Intravenous Injection Of Lipopolysaccharide Research Articles

Synthetic triglyceride containing an arachidonic acid branch (8A8) prevents lipopolysaccharide-induced liver injury

Aims In this study, we investigated the effect of synthetic triglyceride containing an arachidonic acid branch (8A8) on lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-α and nitric oxide (NO) in macrophages, and LPS-induced liver injury in the rat. Main methods RAW264.7 macrophages were co-incubated with 8A8 and LPS (100 ng/ml), and TNF-α mRNA/protein levels, nuclear factor (NF)-κB DNA binding activity, expression of inducible-type NO synthase (NOS2), and NO 2 production were measured. Male Wistar rats were given a single intraperitoneal injection of 8A8 prior to an intravenous injection of LPS (5 mg/kg), and liver histology, apoptotic cell death, serum TNF-α levels, and hepatic TNF-α mRNA were then evaluated. Key findings LPS-induced increases in TNF-α production in RAW264.7 macrophages were blunted by 8A8 in a dose-dependent manner, with 40% inhibition at 100 ppm. Further, 8A8 dose-dependently prevented LPS-induced increases in TNF-α mRNA levels, as well as NF-κB DNA binding activities, in RAW264.7 macrophages. LPS-induction of NOS2 and NO 2 release from these cells was also decreased by 8A8 in a dose-dependent manner. In vivo, LPS-induced liver injury, including hepatocyte apoptosis, was largely prevented when 8A8 (100 µl/kg) was given 30 min prior to LPS. Indeed, 8A8 blunted increases in both serum TNF-α and hepatic TNF-α mRNA levels significantly. Significance LPS-induced liver injury was prevented by 8A8 most likely through the inhibition of TNF-α and NO production from hepatic macrophages, suggesting a potential usefulness of 8A8 as an immuno-modulating nutrient for prevention/treatment of endotoxin-related organ injuries including alcoholic liver disease and non-alcoholic steatohepatitis (NASH).

Systemic inflammation increases cancer cell adhesion to hepatic sinusoids by neutrophil mediated mechanisms

Interactions between endothelial selectins and selectin ligands expressed on tumor cells have been implicated in the binding of circulating metastatic cancer cells to the vascular endothelium during extravasation. Moreover, there is mounting evidence that inflammatory environments can accelerate the progression of metastasis by neutrophil mediated mechanisms. In this study, a physiologically relevant in vivo model of early metastasis coupled with intravital microscopy was used to visualize the trafficking of tumor cells within the liver vasculature in real time. Using GFP-labeled Lewis lung carcinoma subline H-59 cells, we show here that disrupting the interactions between endothelial selectins and tumor cell selectin ligands diminished tumor cell recruitment to the liver. Furthermore, systemic inflammation induced by intravenous injection of lipopolysaccharide significantly enhanced the metastatic potential of these lung carcinoma cells by increasing their propensity to adhere to the liver sinusoidal endothelium. Confocal microscopy revealed frequent colocalization of cancer cells with neutrophils and neutrophil depletion in vivo significantly attenuated the lipopolysaccharide-induced increase in H-59 cell adhesion. Although direct selectin-selectin ligand interactions contributed significantly to tumor cell adhesion to sinusoidal endothelial cells, we show here that in addition, interactions between adherent neutrophils within the inflamed sinusoids and circulating tumor cells may further increase tumor cell arrest in the liver.

Effects of the treatment with different fluids on alveolar epithelium barrier in rats with acute lung injury

To observe the effects of different fluids on alveolar epithelium barrier in rats with acute lung injury (ALI). Thirty-six Sprague-Dawley (SD) rats were randomly assigned into six groups with 6 rats in each group. ALI was induced by intravenous injection of lipopolysaccharide(LPS). Rats in all treatment groups were given different fluids and sacrificed after 4 hours. Evans blue dye (EBD) was injected via the femoral vein 30 minutes before death. Tracheobronchial tree was washed with normal saline (NS) after death, and broncho-alveolar lavage fluid (BALF) was collected. Leakage of EBD from blood into BALF (alveolar epithelial permeability) and wet/dry (W/D) ratio were measured. The mRNA expression of surfactant protein-C (SP-C) was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Alveolar epithelium apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL). Lung injury was evaluated by Smith lung injury score. (1) Lung injury scores in LPS and NS groups were significantly higher than in control group (both P < 0.05). Compared with NS group, lung injury scores were significantly lower in 5% albumin (ALB) and 6% hydroxyethyl starch 130/0.4 (HES) groups (both P < 0.05), and there was no significant difference in 4% succinylated gelatin (GEL) group (P > 0.05). No significant difference was found among the latter three groups. (2) W/D ratio in LPS and NS groups were significantly higher than that in control group (both P < 0.05). Compared with NS group, W/D ratios were lower in ALB, HES and GEL groups (all P < 0.05). But it showed no significant difference among the latter three groups. (3) Alveolar epithelial permeability in LPS and NS groups were remarkably higher than that in control group (both P < 0.05). Compared with NS group, the alveolar epithelial permeability were significantly lower in ALB, HES and GEL groups (all P < 0.05). The alveolar epithelial permeability in the latter two groups were significant lower than that in ALB group (both P < 0.05) but higher than that of control group. (4) The SP-C mRNA expression in LPS, NS, ALB and GEL groups were lower than that in control and HES groups (all P < 0.05), but there was no difference between control and HES groups (P > 0.05). (5) Apoptosis index (AI) of alveolar epithelial cell in all the treatment groups were significantly higher than that in control group (all P < 0.05). Compared with NS group, AI were noticeably lower in ALB and HES groups (both P < 0.05). Compared with NS, colloid can probably improve the alveolar epithelial permeability and protect the barrier function.

Ghrelin Hyporesponsiveness Contributes to Age-Related Hyperinflammation in Septic Shock

To test the hypothesis that hyporesponsiveness to ghrelin due to reduced growth hormone (GH) contributes to the aging-related hyperinflammatory state in sepsis. Sepsis and septic shock are a serious problem, particularly in the geriatric population. Ghrelin is an endogenous ligand for the GH secretagogue receptor 1a (GHSR1a, ie, ghrelin receptor). The decline in GH with age is directly associated with many adverse changes that occur with aging. However, the role of GH, ghrelin, and GHSR1a in the age-associated vulnerability to sepsis remains unknown. Male Fischer 344 rats (young: 3 months; aged: 24 months) were used. Plasma GH levels, ghrelin receptor expression, and neuronal activity in the parasympathostimulatory nuclei of the brain stem in normal young and aged animals were measured. Endotoxemia was induced by intravenous injection of lipopolysaccharide (LPS, 15 mg/kg BW). While LPS-induced release of proinflammatory cytokines from macrophages isolated from aged rats decreased, LPS injection resulted in an in vivo hyperinflammatory state. GH levels were lower in aged rats, which was associated with lower expression of GHSR1a in the dorsal vagal complex and a decrease in parasympathostimulatory neuronal activity. GHSR1a antagonist elevated LPS-induced cytokine release in young rats. GH increased GHSR-1a expression in the dorsal vagal complex in aged rats. Coadministration of ghrelin and GH, but not ghrelin alone or GH alone, markedly reduced cytokine levels and organ injury after endotoxemia in aged rats, which was associated with significantly elevated parasympathostimulatory neuronal activity. These findings suggest that the reduced central (brain) responsiveness to ghrelin due to the decreased GH, plays a major role in producing the hyperinflammatory state, resulting in severe organ injuries and high mortality after endotoxemia in aged animals. Ghrelin and GH can be developed as a novel therapy for sepsis in the geriatric population.

Recombinant human soluble thrombomodulin decreases the plasma high-mobility group box-1 protein levels, whereas improving the acute liver injury and survival rates in experimental endotoxemia

In addition to the hyperactivation of the inflammatory cytokines, high-mobility group box-1 protein (HMGB1), recently identified as a lethal late-phase mediator is suspected to be closely correlated with the development of sepsis. Therefore, the therapeutic efficacy of recombinant human soluble thrombomodulin (ART-123) administration on the production of inflammatory cytokines and the plasma level of HMGB1 was investigated in experimental endotoxemia. Prospective, comparative, experimental study. Laboratory animal research center at a university. Male Sprague-Dawley rats (250-300 g). Endotoxemia was induced in rats by a bolus intravenous injection of lipopolysaccharide (LPS) at a dosage of 4 mg/kg (LPS group). ART-123 (1 mg/kg) was administered as a bolus injection 30 minutes before or 4 hours after injection of LPS (ART-123 pretreated/treated group). As a control, an equal volume of physiologic saline was administered instead of LPS and ART-123 (control group). Rats were randomly divided into ART-123 pretreated group, ART-123 treated group, and LPS group, respectively. After the injection of LPS, the levels of inflammatory cytokines and thrombin-antithrombin III complex, plasma HMGB1 concentrations, liver immunohistochemical and histopathologic characteristics, liver dysfunction, and survival rate were examined. The increased levels of inflammatory cytokines and plasma HMGB1 induced by LPS in this rat model were improved by the administration of ART-123; additionally, reduced liver dysfunction and increased survival rate were observed. This study demonstrated that ART-123 inhibits the expression of inflammatory cytokines and decreases the plasma HMGB1 levels in experimental endotoxemia. In addition, ART-123 administration markedly reduced liver dysfunction and mortality even with delayed treatment of ART-123. The use of ART-123 may therefore be a beneficial treatment for septic patients.

Effects of catecholamines on the splanchnic perfusion in rabbit model of septic shock

Objective To assess the effects of dopamine,dobutamine and norepinephrine on the P(g-a)CO2 and superior mesenteric blood flow in septic shock.Methods Rabbit septic shock model was established by challenging with intravenous injection of lipopolysaccharides from Escherichia coil(2 mg/kg).The rabbits with septic shock were randomly assigned to 3 groups-dopamine group(n = 8),dobutamine group(n = 8) and norepinephrine group(n = 8).Apart from volume resuscitation with normal saline solution [20 ml/(kg· h)]，dopamine[5μg/(kg·min)],dobutamine[(5μg/(kg·min)]and norepinephrine [(1μg/(kg·min)]were infused in dopamine group,dobutamine group and norepinephrine group respeclively.Cardiac index(CI) and superior mesenteric blood flow index(SMBFI) were continuously monitored by doppler flowrneter.Gastric mucosal PCO2 was evaluated by gas tonometry every 10 min.Arterial and venous blood gas analyses and lactate levels were measured every 1 h.Results MAP,CI,and SMBFI significandy decreased and P(g-a) CO2 increased after lipopolysaccharides infusion in three groups.After 2-hour treatment,MAP in norepinephrine group[(70 +3) mm Hg]was higher than that of dopamine group[(66±4) mm Hg]and dobutamine group[(65±4) mm Hg](P ＜0.05).SMBFI in norepinephrine group [(18.7±2.9) ml/(kg·min)]was higher than that of dopamine group[(16.2±1.6) ml/(kg·min)]and dobutamine group[(15.8±1.9) ml/(kg·min)](P＜0.05).P(g-a) CO2 in norepinephrine group [(30±6) mm Hg]was lower than that of dobutamine group[(23±5)mm Hg](P＜0.05).Condnsion As an adjuvant therapy of volume resuscitation,norepinephrine is more effective than low dose dopamine and dobutamine in improving splanchnic perfusion. Key words: Septic shock; Gastric mucosal carbon dioxide tension; Dopamine; Dobutamine; Norepinephrine

Circulating levels of vasoactive peptides in patients with acute bacterial meningitis

The underlying mechanisms for cerebral blood flow (CBF) abnormalities in acute bacterial meningitis (ABM) are largely unknown. Putative mediators include vasoactive peptides, e.g. calcitonin-gene related peptide (CGRP), vasoactive intestinal peptide (VIP), and endothelin-1 (ET-1), all of which may be affected by therapeutic interventions used in the intensive care unit. We measured arterial levels as well as the net cerebral flux of these peptides in patients with ABM, and in healthy volunteers undergoing interventions relevant to intensive care. Seven patients with severe ABM and sepsis and fifteen healthy volunteers were included after informed consent. The net cerebral fluxes of vasoactive peptides were measured by the Kety-Schmidt technique in ABM patients (baseline study only), as well as in volunteers at baseline, during voluntary hyperventilation, after an intravenous injection of lipopolysaccharide (LPS), and during norepinephrine infusion. The arterial levels of CGRP, but not of VIP or ET-1, were elevated in patients with ABM, but no net cerebral flux was present. CGRP levels decreased during hyperventilation and after LPS injection. No net cerebral flux of VIP occurred in any group at any time. A cerebral efflux of ET-1, which occurred in volunteers at baseline, was neither present in volunteers after LPS injection nor in patients with ABM. The arterial concentration of the vasodilatory peptide, CGRP, but of neither VIP nor the vasoconstrictor ET-1, is elevated in patients with ABM and sepsis. A constitutive cerebral output of ET-1 appears to be present in healthy humans, but is abolished after LPS injection.

Effects of Niupo Zhibao Pellet on high-mobility group box-1 protein expression in lung tissues of endotoxin shock rats

To observe the effects of Niupo Zhibao Pellet, a compound traditional Chinese herbal medicine, on high-mobility group box-1 protein (HMGB1) expression in lung tissues of rats with endotoxin shock. Thirty SPF Sprague-Dawley rats were randomly divided into control group, lipopolysaccharide (LPS) group and Niupo Zhibao Pellet group. Rats in Niupo Zhibao Pellet group were consecutively administered 7 days with 3 mL (1 g/L) Niupo Zhibao Pellet saline suspension every day by intragastric administration. Endotoxin shock was induced in rats of the LPS and Niupo Zhibao Pellet groups by intravenous injection of LPS (1.5 mg/kg) and intraperitoneal injection of D-galactosamine (100 mg/kg). Expression of HMGB1 in lung tissues was measured by immunohistochemical method with diaminobenzidine (DAB) coloration, fluorescein isothiocyanate (FITC) labeling, and by Western blotting. Expression of HMGB1 in lung tissues in the LPS group was increased and that in Niupo Zhibao Pellet group was higher than that in the LPS group and the control group. HMGB1 was presented in the cytoplasm of positive cells in the LPS group, but in the nucleus of positive cells in the Niupo Zhibao Pellet group. However, HMGB1 was little expressed in the lung tissues of normal rats. Niupo Zhibao Pellet can increase HMGB1 expression and locate HMGB1 in the nucleus but not the cytoplasm, which may be one of its mechanisms in reducing endotoxin shock.

EF6265, a novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor, protects against sepsis-induced organ dysfunction in rats*

Although thrombin-activatable fibrinolysis inhibitor (TAFI) has been implicated as a negative regulator of fibrinolysis, its pathophysiological significance remains to be unveiled. We performed the pharmacologic study to assess the effect of EF6265, a specific inhibitor of activated form of TAFI (TAFIa) on sepsis-induced organ dysfunction models. A controlled, in vivo laboratory study. Company research laboratory. Wistar and Sprague-Dawley rats. Endotoxemia and sepsis models were induced by intravenous injection of lipopolysaccharide and Pseudomonas aeruginosa, respectively. In the endotoxemia model, posttreatment (1 hour) with EF6265 reduced fibrin deposits in the kidney and liver accompanied by no significant changes in platelet count and fibrinogen concentration in plasma. This compound also significantly decreased levels of plasma lactate dehydrogenase and aspartate aminotransferase, markers of organ dysfunction. In the sepsis model, EF6265, simultaneously administered with ceftazidime (CAZ) 2 hours after Pseudomonas aeruginosa injection, showed no influence on the antibiotic activity of CAZ. Meanwhile, it dramatically potentiated the interleukin-6-reducing effect of CAZ in plasma, suggesting that inhibition of TAFIa leads to the reduction in systemic inflammatory response associated with bacterial infection. This combined treatment also lowered plasma lactate dehydrogenase and blood urea nitrogen more potently than single treatment with CAZ. These results clearly suggest that TAFI plays an important role in the deterioration of organ dysfunction in sepsis and the inhibitor of TAFIa protects against sepsis-induced tissue damage through regulation of fibrinolysis and inflammation.

Penehyclidine hydrochloride attenuates LPS-induced acute lung injury involvement of NF-κB pathway

To investigate the protective effects of penehyclidine hydrochloride (PHC) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the underlying molecular mechanism. ALI was induced by intravenous injection of LPS (5 mg/kg). Male Sprague–Dawley (SD) rats challenged with or without LPS were pretreated with varied doses of PHC 0.5 h before injection of LPS or saline. Blood gas in arterial blood, lung weight gain, bronchoalveolar lavage fluid (BALF), and neutrophils sequestration were examined 6 h after administration of LPS. Pathological changes of lung tissue were measured by light microscopy. Phosphorylation of mitogen-activated protein kinase (MAPK) family and NF-κB were detected by western blot. All animals demonstrated drops in arterial oxygen tension (PaO 2) after LPS application, which were significantly reversed by PHC pretreatment. Administration of PHC reduced lung water gain, bronchoalveolar lavage protein content, infiltration of neutrophils, malondialdehyde (MDA) content, and lactate dehydrogenase (LDH) activity and enhanced superoxide dismutase (SOD) activity. Histopathological study also indicated that PHC treatment markedly attenuated lung histopathological changes, alveolar hemorrhage, and inflammatory cells infiltration with evidence of decreasing of myeloperoxidase (MPO) activity. Furthermore, p38MAPK, ERK, and NF-κB were activated in 6 h after LPS treatment, which could be blunted by PHC, while JNK remained unchanged. These findings confirmed significant protection by PHC against LPS-induced lung vascular leak and inflammation and implicated inhibition of p38MAPK activation signaling a potential role for PHC in the management of ALI.

Study on expression of liver X receptor-alpha in rat lung tissue in acute lung injury

To observe changes in liver X receptor-alpha (LXR alpha) in acute lung injury (ALI) in rats induced by lipopolysaccharide (LPS) to explore mechanism of LXR alpha in pathogenesis of ALI. Forty-eight Wistar rats were randomly divided into two groups. ALI model was reproduced by intravenous injection of LPS (5 mg/kg), and control group was injected with normal saline (2.5 ml/kg). At 1, 2, 4, 8 hours after ALI, artery blood gas analysis, lung tissue wet/dry weight (W/D) ratio, myeloperoxidase (MPO) activity, lung histopathologic changes were observed. The expressions of LXR alpha and tumor necrosis factor-alpha (TNF-alpha) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). TNF-alpha content was measured with enzyme linked immunosorbent assay (ELISA). LXR alpha protein in lung tissues was assessed by immunohistochemistry. Compared with the control group, in ALI rats at different time points, partial pressure of oxygen in arterial blood (PaO(2)) decreased significantly, lung W/D weight ratio and MPO activity increased significantly (all P<0.05), histopathology of lung revealed signs of injury. After injury, expression of LXR alpha mRNA in lung tissue decreased markedly, and expression of TNF-alpha mRNA in lung tissue increased markedly (all P<0.05). TNF-alpha increased markedly in lung homogenate and blood serum at the same period, and TNF-alpha reached peak value at 4 hours. Immunohistochemical staining of LXR alpha showed that lung tissues of normal rats express LXR alpha significantly, however, after injury, expression of LXR alpha in lung tissue decreased markedly (all P<0.05). Lung tissues of normal rats express LXR alpha. The decreased LXR alpha mRNA and protein expressions in the lung tissue of rats with ALI caused by LPS may be associated with the occurrence of ALI.

CDNA cloning and expression of a novel group IB phospholipase A2 in the intestine of the red sea bream, Pagrus (Chrysophrys) major

A cDNA encoding a novel phospholipase A2 (PLA2), which was named IN PLA2, was cloned from the intestine of the red sea bream. The amino acid sequence of IN PLA2 showed 49–75% homology with those of red sea bream group IB sPLA2, hepatopancreas DE-1 and DE-2 PLA2, and gill G-3 PLA2. IN PLA2 consists of a prepropeptide of 24 amino acid residues, followed by a mature protein. IN PLA2 contains 14 cysteines, and includes Cys11, the calcium binding loop and the pancreatic loop that are commonly conserved in group IB sPLA2 enzymes. In addition, IN PLA2 is a cationic protein with a pI of 8.52. Therefore, IN PLA2 was identified as a novel group IB sPLA2 isoform in red sea bream. IN PLA2 mRNA was found by northern blot analysis to be expressed mainly in the pyloric caeca and the intestine, and was detected in the goblet cells of the intestine by in situ hybridization. The expression level of IN PLA2 mRNA was elevated by intravenous injection of lipopolysaccharide—the outer-membrane component of Gram-negative bacteria. These results suggest that IN PLA2 is secreted from the goblet cells of the intestine in response to stimulus such as bacterial infection, and that it contributes to antimicrobial defense in addition to the digestion of dietary phospholipids in the gastrointestinal tract.

Time course of angiopoietin-2 release during experimental human endotoxemia and sepsis

IntroductionEndothelial activation leading to vascular barrier breakdown denotes a devastating event in sepsis. Angiopoietin (Ang)-2, a circulating antagonistic ligand of the endothelial specific Tie2 receptor, is rapidly released from Weibel-Palade and has been identified as a non-redundant gatekeeper of endothelial activation. We aimed to study: the time course of Ang-2 release during human experimental endotoxemia; the association of Ang-2 with soluble adhesion molecules and inflammatory cytokines; and the early time course of Ang-2 release during sepsis in critically ill patients.MethodsIn 22 healthy volunteers during a 24-hour period after a single intravenous injection of lipopolysaccharide (LPS; 4 ng/kg) the following measurement were taken by immuno luminometric assay (ILMA), ELISA, and bead-based multiplex technology: circulating Ang-1, Ang-2, soluble Tie2 receptor, the inflammatory molecules TNF-alpha, IL-6, IL-8 and C-reactive protein, and the soluble endothelial adhesion molecules inter-cellular adhesion molecule-1 (ICAM-1), E-selectin, and P-selectin. A single oral dose of placebo or the p38 mitogen activated protein (MAP) kinase inhibitor drug, RWJ-67657, was administered 30 minutes before the endotoxin infusion. In addition, the course of circulating Ang-2 was analyzed in 21 septic patients at intensive care unit (ICU) admission and after 24 and 72 hours, respectively.ResultsDuring endotoxemia, circulating Ang-2 levels were significantly elevated, reaching peak levels 4.5 hours after LPS infusion. Ang-2 exhibited a kinetic profile similar to early pro-inflammatory cytokines TNF-alpha, IL-6, and IL-8. Ang-2 levels peaked prior to soluble endothelial-specific adhesion molecules. Finally, Ang-2 correlated with TNF-alpha levels (r = 0.61, P = 0.003), soluble E-selectin levels (r = 0.64, P < 0.002), and the heart rate/mean arterial pressure index (r = 0.75, P < 0.0001). In septic patients, Ang-2 increased in non-survivors only, and was significantly higher compared with survivors at baseline, 24 hours, and 72 hours.ConclusionsLPS is a triggering factor for Ang-2 release in men. Circulating Ang-2 appears in the systemic circulation during experimental human endotoxemia in a distinctive temporal sequence and correlates with TNF-alpha and E-selectin levels. In addition, not only higher baseline Ang-2 concentrations, but also a persistent increase in Ang-2 during the early course identifies septic patients with unfavorable outcome.

Host Inactivation of Bacterial Lipopolysaccharide Prevents Prolonged Tolerance Following Gram-Negative Bacterial Infection

A transient state of tolerance to microbial molecules accompanies many infectious diseases. Such tolerance is thought to minimize inflammation-induced injury, but it may also alter host defenses. Here we report that recovery from the tolerant state induced by Gram-negative bacteria is greatly delayed in mice that lack acyloxyacyl hydrolase (AOAH), a lipase that partially deacylates the bacterial cell-wall lipopolysaccharide (LPS). Whereas wild-type mice regained normal responsiveness within 14 days after they received an intraperitoneal injection of LPS or Gram-negative bacteria, AOAH-deficient mice had greatly reduced proinflammatory responses to a second LPS injection for at least 3 weeks. In contrast, LPS-primed Aoah- knockout mice maintained an anti-inflammatory response, evident from their plasma levels of interleukin-10 (IL-10). LPS-primed Aoah-knockout mice experiencing prolonged tolerance were highly susceptible to virulent E. coli challenge. Inactivating LPS, an immunostimulatory microbial molecule, is thus important for restoring effective host defenses following Gram-negative bacterial infection in animals.

Activation of the PGI 2 /IP System Contributes to the Development of Circulatory Failure in a Rat Model of Endotoxic Shock

Prostacyclin levels are increased in septic patients and several animal models of septic shock, and selective inhibition of cyclooxygenase-2 improved cardiovascular dysfunction in rats treated with lipopolysaccharide (LPS). Here, we examine the specific role of prostacyclin and of the receptor for prostacyclin (IP) in the development of LPS-induced circulatory failure. Intravenous injection of LPS (10 mg/kg) into male Sprague-Dawley rats caused a strong increase in plasma prostacyclin levels, which was paralleled by a decrease in blood pressure and an increase in heart rate. Moreover, LPS injection increased the mRNA expression of the IP receptor in the heart, aorta, lung, liver, adrenal glands, and kidneys. Cotreatment with the IP antagonist CAY-10441 (1, 10, 30, and 100 mg/kg) dose-dependently moderated the LPS-induced changes in mean arterial blood pressure, heart rate, cardiac output, and systemic vascular resistance. The development of cardiovascular failure was ameliorated by CAY-10441 in spite of the typical LPS-induced increases in plasma levels of cytokines and NO. In vitro, cytokines dose- and time-dependently induced IP expression in rat vascular smooth muscle cells. Incubation of cells with the stable IP agonist iloprost in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-mehylxanthine resulted in higher cAMP levels in cytokine-treated cells compared with untreated cells. Taken together, our data demonstrate a prominent role of the prostacyclin/IP system in the development of LPS-induced cardiovascular failure.

Role of Shenfu Injection (参附注射液) in rats with systemic inflammatory response syndrome

To investigate the role of Shenfu Injection (SFI) in rats with systemic inflammatory response syndrome (SIRS). The SIRS rat model was induced by the intravenous injection of lipopolysaccharide (LPS). Forty-five male Wistar rats were randomly divided into 3 groups, the sham operative control group (control group, n=5), the SIRS model group (model group, n=20) and the SFI treatment group (SFI group, n=20). LPS was injected through the external jugular vein (12 mg/kg, 6 mg/mL) to all rats except for those in the control group, and SFI (10 mL/kg) was given to those in the SF group only once through intraperitoneal injection, while the normal saline (10 mL/kg) was given to those in the model group. For those in the control group, normal saline was given through the external jugular vein (2 mL/kg) and intraperitoneal injection (10 mL/kg). Then, rats in the model group and SFI group were divided into 4 subgroups according to the time points, i.e., 1 h, 2 h, 4 h and 6 h subgroups, 5 rats in each group. The activity of nuclear factor of kappa B (NF-kappa B) of in blood mononuclear cells and the plasma levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin 6-(IL-6) were determined using enzyme-linked immunoabsordent assay (ELISA) at 1 h, 2 h, 4 h and 6 h after modeling. Histopathologic changes of the lung and liver were observed under a light microscope. Compared with the control group, the activity of NF-kappa B in mononuclear cells and the plasma level of TNF-alpha were obviously increased at each time points (all P<0.01), reaching the peaks at 2 h after modeling. The plasma level of IL-6 increased gradually as time went by in the model group (P<0.01). Pathological examination showed pulmonary alveoli hemorrhage, edema and inflammatory cell infiltration in the lung tissue, and angiotelectasis, congestion, and local necrosis in the liver tissue in the model group. Compared with the model group, the activity of NF-kappa B and the levels of TNF-alpha and IL-6 in plasma decreased significantly in the SFI group (P<0.01), and the pathological injury in the lungs and liver was significantly alleviated. SFI plays a protective role by inhibiting the activity of NF-kappaB, and reducing the expressions of TNF-alpha and IL-6 in SIRS rats.

High Mobility Group Box 1 Induces a Negative Inotropic Effect on the Left Ventricle in an Isolated Rat Heart Model of Septic Shock A Pilot Study

Sepsis can be exacerbated by an inappropriate immune response and the severe impact of this disease on the cardiovascular system is well documented. High mobility group box 1 (HMGB1) protein is an important mediator in the pathogenesis of sepsis and its role in cardiovascular system dysfunction was investigated in an lipopolysaccharide (LPS)-induced rat model of sepsis. Twelve hours after intravenous bolus injections of LPS (5 mg/kg), rats were killed and heart samples were harvested. Immunoblot analysis was performed to assess expression levels of HMGB1 in cardiac myocytes. Left ventricular developed pressure (LVDP) served as a measure of systolic function. LPS administration was associated with an increase in the expression of HMGB1 in cardiac myocytes and a decrease in cardiac function. Hearts from the LPS-treated rats were also perfused with recombinant HMGB1 and cardiac function measured. The dose-dependent effects observed with elevated HMGB1 included decreased LVDP, decreased left ventricular (LV) + dP/dt(max), decreased absolute value of LV- dP/dt(min), and increased LV end-diastolic pressure. HMGB1 stimulation produces a negative inotropic effect during septic shock, suggesting an important role for this molecule in cardiovascular system dysfunction during sepsis.

Systemic and local high mobility group box 1 concentrations during severe infection

Objective: High mobility group box 1 (HMGB1) has been implicated as a late mediator in sepsis. We here sought to determine the extent of HMGB1 release in patients with sepsis stratified to the three most common infectious sources and to determine HMGB1 concentrations at the site of infection during peritonitis or pneumonia. Design: Observational studies in patients and healthy humans challenged with lipopolysaccharide. Setting: Three intensive care units and one clinical research unit. Patients and Subjects: Three patient populations were studied: 1) 51 patients with sepsis due to pneumonia (n = 29), peritonitis (n = 12), or urinary tract infection (n = 10); 2) 17 patients with peritonitis; and 3) four patients with community-acquired pneumonia. In addition, eight healthy subjects were studied after intravenous injection of lipopolysaccharide (4 ng/kg). Interventions: One population of healthy volunteers received lipopolysaccharide intravenously. Measurements and Main Results: Patients with severe sepsis due to pneumonia displayed elevated circulating HMGB1 concentrations at both days 0 and 3 after inclusion. Patients with sepsis due to peritonitis had elevated HMGB1 levels at day 0 but not at day 3, whereas urinary tract infection was associated with a delayed HMGB1 response, with elevated levels only at day 3. HMGB1 concentrations did not differ between survivors and nonsurvivors and were not correlated to either disease severity or concurrently measured cytokine levels. In line with these observations, although intravenous lipopolysaccharide injection clearly elevated plasma cytokine levels, HMGB1 remained undetectable. In patients with peritonitis, HMGB1 concentrations in abdominal fluid were more than ten-fold higher than in concurrently obtained plasma. In pneumonia patients, HMGB1 levels were higher in bronchoalveolar lavage fluid obtained from the site of infection than in lavage fluid from healthy controls. Conclusions: In severe sepsis, the kinetics of HMGB1 release may differ depending on the primary source of infection. In patients with severe infection, HMGB1 release may predominantly occur at the site of infection.

Systemic and local high mobility group box 1 concentrations during severe infection

High mobility group box 1 (HMGB1) has been implicated as a late mediator in sepsis. We here sought to determine the extent of HMGB1 release in patients with sepsis stratified to the three most common infectious sources and to determine HMGB1 concentrations at the site of infection during peritonitis or pneumonia. Observational studies in patients and healthy humans challenged with lipopolysaccharide. Three intensive care units and one clinical research unit. Three patient populations were studied: 1) 51 patients with sepsis due to pneumonia (n = 29), peritonitis (n = 12), or urinary tract infection (n = 10); 2) 17 patients with peritonitis; and 3) four patients with community-acquired pneumonia. In addition, eight healthy subjects were studied after intravenous injection of lipopolysaccharide (4 ng/kg). One population of healthy volunteers received lipopolysaccharide intravenously. Patients with severe sepsis due to pneumonia displayed elevated circulating HMGB1 concentrations at both days 0 and 3 after inclusion. Patients with sepsis due to peritonitis had elevated HMGB1 levels at day 0 but not at day 3, whereas urinary tract infection was associated with a delayed HMGB1 response, with elevated levels only at day 3. HMGB1 concentrations did not differ between survivors and nonsurvivors and were not correlated to either disease severity or concurrently measured cytokine levels. In line with these observations, although intravenous lipopolysaccharide injection clearly elevated plasma cytokine levels, HMGB1 remained undetectable. In patients with peritonitis, HMGB1 concentrations in abdominal fluid were more than ten-fold higher than in concurrently obtained plasma. In pneumonia patients, HMGB1 levels were higher in bronchoalveolar lavage fluid obtained from the site of infection than in lavage fluid from healthy controls. In severe sepsis, the kinetics of HMGB1 release may differ depending on the primary source of infection. In patients with severe infection, HMGB1 release may predominantly occur at the site of infection.

Plasma Nitric Oxide Concentrations in Broilers After Intravenous Injections of Lipopolysaccharide or Microparticles

Nitric oxide is a potent vasodilator synthesized from l-Arg by NO synthase (NOS). Constitutive NOS in endothelial cells (eNOS) produces transient bursts of NO in low but physiologically effective levels. Activated monocytes and macrophages express inducible NOS (iNOS), which produces copious quantities of NO. Previous studies showed that NO attenuates pulmonary hypertensive responses induced by i.v. injections of lipopolysaccharide (LPS) or cellulose microparticles (MP). The present study determined whether changes in plasma NO concentrations could be used to assess the time course of NO production in response to LPS or MP injections. Broilers were injected i.v. with 1 mL of PBS (control), 1 mL of LPS (1 mg/mL), or 0.4 mL of MP (0.02 g/mL). Plasma samples were collected from 10 different broilers per group at 15, 30, 45, and 60 min and at 2, 3, 4, 5, 6, 8, 10, and 12 h postinjection. Total plasma NO concentrations were analyzed by nitrate + nitrite assay. After PBS or MP injection, plasma NO levels did not change throughout the 12-h period. Nitric oxide measured in the plasma increased in LPS-injected broilers from 4.8 ± 0.8 μM at 15 min to 46.6 ± 5.7 μM by 4 h postinjection, reached peak levels of 85.1 ± 10.6 μM at 5 h, and returned to baseline levels similar to PBS-injected broilers by 12 h postinjection. We conclude that LPS triggered widespread iNOS expression by circulating monocytes and macrophages, resulting in copious NO production as reflected by significant increases in total plasma NO. Proportionally few monocytes and macrophages responded to MP entrapped in pulmonary arterioles. Consequently, NO produced by iNOS in activated leukocytes or by eNOS in the pulmonary vasculature had a minimal impact on total plasma NO. Total plasma NO from broilers did reflect the time course of massive iNOS activation in response to LPS, but biologically relevant quantities of NO produced by iNOS and eNOS activated during the local inflammatory response to entrapped MPs were too low to affect total plasma NO concentrations.