy in ameliorating burn shock development.
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Actinas/metabolismo , Queimaduras/metabolismo , Queimaduras/patologia , Proteínas de Ligação a Calmodulina/metabolismo , Permeabilidade da Membrana Celular , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Queimaduras/sangue , Linhagem Celular , Células Cultivadas , Ativação Enzimática , Humanos , Fosforilação , Veias UmbilicaisRESUMO
OBJECTIVE: To investigate the influence of microtubule depolymerization of myocardial cells on distribution and activity of mitochondria, and energy metabolism of cells in adult rats. METHODS: Myocardial cells of SD adult rats and SD suckling rats were isolated and cultured. They were divided into adult and suckling rats control groups (AC and SC, normally cultured without any stimulating factor), adult and suckling rats microtubule depolymerization agent groups (AMDA and SMDA, cultured with 8 micromol/L colchicine containing nutrient solution for 30 minutes) according to the random number table. (1) The expression of polymerized beta tubulin in myocardial cells of adult and suckling rats was detected with Western blot. (2) Myocardial cells of rats in AC and AMDA groups were collected. The expression of cytochrome c was detected with Western blot. Distribution of voltage-dependent anion channels (VDAC) and polymerized beta tubulin in myocardial cells were observed with immunofluorescent staining. Mitochondrial inner membrane potential was determined with immunocytochemical method. Activity of myocardial cells was detected with MTT method. Contents of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) and energy charge of cells were determined with high performance liquid chromatography. RESULTS: (1) The expression of polymerized beta tubulin:in AMDA group it was 0.52 + or - 0.07, which was obviously lower than that (1.25 + or - 0.12) in AC group (F = 31.002, P = 0.000); in SMDA group it was 0.76 + or - 0.12, which was significantly lower than that (1.11 + or - 0.24) in SC group (F = 31.002, P = 0.000), but was obviously higher than that in AMDA group (F = 31.002, P = 0.009). (2) The expression of cytochrome c in AC group was 0.26 + or - 0.03, which was obviously lower than that (1.55 + or - 0.13) in AMDA group (t = -24.056, P = 0.000). (3) Immunofluorescent staining result: in AC group, microtubules of myocardial cells were in linear tubiform, distributed in parallel with myocardial fiber; VDAC staining result showed that mitochondria were in granular form, distributed in the same direction as microtubules. In AMDA group, the normal distribution regularity of microtubules was destroyed, with weakened immune fluorescence intensity, microtubules structure indistinct, continuity lost, rough in appearance, and the distribution of mitochondria became disrupted. (4) Mitochondrial inner membrane potential in AC group fluorescent intensity was 1288 + or - 84, which was obviously higher than that (331 + or - 27) in AMDA group (t = 26.508, P = 0.000). (5) Cellular activity: in AC group absorbance value was 1.75 + or - 0.11, which was obviously lower than that (0.81 + or - 0.07) in AMDA group (t = 17.348, P = 0.000). (6) Energy metabolism: compared with those in AC group, content of ATP decreased, contents of ADP and AMP increased, and ATP/ADP value and energy charge decreased in AMDA group. CONCLUSIONS: Microtubules and mitochondria distribute in the same direction in normal myocardial cells in adult rats. After microtubule depolymerization, mitochondria are arranged in disorder fashion; cytochrome c leaks from mitochondria; mitochondrial membrane potential, energy supply, and cellular activity decrease in the myocardial cells.
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Metabolismo Energético , Microtúbulos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , Masculino , Potencial da Membrana Mitocondrial , Ratos , Ratos Sprague-Dawley , Tubulina (Proteína)/metabolismoRESUMO
OBJECTIVE: To investigate the therapeutic effects of Enalaprilat on the myocardial kinetics in rats at early stage of severe scald. METHODS: Eighty-four SD rats were inflicted with 30% TBSA full-thickness scald, and randomly divided into scald (S, with intraperitoneal injection of isotonic saline according to Parkland formula, n=30), L (n=30), M (n=12) and H (n=12) groups. The rats in L,M,H groups were intraperitoneally injected with 1,2,4 mg/kg Enalaprilat. Other 6 healthy rats were enrolled into study as control (C group). The myocardial kinetic parameters including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), +/- dp/dt max and the levels of A II in myocardium were observed at 1,3,6,12 and 24 post scald hour (PBH) in L and S groups,and at 6,12 PBH in M and H groups. The above indices in C group were also examined. RESULTS: The levels of LVSP, LVEDP, +/- dp/dt max in C group were higher than those in other groups during 3-24 PBH (P < 0.05 or P < 0.01), while those in L,M,H groups were obviously higher than those in S group (P < 0.05 or P < 0.01). The level of +/- dp/dt max in H group at 6,12 PBH were obviously lower than those in L and M groups. The level of A II in S group at 1 PBH was (53.0 +/- 2.6) pg/200 mg, which was significantly higher than thatin C group [(14.8 +/- 0.7) pg/200 mg, P < 0.05 or P < 0.01]; it peaked at6 PBH and lowered afterwards, and they were significantly higher than that in C group at 24 PBH (P < 0.01). The levels of A II in L group during 3-24 PBH were obviously higher than those in C group (P < 0.01), which were also lower than those in S group. The level of A II in S group was significantly higher than in L,M,H groups at 6 PBH [(145.2 +/- 14.5) pg/200 mg. vs. (65.1 +/- 0.9) pg/200 mg, (53.6 +/- 1.1) pg/200 mg, (34.2 +/- 0.9) pg/200 mg, respectively, P < 0.01]. CONCLUSION: Myocardium can be obviously damaged at early stage after severe scald,cardiac function is impaired. Enalaprilat injection (especially at low dose) can significantly ameliorate the myocardial kinetics indices, and it seems to exert a protective effect on cardiac function.
Assuntos
Queimaduras/fisiopatologia , Enalaprilato/farmacologia , Miocárdio/patologia , Animais , Queimaduras/tratamento farmacológico , Relação Dose-Resposta a Droga , Enalaprilato/uso terapêutico , Ratos , Ratos Sprague-Dawley , Remodelação VentricularRESUMO
OBJECTIVE: To investigate and compare the protective effects of Astragaloside IV (AST) and Quercetin (QUE) on rat myocardial cells after their exposure to hypoxia, and to determine their dose-effect relationship. METHODS: Myocardial cells from fetal SD rat were cultured in vitro and divided into 7 groups: i.e. A (hypoxia), B (hypoxia and 100 mg/L of QUE), C (hypoxia and 50 mg/L of QUE), D (hypoxia and 25 mg/L of QUE), E (hypoxia and 50.0 mg/L of AST), F (hypoxia and 25.0 mg/L of AST), G (hypoxia and 12.5 mg/L AST) H(hypoxia and 10 mg/L of VitE) groups. Different doses of AST and QUE were added into the culture media cells in each group before the myocardial cells receiving hypoxia for 12 hrs. The number of viable cells (CCK-8) and the content of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), active oxygen (ROS, with detection only in A, C, F and H groups) were determined after hypoxia. RESULTS: The amount of LDH, MDA, ROS (C, F groups) in group B - G decreased significantly compared with those of group A, while the number of viable cells and the SOD content increased significantly. The protective effects were better in group B - G than that of the group H. With the same dosage, levels of LDH, CCK-8 in AST-treated groups were significantly lower than those in QUE-treated group (the number of viable cells in group C, F was 0.454 +/- 0.018, 0.471 +/- 0.017, and the content of lactate dehydrogenase was 2800 +/- 9,2312 +/- 52). There were no significant differences in MDA, SOD and ROS levels between AST and QUE treated groups (ROS in C and F groups were 16.0 +/- 5.3 vs 22.4 +/- 8.7, P > 0.05). CONCLUSION: AST and QUE might be beneficial in the protection of myocardial cells against hypoxia because of attenuation of oxidative damage. The protective effects of both AST and QUE are better than that of VitE, and that of AST is better than QUE as shown by a decrease in the amount of LDH and increase in the number of viable cells with the same dosage, but no obvious difference is shown between them in attenuating oxidative damage.
Assuntos
Miócitos Cardíacos/efeitos dos fármacos , Quercetina/farmacologia , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Hipóxia Celular , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To investigate the preventive and therapeutic effects of enalapril maleate (Enalaprilat) (E) on myocardial damage in early stage after burns. METHODS: A total of 60 SD rats were subjected to 30% TBSA III degree scald injury, and randomly divided into scald group (with conventional fluid transfusion after scald) and ENA group (with intraperitoneal injection of 1 mg/kg Enalaprilat after scald). Normal control consisted of 6 rats. Plasma levels of cTnI and CK-MB were determined in all the groups at 1, 3, 6, 12, 24 post-scald hours (PSH) by enzyme linked immunosorbent assay. The pathological changes in myocardium were observed at the same time-points. RESULTS: (1) The serum level of cTnI and CK-MB in scald group were significantly higher than that of normal controls at each time-point (P < 0.01). The serum level of cTnI and CK-MB in ENA group were (1.32 +/- 0.12 microg/L to 2.47 +/- 0.22 microg/L) and (438 +/- 68 U/L to 5569 +/- 322 U/L), respectively, which were obviously lower than those in B group (6.42 +/- 0.96 microg/L to 15.10 +/- 3.69 microg/L) and (2556 +/- 74 U/L to 8047 +/- 574 U/L, P < 0.05 or P < 0.01) at different time-points. (2) Compared with normal controls, cloudy swelling, stromal blood vessel dilatation and congestion inflammatory cell infiltration were observed in scald group, but these pathological changes were less marked in ENA group. CONCLUSION: Severe myocardial damage in rat occurred early after burns. Enalaprilat injection can markedly alleviate myocardial damage.