Endotoxemic Shock Research Articles

REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation.

Regulated in development and DNA damage responses 1 (REDD-1), an inhibitor of mammalian target of rapamycin (mTOR), is induced by various cell stressors, including LPS, a major player in the pathogenesis of endotoxemic shock. However, the pathologic role of REDD-1 in endotoxemia is largely unknown. We found that LPS increased REDD-1 expression, nuclear transcription factor-κB (NF-κB) activation, and inflammation and that these responses were suppressed by REDD-1 knockdown and in REDD-1+/- macrophages. REDD-1 overexpression stimulated NF-κB-dependent inflammation without additional LPS stimulation. REDD-1-induced NF-κB activation was independent of 2 classic IKK-dependent NF-κB pathways and the mTOR signaling pathway; however, REDD-1, particularly its C-terminal region (178-229), interacted with and sequestered IκBα, to elicit atypical NF-κB activation during the delayed and persistent phases of inflammation after stimulation. Moreover, REDD-1 knockdown mitigated vascular inflammation and permeability in endotoxemic mice, resulting in decreases in immune cell infiltration, systemic inflammation, caspase-3 activation, apoptosis, and consequent mortality. We further confirmed the inflammatory and cytotoxic effects of REDD-1 in endotoxemic REDD-1+/- mice. Our data support the likelihood that REDD-1 exacerbates endotoxemic inflammation via atypical NF-κB activation by sequestering IκBα.-Lee, D.-K., Kim, J.-H., Kim, J., Choi, S., Park, M., Park, W., Kim, S., Lee, K.-S., Kim, T., Jung, J., Choi, Y. K., Ha, K.-S., Won, M.-H., Billiar, T. R., Kwon, Y.-G., Kim, Y.-M. REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation.

Novel Function of Isoamylamine Improves Survival in Endotoxemic Mice by Ameliorating Coagulopathy and Attenuating MMP-9 Expression Through p-ERK/p-p38 Signaling at Early Stage.

When a host suffers endotoxemic shock or septic shock, it results in many symptoms including disseminated intravascular coagulation (DIC). Septic shock (SS) causes coagulation time to decrease and then gradually increase, finally becoming prolonged and giving rise to DIC. Isoamylamine (IA) is one of the main components of grape products and can improve the survival rate of endotoxin lipopolysaccharide (LPS)-induced endotoxemic shock. The aim of this study was to elucidate if IA ameliorates coagulopathy in the early phase of LPS-induced damage. We studied the effects of IA on the coagulation system of extrinsic (prothrombin time [PT]) and intrinsic (activated partial thromboplastin time [aPTT]) pathways. PT and aPTT were tested in plasma drawn from mice following intraperitoneal (IP) injection of 1 mL of 1,000 ppm IA after LPS administration. Shortened PT was ameliorated by 1,000 ppm IA 1 h after LPS administration, but there was no effect on aPTT. In conclusion, IA 1,000 ppm partially intervenes in the early phase of LPS-induced damage, shortening plasma PT 1 h after LPS treatment in mice. Furthermore, we found 1,000 ppm IA also could attenuate MMP-9 expression through p-ERK/p-p38 signaling in mice hepatocyte extracts. This study focused on the effects of IA on blood coagulation function and inflammatory proteins. In the current situation of absence of effective treatment for SS, IA can increase survival rate and may offer another choice of patient avoiding causing death during endotoxemic shock.

Lanosterol Modulates TLR4-Mediated Innate Immune Responses in Macrophages

Macrophages perform critical functions in both innate immunity and cholesterol metabolism. Here, we report that activation of Toll-like receptor 4 (TLR4) in macrophages causes lanosterol, the first sterol intermediate in the cholesterol biosynthetic pathway, to accumulate. This effect is due to type I interferon (IFN)-dependent histone deacetylase 1 (HDAC1) transcriptional repression of lanosterol-14α-demethylase, the gene product of Cyp51A1. Lanosterol accumulation in macrophages, because of either treatment with ketoconazole or induced conditional disruption of Cyp51A1 in mouse macrophages invitro, decreases IFNβ-mediated signal transducer and activator of transcription (STAT)1-STAT2 activation and IFNβ-stimulated gene expression. These effects translate into increased survival to endotoxemic shock by reducing cytokine secretion. In addition, lanosterol accumulation increases membrane fluidity and ROS production, thus potentiating phagocytosis and the ability to kill bacteria. This improves resistance of mice to Listeria monocytogenes infection by increasing bacterial clearance in the spleen and liver. Overall, our data indicate that lanosterol is an endogenous selective regulator of macrophage immunity.

The impact of hydrocortisone treatment on neutrophil gelatinase-associated lipocalin release in porcine endotoxemic shock

BackgroundA key feature of sepsis is systemic inflammatory activation that could be counteracted by steroids. In this experimental model of systemic inflammation, we sought to investigate whether septic neutrophil activation, evaluated by the plasma levels of neutrophil gelatinase-associated protein (NGAL), is modulated by the timing of hydrocortisone treatment.MethodsSixteen anesthetized pigs were allocated to one of four equally sized groups. Three of these groups received endotoxin at 2 μg × kg−1 × h−1 for 6 h so as to induce endotoxemic shock. Hydrocortisone (5 mg × kg−1) was administered intravenously before endotoxemic challenge, or at the onset of endotoxemic shock. Endotoxemic pigs not receiving hydrocortisone and non-endotoxemic pigs served as control groups. Physiologic variables, hematology, and biochemistry, including plasma NGAL, were measured repeatedly.ResultsHydrocortisone treatment prior to endotoxemia attenuated some inflammatory, hematological, circulatory, and metabolic manifestations of shock (i.e., higher white blood cell count, higher mean arterial pressure, lower heart rate and mean pulmonary arterial pressure, higher left ventricular stroke work index, higher base excess). Endotoxemic shock increased plasma NGAL (p < 0.001). In pigs given hydrocortisone before the endotoxin infusion, plasma NGAL was lower as compared to those given hydrocortisone at endotoxemic shock (p < 0.05). Plasma NGAL levels correlated inversely to neutrophil granulocyte counts (rho = −0.65) but not to urine output (rho = −0.10) at the end of the experiment.ConclusionsThe increase in plasma NGAL is counteracted by hydrocortisone administration prior to endotoxemia; concomitantly, this treatment was associated with less expressed circulatory derangement. Urine NGAL did not differ between the groups, suggesting that the NGAL response was not primarily related to kidney injury.

Progranulin Controls Sepsis via C/EBPα-Regulated Il10 Transcription and Ubiquitin Ligase/Proteasome-Mediated Protein Degradation.

Progranulin (PGRN) is a widely expressed, pleiotropic protein that is involved in diverse biological processes, including cellular proliferation, neuron development, and wound healing. However, the role of PGRN in the regulation of pathogen-induced systemic inflammation and the mechanisms involved have not been established. In this study, we show that PGRN-deficient mice display heightened mortality in models of polymicrobial sepsis and endotoxinemia, with increased tissue levels of inflammatory cytokines and reduced IL-10 production. Conversely, administration of rPGRN decreases the susceptibility of PGRN-deficient mice to LPS-induced endotoxemic shock and augments IL-10 production by LPS-activated macrophages in a TNFR-dependent manner. Molecular analysis reveals a direct role of the transcription factor C/EBPα in PGRN-regulated IL-10 expression. C/EBPα-deficient macrophages produce less IL-10 in response to LPS. Furthermore, mice deficient in C/EBPα in hematopoietic cells are highly vulnerable to LPS-induced septic shock. Lastly, the defective IL-10 production by PGRN-deficient cells is primarily due to reduced C/EBPα protein stability via the E3 ubiquitin-conjugating enzyme E6AP and proteasome-mediated degradation. To our knowledge, this study provides the first evidence that PGRN is a nonredundant regulator of systemic inflammation via modulating the levels and activity of C/EBPα, IL-10, and the ubiquitin-proteasome proteolysis pathway. The results bear strong and profound implications for PGRN insufficiency and its mutation-associated systemic and organ-specific inflammatory human diseases.

Effects of N-acetylcysteine (NAC) supplementation in resuscitation fluids on renal microcirculatory oxygenation, inflammation, and function in a rat model of endotoxemia.

BackgroundModulation of inflammation and oxidative stress appears to limit sepsis-induced damage in experimental models. The kidney is one of the most sensitive organs to injury during septic shock. In this study, we evaluated the effect of N-acetylcysteine (NAC) administration in conjunction with fluid resuscitation on renal oxygenation and function. We hypothesized that reducing inflammation would improve the microcirculatory oxygenation in the kidney and limit the onset of acute kidney injury (AKI).MethodsRats were randomized into five groups (n = 8 per group): (1) control group, (2) control + NAC, (3) endotoxemic shock with lipopolysaccharide (LPS) without fluids, (4) LPS + fluid resuscitation, and (5) LPS + fluid resuscitation + NAC (150 mg/kg/h). Fluid resuscitation was initiated at 120 min and maintained at fixed volume for 2 h with hydroxyethyl starch (HES 130/0.4) dissolved in acetate-balanced Ringer’s solution (Volulyte) with or without supplementation with NAC (150 mg/kg/h). Oxygen tension in the renal cortex (CμPO2), outer medulla (MμPO2), and renal vein was measured using phosphorimetry. Biomarkers of renal injury, inflammation, and oxidative stress were assessed in kidney tissues.ResultsFluid resuscitation significantly improved the systemic and renal macrohemodynamic parameters after LPS. However, the addition of NAC further improved cortical renal oxygenation, oxygen delivery, and oxygen consumption (p < 0.05). NAC supplementation dampened the accumulation of NGAL or L-FABP, hyaluronic acid, and nitric oxide in kidney tissue (p < 0.01).ConclusionThe addition of NAC to fluid resuscitation may improve renal oxygenation and attenuate microvascular dysfunction and AKI. Decreases in renal NO and hyaluronic acid levels may be involved in this beneficial effect. A therapeutic strategy combining initial fluid resuscitation with antioxidant therapies may prevent sepsis-induced AKI.

Fully Balanced Fluids do not Improve Microvascular Oxygenation, Acidosis and Renal Function in a Rat Model of Endotoxemia

The expectation of fluid therapy in patients with septic shock is that it corrects hypovolemia, with the aim of restoring tissue perfusion and oxygenation and organ function. This study investigated whether different types of resuscitation fluids were effective in improving renal microcirculatory oxygenation, acidosis, oxidative stress, and renal function in a rat model of endotoxemic shock. Five groups of rats were used: a sham group, a lipopolysaccharide (LPS) group, and three LPS groups that received 30 mL/kg/h of 0.9% sodium chloride (0.9% NaCl), a new bicarbonate buffered crystalloid solution closely resembling the composition of plasma (FB-Cxt) or a hydroxyethyl starch-ringer acetate solution. Systemic hemodynamic variables, renal blood flow, microvascular oxygenation, oxidative/nitrosative stress, and renal function were measured. LPS-induced shock was only partially resolved by fluid administration. Animals became arterially hypotensive despite adequate central venous pressure. Hydroxyethyl starch-ringer acetate was more effective at improving arterial pressures and renal blood flow than 0.9% NaCl or FB-Cxt. Fluids had marginal effects on pH and HCO3 levels irrespective of the buffer, or on renal μPO2 and dysfunction. Colloids increased the markers of renal oxidative stress (P < 0.001), whereas unbalanced crystalloids increased the markers of nitrosative stress during sepsis (P < 0.01). Endotoxemia-induced acidosis and decreases in renal μPO2 or renal injury were not corrected solely by fluid resuscitation, irrespective of the buffer of the fluid. Our study supported the idea that fluids must be supplemented by other compounds that specifically correct renal inflammation and oxygenation to be effective in resolving septic shock-induced renal failure.

0011 : In early phase of septic shock, O-GlcNAc improves cardiovascular function without modification of autophagic or inflammatory pathways

O-GlcNAcylation, a post-translational modification, is the end product of the hexosamine biosynthetic pathway (HBP). Acute septic shock is characterized by a systemic inflammation associated with a cardiovascular dysfunction. The aim of this study was to determine if the increase in total protein OGlcNAcylation at the early phase of septic shock could improve cardiovascular function and the autophagic or inflammatory pathways were involved in this effect. To induce an endotoxemic shock, rats (n=6-8) received iv either lipopolysaccharide (LPS, 5 mg/kg) or saline (CTRL). After 1 h, fluid resuscitation (FR, 15 ml/kg of colloid, iv ) was associated or not with HBP substrate: glucosamine (GlcN, 180 mg/kg) or an O-GlcNAcase inhibitor (NButGT, 10 mg/kg). Two hours later, echography and mean arterial pressure (MAP) measurement were performed. Then, hearts and blood samples were collected to evaluate biological parameters, inflammation and autophagy. LPS rats developed an hypotension which was restored by FR, GlcN and NButGT. They have also a systolic dysfunction with a trend toward an improvement by NButGT and GlcN (ejection fraction: CTRL 80±3, LPS 66±3*, LPS-FR 69±2, NButGT 74±2, GlcN 75±2%, *: p<0.01 vs CTRL). Plasmatic troponin T and lactate were significantly increased by 31 and 1.7-fold respectively in LPS rats, and were normalised under NButGT and GlcN conditions. Plasmatic and cardiac cytokines (IL-6 and TNFα) were increased in LPS rats without modification wathever treatments. Autophagic actors like Beclin-1, LC3 and Lamp-2 were not modified by LPS or any treatments. Our results suggest that O-GlcNAc increase at the early phase of septic shock improves cardiovascular function without involvement of autophagic or inflammatory pathways. We are undergoing to determine which pathways are involved in the beneficial effects induced by the O-GlcNAc increase. The author hereby declares no conflict of interest

Induction of endothelium-dependent constriction of mesenteric arteries in endotoxemic hypotensive shock.

Effective management of hypotension refractory to vasoconstrictors in severe sepsis is limited. A new strategy to ameliorate endotoxemic hypotension by inducing endothelium-dependent constriction of large arteries was assessed. Endotoxemia in rats was induced by injection of LPS (10 mg·kg(-1), i.v.). Haemodynamics were measured in vivo, reactivity of isolated mesenteric arteries by myography and expression of proteins and enzyme activities by immunohistochemistry, biochemistry and molecular biology. Six hours after LPS, the hypotension was promptly reversed following injection (i.v. or i.p.) of oroxylin-A (OroA) . In isolated LPS-treated but not normal mesenteric arteries, OroA (1-10 μM) induced endothelium-dependent, sustained constriction, blocked by endothelin-1 (ET-1) receptor antagonists. OroA further enhanced LPS-induced expression of endothelin-converting enzyme, ET-1 mRNA and proteins and ET-1 release, OroA also enhanced phosphorylation of Rho-associated protein kinase (ROCK) and reversed LPS-induced suppression of RhoA activities in smooth muscle of arteries with endothelium. Activated- phosphorylation of smooth muscle ROCK was blocked by ET-1-receptor antagonists and ROCK inhibitors. Moreover, OroA post-treatment suppressed, via inhibiting NF-κB activation, inducible NOS expression and circulating NO. Reversal of endotoxemic hypotensive by OroA was due to release of endothelial ET-1, upregulated by LPS, from mesenteric arteries, inducing prompt and sustained vasoconstriction via activation of vascular smooth muscle RhoA/ROCK-pathway. In late endotoxemia, OroA-induced vasoconstriction was partly due to decreased circulating NO. Activation of endothelium-dependent constriction in large resistance arteries and suppression of systemic inflammation offer new strategies for acute management of endotoxemic hypotensive shock.

0347: Beneficial cardiovascular effects of O-GlcNAc stimulation in early phase of septic shock

O-GlcNAcylation, a post-translational modification, is the end product of the hexosamine biosynthetic pathway (HBP). Recent studies showed beneficial effects of its increase in acute pathologies, such as hemorrhagic shock. We postulated that increase in total protein OGlcNAcylation at the early phase of septic shock, a systemic inflammation associated with a cardiovascular dysfunction, could improve cardiovascular function and reduce mortality. To induce an endotoxemic shock, rats (n=6-8) received iv either lipopolysaccharide (LPS, 5mg/kg) or saline (CTRL). After 1 h, fluid resuscitation (FR,15mL/kg of colloid, iv) was associated or not with HBP substrate: glucosamine (GlcN, 180mg/kg) or an O-GlcNAcase inhibitor (NButGT, 10mg/kg). Two hours later, echography and mean arterial pressure (MAP) evaluation were performed; blood samples and heart were then collected to evaluate biological parameters (lactate, troponin T) and total O-GlcNAcylation by western-blot. In vivo studies showed an hypotension in LPS restored by FR and treatments (MAP: CTRL 87±1, LPS 73±5*, LPS-FR 93±6#, NButGT 83±5, GlcN 92±4mmHg, *: p<0.01 vs CTRL, #: p<0.01 vs LPS) and a systolic dys-function in LPS with a trend toward an improvement by NButGT and GlcN (ejection fraction: CTRL 80±3, LPS 66±3*, LPS-FR 69±2, NButGT 74±2, GlcN 75±2%, *: p<0.01 vs CTRL). NButGT and GlcN efficiently increased total O-GlcNAc (200 and 300% respectively vs LPS-FR). This effect was associated with a reduced cardiomyocyte insult and tissue dysoxia. At the early phase of septic shock NButGT and GlcN induce an increase in cardiac O-GlcNAcylation leading to improve tissue oxygenation and to reduce cardiac injury. These treatments trend to increase the cardiovascular function. Yet, coming studies will evaluate the effect of NButGT and GlcN on mortality.

Cardiac Effects of Echinocandins in Endotoxemic Rats.

Echinocandins are known as effective and safe agents for the prophylaxis and treatment of different cohorts of patients with fungal infections. Recent studies revealed that certain pharmacokinetics of echinocandin antifungals might impact clinical efficacy and safety in special patient populations. The aim of our study was to evaluate echinocandin-induced aggravation of cardiac impairment in septic shock. Using an in vivo endotoxemic shock model in rats, we assessed hemodynamic parameters and time to hemodynamic failure (THF) after additional central-venous application of anidulafungin (2.5 mg/kg of body weight [BW]), caspofungin (0.875 mg/kg BW), micafungin (3 mg/kg BW), and control (0.9% sodium chloride). In addition, echinocandin-induced cytotoxicity was evaluated in isolated rat cardiac myocytes. THF of the animals in the caspofungin group (n = 7) was significantly reduced compared to that in the control (n = 6) (136 min versus 180 min; P = 0.0209). The anidulafungin group (n = 7) also showed a trend of reduced THF (136 min versus 180 min; log-rank test P = 0.0578). Animals in the micafungin group (n = 7) did not show significant differences in THF compared to those in the control. Control group animals and also micafungin group animals did not show altered cardiac output (CO) during our experiments. In contrast, administration of anidulafungin or caspofungin induced a decrease in CO. We also revealed a dose-dependent increase of cytotoxicity in anidulafungin- and caspofungin-treated cardiac myocytes. Treatment with micafungin did not cause significantly increased cytotoxicity. Further studies are needed to explore the underlying mechanism.

Different effects of propofol and dexmedetomidine on preload dependency in endotoxemic shock with norepinephrine infusion

BackgroundTo clarify whether propofol (PROP) and dexmedetomidine (DEX) differentially affect preload dependency in an endotoxemic model based on evaluations of the systemic vascular system and cardiac function. MethodsAnimals were prepared under PiCCO monitoring (BL), and endotoxemic shock was induced using an intravenous bolus of lipopolysaccharide (055:B5) in 16 New Zealand ketamine-anesthetized rabbits. After fluid resuscitation and norepinephrine infusion (SD0), the animals were randomized to PROP (n = 8) or DEX (n = 8) sedation at two incremental doses (SD1 and SD2). The mean arterial pressure and the central venous pressure were monitored. Pulse pressure variation (PPV) was assessed to evaluate preload dependency. Global end-diastolic volume, vascular resistance, mean systemic filling pressure, and cardiac function index were assessed at each time point. ResultsPPV progressively and significantly increased with increasing infusion rates of PROP (SD1 versus SD0, P < 0.01; SD2 versus SD0, P < 0.001; and SD2 versus SD1, P = 0.024) but not DEX. PPV was higher at SD1 and SD2 in the PROP group than in the DEX group (P < 0.001). PROP increased the heart rate without affecting cardiac contractility or vascular resistance. In contrast, DEX decreased heart contractility and increased vascular resistance at the highest dose. However, neither drug affected mean arterial pressure, central venous pressure, mean systemic filling pressure, global end-diastolic volume, or venous return. ConclusionsPROP more effectively increased PPV than DEX in an endotoxemic shock model after fluid resuscitation during norepinephrine infusion. DEX, but not PROP, at the highest dose influenced vascular resistance and heart contractility.

Increased β2-adrenergic vasorelaxation at the early phase of endotoxemic shock in rats

Background and purposeThe early management of the cardiovascular dysfunction of septic shock is critical as it is associated with a poor outcome. Although the use of catecholamines is a common therapy in this syndrome, no data are available on the involvement of β-adrenoceptor (β-AR) subtypes and only few studies report an alteration of β-adrenergic-induced vasodilation in septic shock. The purpose of the study was to evaluate vascular β1, β2 and β3-AR expression and function in an endotoxemic rat model. Experimental approachEndotoxemia was induced in rats by intravenous injection of lipopolysaccharide (LPS). β1, β2 and β3-AR mRNA expression was evaluated by RT-PCR in aorta and vascular β1, β2 and β3-AR responses were determined on conducting (aorta) and/or resistance (mesenteric and renal) arteries by constructing relaxation curves in response to different β-AR agonists. ResultsThe maximal effect of isoproterenol decreased by 31 to 61% in the three vascular beds of LPS-treated rats compared to controls. In aortas from LPS-treated rats, β1 and β3-AR mRNA expression was decreased and associated to a reduced β1 and β3-induced vasodilation. Conversely, albeit β2-AR mRNA was unchanged, the maximal β2-AR-induced vasodilation increased by 49% in aortas from LPS-treated rats compared to controls. This increase was not affected by endothelium removal but was abolished in the presence of a β2-AR antagonist or an adenylate cyclase inhibitor. ConclusionsIn endotoxemia, β2-AR vasodilation was increased by a potential recruitment of β2-AR located on smooth muscle cells. This study suggests that vascular β2-AR should be a putative new therapeutic target in septic shock.

Renal Effects of Saline-based 10% Pentastarch versus 6% Tetrastarch Infusion in Ovine Endotoxemic Shock: Erratum

Renal Effects of Saline-based 10% Pentastarch <i>versus</i> 6% Tetrastarch Infusion in Ovine Endotoxemic Shock: Erratum

PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock

AbstractNeutrophil extracellular traps (NETs), consisting of nuclear DNA with histones and microbicidal proteins, are expelled from activated neutrophils during sepsis. NETs were shown to trap microbes, but they also fuel cardiovascular, thrombotic, and autoimmune disease. The role of NETs in sepsis, particularly the balance between their antimicrobial and cytotoxic actions, remains unclear. Neutrophils from peptidylarginine deiminase 4-(PAD4−/−) deficient mice, which lack the enzyme allowing for chromatin decondensation and NET formation, were evaluated. We found that neutrophil functions involved in bacterial killing, other than NETosis, remained intact. Therefore, we hypothesized that prevention of NET formation might not have devastating consequences in sepsis. To test this, we subjected the PAD4−/− mice to mild and severe polymicrobial sepsis produced by cecal ligation and puncture. Surprisingly, under septic conditions, PAD4−/− mice did not fare worse than wild-type mice and had comparable survival. In the presence of antibiotics, PAD4-deficiency resulted in slightly accelerated mortality but bacteremia was unaffected. PAD4−/− mice were partially protected from lipopolysaccharide-induced shock, suggesting that PAD4/NETs may contribute to the toxic inflammatory and procoagulant host response to endotoxin. We propose that preventing NET formation by PAD4 inhibition in inflammatory or thrombotic diseases is not likely to increase host vulnerability to bacterial infections.

Punicalagin Induces Nrf2/HO-1 Expression via Upregulation of PI3K/AKT Pathway and Inhibits LPS-Induced Oxidative Stress in RAW264.7 Macrophages.

Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in potentiating macrophage activation, causing excessive inflammation, tissue damage, and sepsis. Recently, we have shown that punicalagin (PUN) exhibits anti-inflammatory activity in LPS-stimulated macrophages. However, the potential antioxidant effects of PUN in macrophages remain unclear. Revealing these effects will help understand the mechanism underlying its ability to inhibit excessive macrophage activation. Hemeoxygenase-1 (HO-1) exhibits antioxidant activity in macrophages. Therefore, we hypothesized that HO-1 is a potential target of PUN and tried to reveal its antioxidant mechanism. Here, PUN treatment increased HO-1 expression together with its upstream mediator nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). However, specific inhibition of Nrf2 by brusatol (a specific Nrf2 inhibitor) dramatically blocked PUN-induced HO-1 expression. Previous research has demonstrated that the PI3K/Akt pathway plays a critical role in modulating Nrf2/HO-1 protein expression as an upstream signaling molecule. Here, LY294002, a specific PI3K/Akt inhibitor, suppressed PUN-induced HO-1 expression and led to ROS accumulation in macrophages. Furthermore, PUN inhibited LPS-induced oxidative stress in macrophages by reducing ROS and NO generation and increasing superoxide dismutase (SOD) 1 mRNA expression. These findings provide new perspectives for novel therapeutic approaches using antioxidant medicines and compounds against oxidative stress and excessive inflammatory diseases including tissue damage, sepsis, and endotoxemic shock.

Aging and the intramyocardial inflammatory response.

The sepsis-induced intramyocardial inflammatory response results in decreased ventricular function and myocardial damage. Chemokines such as monocyte chemoattractant protein-1 causally contribute to retention of intramyocardial mononuclear leukocytes and subsequent ventricular dysfunction during endotoxemic shock in mice and, importantly, this effect is age dependent. It is therefore useful to consider where monocyte chemoattractant protein-1 fits in the complex pathway leading to ventricular dysfunction during sepsis, why this might be an age-dependent effect, and what this implies for care of older sepsis patients.

Study of acute hemocoagulation changes in a porcine endotoxemic shock model using thrombelastography

Disseminated intravascular coagulation and fibrinolysis have been associated with lipopolysaccharide (LPS)-induced endotoxemic sepsis. It has been well established by point-of-care (POC) thrombelastography (TEG) that pigs have a hemocoagulation pathophysiology that resembles humans. We evaluated the use of TEG during the development of coagulation abnormalities in a porcine model of endotoxemia. After approval by the Animal Welfare Committee, pigs were instrumented to record hemodynamic variables. Ten days after surgical instrumentation, LPS (50 μg/kg) was infused intravenously over a period of 45 minutes in conscious animals. Hemodynamic parameters were recorded before and for 6 hours after LPS infusion was completed. Simultaneously, blood samples were analyzed using TEG to measure reaction time (R), clotting time (K), alpha angle (α), maximum amplitude (MA), coagulation index (CI), percent lysis at 30 minutes, and percent lysis at 60 minutes. LPS induced profound hemodynamic changes associated with the induced endotoxemia. Concomitantly, a progressive consumption coagulopathy characterized by significant increases in R and K and decreases in α, MA, and CI developed. The overall hemocoagulation profile of the 3 nonsurviving animals (27%) was significantly different than that of the survivors. Fibrinolysis was not detected during the 6-hour evaluation period. All stages of clot formation were affected as demonstrated by TEG (increased R and K, decreased α and MA). Our results suggest that TEG is a rapid method for assessing coagulation abnormalities in early stages of endotoxemia in pigs. TEG could have significant clinical applications as a rapid POC method in human patients with sepsis.

An extracorporeal blood-cleansing device for sepsis therapy.

Here we describe a blood-cleansing device for sepsis therapy inspired by the spleen, which can continuously remove pathogens and toxins from blood without first identifying the infectious agent. Blood flowing from an infected individual is mixed with magnetic nanobeads coated with an engineered human opsonin--mannose-binding lectin (MBL)--that captures a broad range of pathogens and toxins without activating complement factors or coagulation. Magnets pull the opsonin-bound pathogens and toxins from the blood; the cleansed blood is then returned back to the individual. The biospleen efficiently removes multiple Gram-negative and Gram-positive bacteria, fungi and endotoxins from whole human blood flowing through a single biospleen unit at up to 1.25 liters per h in vitro. In rats infected with Staphylococcus aureus or Escherichia coli, the biospleen cleared >90% of bacteria from blood, reduced pathogen and immune cell infiltration in multiple organs and decreased inflammatory cytokine levels. In a model of endotoxemic shock, the biospleen increased survival rates after a 5-h treatment.

Fludrocortisone and Hydrocortisone, Alone or in Combination, on In Vivo Hemodynamics and In Vitro Vascular Reactivity in Normal and Endotoxemic Rats

Hydrocortisone enhances the pressor response to catecholamines in healthy volunteers and septic shock patients. Similar data do not exist for fludrocortisone. We assessed the effects of single administrations of fludrocortisone and hydrocortisone on systolic blood pressure until 2 hours after treatments injection and on in vitro vascular contraction of mesenteric artery rings to phenylephrine at 3 hours, in normal and endotoxemic rats. Intravenous fludrocortisone (5 and 20 μg/kg) and hydrocortisone (4 and 20 mg/kg) were administered in 16 groups (8 without and 8 with lipopolysaccharide-induced endotoxemic shock) of 10 Wistar rats according to four 2 × 2-factorial designs. Fludrocortisone and hydrocortisone similarly increased systolic blood pressure (P < 0.001 for both) but more in endotoxemic than in normal animals. Fludrocortisone and hydrocortisone significantly modified contractile response to phenylephrine (P = 0.039 and P = 0.007, respectively). At dose 1, fludrocortisone had no effect and hydrocortisone decreased contraction, whereas, at dose 2, both fludrocortisone and hydrocortisone increased contraction, especially in endotoxemic rats and with additive effect. Our results show that single intravenous administrations of fludrocortisone and hydrocortisone increase blood pressure and contractile response of mesenteric arteries to phenylephrine. The magnitude of these effects depends on dose and pathophysiological conditions and is higher in endotoxemic than in normal rats.