RESUMO
BACKGROUND/AIMS: The phosphatidylinositol-3-kinase -AKT (PI3K-AKT) is an important intracellular signal pathway in regulating cell proliferation, differentiation and apoptosis. In previous studies, we've demonstrated that PI3K-AKT pathway protects cardiomyocytes from ischemic and hypoxic apoptosis through mitochondrial function. However, the molecular mechanisms underlying hypoxia-induced cardiomyocyte apoptosis via PI3K-AKT pathway remain ill-defined. Here, we addressed this question. METHODS: Cardiomyocytes were exposed to hypoxia, with/without different inhibitors and then protein levels were assessed by Western blotting. RESULTS: We found that the PI3K-AKT pathway was activated in cardiomyocytes that were exposed to hypoxia. Moreover, the phospho-AKT (pAKT) translocated from cytosol to mitochondria via mitochondrial adenosine triphosphate-dependent potassium (mitoKATP), leading to an increase in cytochrome c oxidase (CcO) activity to suppress apoptosis. On the other hand, the mitoKATP specific blocker, 5-hydroxydecanote (5-HD), or suppression of CcO using siRNA, inhibited the pAKT mitochondrial translocation to maintain the CcO activity, resulting in mitochondrial dysfunction and cellular apoptosis induced by hypoxia. CONCLUSION: These findings suggest that the anti-apoptotic effect of the PI3K-AKT pathway through pAKT translocation to mitochondrial via mitoKATP may be conducted through modification of CcO activity.
Assuntos
Apoptose , Hipóxia Celular , Fosfatidilinositol 3-Quinases/metabolismo , Canais de Potássio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Células Cultivadas , Cromonas/farmacologia , Ácidos Decanoicos/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidroxiácidos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Morfolinas/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Canais de Potássio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Microtubules of cardiac myocytes depolymerize after a hypoxic insult or treatment with colchicine. However, little attention has been paid to quantifying changes in microtubule distribution when using fluorescent images. We converted fluorescence images of labeled microtubules in H9C2 cardiac myocytes to grayscale images, then filtered the images to remove any noise, and used grayscale histograms to quantify features of the images. The results show that parameters such as the mean, variance, skewness, kurtosis, energy, and entropy can be used to quantitatively describe the distribution of microtubules in cells. Quantitative characteristics of microtubule distribution were similar after culturing cells under hypoxic conditions or after treatment with colchicine. These results parallel those described for neonatal rat cardiac myocytes following ischemia and hypoxia. In addition, we provide a method for internal segmentation of the cells, which revealed that microtubular depolymerization was more evident near the cell membrane following hypoxia or colchicine treatment.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência , Microtúbulos/metabolismo , Miócitos Cardíacos/química , Animais , Linhagem Celular , Colchicina/metabolismo , Hipóxia , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Coloração e Rotulagem/métodos , Moduladores de Tubulina/metabolismoRESUMO
Microtubule analysis is of significant value for a better understanding of normal and pathological cellular processes. Although immunofluorescence microscopic techniques have proven useful in the study of microtubules, comparative results commonly rely on a descriptive and subjective visual analysis. We developed an objective and quantitative method based on image processing and analysis of fluorescently labeled microtubular patterns in cultured cells. We used a multi-parameter approach by analyzing four quantifiable characteristics to compose our quantitative feature set. Then we interpreted specific changes in the parameters and revealed the contribution of each feature set using principal component analysis. In addition, we verified that different treatment groups could be clearly discriminated using principal components of the multi-parameter model. High predictive accuracy of four commonly used multi-classification methods confirmed our method. These results demonstrated the effectiveness and efficiency of our method in the analysis of microtubules in fluorescence images. Application of the analytical methods presented here provides information concerning the organization and modification of microtubules, and could aid in the further understanding of structural and functional aspects of microtubules under normal and pathological conditions.
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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.
Assuntos
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.
Assuntos
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
BACKGROUND: Ischemic/hypoxic myocardial damage and functional impairment of the myocardium occurs immediately after major burns. This experimental study investigated whether the prompt cardiac dysfunction initiates hepatic, renal, and intestinal injuries soon after a severe burn. METHODS: Wistar rats were randomized to a sham burn group, a burn group (subjected to 30% total body surface area third-degree burn) that was subdivided into two groups: a simple burn group, observed at 0.5 hour, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours postburn and a group medicated with propranolol (a cardiac inhibitor), cedilanid (a cardiotonic agent), enalaprilat (an angiotensin converting enzyme inhibitor), and cedilanid plus enalaprilat injected at 0.5 hour postburn and observed at 6 hours later. Serum cardiac troponin I, total bile acid, beta2-microglobulin concentrations, and diamine oxidase activity were measured to reflect the severity of cardiac, hepatic, renal, and intestinal injuries that were confirmed by histopathologic observations. Cardiac function and organs' blood flow were also recorded. RESULTS: Histopathologic changes and serum cardiac troponin I increase occurred significantly earlier than the other organs, and the organ damage developments followed a similar pattern. Myocardial injury was significantly aggravated in rats treated with propranolol, with further decreases in myocardial function, blood flow to the liver, kidneys, and intestines significantly decreased, and injuries were aggravated. In contrast, these conditions were greatly improved in the rats treated with enalaprilat, cedilanid, or with both. CONCLUSION: The prompt cardiac dysfunction has some initiating effects on ischemic/hypoxic injury to organs such as the liver, kidneys, and intestines soon after a severe burn.
Assuntos
Queimaduras/complicações , Intestinos/patologia , Rim/patologia , Miocárdio/patologia , Animais , Queimaduras/patologia , Queimaduras/fisiopatologia , Débito Cardíaco , Circulação Coronária , Mucosa Intestinal/metabolismo , Intestinos/irrigação sanguínea , Rim/irrigação sanguínea , Rim/metabolismo , Fígado , Masculino , Contração Miocárdica , Miocárdio/metabolismo , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional , Fatores de TempoRESUMO
Hypoxia-inducible factor (HIF)-1alpha is a key regulator of anaerobic energy metabolism. We asked the following question: Does the breakdown of microtubular structures influence glycolysis in hypoxic cardiomyocytes by regulating HIF-1alpha? Neonatal rat cardiomyocytes were cultured under hypoxic conditions, while microtubule-stabilizing (paclitaxel) and -depolymerizing (colchicine) agents were used to change microtubular structure. Models of high microtubule-associated protein 4 (MAP4) expression and RNA interference of microtubulin expression were established. Microtubular structural changes and intracellular HIF-1alpha protein distribution were observed with laser confocal scanning microscopy. Content of key glycolytic enzymes, viability, and energy content of cardiomyocytes were determined by colorimetry and high-performance liquid chromatography. HIF-1alpha protein content and mRNA expression were determined by Western blotting and real-time PCR, respectively. Low doses of microtubule-stabilizing agent (10 mumol/l paclitaxel) and enhanced expression of MAP4 stabilized the reticular microtubular structures in hypoxic cardiomyocytes, increased the content of key glycolytic enzymes, ameliorated energy supply and enhanced cell viability, and upregulated HIF-1alpha protein expression and endonuclear aggregation. In contrast, the microtubule-depolymerizing agent (10 mumol/l colchicine) or reduced microtubulin expression had adverse affects on the same parameters, in particular, HIF-1alpha protein content and endonuclear aggregation. We conclude that microtubular structural changes influence glycolysis in the early stages of hypoxia in cardiomyocytes by regulating HIF-1alpha content. Stabilizing microtubular structures increases endonuclear and total HIF-1alpha expression, content of key glycolytic enzymes, and energy supply. These findings provide potential therapeutic targets for ameliorating cell energy metabolism during early myocardial hypoxia.
Assuntos
Núcleo Celular/metabolismo , Glicólise/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Microtúbulos/fisiologia , Miócitos Cardíacos/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colchicina/farmacologia , Relação Dose-Resposta a Droga , Metabolismo Energético/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Microtúbulos/efeitos dos fármacos , Modelos Animais , Miócitos Cardíacos/citologia , Paclitaxel/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Moduladores de Tubulina/farmacologiaRESUMO
1. After a severe burn, a marked decrease in myocardial blood flow results in ischaemic and hypoxic injury, which subsequently leads to apoptosis or necrosis. Phosphatidylinositol 3-kinase (PI3-K)/Akt is an important intracellular signal transduction molecule that regulates cell proliferation, differentiation, glucose metabolism and migration. However, the function and mechanisms of the PI3-K-Akt pathway in cardiomyocyte apoptosis after a burn remain unclear. 2. In the present study, an in vivo rat model of burn injury and an in vitro hypoxic model using rat cardiomyocytes were established. In burned rats, the expression of PI3-K and phosphorylated (p-) Akt expression increased, as did myocardial apoptosis. Inhibition of the PI3-K-Akt pathway with 1.4 mg/kg LY294002 caused a significant increase in the myocardial apoptotic index compared with hypoxia alone in the in vivo model. 3. Cardiomyocytes cultured under hypoxic conditions exhibited increased apoptosis, decreased cell viability, enhanced caspase 3 activity, a decreased mitochondrial membrane potential, increased cytoplasmic calcium transients and increased p53 and Bax mRNA expression. Pretreatment with 50 mumol/L LY294002 significantly enhanced all these negative indicators compared with hypoxia alone. In contrast, pretreatment of cells with 200 ng/mL insulin-like growth factor-1, an activator of PI3-K-Akt, significantly ameliorated the effects of hypoxia, although control levels were not reached. 4. These findings indicate that activation of the PI3-K-Akt pathway induced by ischaemia and hypoxia after a severe burn can protect cardiomyocytes from apoptosis. This anti-apoptotic effect is most likely mediated via the mitochondria and changes in p53 and Bax gene expression, intracellular [Ca(2+)] and caspase 3 activity.
Assuntos
Apoptose , Queimaduras/complicações , Mitocôndrias Cardíacas/fisiologia , Isquemia Miocárdica/prevenção & controle , Miócitos Cardíacos , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Queimaduras/enzimologia , Queimaduras/patologia , Queimaduras/fisiopatologia , Cálcio/metabolismo , Caspase 3/metabolismo , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Morfolinas/farmacologia , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/etiologia , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinases/biossíntese , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/biossínteseRESUMO
OBJECTIVE: To investigate the preventive effect of ulinastatin on shock in the heart after burn. METHODS: In an open prospective clinical study 34 adults with burns >50% total body surface area were randomly divided into control (B) and ulinastatin-treated (U) groups. All underwent routine treatment, and group U received 100,000U ulinastatin intravenously three times a day for 1 week. In an animal experiment, 72 healthy rats underwent equivalent burn, similar division into groups B and U, and resuscitation according to Parkland's formula. Rats in group U received ulinastatin (40,000U/kg) immediately after burn. Myocardial pathomorphology, plasma cTnI, CK-MB and PMNE, myocardial MDA, TNF-alpha, IL-10 and caspase-3 activity and cardiocyte apoptosis were determined. RESULTS: Plasma cTnI, CK-MB, and PMNE were higher in clinical group B than group U. In the animal experiment, plasma cTnI, CK-MB, myocardial MDA, TNF-alpha, IL-10 and caspase-3 activity, and apoptotic index and myocardial pathomorphological changes were significantly less in group U than in group B, save IL-10. CONCLUSION: The clinical and experimental data showed that ulinastatin relieved myocardial damage from severe burn. The mechanism might involve modulation of the anti- and pro-inflammatory balance and lipid peroxidation, and decreased myocardiocyte apoptosis.
Assuntos
Queimaduras/complicações , Cardiotônicos/uso terapêutico , Glicoproteínas/uso terapêutico , Choque Cardiogênico/prevenção & controle , Adulto , Animais , Apoptose/efeitos dos fármacos , Queimaduras/sangue , Queimaduras/patologia , Cardiotônicos/administração & dosagem , Modelos Animais de Doenças , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Feminino , Glicoproteínas/administração & dosagem , Humanos , Mediadores da Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/sangue , Isquemia Miocárdica/etiologia , Isquemia Miocárdica/patologia , Isquemia Miocárdica/prevenção & controle , Estudos Prospectivos , Ratos , Ratos Sprague-Dawley , Choque Cardiogênico/sangue , Choque Cardiogênico/etiologia , Choque Cardiogênico/patologia , Troponina I/sangue , Adulto JovemRESUMO
Acute burn injuries are among the most devastating forms of trauma and lead to significant morbidity and mortality. Appropriate fluid resuscitation after severe burn, specifically during the first 48 hours following injury, is considered as the single most important therapeutic intervention in burn treatment. Although many formulas have been developed to estimate the required fluid amount in severe burn patients, many lines of evidence showed that patients still receive far more fluid than formulas recommend. Overresuscitation, which is known as "fluid creep," has emerged as one of the most important problems during the initial period of burn care. If fluid titration can be personalized and automated during the resuscitation phase, more efficient burn care and outcome will be anticipated. In the present study, a dynamic urine output based infusion rate prediction model was developed and validated during the initial 48 hours in severe thermal burn adult patients. The experimental results demonstrated that the developed dynamic fluid resuscitation model might significantly reduce the total fluid volume by accurately predicting hourly urine output and has the potential to aid fluid administration in severe burn patients.
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Queimaduras/tratamento farmacológico , Hidratação , Ressuscitação , Adolescente , Adulto , Pressão Sanguínea/efeitos dos fármacos , Queimaduras/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
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
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.
Assuntos
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 influence of microtubule intervention drugs on glycolytic key enzymes in myocardial cells after hypoxia. METHODS: The primary passage of cultured myocardial cells from neonatal rats were divided into A group (with hypoxia), B group (with hypoxia and administration of l0 micromol/L colchicine), C group (with hypoxia and administration of 5 micromol/L taxol), D group (with hypoxia and administration of 10 micromol/L taxol), E group (with hypoxia and administration of 15 micromol/L taxol). The morphology of microtubule was observed with laser scanning microscope (LSM). The cell vitality was assayed by cell counting kit (CCK). The activities of hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were assayed with colorimetry. RESULTS: In group B and E, the microtubule structure was damaged heavily, and the cell vitality was decreased significantly [The cell vitality was (89.99 +/- 3.47)% in B group and (84.56 +/- 6.61)% in E group, respectively, at 1.0 post hypoxia hour (PHH), and hoth values were obviously lower than that in A group (97.44 +/- 1.76)%, P < 0.01]. The HK, PK and PFK activities decreased obviously. The activities of HK, PK and PFK in group C were similar to those of the A group. Compared with that in other groups, the degree of damage of microtubule structure in D group was milden. The activities of HK, PK and PFK in D group during 0.5 - 6.0 PHH were significantly higher than those in A group. The activity of LDH in each group was increased after hypoxia. CONCLUSION: Proper concentration of microtubule-stabilizing drugs can alleviate the damages to microtubule structure, and enhance the activity of glycolytic key enzymes of myocardial cells at early stage of hypoxia.
Assuntos
Glicólise/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , Hexoquinase/metabolismo , L-Lactato Desidrogenase/metabolismo , Microtúbulos/metabolismo , Miócitos Cardíacos/enzimologia , Fosfofrutoquinase-1/metabolismo , Piruvato Quinase/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To investigate the effects of glycine on apoptosis in murine cardiomyocyte suffering from ischemia and hypoxia. METHODS: The primary passage of cultured cardiomyocytes from neonatal rats were subjected to ischemia and hypoxia, and the cells were divided into IH (without other treatment), and G (with treatment of 5 mmol/L glycine) groups. Normal murine cardiomyocytes served as control (C group). Cardiomyocytes were cultured for 6 hours in vitro. Apoptosis, mitochondrial membrane potential and its distribution, the condition of mitochondria permeability transition pore (mPTP) were observed with expression of fluorescence intensity. The activity of caspase-3 was observed by Laser Scanning staining. RESULTS: (1) Apoptosis: the fluorescence intensity in IH group was obviously higher than that in G and C groups (P < 0.01). (2) Mitochondrial membrane potential: the fluorescence intensity in IH group was 32 +/- 7, which was obviously lower than that in G and C groups (52 +/- 4, 73 +/- 4, respectively, P < 0.01). (3) The condition of mPTP: the intensity in IH group was 27 +/- 4, which was obviously lower than that in G and C groups (62 +/- 8, 90 +/- 7, respectively, P < 0.01). (4) The activity of caspase-3: the activity of caspase-3 in IH group was obviously higher than that in G and C groups (P < 0.01). CONCLUSION: Glycine can inhibit apoptosis in cardiomyocytes subjected to ischemia and hypoxia,and the effect may be attributable to changes in mitochondrial membrane potential, lessening opening of mPTP, alleviation of calcium overload , and decrease in activity of caspase-3.
Assuntos
Apoptose , Glicina/farmacologia , Isquemia/metabolismo , Miócitos Cardíacos/citologia , Animais , Caspase 3/metabolismo , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
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 the influence of human C-type natriuretic peptide (hCNP) on proliferation of vascular endothelial cells (HUVECs). METHODS: Reconstructed pcDNA3.1 (+)/hCNP was transfected into HUVECs with polyethylenimine and its plasmid expression was examined with RT-PCR, immunohistochemistry and Western blot. MTT method was used to determine the effect of expressed protein on proliferation of HUVECs. pcDNA3.1 (+)/hCNP transfection was used for control. RESULTS: The proliferation of HUVEC 48 h after pcDNA3.1 (+)/hCNP transfection was (0.301 +/- 0.096), which was obviously higher than that with pcDNA3.1 (+) transfection (0.164 +/- 0.012). Reconstructed pcDNA3.1 (+)/hCNP might be expressed in HUVECs effectively and its protein expression was capable of promoting HUVECs proliferation markedly. CONCLUSION: The successive expression of reconstructed pcDNA3.1 (+)/hCNP and the promoting activity of its expressed protein on HUVECs lay the foundation potential therapeutic value of C-type natriuretic peptide.
Assuntos
Proliferação de Células , Células Endoteliais/citologia , Peptídeo Natriurético Tipo C/genética , Linhagem Celular , Humanos , Plasmídeos , RNA Mensageiro/genética , TransfecçãoRESUMO
OBJECTIVE: To investigate the influence of microtubule intervention drugs on the energy metabolism of myocardial cells after hypoxia. METHODS: The primary passage of cultured myocardial cells from neonatal rats were divided into A (with hypoxia), B (with hypoxia and administration of 10 micromol/ml colchicine), C (with hypoxia and administration of 5 micromol/ml taxol), D (with hypoxia and administration of 10 micromol/ml taxol) and E (with hypoxia and administration of 15 micromol/ml taxol) groups. The creatine kinase (CK) activity and contents of ATP and ADP were assayed with colorimetry and HPLC, respectively, and the vitality of myocardial cells were determined by trypan blue method at 0.5, 1.0, 3.0, 6.0, 12.0, 24.0 post-hypoxia hours (PHH). RESULTS: The mortality was obviously higher in B and E groups than those in A group( P < 0.05) at each time-points, but that in C and D groups were markedly lower than those in A group during 6.0 to 24.0 PHH (P < 0.01). The CK activity was significantly higher in B group than that in A group during 1.0 to 24.0 PHH, while that in E group was evidently higher, but it was lower in C and D groups than that in A group at each time-points (P < 0.05 or 0.01). The ATP contents in C group during 0.5 to 6.0 PHH were [(49.9 +/- 2.8), (40.7 +/- 2.0), (25.8 +/- 1.9), (19.1 +/- 1.2) microg/10(6) cells, respectively], which were obviously higher than those in A group [(42.9 +/- 5.8), (29.5 +/- 1.8), (18.2 +/- 0.9), (14.1 +/- 0.7) microg/10(6) cells, respectively, P < 0.05 or P < 0.01, and those in E group at each time-point were significantly lower than those in A and D groups (P < 0.01). The changes in the contents of ADP were on the contrary to the above. CONCLUSION: Microtubule-destabilizing drugs and high concentration microtubule-stabilizing drugs can sharply decrease ATP content in myocardiocytes under hypoxic conditions, while suitable amount of microtubule-stabilizing drugs can protect myocardiocytes by promoting its energy production.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , Colchicina/farmacologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Paclitaxel/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To investigate the role of p38 mitogen activated protein kinase ( p38 MAPK) in the regulation of cytosolic phospholipase A2 ( cPLA2 ) expression and degradation of membrane phospholipids in myocardium in early stage of burn rats. METHODS: Wistar rats were randomized into normal group (n = 8), burn(n =40) , burn and SB203580(n = 16), burn and isotonic saline( n = 16) groups, with 8 rats at each time-points. There were 5 time-points in burn group, and 2 time-points in other groups. The rats in the latter 3 groups were inflicted with 40% TBSA full-thickness burns, and those in burn and SB203580, burn and isotonic saline groups were administered with SB203580 (p38 MAPK inhibitor) or isotonic saline, respectively. The levels of cPLA2 mRNA and membrane phospholipids in myocardium were detected with RT-PCR. In the same experiment, the effect of SB203580 on cPLA2 expression in rat myocardial cells was determined after hypoxia and burn serum treatment in vitro. RESULTS: The level of myocardial cPLA2 mRNA in burn group at each time-point was obviously higher than those in normal group (0. 280 +/- 0. 020) , and it reached the peak value at 3 PBH. In contrast, the level of cardiac membrane phospholipids was lowered immediately after burns, and it reached the lowest level at 6 PBH [(0. 052 +/- 0. 017) mg phosphorus/mg protein]. Herein, a significant negative correlation was showed between the levels of cPLA2 mRNA and cardiac membrane phospholipids ( r = - 0. 53, P < 0. 05). Administration of SB203580, however, inhibited the increased activity of p38 MAP kinase, suppressed the upregulation of cPLA2(72% and 51% of those in burn and saline group, P <0. 01) , and markedly increased the levels of membrane phospholipids in myocardium at 6 and 12 PBH. In addition, treatment of cardiac myocytes with SB203580 also abolished the upregulation of cPLA2 mRNA elicited by hypoxia and burn serum challenge. CONCLUSION: p38 MAP kinase play an important role in the burn-induced degradation of cardiac membrane phospholipids in rat through the upregulation of myocardial expression of cPLA2 mRNA in the myocardial cells.
Assuntos
Queimaduras/metabolismo , Miócitos Cardíacos/metabolismo , Fosfolipases A2/metabolismo , Fosfolipídeos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Modelos Animais de Doenças , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
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.
Assuntos
Queimaduras/tratamento farmacológico , Enalapril/uso terapêutico , Miócitos Cardíacos/metabolismo , Animais , Queimaduras/sangue , Queimaduras/patologia , Creatina Quinase Forma MB/sangue , Miócitos Cardíacos/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Troponina I/sangueRESUMO
OBJECTIVE: To investigate the alleviation of myocardial injury of rats after early escharectomy en masse of severe burns, and to explore its molecular mechanism. METHODS: Totally 66 SD rats were randomly divided into normal control (n=6), non-escharectomy (NE, n=30) and escharectomy (E, n=30, with total escharectomy 20 minutes after burns ) groups. The rats in the NE and E groups were inflicted with 30% TBSA full-thickness scald. The content of ATP in mitochondria, troponin I (Tn I) in serum and 4.8-kb deletion of myocardial mitochondrial DNA (mtDNA) of the rats in each group were determined at 1, 3, 6, 12 and 24 post-scald hours (PSH). RESULTS: (1) The content of ATP in myocardial mitochondria was decreased in both E and NE groups, but it was obviously increased at 1 and 6 PSH (0.90 +/- 0.27 microg/mg 0.66 +/- 0.19 microg/mg) in E group when compared with those in NE group (0.74 +/- 0.18 microg/mg, 0.46 +/- 0.21 microg/mg, P < 0.05). (2) There was no obvious change in the serum content of Tn I in E group at 1 and 3 PSH, but the respective content in 1, 3 and 6 PSH was markedly lower than those in NE group (P < 0.05). (3) The 4.8 kb deletion of myocardial mtDNA was found at 1, 3, 24 PSH in NE group, while it was observed only at 1, 12 PSH in E group. The partial and whole deletion rate in E group was lower than that in NE group. CONCLUSION: Early escharectomy en masse can significantly alleviate the myocardial injury after burns,which might be related to its effect in lowering the deletion rate of myocardial mtDNA at early postburn stage.