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The ability to effectively detect bacterial infection in human tissues is important for the timely treatment of the infection. However, traditional techniques fail to visualize bacterial species adhered to host cells

Stress ulceration is a common complication in critically ill patients, but the mechanisms involved are poorly understood. In this study we investigated the temporal activation of the redox-sensitive transcription factor nuclear factor-kappaB and its roles in an experimental model of cold immobilization stress-induced gastric mucosal lesions.Prospective, controlled, and randomized animal study.University research laboratory.Male Sprague-Dawley rats.The rats were subjected to cold immobilization stress for a total of 6 hrs. The temporal profiles of nuclear factor-kappaB activation and expression of tumor necrosis factor-alpha, interleukin-1beta, cytokine-induced neutrophil chemoattractant-1 (CINC-1), intercellular adhesion molecule-1 (ICAM-1), and inducible nitric oxide synthase (iNOS) were determined in the gastric corpus mucosa of stressed rats. To study the roles of nuclear factor-kappaB activation, rats received an intravenous bolus of a specific nuclear factor-kappaB inhibitor Bay 11-7082 (20 mg/kg) 1 hr before stress. For antioxidant administration, rats were treated with intravenous injection of a free radical scavenger pyrrolidine dithiocarbamate (50, 100, and 200 mg/kg) 1 hr before stress.Exposure of rats to 6 hrs of stress led to a rapid and persistent activation of nuclear factor-kappaB, which was associated with transient degradation of inhibitory protein IkappaBalpha and slower but sustained degradation of IkappaBbeta. Nuclear factor-kappaB activation preceded the induction of tumor necrosis factor-alpha, interleukin-1beta, CINC-1, ICAM-1, and iNOS messenger RNAs, all of which were linearly increased with the duration of stress. Bay 11-7082 selectively blocked the stress-induced nuclear factor-kappaB activation and up-regulation of tumor necrosis factor-alpha, interleukin-1beta, CINC-1, ICAM-1, and iNOS messenger RNAs. Inhibition of expression of these proinflammatory genes prevented the increases in myeloperoxidase activity (an indicator of neutrophil infiltration) in gastric mucosa and the development of gastric damage. Pyrrolidine dithiocarbamate dose-dependently inhibited the stress-induced nuclear factor-kappaB pathway activation and consequential proinflammatory gene expression, neutrophil infiltration, and gastric damage, suggesting the involvement of reactive oxygen species in these processes.Sustained activation of nuclear factor-kappaB by reactive oxygen species is an important in vivo mechanism mediating stress-induced gastric inflammatory damage in rats.

This study was made to evaluate the effect of SB203580, a specific p38 MAP kinase inhibitor, on burn-induced hepatic injury as well as the activation of nuclear factor (NF)-kappaB in severely burned rats. Sprague-Dawley rats were divided into three groups: (1) sham group, rats underwent sham burn; (2) burn group, rats given third-degree burns over 30% total body surface area (TBSA) and treated with vehicle plus lactated Ringer solution for resuscitation 4 ml/(kg% TBSA); and (3) burn plus SB203580 group, rats given burn injury and fluid resuscitation plus SB203580 (10 mg/kg i.v., 15 min and 12 h after burn). Hepatocellular injury (measured by serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) and hepatocellular function (determined by the indocyanine green dye retention rate (ICG R15)) were assessed at 24 h post-burn. Liver histologic changes were also analyzed. Burn trauma resulted in increased serum aminotransferases concentrations, decreased ICG R15, elevated serum tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta levels and hepatic TNF-alpha and IL-1beta mRNA expressions, and worsen histologic condition. The level of Nuclear Factor (kappa) inhibitor (IkappaBalpha) in liver was decreased and DNA-binding activity of Nuclear Factor-kappaB (NF-kappaB) was increased after thermal injury. p38 MAP kinase was more significantly activated in liver harvested from burn rats than from shams. SB203580 inhibited the activation of p38 MAP kinase, reduced the levels of TNF-alpha and IL-1beta, and prevented burn-mediated liver injury. Both the IkappaBalpha level and NF-kappaB activity in the liver following burns was not affected by administration with SB203580. These findings suggest that (1) p38 MAP kinase activation is one important aspect of the signaling event that may mediate the release of TNF-alpha and IL-1beta and contributes to burn-induced liver injury and (2) p38 MAP kinase does not influence the activation of NF-kappaB directly in the liver of severely burned rats.

This study was undertaken to evaluate the effect of SB203580, a specific p38 mitogen-activated protein (MAP) kinase inhibitor, on burn-induced lung injury as well as the release of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in rats to characterize the role of p38 MAP kinase in lung injury after burn trauma. Sprague-Dawley rats were divided into three groups: 1) sham group, or rats who underwent sham burn; 2) control group, or rats given third-degree burns over 30% total body surface area (TBSA) and lactated Ringer solution for resuscitation; and 3) SB203580 group, or rats given burn injury and lactated Ringers solution with SB203580 inside for resuscitation. Pulmonary injury was assessed at 24 h by pulmonary capillary permeability determined with fluorescein isothiocyanate-labeled albumin and lung histologic analysis. TNF-alpha and IL-1beta protein in bronchoalveolar lavage fluid and serum were measured by enzyme-linked immunosorbent assay and p38 MAP kinase was activity determined in lung by Western blot analysis. These studies showed that significant activation of p38 MAP kinase at 24 h postburn compared with control. Burn trauma resulted in increased pulmonary capillary leakage permeability, elevated levels of TNF-alpha and IL-1beta in bronchoalveolar lavage fluid and serum, and worsened histologic condition. SB203580 inhibited the activation of p38 MAP kinase, reduced the levels of TNF-alpha and IL-1beta, and prevented burn-mediated lung injury. These data suggest that p38 MAP kinase activation is one important aspect of the signaling event that may mediate the release of TNF-alpha and IL-1beta and contributes to burn-induced lung injury.

Cutaneous burn trauma causes functional inhibition of the heart, but the mechanism is unclear. Using a high dissociation constant (K(D)) calcium indicator TF-BAPTA and 19F MR spectroscopy, the relationship between the changes of cytosolic free calcium and cardiac function after burn trauma was examined. Sprague-Dawley rats received scald (43% TBSA) or sham burns. Twenty-four hours later, the hearts were excised and perfused by the Langendorff method with a modified phosphate-free Krebs-Henseleit bicarbonate buffer. Left ventricular developed pressure (LVDP) was recorded through a catheter attached to an intraventricular balloon. At the same time, 31P and 19F nuclear magnetic resonance (NMR) spectroscopy was perforined before and after TF-BAPTA loading. LVDP of the heart from burned rats was 40% less than in sham burn rats (65+/-6 vs 110+/-12 mmHg, P0.01). Cytosolic free calcium increased about four-fold in those hearts from the burn group compared to the sham burn group (0.807+/-0.192 vs 3.891+/-0.929 microM). Loading TF-BAPTA in those hearts only caused about a 15-20% decrease in LVDP. PCr/Pi ratio also decreased significantly with this loading, but ATP signals were not affected. In conclusion, the inhibition of cardiac contractility caused by burn trauma correlated with the overload of cytosolic free calcium in the heart.

Acute lung injury (ALI) and adult respiratory distress syndrome (ARDS) are a spectrum of lung diseases characterized by a severe inflammatory process resulting in diffuse alveolar damage and variable degrees of ventilation-perfusion mismatch, severe hypoxemia, and poor lung compliance. ALI and ARDS

Macrophages are heterogeneous cell populations that are present in all tissues. Macrophages can be divided into classically activated inflammatory macrophages (M1) and alternatively activated anti-inflammatory macrophages (M2). It has been generally accepted that M1 macrophages are polarised in an inflammatory environment to produce pro-inflammatory cytokines, whilst M2 macrophages are involved in anti-inflammation and aid tissue repair in wound healing. Bacterial endotoxin (lipopolysaccharide; LPS) is a potent factor in infection, which induces M1 macrophages resulting in higher levels of iNOS, TNFα and IL-12p70 which dictate inflammatory T cell responses. M2 macrophages can be transformed into M1 macrophages following LPS stimulation to promote inflammation. Candida albicans is a commensal fungal microorganism, which has been suggested to induce immune tolerance; however, the mechanism of C. albicans-induced immune tolerance has not been investigated in detail. IL-35 is a recently identified anti-inflammatory cytokine which is a heterodimeric protein consisting of the Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 shares the protein subunit p35, with IL-12p70. IL-12p70 is the most potent cytokine to induce Th1 responses during inflammation. In this study, we demonstrate that heat-killed C. albicans (HKC) strongly suppressed LPS-induced IL-12p70 production in M2 macrophages. Candida albicans induced a high level of EBI3 expression in M2 macrophages, which served as a mechanism for IL-12p70 suppression by competitive binding of the common protein subunit (p35) of IL-35 and IL-12p70. To demonstrate that EBI3 expression had the ability to block IL-12p70 production intracellularly, a Chinese Hamster Ovary (CHO) cell line with biscistronic expression of IL-12p40 and p35 was constructed, followed by ectopic over-expression of EBI3. The over-expression of EBI3 in the IL-12p70 producing cell line effectively suppressed IL-12p70 production. IL-35 secretion was also detected in the cell line, with suppressed IL-12p70 production by immune-precipitation Western blotting. However, this secretion was not evident in M2 macrophages following stimulation by HKC. This can be explained by the constitutive expression of IL-35 receptors (gp130 and IL-12Rβ2) in M2 macrophages for cytokine consumption. Our results have indicated that C. albicans can suppress host inflammatory responses in mucosal skin by suppressing LPS-induced IL-12p70 production. Lower IL-12p70 production may avoid an unnecessary Th1 response in order to retain immune tolerance, which may be one of the mechanisms by which C. albicans achieves a successful commensal lifestyle without having a detrimental effect on the host's health.

In vivo 31P- and 23Na-magnetic resonance spectroscopy was used to measure phosphorus metabolites, intracellular pH, cytosolic free Mg2+, and intracellular Na+ in the liver of rats 24 h after 40% total body surface area full-thickness burn injury. Studies were performed during infusion of thulium (III) 1,4,7,10-tetraazacyclododecane N,N′,N″,N″′-tetra(methylenephosphonate), which served as the Na+ shift agent. Compared with the sham-burn group, there was a significant increase in hepatic intracellular Na+ along with a decrease in intracellular pH and free Mg2+. The ratio of intra- to extra-cellular Na+ increased, indicating a decreased Na+ gradient that may determine the hepatic transmembrane potential difference. Hepatic beta-ATP/P(i) also significantly decreased, which suggests that either ATP utilization is significantly accelerated or ATP synthesis is inhibited after the thermal injury. Of the cations measured (Na+, Mg2+, H+), the change in intracellular Na+ was most dramatic. This study demonstrates that major burn injury may cause profound changes in hepatic bioenergetics and ionic metabolism 24 h after injury and that intracellular Na+ may be a sensitive indicator of hepatic dysfunction 24 h after injury. Because these animals tolerated the shift reagent, thuliumIII) 1,4,7,10-tetraazacyclododecane N,N′,N″,N″′-tetra(methylenephosphonate), nuclear magnetic resonance spectroscopy may prove valuable in monitoring intracellular cations in the liver after major injury.

1. Hydrogen is a colourless, odourless, tasteless and flammable gas. Hydrogen is considered a physiologically inert gas and is often used in deep sea diving medicine. In mammals, endogenous hydrogen is produced as a result of the fermentation of non-digestible carbohydrates by intestinal bacteria and it is absorbed into the systemic circulation. 2. Recent evidence indicates that hydrogen is a potent anti-oxidative, anti-apoptotic and anti-inflammatory agent and so may have potential medical application. The present review evaluates the concept of 'hydrogen resuscitation', based on knowledge that hydrogen treatment effectively protects cells, tissues and organs against oxidative injury and helps them recover from dysfunction. 3. Hydrogen therapy can be delivered by inhalation, the administration of hydrogen-enriched fluid or by approaches that affect endogenous hydrogen production. 4. Studies have shown that hydrogen resuscitation has cytoprotective effects in different cell types and disease models, including ischaemia-reperfusion injury, inflammation, toxicity, trauma and metabolic disease. The underlying mechanism may be the selective elimination of hydroxyl radicals, although other mechanisms may also be involved (e.g. hydrogen functioning as a gaseous signalling molecule). 5. Hydrogen resuscitation may have several potential advantages over current pharmacological therapies for oxidative injuries. However, more work is needed to identify the precise mechanism underlying the actions of hydrogen and to validate its therapeutic potential in the clinical setting.

To study the effect of hydrogen-rich saline on blood pressure and antioxidant ability of lung tissue in scalded rats following delayed resuscitation.The hydrogen-rich saline was prepared (hydrogen-saturated normal saline with hydrogen concentration of 0.6 mmol/L). Twenty SD rats were divided into hydrogen-rich saline group (HS) and normal saline group (NS) according to the random number table, with 10 rats in each group. All the rats were subjected to 30% total body surface area (TBSA) full-thickness scald. Rats in HS and NS groups were infused with hydrogen-rich saline or normal saline with one half of the total fluid replacement volume as calculated according to the Parkland formula (4 mL×kg(-1)×%TBSA(-1)) at post scald hour (PSH) 7 and one-quarter of the volume at PSH 9 and 17 respectively. The general condition of rats during the experiment was observed. The systolic pressure of rats was measured at PSH 6 and 24. All rats were sacrificed at PSH 24 to collect lung tissue for detecting superoxide dismutase (SOD) inhibition ratio and malondialdehyde (MDA) level. Data were processed with t test.All rats survived through the experiment. The systolic pressure of rats in HS group and NS group was respectively (87 ± 4) mm Hg (1 mm Hg = 0.133 kPa) and (86 ± 5) mm Hg at PSH 6, and the values were close (t = 0.213, P = 0.834); however the systolic pressure at 24 h was higher in HS group than in NS group [(124 ± 7) mm Hg vs. (115 ± 6) mm Hg, t = 2.958, P = 0.008]. SOD inhibition ratio of lung tissue in HS group [(0.465 ± 0.014)%] was higher than that in NS group [(0.358 ± 0.021)%, t = 11.767, P = 0.000]. MDA level of lung tissue in HS group [(922 ± 196) pmol/mg] was lower than that in NS group [(1118 ± 212) pmol/mg, t = -2.142, P = 0.046].Delayed resuscitation for scalded rats with hydrogen-rich saline is helpful in the recovery of systolic pressure, and it can ameliorate lung tissue injury caused by reperfusion through enhancing the effect of antioxidase.

Glucocorticoid (GC) insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor (GR). Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins (hsp) especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro.The affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting.Similar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats (30% total body surface area immersion scald) and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols.The primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity.

It has been reported by us and other groups that the expression of small GTP binding protein RhoB can be induced by genotoxic stressors and glucocorticoid (GC), a stress hormone that plays a key role in stress response. Until now stress-induced genes that confer cytoprotection under stressed conditions are largely unknown. In this study, we investigated the effects and mechanism of non-genotoxic stressors, including scalding in vivo and heat stress in vitro on the expression of RhoB. We found for the first time that both scalding, which could induce typical neuroendocrine responses of acute stress and cellular heat stress significantly increased the expression of RhoB at mRNA and protein levels. Moreover, in vitro experiments in human lung epithelial cells (A549) showed that induction of RhoB by heat stress was in a glucocorticoid receptor (GR)-independent manner and through multiple pathways including stabilization of RhoB mRNA and activation of p38 MAPK. Further experiments demonstrated that up-regulation of RhoB significantly inhibited heat stress-induced apoptosis and elevated transcriptional activity of NF-κB, but did not affect the expression of Hsp70 in A549 cells. In conclusion, we showed for the first time that RhoB was up-regulated by scalding in vivo and heat stress in vitro and played an important cytoprotective role during heat stress-induced apoptotic cell death.

Introduction Early diagnosis and treatment for thermal injury with septic complications continue to be a serious clinical problem. In this study, plasma biomarkers of rats in the burn and/or septic models were investigated with a metabolomic method. Methods Rat plasma samples were analyzed by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF-MS). Multivariate analysis, the principal components analysis (PCA), was used to validate metabolic changes. In addition, another multivariate method, the orthogonal partial least-squares analysis (OPLS), was used to profile potential biomarkers in models. Results Nine characteristic metabolites, including hypoxanthine, indoxyl sufate, glucuronic acid, gluconic acid, proline, uracil, nitrotyrosine, uric acid, and trihydroxy cholanoic acid were identified in models of thermal injury and/or sepsis. Conclusion These biomarkers were mainly involved in oxidative stress and tissue damage, and might supply evidence for distinguishing burned septic patients from non-septic ones.

The time course of changes in the levels of acute-phase-reactant (APR) mRNAs in different tissues of rats with a 10% or a 60% total-body-surface-area (TBSA) burn and the relationship between the induction of APRs and the host's tolerance to thermal injury were studied. The acute phase response in a LPS-induced inflammation model and a burn-plus-LPS model were compared. The results of this study indicated that (1) the major site of APR synthesis is the liver; (2) even a small surface burn injury can elicit a rapid acute phase response, but the intensity of APR expression increases with the severity of the burn; (3) the down regulation of albumin mRNA, which is characteristic of the acute phase response, does not occur even though transferrin (Trf) mRNA levels are significantly decreased; (4) the resistant strain of inbred rats showed higher levels of alpha 1-antitrypsin (AT) mRNA before and after burn injury, indicating its contribution to the host's tolerance to thermal injury; (5) the increases in alpha 1-acid glycoprotein (AGP) and AT expressions are limited in the burn-plus-LPS rat model compared with either the burn model or LPS-stimulated model alone.

To investigate the epithelial growth factor (EGF) expression of EGF gene-transfected keratinocytes and its effect on cell proliferation after grafting.Newborn Balb/c mouse keratinocytes and gene transfected keratinocytes were seeded on the surface of acellular dermal matrix and cocultured in different ratios as follows: 1:1, 1:3, or 1:5 1 week after culture. The composite skin was grafted onto the full-thickness wound in Balb/c mouse. Specimen was harvested at interval after grafting and underwent the immunohistochemistry staining for EGF and proliferating cell nuclear antigen (PCNA).Immunohistochemical staining showed EGF was expressed in the newly generated epidermis 1-2 week after grafting of the composite skin comprising Balb/c mouse keratinocytes and gene-transfected keratinocytes (at the ratio of 1:5). One week after surgery, Anti-PCNA positive basal cells were more than that in composite skin containing Balb/c mouse keratinocytes alone (P0.01).The gene-transfected keratinocytes expresses EGF and promotes the proliferation of keratinocytes in the early stage after transplantation.

Organ failure from ischemic injury is common in deaths that are due to burn when fluid resuscitation is not performed. Organ perfusion after a delay in resuscitation, however, may induce or even accelerate ischemic organ damage. To study this phenomenon, 40 rats were classified (n = 10) to serve as normal control, burn with no resuscitation, burn with early fluid resuscitation, and burn with delayed resuscitation groups. A modified Walker burn model was used to inflict 50% total body surface area scald burns on the rats. Cellular energy metabolism and tissue water content of several vital organs were measured at 8 hours after burn injury. Adenosine triphosphate, total adenine nucleotides, and energy charge in liver, heart, and kidney tissues were significantly lower (p less than 0.05) with delayed fluid resuscitation compared with early resuscitation. Furthermore, in heart and kidney tissues adenosine triphosphate, total adenine nucleotides, and energy charge were significantly lower in the delayed resuscitation group compared with the group that received no fluid resuscitation. This indicates that heart and kidney tissue are more viable at 8 hours after burn injury, with no fluid resuscitation compared with delayed resuscitation. Water content of lung and muscle tissue were significantly lower (p less than 0.05) in the burn group that received no fluid resuscitation compared with that in early and delayed resuscitation groups. Water content of muscle was significantly greater with delayed resuscitation compared with the early resuscitation group. Results indicate that delayed fluid resuscitation in cases of burn shock may disrupt the cellular energy metabolism in some vital organs and cause skeletal muscle edema.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the role and mechanism of c-Jun N-terminal kinase (JNk) inhibitor (SP600125) in amelioration of insulin resistance after scald.Twenty-four Sprague-Dawley rats were randomized into sham (the process of scald was mimicked by water at room temperature) , scald, scald and SP600125 groups. The rats were inflicted with 30% TBSA full-thickness scald in the latter two groups. Euglycemic-hyperinsulinemic glucose clamp experiment was carried out 4 days after scald. SP600125 was administered to the rats in scald and SP600125 2 hrs before Euglycemic-hyperinsulinemic glucose clamp was performed. Changes in the phospho-Serine307 and phospho-tyrosine of IRS-1 activity, as well as expression of phospho-JNK in muscles were determined.Euglycemic-Hyperinsulinemic Glucose Clamps experiment showed that the infusion rate of 100 g/L glucose in sham, scald, scald and SP600125 groups were (12. 33 +/-0. 42) , (6. 61 +/-0. 27) , (11. 11 +/-0. 68) mgx kg(-1) x min(-1) , respectively ( P0.01). The level of IRS-1 Serine307 phosphorylation and JNK activity in muscles were significantly increased, while insulin-induced tyrosine phosphorylation of IRS-1 decreased markedly after scald. Compared with scald group, the level of IRS-1 Serine307 phosphorylation and JNK activity in scald and SP600125 group were decreased but tyrosine phosphorylation was elevated.SP600125 can partially ameliorate insulin resistance after scald by inhibition of JNK activation, and decrease the level of IRS-1 phospho-serine307.

To investigate the role of c-Jun NH (2)-terminal kinase (JNk) in insulin resistance after burn and its mechanism.Twenty-four Sprague-Dawley rats were randomized to control, burn and burn + anisomycin groups. The rats in control group received sham burn trauma, and burn and burn + anisomycin groups received 30% total body surface area (TBSA) full thickness burn injury. Anisomycin (5 mg/kg) together with 250 microl dimethyl sulfoxide (DMSO) was injected to the rats in anisomycin group intravenously, and only 250 microl DMSO in the other two groups. Euglycemic-hyperinsulinemic glucose clamps was performed 2 hours after the injection. The changes of phospho-serine 307, phospho-tyrosine of insulin receptor substrate (IRS)-1 and phospho-JNK in muscle tissues were determined and compared using immunoprecipitation and Western blot analysis or immunohistochemistry in the three groups.The infusing rates of total 10% glucose (mg x kg(-1) x min(-1)) in control, burn and burn + anisomycin group were 12.3 +/- 0.4, 6.6 +/- 0.3, 6.5 +/- 0.4, respectively. The level of IRS-1 Serine 307 phosphorylation and phospho-JNK in muscle increased significantly, while insulin-induced tyrosine phosphorylation of IRS-1 decreased markedly after burn.The activation of JNK elevates the level of IRS-1 phospho-serine 307 and might play a role in insulin resistance after burn in rats.

To investigate the time course of nuclear factor-kappaB (NF-kappaB) activation in the process of stress ulcer formation.Model of stress ulcer was reproduced by subjecting male Sprague-Dawley rats to water-immersion restraint (WIR) stress. At indicated time after the beginning of WIR stress, animals were sacrificed and cytoplasmic and nuclear protein and total RNA were prepared from gastric corpus mucosal tissues. DNA-binding activity of NF-KB was assessed as an index of NF-kappaB activation with electrophoretic mobility shift assay. Degradation of IkappaBalpha and IkappaBbeta, the inhibitory proteins of NF-kappaB, was analyzed by Western blot analysis. Expression of NF-kappaB dependent genes including tumor necrosis factor-alpha (TNF-alpha) , interleukin-1beta (IL-1beta), cytokine-inducible neutrophil chemoattractant-1 ( CINC-1), intercellular adhesion molecule-1 (ICAM-1), and inducible nitric oxide synthase (iNOS) was detected with Northern blot analysis.WIR stress induced a rapid biphasic activation of gastric mucosal NF-kappaB within 15 min of the beginning of stress, peaking at 45 min and 360 min. Compared with baseline, NF-kappaB activation by stress was increased (10.6 +/- 1.3) and (8.9 +/- 1.2) fold at 45 min and 360 min, respectively (P0.01). Antibody supershift assays revealed that p50/p65 heterodimer was the major active component of mucosal NF-kappaB. Western blot analysis showed that degradation of IkappaBalpha and IkappaBbeta occurred at first and second wave of NF-kappaB activation. Corresponding with the rapid and persistent activation of NF-kappaB, the levels of TNF-alpha, IL-1beta, CINC-1 and ICAM-1 mRNA in gastric mucosa were markedly increased 15 to 30 min after stress, respectively. Up-regulation of iNOS mRNAs was observed 30 to 90 min after stress, and the expression of all of these genes was increased consistently until the end of stress.NF-kappaB activation is an early event and may play an important role in proinflammatory gene over-expression in rat gastric mucosa during WIR stress.

To investigate The modulating role of p38 mitogen-activated protein kinase (MAPK) in the expression of tumor necrosis factor-alpha in hepatic cells and its role in hepatic injury in severely burned rats.Twenty-four adult healthy male SD rats were randomly divided into three groups (8 rats in each group): sham group, burn group, and burn with SB203580 group. A rat model of full-thickness burn injury covering 30% total body surface area (TBSA) was reproduced. The specific inhibitor of p38MAPK (SB203580 in 10 mg/kg) was given to the rats in the burn with SB203580 group at 15 minutes and 12 hours after burn. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured at 24 postburn hours (PBHs). The TNF-alpha mRNA expression in the liver was determined by real-time reverse transcription polymerase chain reaction, and the expression levels of p38MAPK and phosphor-p38MAPK in the liver were determined by Western blot analysis.The serum levels of AST and ALT, and the expression of TNF-alpha mRNA in liver cells were significantly higher in burn group than those in sham and SB203580 groups (P0.05 or 0.01), but there was no difference between the two latter groups. It was indicated by Western blot results that there was no difference of p38MAPK expression in rat liver among the three groups (P0.05). The phospho-p38MAPK expression ratio among sham, burn and burn with SB203580 groups was 1.00:3.90:1.10. The phospho-p38MAPK expression was significantly lower in burn with SB203580 group than that in burn group (P0.01), but there was no significant difference compared with that in sham group (P0.05).The postburn activated p38MAPK in rat liver after severe burn injury enhances the expression of TNF-alpha mRNA and participates in the development of postburn hepatic injury.

To investigate the changes in the distribution and chemical states of the hepatic intra- and extra-cellular sodium ion in the rats with severe burns, so as to provide guidance for fluid resuscitation at early postburn stage.Nineteen adult male Sprague-Dawley (SD) rats were employed in the study and were randomly divided into control (n = 12) and burn (n = 7) groups. The changes in the longitudinal (T1) and transverse (T2) relaxation times of hepatic intra-cellular and extra-cellular sodium in the two groups were studied with 23Na NMR spectroscopy and a shift reagent.After infusion of the shift reagent,the extra-cellular sodium content in rat liver decreased by 17%, with obvious increase in fast T2 component (P0.01), indicating an increase in the fraction of Na+ binding sites in the extra-cellular space. The characteristics of relaxation of intra-cellular sodium remained unchanged despite a 57% increment in intra-cellular sodium content.The deficiency of sodium as a permeable molecule might be related to the postburn movement of hypertonic sodium from extra-cellular to intra-cellular space. The results indicated that it is reasonable to administer high concentration of sodium in fluid resuscitation during the first 24 postburn hours.

To determine whether the activation of p38 mitogen-activated protein kinase (MAPK) is involved in the pathogenesis of stress ulcer.Model of stress ulcer was established with the treatment of rats with water-immersion restraint (WIR) stress. Ulcer index (UI) was macroscopically evaluated as a parameter of gastric mucosal lesions. Expression of phospho- and pan-p38 in gastric mucosa was detected using Western blot analysis. Tumor necrosis factor-alpha (TNF-alpha) and Interleukin 1beta (IL-1beta) gene expressions were analyzed by Northern blot analysis. As indicated in some experiments, rats were pretreated with intravenous injection of the specific p38 MAPK inhibitor CNI-1493 prior to WIR stress and then the changes of UI and TNF-alpha and IL-1beta mRNA expression were examined.The p38 MAPK was persistently activated in the gastric mucosa of rats with WIR stress, with maximal activation after 1 h of stress [(6.8 +/- 3.2) fold of baseline levels, P0.01]. Inhibition of p38 MAPK activation with CNI-1493 led to a marked decrease in UI in WIR stress rats. Similarly, the increased gene expression of proinflammatory cytokines TNF-alpha and IL-1beta in gastric mucosa induced by WIR stress were significantly diminished by p38 MAPK inhibition.p38 MAPK might have an important role in the pathogenesis of stress ulcer.

To investigate the role of p38 mitogen-activated protein kinase (MAPK) signal transduction pathway in the Kupffer cells production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in severely burns rats.Male health adult Sprague-Dawley rats were randomized into four groups: sham burn rats given vehicle, sham burn rats given the p38 MAP kinase inhibitor SB203580, rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24 h after burn or sham burn and Kupffer cells (KCs) were isolated. After 18 h incubation, KCs next were stimulated with 50 ng/ml of LPS for 18 h. After stimulation, supernatants were removed for analysis of TNF-alpha and IL-1beta levels by ELISA. The TNF-alpha and IL-1beta mRNA expressions (by quantitative real-time RT-PCR) and the activities of p38 MAPK and JNK (by Western blot analysis) in KCs were examined.Eighteen hours after 50 ng/ml LPS stimulation, KCs from burn rats released significantly higher levels of TNF-alpha and IL-1beta than did shams. The mRNA levels of TNF-alpha and IL-1beta in KCs increased significantly postburn. Western blot analysis suggested that expression of phosphorylated p38 MAPK and JNK were increased in KCs harvested from burn group after stimulation with LPS compared with those from sham group. In vivo administration of SB203580 markedly suppressed both the release of TNF-alpha and IL-1beta and the mRNA expressions of TNF-alpha and IL-1beta in KCs from both sham and burn rats. p38 MAPK activity in KCs was abolished by administration with SB203580, whereas JNK was not.p38 MAPK signal transduction pathway mediates KCs production of proinflammatory cytokines TNF-alpha and IL-1beta in severely burned rats.

To investigate the role of p38 mitogen-activated protein kinase (MAPK) signal transduction pathway in the production of the proinflammatory factors such as tumor necrosis factor (TNF-alpha) and interleukin 1beta (IL-1beta) in lungs and in the pulmonary endothelial cell injury in severely scalded rats.Forty eight adult healthy SD rats were randomly divided into three groups with 16 rats in each group, i.e. sham, burn and burn with SB203580 treatment groups. The changes in the TNF-alpha and IL-1beta contents in serum and bronchoalveolar lavage fluid (BALF), the von Willebrand factor (vWF) contents in plasma and pulmonary microvessels and pulmonary activating protein (AP-1) activity were determined at 24 postburn hours (PBH).Compared with those in sham group, the TNF-alpha and IL-1beta contents in serum and BALF and the vWF content in plasma (194.2% +/- 28.3% vs 93.2% +/- 14.3%) at 24 PBH in burn group increased significantly (P0.01), whereas vWF content in pulmonary microvessel decreased obviously (1.1 +/- 0.3 vs 3.3 +/- 0.4, P0.01). In addition, the pulmonary AP-1 activity also increased at 24 PBH. Nevertheless, all the above indices improved obviously in burn with SB203580 (inhibitor of p38 MAPK signal transduction pathway) treatment group when compared with those in burn group.AP-1 might mediate the production of proinflammatory factors, such as TNF-alpha and IL-1beta in lungs leading to pulmonary vascular endothelial injury, after being activated by activated p38 MAPK.

To investigate the role of p38 mitogen-activated protein kinase (MAPK) signal transduction pathway in the acute lung injury of severely burned rats.Forty-eight adult healthy rats were randomly divided into three groups: sham group, burn control group, and burn + SB203580 group. A third-degree burns over 30% total body surface area rat model was used and pulmonary capillary permeability, lung water content, pulmonary histology and p38 MAPK activity were measured at 24 hours postburn.Burn trauma resulted in increased pulmonary capillary leakage permeability (42.5 +/- 4.7 vs. 12.1 +/- 1.4, P0.01), elevated lung water content (P0.05), and worsen histologic condition. There was a significant activation of p38 MAPK at 24 hours postburn compared with control. SB203580 inhibited the activation of p38 MAPK, reduced the pulmonary capillary leakage permeability (24.7 +/- 2.9 vs. 42.5 +/- 4.7, P0.01), decreased lung water content, and prevented burn-mediated lung injury.The activation of p38 MAPK is one important aspect of the signaling event that contributes to burn-induced lung injury.

This study was designed to investigate the role of p38 mitogen-activated protein (MAP) kinase on Kupffer cells (KCs) secretion of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and hepatic injury following burn trauma. Sprague-Dawley rats were randomized into four groups: (1) sham burn rats given vehicle, (2) sham burn rats given the p38 MAP kinase inhibitor SB203580 (10mg/kg i.v., 15min and 12h after sham burn), (3) rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and (4) burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24h post-burn to examine plasma aspartate transaminase (AST) and alanine transaminase (ALT) and KCs were isolated. The KCs secretion of TNF-alpha and IL-1beta and p38 MAP kinase activity (by Western blot analysis) were also examined. These studies showed by more significant activation of p38 MAP kinase in KCs harvested from burn rats than from shams. Burn trauma resulted in hepatic dysfunction and promoted KCs secretion of TNF-alpha and IL-1beta. SB203580 inhibited p38 MAP kinase activity, reduced KCs secretion of proinflammatory cytokines, and alleviated burn-mediated hepatic dysfunction. These data suggest p38 MAP kinase activation is one important aspect of the signaling event that may mediate the KCs secretion of proinflammatory cytokines TNF-alpha and IL-1beta following burn trauma.

To study whether nimodipine, a dihydropyridine-type calcium channel blocker, can inhibit the production of interleukin-1beta(IL-1beta), interleukin-6(IL-6) by Kupffer cells(KC) and down-regulate its level of plasma after severe burn injury.KC of normal rats were isolated with portal vein catheter, intrahepatic digestion and density gradient centrifugation. Intracellular calcium concentration ([Ca2+]i) in individual KC after stimulated with postburn serum was assessed fluorometrically with microspectrofluorimeter. Level of IL-1beta and IL-6 in the supernatant of KC cultured with postburn serum were detected by enzyme-linked immunosorbent assay(ELISA). SD rats underwent 30% total body surface area(TBSA) full thickness burn 6 hours later, KCs was isolated and their mRNA were extracted. Level of IL-1beta mRNA and IL-6 mRNA were detected by ribonuclease protection assay(RPA). Levels of plasma IL-1beta and IL-6 were also detected. Role of nimodipine on above-mentioned effects were observed.Compared with that of control group, levels of [Ca2+]i of KCs and IL-1beta and IL-6 supernatant in burn group increased significantly(all P0.01). At present of 1 micromol/L nimodipine, however, the [Ca2+]i, IL-1beta, IL-6 values decreased significantly(all P0.01). The level of plasma cytokines and KC mRNA in burn group also increased significantly. After intravenously injection with nimodipine (40 microg x kg(-1) x h(-1)), the numerical values decreased significantly(all P0.01).Kupffer cells of rats are activated to secret IL-1beta and IL-6 after severe burn injury and this process is realized through calcium ion signal transduction channel. Nimodipine can inhibit IL-1beta and IL-6 production of KC by preventing its mRNA transcription, down-regulating its level of plasma.

To investigate the outcome and histological changes of transplantation of acellular xeno-dermis combined with suspended keratinocytes.Forty-two nude mice with full-thickness skin defect on the back were randomly divided into 2 groups, then acellular xeno-dermisand and suspended keratinocytes were adopted to cover the skin defect in the experimental group, pure suspended keratinocytes in the control group. The area of wound healing was calculated 2, 3 and 5 weeks after transplantation, and the rates of wound contraction were also calculated, and biopsy for histological examination was performed 3, 6 and 12 weeks after transplantation.Compared with the experimental group, the control group showed delayed wound healing (P0.05), intensive wound contraction (P0.05), poor durability, elasticity, and cosmetic appearances as well as disordered collagen fibers. In contrast, it was observed that the proliferation of collagen fibers was regularly organized, with no obvious acute immuno-rejection responses in the experimental group.The composite transplantation of acellular xenodermis and suspended keratinocytes could promote the wound healing with a satisfactory outcome.

In vivo longitudinal (T1) and transverse (T2) relaxation times of hepatic intracellular and extracellular sodium were studied in rats with sham burn or burn injury with 23Na NMR spectroscopy and shift reagent. Burn injury decreases hepatic extracellular sodium content by 17% compared with sham burn, whereas it increases the percent of the fast T2 component of extracellular sodium, suggesting an increase in the fraction of bound Na+/- sites in the extracellular space. It is interesting that the relaxation characteristics of intracellular sodium remained unchanged despite a 57% increase in intracellular sodium content, suggesting the increase in intracellular free sodium is matched by either a proportional increase in intracellular bound sodium or an uncovering of ordered domain sites that can preferentially orient rapidly exchanging sodium ions. This study also demonstrated that spin lattice (T1) relaxation rates or the percent contribution of the fast/slow T2 components of the combined intracellular/extracellular 23Na signal (before the infusion of shift reagent) may also be sensitive to changes in intracellular sodium levels during pathologic changes.

To test the hypothesis that energy deficits and intracellular ion derangements may be the cellular basis for intrinsic myocardial dysfunction in rats after burn trauma, we examined ATP metabolism, intracellular pH, sodium, and mechanical performance simultaneously in perfused beating hearts from sham burn or burned rats (43% TBSA, 3 degrees scald burn, resuscitated for 24 hr with lactated Ringer's solution, Parkland formula). Intracellular calcium was also measured in myocytes harvested from parallel groups of sham burn and burn resuscitated rats. Burn trauma caused a 46% decrease in left ventricular developed pressure, a 69% decrease in +dP/dtmax, and a 72% decrease in -dP/dtmax. Intracellular to external standard sodium ratio increased (+58%) from 0.318 +/- 0.027 to 0.500 +/- 0.048 (P0.05), and intracellular calcium increased (+67%) from 206 +/- 13 to 445 +/- 37 nM (P0.01). Burn hearts exhibited decreased functional response to isoproterenol challenge compared to sham burn controls, but energy metabolism was similar in all hearts, regardless of burn injury. Our data suggest that burn trauma alters intracellular cardiomyocyte calcium and sodium homeostasis, and ionic derangements are not related to either altered intracellular pH or high energy phosphate deficits.

Metabolic factors that influence the transition form reversible to irreversible ischemic injury were studied in the rat liver in vivo with 31P-nuclear magnetic resonance (NMR) spectroscopy. Hepatic ischemia for 15, 35, or 65 min was produced by occlusion of the hepatic artery and portal vein in rats. Ischemia caused a rapid decrease in the ATP concentration ([ATP])-to-P(i) concentration ratio and pH within 5 min, but there was little change in these variables detectable by 31P-NMR with longer periods of ischemia. After reperfusion, the [ATP] and P(i) concentration returned toward normal values in livers exposed to 15 or 35 min of ischemia, but 65 min of ischemia were associated with only modest recovery in [ATP], and the [ATP] later decreased. Because the 31P-NMR spectrum was similar after brief compared with prolonged ischemia, it appears that neither ATP depletion, P(i) accumulation, nor acidosis predicts metabolic recovery. Hepatic intracellular NA+ was also measured in separate groups of animals by 23Na-NMR in the presence of a shift agent, thulium (III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylene-phosphonate) (TmDOTP5-), and by atomic absorption spectroscopy. Under baseline conditions, the concentration of intracellular Na+ was 15.2 mM by atomic absorption spectroscopy and 16.5 mM by 23Na-NMR. Although the 31P-NMR spectrum responded very rapidly to the onset of ischemia, intracellular Na+ concentration measured by 23Na-NMR increased gradually but steadily at approximately 1.0 mM/min during early (up to 15 min) ischemia. These observations demonstrate that a rise in intracellular Na+ does occur early ischemia, that TmDOTP5- can be applied in vivo for analysis of intracellular Na+ in the ischemic liver, and that 31P-NMR spectroscopy is very sensitive to early ischemic injury.

Delayed fluid resuscitation during burn shock is thought to compromise the integrity of gut mucosa and allow enteric bacteria to cross the luminal wall and infect other sterile organ systems. Superoxide dismutase, a free-oxygen radical scavenger; leupeptin, a protease inhibitor; and verapamil, a calcium channel blocker, were studied to evaluate their efficacy in maintaining cellular integrity in the gut of thermally burned rats whose fluid resuscitation had been delayed. Fifty male rats weighting 280 to 320 gm were given a full-thickness scald burn covering 50% total body surface area. Ten received early fluid resuscitation beginning half an hour after burn, and 40 received fluid resuscitation delayed by 6 hours. Those receiving delayed resuscitation were given superoxide dismutase (n = 10), leupeptin (n = 10), verapamil (n = 10), or a placebo of normal saline solution (n = 10) at the time of fluid resuscitation. Ileal mucosa samples were harvested, and adenosine triphosphate, diphosphate, and monophosphate were measured. Adenosine triphosphate, total nucleotides, and energy charge potential were significantly lower in the placebo group without therapy compared with those of the early resuscitation group. Superoxide dismutase and leupeptin therapy prevented this drop in cellular energy. Total water content was significantly increased in the placebo group compared with that of the early resuscitation group; superoxide dismutase was able to prevent this increase. Data indicate that intestinal reperfusion injury in burned rats can be effectively modulated with superoxide dismutase or leupeptin therapy.