ABSTRACT
This study aimed to investigate the protective effect and its underlying mechanism of n-butanol extract of Pulsatilla Decoction(BEPD) containing medicinal serum on vaginal epithelial cells under Candida glabrata stimulation via the epidermal growth factor receptor/mitogen activated protein kinase( EGFR/MAPK) pathway based on transcriptomics. A vulvovaginal candidiasis(VVC) mouse model was established first and transcriptome sequencing was performed for the vaginal mucosa tissues to analyze the gene expression differences among the control, VVC model, and BEPD intervention groups. Simultaneously, BEPD-containing serum and fluconazole-containing serum were prepared. A431 cells were divided into the control, model, blank serum, fluconazole-containing serum, BEPD-containing serum, EGFR agonist and EGFR inhibitor groups. Additionally, in vitro experiments were conducted using BEPD-containing serum, fluconazole-containing serum, and an EGFR agonist and inhibitor to investigate the intervention mechanisms of BEPD on C. glabrata-induced vaginal epithelial cell damage. Cell counting kit-8(CCK-8) assay was utilized to determine the safe concentrations of C. glabrata, drug-containing serum, and compounds on A431 cells. Enzyme-linked immunosorbent assay(ELISA)was employed to measure the expression levels of interleukin(IL)-1ß, IL-6, granulocyte-macrophage colony-stimulating factor(GMCSF), granulocyte CSF(G-CSF), chemokine(C-X-C motif) ligand 20(CCL20), and lactate dehydrogenase(LDH). Gram staining was used to evaluate the adhesion of C. glabrata to vaginal epithelial cells. Flow cytometry was utilized to assess the effect of C.glabrata on A431 cell apoptosis. Based on the transcriptomics results, immunofluorescence was performed to measure the expressions of p-EGFR and p-ERK1/2 proteins, while Western blot validated the expressions of p-EGFR, p-ERK1/2, p-C-Fos, p-P38, Bax and Bcl-2 proteins. Sequencing results showed that compared with the VVC model, BEPD treatment up-regulated 1 075 genes and downregulated 927 genes, mainly enriched in immune-inflammatory pathways, including MAPK. Mechanistically, BEPD significantly reduced the expression of p-EGFR, p-ERK1/2, p-C-Fos and p-P38, as well as the secretion of IL-1ß, IL-6, GM-CSF, G-CSF and CCL20, LDH release induced by C. glabrata, and the adhesion of C. glabrata to A431 cells, suggesting that BEPD exerts a protective effect on vaginal epithelial cells damaged by C. glabrata infection by modulating the EGFR/MAPK axis. In addition, BEPD downregulated the pro-apoptotic protein Bax expression and up-regulated the anti-apoptotic protein Bcl-2 expression, leading to a reduction in C. glabrata-induced cell apoptosis. In conclusion, this study reveals that the intervention of BEPD in C. glabrata-induced VVC may be attributed to its regulation of the EGFR/MAPK pathway, which protects vaginal epithelial cells.
Subject(s)
Candida albicans , Epithelial Cells , ErbB Receptors , Pulsatilla , Vagina , Female , ErbB Receptors/genetics , ErbB Receptors/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Vagina/microbiology , Vagina/drug effects , Candida albicans/drug effects , Mice , Humans , Animals , Pulsatilla/chemistry , Transcriptome/drug effects , 1-Butanol/chemistry , Drugs, Chinese Herbal/pharmacology , MAP Kinase Signaling System/drug effects , Candidiasis, Vulvovaginal/drug therapy , Candidiasis, Vulvovaginal/microbiology , Protective Agents/pharmacology , Protective Agents/chemistry , Mitogen-Activated Protein Kinases/metabolism , Mitogen-Activated Protein Kinases/genetics , Candida glabrata/drug effects , Candida glabrata/geneticsABSTRACT
Step error calculation of numerical control (NC) machining tool path is a premise for generating high-quality tool path and promoting its application. At present, iterative methods are generally used to calculate step error, and the computation time increases when accuracy improves. Neural networks can be calculated on GPUs and cloud platforms, which is conducive to reducing computation time and improving accuracy through continuous learning. This article innovatively introduces a BP neural network model to predict step error values. Firstly, the core parameters required for step error calculation are taken as the data samples to construct the neural network model, and map to the same scale through Z-score normalization to eliminate the adverse effects of singular parameters on the calculation results. Then, considering only a small number of parameters determine theoretical values of step error, the Dropout technique can drop hidden layer neurons with a certain probability, which is helpful to avoid overfitting and used in the neural network model design. In the neural network model training, this paper adds the Stochastic Gradient Descent with Momentum (SGDM) optimizer to the back propagation of network training in order to improves the network' stability and accuracy. The proposed neural network predicts step error of samples from three surface models, the results show that the prediction error decreases as sample training increases. After trained by 15% of the surface samples, the neural network predicts the step errors of the remaining samples. Compared with theoretical values, more than 99% of the predicted values have an absolute error less than 1 µm. Moreover, the cost time is only one-third of the geometric method, which verifies the effectiveness and efficiency of our method.
ABSTRACT
A phytochemical study on the underground parts of Hosta ventricosa yielded one new spirostanol saponin (1), two new furostanol saponins (2 and 3), and one new pregnane glycoside (4), along with three known compounds (5â7). Their structures were elucidated on the basis of chemical and spectroscopic analysis. All isolated compounds were evaluated for their cytotoxic effects against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW-480). Compounds 1, 2, and 5â7 showed cytotoxic activities with IC50 values of 3.21-17.06 µM.
Subject(s)
Antineoplastic Agents, Phytogenic , Hosta , Saponins , Antineoplastic Agents, Phytogenic/pharmacology , Glycosides/pharmacology , Humans , Molecular Structure , Saponins/pharmacologyABSTRACT
Bulblet development is a problem in global lily bulb production and carbohydrate metabolism is a crucial factor. Micropropagation acts as an efficient substitute for faster propagation and can provide a controllable condition to explore bulb growth. The present study was conducted to investigate the effects of humic acid (HA) on bulblet swelling and the carbohydrate metabolic pathway in Lilium Oriental Hybrids 'Sorbonne' under in vitro conditions. HA greatly promoted bulblet growth at 0.2, 2.0, and 20.0 mg/L, and pronounced increases in bulblet sucrose, total soluble sugar, and starch content were observed for higher HA concentrations (≥2.0 mg/L) within 45 d after transplanting (DAT). The activities of three major starch synthetic enzymes (including adenosine 5'-diphosphate glucose pyrophosphorylase, granule-bound starch synthase, and soluble starch synthase) were enhanced dramatically after HA application especially low concentration HA (LHA), indicating a quick response of starch metabolism. However, higher doses of HA also caused excessive aboveground biomass accumulation and inhibited root growth. Accordingly, an earlier carbon starvation emerged by observing evident starch degradation. Relative bulblet weight gradually decreased with increased HA doses and thereby broke the balance between the source and sink. A low HA concentration at 0.2 mg/L performed best in both root and bulblet growth. The number of roots and root length peaked at 14.5 and 5.75 cm, respectively. The fresh bulblet weight and diameter reached 468 mg (2.9 times that under the control treatment) and 11.68 mm, respectively. Further, sucrose/starch utilization and conversion were accelerated and carbon famine was delayed as a result with an average relative bulblet weight of 80.09%. To our knowledge, this is the first HA application and mechanism research into starch metabolism in both in vitro and in vivo condition in bulbous crops.
Subject(s)
Carbohydrate Metabolism/drug effects , Humic Substances , Lilium/drug effects , Lilium/growth & development , Lilium/metabolism , Plant Roots/drug effects , Plant Roots/growth & developmentABSTRACT
(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.
Subject(s)
Autophagy/physiology , NAD/metabolism , NAD/physiology , Animals , Homeostasis , Humans , Poly(ADP-ribose) PolymerasesABSTRACT
F-actin rearrangement is an early event in burn-induced endothelial barrier dysfunction. HSP27, a target of p38 MAPK/MK2 pathway, plays an important role in actin dynamics through phosphorylation. The question of whether HSP27 participates in burn-related endothelial barrier dysfunction has not been identified yet. Here, we showed that burn serum induced a temporal appearance of central F-actin stress fibers followed by a formation of irregular dense peripheral F-actin in pulmonary endothelial monolayer, concomitant with a transient increase of HSP27 phosphorylation that conflicted with the persistent activation of p38 MAPK/MK2 unexpectedly. The appearance of F-actin stress fibers and transient increase of HSP27 phosphorylation occurred prior to the burn serum-induced endothelial hyperpermeability. Overexpressing phospho-mimicking HSP27 (HSP27(Asp)) reversed the burn serum-induced peripheral F-actin rearrangement with the augmentation of central F-actin stress fibers, and more importantly, attenuated the burn serum-induced endothelial hyperpermeability; such effects were not observed by HSP27(Ala), a non-phosphorylated mutant of HSP27. HSP27(Asp) overexpression also rendered the monolayer more resistant to barrier disruption caused by Cytochalasin D, a chemical reagent that depolymerizes F-actin specifically. Further study showed that phosphatases and sumoylation-inhibited MK2 activity contributed to the blunting of HSP27 phosphorylation during the burn serum-induced endothelial hyperpermeability. Our study identifies HSP27 phosphorylation as a protective response against burn serum-induced endothelial barrier dysfunction, and suggests that targeting HSP27 wound be a promising therapeutic strategy in ameliorating burn-induced lung edema and shock development.
Subject(s)
Actins/metabolism , Burns/blood , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , HSP27 Heat-Shock Proteins/metabolism , Actins/analysis , Adult , Animals , Burns/pathology , Cell Line , Female , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Male , Middle Aged , Permeability , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Rats , Stress Fibers/metabolism , Stress Fibers/pathology , Sumoylation , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.
Subject(s)
Cell Movement/physiology , Keratinocytes/cytology , Keratinocytes/metabolism , Matrix Metalloproteinase 9/metabolism , Tetraspanin 29/metabolism , Animals , Blotting, Western , Cell Line , Cell Proliferation , Cells, Cultured , Humans , Immunohistochemistry , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Mice , Mice, Inbred BALB C , Microscopy, Fluorescence , Real-Time Polymerase Chain Reaction , Tetraspanin 29/geneticsABSTRACT
Shock is the one of the most serious complications during the early stage of burn injury. Early effective fluid resuscitation, enabling the burn patient to pass through the shock stage smoothly and uneventfully, plays a necessary and essential role in the prevention of the subsequent organ complications, reduction of mortality and morbidity, and improvement in life quality. Rapid restoration of blood volume is the fundamental measure to prevent burn shock. In this review, the history and the current status of several important issues related to burn shock resuscitation, including the fluid replacement formula, quality of fluids, and monitoring of physiological parameters, were over viewed. The authors also proposed that a new therapeutic strategy to prevent microvascular permeability should be emphasized and developed in future, which may hopefully act as the most basic approach to prevent burn shock and its related complications.
Subject(s)
Burns/therapy , Fluid Therapy , Shock/therapy , Humans , ResuscitationABSTRACT
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.
Subject(s)
Burns/complications , Cardiovascular Diseases/physiopathology , Kidney Diseases/physiopathology , Psychomotor Agitation/physiopathology , Stress, Physiological/physiology , Animals , Blood Urea Nitrogen , Burns/therapy , Cardiovascular Diseases/etiology , Creatinine/blood , Disease Models, Animal , Fluid Therapy , Hemodynamics/physiology , Inflammation/therapy , Kidney Diseases/etiology , Lactic Acid/blood , Male , RabbitsABSTRACT
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.
Subject(s)
Burns/metabolism , Mitochondria, Heart/metabolism , Mitochondrial Proteins/metabolism , Muscle Proteins/metabolism , Myocardium/metabolism , Peptide Elongation Factor Tu/metabolism , Animals , Burns/pathology , Electron Transport Complex I/metabolism , Gene Expression Regulation , Male , Mitochondria, Heart/pathology , Myocardium/pathology , Oxidative Stress , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolismABSTRACT
OBJECTIVE: To retrospectively analyze the effect of restrictive fluid management strategy (RFMS) on the early pulmonary function and the prognosis of patients with extremely severe and extensive burn. METHODS: Thirteen patients with extremely severe burn hospitalized from June 2010 to November 2011, being treated with RFMS in the fluid reabsorption stage, were enrolled as treatment group. Twenty-six patients with extremely severe burn hospitalized from March 2008 to November 2011, being treated with normal fluid therapy in the fluid reabsorption stage, were enrolled as control group. The match proportion between treatment group and control group was 1:2. Fluid intake, fluid output, fluid balance (the difference between fluid intake and output), and plasma albumin level from post burn day (PBD) 3 to 10, pulmonary oxygenation index on PBD 3, 5, 7, 10, and 14, occurrence of lung and blood stream infections from PBD 7 to 14, and occurrence of acute respiratory distress syndrome (ARDS), occurrence of other organ complications, and mortality within 2 weeks post burn (PBW) were recorded and compared. Measurement data were processed with t test and randomized blocks analysis of variance, enumeration data were processed with Fisher's exact test. RESULTS: Daily fluid intake of patients showed a tendency of decrease in both groups from PBD 3 to 10. Except for that of PBD 4, there was no statistically significant difference between two groups in fluid intake (with F values from 0.072 to 1.939, P values all above 0.05). Daily fluid output of patients showed a tendency of increase in both groups from PBD 3 to 10. It peaked on PBD 10 in control group and PBD 6 in treatment group. The mean daily fluid output was higher in treatment group than in control group from PBD 4 to 9, but without statistically significant difference (with F values from 0.001 to 3.026, P values all above 0.05). Fluid balance lowered in both groups, and it was the lowest on PBD 10 in control group and PBD 6 in treatment group. Fluid balance was lower in treatment group than in control group from PBD 3 to 7, and it showed statistically significant differences on PBD 4, 5, and 6 (with F values from 4.799 to 8.031, P values below 0.05). Plasma albumin level was higher in treatment group than in control group from PBD 3 to 10, with statistically significant differences observed on PBD 4, 9, and 10 (with F values from 5.691 to 10.551, P < 0.05 or P < 0.01). Pulmonary oxygenation index was higher in treatment group than in control group from PBD 3 to 14, with statistically significant differences observed on PBD 7 (respectively 372 ± 78 in treatment group and 291 ± 92 in control group, F = 5.184, P < 0.05) and 14 (respectively 354 ± 39 in treatment group and 283 ± 72 in control group, F = 8.683, P < 0.05). Lung infection and blood stream infection were respectively observed in 1 and 4 patient (s) in treatment group, and 9 and 11 patients in control group from PBD 7 to 14. Occurrence of ARDS, occurrence of other organ complications, and mortality were fewer in treatment group than in control group within PBW 2, though the differences were not statistically significant (P values all above 0.05). CONCLUSIONS: RFMS is a useful strategy in improving early pulmonary oxygenation of patients with extremely severe and extensive burn by promoting the process of fluid reabsorption and rebalance. This strategy may be also beneficial for the prevention of organ complications as well as a better prognosis in severely burned patients.
Subject(s)
Burns/physiopathology , Burns/therapy , Fluid Therapy/methods , Adolescent , Adult , Female , Humans , Lung/physiopathology , Male , Middle Aged , Prognosis , Retrospective Studies , Water-Electrolyte Balance , Young AdultABSTRACT
BACKGROUND: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. METHODOLOGY/PRINCIPAL FINDINGS: In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Microtubules/metabolism , Myocytes, Cardiac/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Hypoxia/physiology , Cells, Cultured , Down-Regulation , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/genetics , Phosphorylation , Rats , Rats, Sprague-Dawley , Signal Transduction , Tubulin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Up-Regulation , Von Hippel-Lindau Tumor Suppressor Protein/genetics , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
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.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Burns/complications , Enalapril/pharmacology , Multiple Organ Failure/etiology , Multiple Organ Failure/prevention & control , Peptidyl-Dipeptidase A/metabolism , Animals , Biomarkers/metabolism , Female , Hemodynamics/drug effects , Kidney/drug effects , Kidney/physiopathology , Liver/drug effects , Liver/physiopathology , Male , Multiple Organ Failure/pathology , Multiple Organ Failure/physiopathology , Organ Specificity , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Dyneins/metabolism , Gene Expression Regulation , Hypoxia/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Mitochondria/metabolism , Voltage-Dependent Anion Channel 1/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Survival , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Microscopy, Confocal/methods , Permeability , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism , Two-Hybrid System TechniquesABSTRACT
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.
Subject(s)
Energy Metabolism , Microtubules/metabolism , Mitochondria, Heart/metabolism , Myocytes, Cardiac/metabolism , Animals , Cells, Cultured , Male , Membrane Potential, Mitochondrial , Rats , Rats, Sprague-Dawley , Tubulin/metabolismABSTRACT
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.
Subject(s)
Cell Nucleus/metabolism , Glycolysis/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/metabolism , Microtubules/physiology , Myocytes, Cardiac/physiology , Adenosine Triphosphate/metabolism , Animals , Cell Survival/drug effects , Cells, Cultured , Colchicine/pharmacology , Dose-Response Relationship, Drug , Energy Metabolism/drug effects , Glycolysis/drug effects , Hypoxia/physiopathology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Microtubules/drug effects , Models, Animal , Myocytes, Cardiac/cytology , Paclitaxel/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Tubulin Modulators/pharmacologyABSTRACT
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.
Subject(s)
Actins/metabolism , Burns/metabolism , Burns/pathology , Calmodulin-Binding Proteins/metabolism , Cell Membrane Permeability , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , p38 Mitogen-Activated Protein Kinases/physiology , Burns/blood , Cell Line , Cells, Cultured , Enzyme Activation , Humans , Phosphorylation , Umbilical VeinsABSTRACT
1. Adenosine A(1) receptors (A(1)R) play an important role in cardioprotection against hypoxic damage and the opening of mitochondrial permeability transition pores (MPTP) is central to the regulation of cell apoptosis and necrosis. However, it is still unclear whether A(1)R open MPTP in hypoxic cardiomyocytes. 2. The present study used primary cardiomyocyte cultures from neonatal rats to investigate the mechanisms of A(1)R activation and the effects of A(1)R on MPTP opening under hypoxic conditions. 3. Hypoxia increased both MPTP opening and the production of reactive oxygen species (ROS), while decreasing cell viability and mitochondrial membrane potential (Deltapsi). The A(1)R agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 500 nmol/L) blocked the increase in MPTP opening and ROS production and maintained cell viability and Deltapsi under hypoxic conditions. 4. The protective effects of CCPA were eliminated by both the protein kinase C (PKC) inhibitor chelerythine (2 micromol/L) and the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) inhibitor 5-hydroxydecanoate (500 micromol/L). Moreover, CCPA significantly increased the PKC content in both total protein and membrane protein of cardiomyocytes. 5-Hydroxydecanoate did not prevent these CCPA-induced increases in PKC. 5. These results demonstrate that CCPA reduces MPTP opening in hypoxic cardiomyocytes, possibly by activating PKC, stabilizing Deltapsi and reducing ROS production following the opening of mitoK(ATP). Consequently, fewer MPTP open.
Subject(s)
Mitochondrial Membrane Transport Proteins/antagonists & inhibitors , Mitochondrial Membrane Transport Proteins/metabolism , Myocytes, Cardiac/metabolism , Receptor, Adenosine A1/metabolism , Adenosine/analogs & derivatives , Adenosine/pharmacology , Animals , Animals, Newborn , Cell Hypoxia/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Mitochondria, Heart/drug effects , Mitochondria, Heart/metabolism , Mitochondrial Permeability Transition Pore , Myocytes, Cardiac/drug effects , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A1/physiologyABSTRACT
OBJECTIVE: To investigate the therapeutic effect and side effects of colistin in treating infections caused by multidrug-resistant (MDR) gram-negative bacillus in patients with severe burn in order to provide the basis for reasonable application of this antibiotic in clinic. METHODS: Nine burn patients suffered from infections caused by MDR gram-negative bacillus admitted to our institute from August 2005 to January 2009 were involved in this study. On the premises that isolated bacteria were only sensitive to colistin or not sensitive to other antibiotics, patients were treated with intravenous drip of colistin (100 x 10(4) - 150 x 10(4) U/d), or intravenous drip combined with administration of the drug into respiratory tract by atomization or instillation (50 x 10(4) - 100 x 10(4) U/d). The bacteriologic and therapeutic effects and side effects (including neurotoxicity and nephrotoxicity, rise in serum levels of creatinine, urea nitrogen and cystatin C were detected and compared before and after administration) of colistin were observed. RESULTS: Out of 9 patients, 7 patients were with bloodstream and pulmonary infections, 1 patient was with bloodstream, pulmonary, and invasive wound infections, and 1 patient was with bloodstream and urinary tract infections. The pathogenic bacteria were proved to be Pseudomonas aeruginosa, Acinetobacter baumannii and Pseudomonas maltophilia. After the administration of colistin, bacteria clearance rate of blood reached 92.3% in 9 patients; isolation rate of MDR gram-negative bacillus of sputum was significantly decreased in 7 patients with pulmonary infection (before treatment 58.2% v.s. after treatment 14.6%, P < 0.01); a complete MDR gram-negative bacillus clearance of urine was observed in 1 patient with urinary tract infection. Colistin was clinically effective in 8 patients but ineffective in 1 patient (effective rate 88.9%). Compared with those before administration, serum levels of creatinine and urea nitrogen were decreased after administration in all patients; no significant difference in serum level of cystatin C among 8 patients was detected, except an obvious elevation in serum level of cystatin C in 1 patient after colistin therapy, and it lowered 1 month after discontinuation. No neurotoxicity or other side effect was observed during medication and 5 days after discontinuation in all patients. CONCLUSIONS: Reasonable application of colistin is a good option for treating infections caused by MDR gram-negative bacillus in patients with severe burn, as no other more effective drug is found.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Burns/drug therapy , Colistin/therapeutic use , Gram-Negative Bacterial Infections/drug therapy , Adult , Anti-Bacterial Agents/adverse effects , Burns/microbiology , Colistin/adverse effects , Drug Resistance, Multiple, Bacterial , Gram-Negative Bacteria/drug effects , Humans , Male , Middle Aged , Treatment OutcomeABSTRACT
OBJECTIVE: To address the features of the fungal infection after burn injury in clinic. METHODS: Three thousand nine hundred and nine burn patients admitted to our institute from Jan. 2003 to Dec. 2006 were involved in this study. Two thousand two hundred and seventy-one samples were harvested for fungal detection by culture from 467 patients suspected to be infected by fungi based on their clinic manifestations. The collected samples included wound tissue, blood, urine, stool, sputum, catheters and others. The antibiotic sensitivity of the identified fungi were determined by routine method. When same kind of fungus was found from different samples taken from one patient, it was recorded as one positive sample. The samples were ranked in an ascending order as wound secretion, stool, urine, sputum and bronchial alveolar lavage fluid, arteriovenous catheter or urinary catheter, blood. Only the positive sample of the highest rank source was recorded as the positive strain of fungus from this particular patient. RESULTS: It was found 61 fungal positive samples from the 2271 samples collected. Out of 467 patients, 38 strains of fungi were detected from 36 burn patients during the investigated period, the incidence was 0.92% (36/3909). The most three commonest types among the identified 38 strains of fungi were Candida tropicalis (42.1%), Candida albicans (31.6%) and Candida famata (T. Famata, 10.5%). The drug sensitivity tests demonstrated that most of the strains detected in this investigation, with the exception of candida glabrata, were sensitive to most of the routine antimycotics agents such as Amphotericin B, Fluconazole, and Itraconazole etc. Among the 36 fungus positive patients, in 18 patients the burn area exceeded 80% TBSA, 12 patients with 50%-79% TBSA, 4 patients with 30%-49% TBSA, and in 2 patients the burn area was smaller than 30% TBSA. It was found most of the fungal infections (77.78%) occurred 2 weeks after burn injury, and 8 of the 36 fungus-infected patients died (the mortality was 22.22%). Conclusions Further examinations are necessary to confirm the diagnosis in burn patients suspected to have fungal infection. Once fungal infections are confirmed, antimycotic therapy must be started immediately.