Cardiac power output accurately reflects external cardiac work over a wide range of inotropic states in pigs.

BACKGROUND: Cardiac power output (CPO), derived from the product of cardiac output and mean aortic pressure, is an important yet underexploited parameter for hemodynamic monitoring of critically ill patients in the intensive-care unit (ICU). The conductance catheter-derived pressure-volume loop area reflects left ventricular stroke work (LV SW). Dividing LV SW by time, a measure of LV SW min- 1 is obtained sharing the same unit as CPO (W). We aimed to validate CPO as a marker of LV SW min- 1 under various inotropic states. METHODS: We retrospectively analysed data obtained from experimental studies of the hemodynamic impact of mild hypothermia and hyperthermia on acute heart failure. Fifty-nine anaesthetized and mechanically ventilated closed-chest Landrace pigs (68 ± 1 kg) were instrumented with Swan-Ganz and LV pressure-volume catheters. Data were obtained at body temperatures of 33.0 °C, 38.0 °C and 40.5 °C; before and after: resuscitation, myocardial infarction, endotoxemia, sevoflurane-induced myocardial depression and beta-adrenergic stimulation. We plotted LVSW min- 1 against CPO by linear regression analysis, as well as against the following classical indices of LV function and work: LV ejection fraction (LV EF), rate-pressure product (RPP), triple product (TP), LV maximum pressure (LVPmax) and maximal rate of rise of LVP (LV dP/dtmax). RESULTS: CPO showed the best correlation with LV SW min- 1 (r2 = 0.89; p < 0.05) while LV EF did not correlate at all (r2 = 0.01; p = 0.259). Further parameters correlated moderately with LV SW min- 1 (LVPmax r2 = 0.47, RPP r2 = 0.67; and TP r2 = 0.54). LV dP/dtmax correlated worst with LV SW min- 1 (r2 = 0.28). CONCLUSION: CPO reflects external cardiac work over a wide range of inotropic states. These data further support the use of CPO to monitor inotropic interventions in the ICU.

Mitochondrial bioenergetics links inflammation and cardiac contractility in endotoxemia.

There is current awareness about the central role of mitochondrial dysfunction in the development of cardiac dysfunction in systemic inflammatory syndromes, especially in sepsis and endotoxemia. The aim of this work was to elucidate the mechanism that governs the link between the severity of the systemic inflammatory insult and mitochondrial function, analysing the consequences on heart function, particularly in cardiac contractile state. Female Sprague-Dawley rats were subjected to low-grade endotoxemia (i.p. injection LPS 0.5 mg kg-1 body weight) and severe endotoxemia (i.p. injection LPS 8 mg kg-1 body weight) for 6 h. Blood NO, as well as cardiac TNF-α and IL-1ß mRNA, were found increased as the severity of the endotoxemia increases. Cardiac relaxation was altered only in severe endotoxemia, although contractile and lusitropic reserves were found impaired in both treatments in response to work-overload. Cardiac ultrastructure showed disorientation of myofibrillar structure in both endotoxemia degrees, but mitochondrial swelling and cristae disruption were only observed in severe endotoxemia. Mitochondrial ATP production, O2 consumption and mitochondrial inner membrane potential decreases were related to blood NO levels and mitochondrial protein nitration, leading to diminished ATP availability and impairment of contractile state. Co-treatment with the NOS inhibitor L-NAME or the administration of the NO scavenger c-PTIO leads to the observation that mitochondrial bioenergetics status depends on the degree of the inflammatory insult mainly determined by blood NO levels. Unravelling the mechanisms involved in the onset of sepsis and endotoxemia improves the interpretation of the pathology, and provides new horizons for novel therapeutic targets.

Metabolic and Inflammatory Effects of an ω-3 Fatty Acid-Based Eucaloric Ketogenic Diet in Mice With Endotoxemia.

BACKGROUND: Dietary strategies can aid in the management of critically ill patients. Very-low-carbohydrate diets have been shown to improve glucose control and the inflammatory response. We aimed to determine the effects of a eucaloric ketogenic diet (EKD) enriched with ω-3 fatty acids (O3KD) on glucose levels and inflammation in mice with endotoxemia. METHODS: Adult mice were fed 1 of 3 diets (control diet [CD], EKD, or O3KD). After 4 weeks, each group received saline or Escherichia coli lipopolysaccharide (LPS) (5 mg/kg) intraperitoneally during the postprandial (PPP) or postabsorptive (PAP) periods. Blood glucose was measured at 0, 15, 30, 60, 90, 120, 180, and 240 minutes. Serum tumor necrosis factor (TNF)-α and interleukin (IL) 6 were measured by enzyme-linked immunosorbent assay. Distribution of serum fatty acids was determined by gas liquid chromatography. Hepatic expression of genes involved in inflammation, as well as glucose and lipid metabolism, were determined by quantitative polymerase chain reaction. RESULTS: During the PPP, glucose curves were comparable among the experimental groups. During the PAP, EKD showed a more pronounced increase in glucose levels at the first hour after LPS challenge compared with the CD-LPS group. During the PAP, IL6 was lower in O3KD-LPS compared with CD-LPS and EKD-LPS groups. These differences disappeared in the PPP. Similarly, TNF-α was lower in the O3KD-LPS group compared with the EKD-LPS group. The O3KD significantly increased the serum levels of the ω-3 eicosapentaenoic and docosahexaenoic acids and decreased the ω-6 arachidonic acid. CONCLUSION: An O3KD leads to reduced inflammation and maintains glucose homeostasis in mice with endotoxemia.

Piceatannol Mediated Modulation of Oxidative Stress and Regeneration in the Liver of Endotoxemic Mice.

Liver plays a pivotal role in host defense mechanisms related to endotoxemia. However, liver dysfunction often occurs in early sepsis. This study investigated the hepatoprotective potential of natural stilbenoid piceatannol (PIC) in lipopolysaccharide (LPS)-induced endotoxemic mice. Swiss Albino mice were divided into four groups: Control (C), LPS administrated (LPS), PIC administrated (PIC), and LPS administrated/PIC preadministrated (LPS+PIC) animals. PIC was administrated intraperitoneally (i.p.) at the dose of 4 mg/kg/day during 7 days. Endotoxemia was induced with a single i.p. administration of LPS at the dose of 4 mg/kg. Superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LP) levels, light microscopic pathology, and genotoxicity were investigated. Proliferating cell nuclear antigen and SQSTM1/p62 immunofluorescence were measured. PIC preadministration restored SOD activity, reduced LP and genotoxicity. However, moderate level of oxidative stress (OS) had been progressed in PIC preadministrated animals depending upon prolonged autophagic response and selective degradation of CAT. Positive OS stimulated liver regeneration by upregulating oval cells' and downregulating hepatocytes' proliferation and resulted in the maintanence of hepatic tissue integrity in PIC preadministrated animals. These results suggested that PIC may be a useful hepatoprotective agent in LPS-induced endotoxemia as a modulator of OS and genotoxicity, as an inducer of autophagy, and as a promoter of liver regeneration.

The colon revisited or the key to wellness, health and disease.

The hypothesis being advanced in this paper is that there is a new medical paradigm emerging from the biomedical research carried out in this century, mainly due to the explosion of the so called "omics" and associated techniques. The main idea is that there is a common pathway from wellbeing and health to chronic disease ("chronopathy") and even to death, which comprises following steps: 1) unhealthy diet, sedentary life style and permanent exposition to xenobiotics and all kinds of noxious stimuli;→2) intestinal dysbiosis;→3) alteration of the intestinal mucus layer (especially that of the colon);→4) disruption of the endothelial tight junctions;→5) metabolic endotoxemia+bacterial translocation;→6) inflammation;→7) exacerbation of the enteric nervous system (ENS) and consequent maladaptation and malfunctioning of the colon;→8) epigenetic manifestations;→9) "chronopathy" and premature death. Therefore, in order to maintain a good health or to improve or even reverse chronic diseases in a person, the main outcome to look for is a homeostatic balance of the intestinal microbiota (eubiosis), most of which is located in the colon. Lynn Margulis was one of the main scientists to highlight the importance of the role played by bacteria not only in the origin of all biological species now present on earth, but also on their role in global homeostasis. Bacteria do not rely on other living beings for their existence, while the latter depend completely on the former. Humans are no exemption, and new evidence emerges each day about the pivotal role of intestinal microbiota in human health, disease and, in general, in its wellbeing. The following facts about intestinal microbiota are nowadays generally accepted: there are about 10 times more bacteria in the gut than human cells in every human being; the microbioma is about 100-150 times bigger that the human genome, and there is a clear link between intestinal microbiota and many of the most common chronic diseases, from obesity and diabetes to depression and Parkinson disease and different kinds of cancer. The main implication of this theory is that we should become a sort of microbiota farmers, that is, we ought to be more conscious of our intestinal microbiota, take care of it and monitor it permanently. Thus, as part of our good life habits (healthy eating, physical exercise), we should probably undergo periodic seasons of fasting and colon cleansing, as it was common in older times.

North American ginseng inhibits myocardial NOX2-ERK1/2 signaling and tumor necrosis factor-α expression in endotoxemia.

Sepsis is a systemic inflammatory response to infection with a high mortality but has no specific treatment despite decades of research. North American (NA) ginseng (Panax quinquefolius) is a popular natural health product with anti-oxidant and anti-inflammatory properties. The aim of the present study was to investigate the effects of NA ginseng on pro-inflammatory cytokine expression and cardiac function in endotoxemia, a model of sepsis. Mice were challenged with lipopolysaccharide (LPS) to induce endotoxemia. Myocardial expression of tumor necrosis factor-alpha (TNF-α), a major pro-inflammatory cytokine that causes cardiac dysfunction, was upregulated in mice with endotoxemia, which was accompanied by increases in NOX2 expression, superoxide generation and ERK1/2 phosphorylation. Notably, pretreatment with NA ginseng aqueous extract (50mg/kg/day, oral gavage) for 5days significantly inhibited NOX2 expression, superoxide generation, ERK1/2 phosphorylation and TNF-α expression in the heart during endotoxemia. Importantly, cardiac function and survival in endotoxemic mice were significantly improved. Additionally, pretreatment with ginseng extract inhibited superoxide generation, ERK1/2 phosphorylation and TNF-α expression induced by LPS in cultured cardiomyocytes. We conclude that NA ginseng inhibits myocardial NOX2-ERK1/2-TNF-α signaling pathway and improves cardiac function in endotoxemia, suggesting that NA ginseng may have the potential in the prevention of clinical sepsis.

Amino acids, independent of insulin, attenuate skeletal muscle autophagy in neonatal pigs during endotoxemia.

BACKGROUND: Sepsis induces loss of skeletal muscle mass by activating the ubiquitin proteasome (UPS) and autophagy systems. Although muscle protein synthesis in healthy neonatal piglets is responsive to amino acids (AA) stimulation, it is not known if AA can prevent the activation of muscle protein degradation induced by sepsis. We hypothesize that AA attenuate the sepsis-induced activation of UPS and autophagy in neonates. METHODS: Newborn pigs were infused for 8 h with liposaccharide (LPS) (0 and 10 µg·kg(-1)·h(-1)), while circulating glucose and insulin were maintained at fasting levels; circulating AA were clamped at fasting or fed levels. Markers of protein degradation and AA transporters in longissimus dorsi (LD) were examined. RESULTS: Fasting AA increased muscle microtubule-associated protein light 1 chain 3 II (LC3-II) abundance in LPS compared to control, while fed AA levels decreased LC3-II abundance in both LPS and controls. There was no effect of AA supplementation on activated protein kinase (AMP), forkhead box O1 and O4 phosphorylation, nor on sodium-coupled neutral AA transporter 2 and light chain AA transporter 1, muscle RING-finger protein-1 and muscle Atrophy F-Box/Atrogin-1 abundance. CONCLUSION: These findings suggest that supplementation of AA antagonize autophagy signal activation in skeletal muscle of neonates during endotoxemia.

Effect of systemic high dose vitamin C therapy on forearm blood flow reactivity during endotoxemia in healthy human subjects.

OBJECTIVE: Acute inflammation induced by administration of Escherichia coli lipopolysaccharide endotoxin (LPS) reduces plasma concentrations of vitamin C and impairs vascular endothelium-derived nitric oxide (NO) bioactivity. We tested the hypothesis that systemically administered high dose vitamin C restores the endogenous anti-oxidant potential and improves NO-dependent vasodilatation in the forearm vasculature. DESIGN & SETTING: 36 male subjects were enrolled in this balanced, placebo controlled cross-over study. Forearm blood flow (FBF) reactivity to acetylcholine (ACh) and glyceryl-trinitrate (GTN), a sensitive test for endothelial function, was assessed at baseline and 4h after LPS-administration (20 IU/kg i.v). The effect of two different doses of intravenous vitamin C (Vitamin C-Injektopas®), 320 mg/kg and 480 mg/kg over 2h, or placebo on forearm vascular function was studied after LPS. MAIN RESULTS: LPS caused transient flu-like symptoms, decreased plasma vitamin C concentrations and reduced the ACh-dependent increase in FBF by up to 76%. Vitamin C at a mean plasma concentration of 3.2 or 4.9 mmol/L restored the response to ACh compared to baseline. CONCLUSION: High dose systemic vitamin C recovers LPS-induced endothelium-dependent vasodilation in the forearm resistance vasculature. This provides a rationale for a further clinical study of the systemic vitamin C effect under inflammatory conditions.

Emodin improves lipopolysaccharide-induced microcirculatory disturbance in rat mesentery.

OBJECTIVE: Sepsis is a systemic inflammatory response syndrome. Emodin is a major ingredient of Rheum Palmatum, a Chinese herb that is widely used in China for treatment of endotoxemia-related diseases. This study intended to examine the effect of Emodin on LPS-induced rat mesenteric microcirculatory disturbance and the underlying mechanisms. METHODS: The male Wistar rats received LPS (5 mg/kg/hr) for 90 min, with or without administration of Emodin (10 mg/kg/hr) by enema 30 min before (pre-treatment) or after (post-treatment) LPS infusion, and the dynamics of mesenteric microcirculation were determined by inverted intravital microscopy. Expression of adhesion molecules and TLR4, NF-κB p65, ICAM-1, MPO, and AP-1 in mesentery tissue was evaluated by flow cytometry and Western-blot, respectively. RESULTS: Pre or post-treatment with Emodin significantly ameliorated LPS-induced leukocyte emigration, reactive oxygen species production and albumin leakage, and the expression of TLR4, NF-κB p65, ICAM-1, MPO and AP-1 in mesentery. CONCLUSIONS: These results demonstrate the beneficial role of Emodin in attenuating the LPS-induced microcirculatory disturbance, and support the use of Emodin for patients with endotoxemia.

Cardiovascular and inflammatory response to cholecystokinin during endotoxemic shock.

Cholecystokinin (CCK) was first described as a gastrointestinal hormone, but its receptors have been located in cardiac and vascular tissues, as well as in immune cells. Our aims were to investigate the role of CCK on lipopolysaccharide (LPS)-induced hypotension and its ability to modulate previously reported inflammatory mediators, therefore affecting cardiovascular function. To conduct these experiments, rats had their jugular vein cannulated for drug administration, and also, the femoral artery cannulated for mean arterial pressure (MAP) and heart rate records. Endotoxemia induced by LPS from Escherichia coli (1.5 mg/kg; i.v.) stimulated the release of CCK, a progressive drop in MAP, and increase in heart rate. Plasma tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), nitrate, vasopressin, and lactate levels were elevated in the endotoxemic rats. The pretreatment with proglumide (nonselective CCK antagonist; 30 mg/kg; i.p.) aggravated the hypotension and also increased plasma TNF-α and lactate levels. On the other hand, CCK (0.4 µg/kg; i.v.) administered before LPS significantly restored MAP, reduced aortic and hepatic inducible nitric oxide synthase (iNOS) production, and elevated plasma vasopressin and IL-10 concentrations; it did not affect TNF-α. Physiological CCK concentration reduced nitrite and iNOS synthesis by peritoneal macrophages, possibly through a self-regulatory IL-10-dependent mechanism. Together, these data suggest a new role for the peptide CCK in modulating MAP, possibly controlling the inflammatory response, stimulating the anti-inflammatory cytokine, IL-10, and reducing vascular and macrophage iNOS-derived nitric oxide production. Based on these findings, CCK could be used as an adjuvant therapeutic agent to improve cardiovascular function.

The safety of Homnawakod herbal formula containing Aristolochia tagala Cham. in Wistar rats.

BACKGROUND: A dried root of Aristolochia tagala Cham. (ATC) is often used in Thai traditional medicine as an antipyretic, anti-inflammatory agent, muscle relaxant, appetite-enhancing agent, and analeptic. Homnawakod, an important herbal recipe, originally contains ATC in its formula, however, some Aristolochia species have been reported to cause nephrotoxicity due to aristolochic acid (AA) and its derivatives, resulting in ATC removal from all formulae. Therefore, this study investigates the chemical profiles of ATC, the original (HNK+ATC) and the present Homnawakod Ayurved Siriraj Herbal Formulary™ (HNK), and investigates whether they could cause nephrotoxicity or aggravate LPS-induced organ injuries in vivo. METHODS: HPLC and LC/MS were used for chemical profile study. Male Wistar rats were randomly divided into groups in which the rats were intragastrically administered distilled water (2 groups), ATC (10 or 30 mg/kg), HNK+ATC (540 or 1,620 mg/kg), or HNK (1,590 mg/kg) for 21 days. A positive control group was administered with single dose 100 mg/kg standard AA-I intragastrically at day 1. Serum creatinine and urea were measured at baseline and at 7, 14 and 21 days of the treatment. On day 22, a model of lipopolysaccharide (LPS)-induced endotoxemia was used. One-way and two-way analyses of variance were performed and a P value of less than 0.05 was considered to be significant. RESULTS: The similarity of the HPLC chromatograms of HNK+ATC and HNK could suggest that the qualities of both formulae are nearly the same in terms of chemical profile. The amount of AA-I found in ATC is 0.24%w/w. All experimental groups exhibited similar levels of serum urea at baseline and 7 and 14 days of the treatment. At 21 days, rats received AA exhibited a significant increase in serum urea, whereas the others did not exhibit such toxicity. On day 22, there were no significant changes in LPS-induced renal and liver dysfunction, or LPS-induced mean arterial pressure (MAP) reduction upon administration of ATC, HNK+ATC, HNK or AA-I. CONCLUSIONS: These results suggest that ATC, HNK+ATC or HNK, at the animal dose equivalent to that used in human, do not cause the acute nephrotoxicity in rats and do not aggravate LPS-induced organ injuries even further.

Potential mechanisms for the emerging link between obesity and increased intestinal permeability.

Recently, increased attention has been paid to the link between gut microbial composition and obesity. Gut microbiota is a source of endotoxins whose increase in plasma is related to obesity and insulin resistance through increased intestinal permeability in animal models; however, this relationship still needs to be confirmed in humans. That intestinal permeability is subject to change and that it might be the interface between gut microbiota and endotoxins in the core of metabolic dysfunctions reinforce the need to understand the mechanisms involved in these aspects to direct more efficient therapeutic approaches. Therefore, in this review, we focus on the emerging link between obesity and increased intestinal permeability, including the possible factors that contribute to increased intestinal permeability in obese subjects. We address the concept of intestinal permeability, how it is measured, and the intestinal segments that may be affected. We then describe 3 factors that may have an influence on intestinal permeability in obesity: microbial dysbiosis, dietary pattern (high-fructose and high-fat diet), and nutritional deficiencies. Gaps in the current knowledge of the role of Toll-like receptors ligands to induce insulin resistance, the routes for lipopolysaccharide circulation, and the impact of altered intestinal microbiota in obesity, as well as the limitations of current permeability tests and other potential useful markers, are discussed. More studies are needed to reveal how changes occur in the microbiota. The factors such as changes in the dietary pattern and the improvement of nutritional deficiencies appear to influence intestinal permeability, and impact metabolism must be examined. Also, additional studies are necessary to better understand how probiotic supplements, prebiotics, and micronutrients can improve stress-induced gastrointestinal barrier dysfunction and the influence these factors have on host defense. Hence, the topics presented in this review may be beneficial in directing future studies that assess gut barrier function in obesity.

Preventive and therapeutic effects of quercetin on lipopolysaccharide-induced oxidative stress and vascular dysfunction in mice.

Quercetin, a dietary antioxidant flavonoid, possesses strong anti-inflammatory and cytoprotective activities. The effects were investigated in an animal model of lipopolysaccharide (LPS)-induced endotoxaemia and vascular dysfunction in vivo. Male ICR mice were injected with LPS (10 mg/kg; i.p.). Quercetin (50 or 100 mg/kg) was intragastrically administered either before or after LPS administration. Fifteen hours after LPS injection, mice were found in endotoxaemic condition, as manifested by hypotension, tachycardia, and blunted vascular responses to vasodilators and vasoconstrictor. The symptoms were accompanied by increased aortic iNOS protein expression, decreased aortic eNOS protein expression, marked suppression of cellular glutathione (GSH) redox status, enhanced aortic superoxide production, increased plasma malodialdehyde and protein carbonyl, and elevated urinary nitrate/nitrite. Treatment with quercetin either before or after LPS preserved the vascular function, as blood pressure, heart rate, vascular responsiveness were restored to near normal values, particularly when quercetin was given as a preventive regimen. The vascular protective effects were associated with upregulation of eNOS expression, reduction of oxidative stress, and maintained blood GSH redox ratio. Overall findings suggest the beneficial effect of quercetin on the prevention and restoration of a failing eNOS system and alleviation of oxidative stress and vascular dysfunction against endotoxin-induced shock in mice.

Citrulline a more suitable substrate than arginine to restore NO production and the microcirculation during endotoxemia.

BACKGROUND: Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia. METHODOLOGY/PRINCIPAL FINDINGS: To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg • g bodyweight(-1) • h(-1)), combined with either L-Citrulline (6.25 mg • h-1), L-Arginine (6.25 mg • h(-1)), or L-Alanine (isonitrogenous control; 12.5 mg • h(-1)) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot. CONCLUSION/SIGNIFICANCE: L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

Electroacupuncture improves survival in rats with lethal endotoxemia via the autonomic nervous system.

BACKGROUND: Recent advances have indicated a complex interplay between the autonomic nervous system and the innate immune system. Targeting neural networks for the treatment of sepsis is being developed as a therapeutic strategy. Because electroacupuncture at select acupoints can modulate activities of the autonomic nervous system, we tested the hypothesis that electroacupuncture at specific acupoints could modulate systemic inflammatory responses and improve survival via its impact on the autonomic nervous system in a rat model of sepsis. METHODS: Sprague-Dawley male rats received electroacupuncture for 45 min before and at 1, 2, or 4 h after a lethal dose of intraperitoneal lipopolysaccharide injection (6 mg/kg). Outcomes included survival and systemic cytokine responses. Also, the possible roles of neural circuitry, including the hypothalamic-pituitary-adrenal axis and the autonomic nervous system, were evaluated. RESULTS: Electroacupuncture pretreatment at the Hegu acupoints significantly attenuate systemic inflammatory responses and improve survival rate from 20% to 80% in rats with lethal endotoxemia. Such a site-specific effect requires the activation of muscarinic receptors in the central nervous system, but not increasing central sympathetic tone. In the periphery synergistic, rather than independent, action of the sympathetic and parasympathetic systems is also necessary. CONCLUSIONS: Electroacupuncture pretreatment has a dramatic survival-enhancing effect in rats with lethal endotoxemia, which involves the activation of efferent neural circuits of the autonomic nervous system (e.g., cholinergic antiinflammatory pathway). This approach could be developed as a prophylactic treatment for sepsis or perioperative conditions related to excessive inflammation.

Glibenclamide reduces proinflammatory cytokines in an ex vivo model of human endotoxinaemia under hypoxaemic conditions.

AIMS: In vivo application of the K(ATP)-channel blocker glibenclamide can reverse endotoxin-induced hypotension, vascular hyporeactivity and shock in experimental animals. The hypothesis of the present study is, that the drug effects might not only be based on direct inhibition of K(ATP)-channels of vascular smooth muscle cells, but might also reflect reduction of shock-induced excess proinflammatory cytokines and procoagulatory molecules produced in the blood monocytes. MAIN METHODS: Human whole blood (normoxaemic or hypoxaemic) supplemented ex vivo with 100 ng/ml LPS was used to assess glibenclamide (3-100 µM) effects on IL-1 beta, IL-6, TNF-alpha, tissue factor, and plasminogen-activator-inhibitor-2 (PAI-2). Co-incubations with monocytes and erythrocytes and cytosolic calcium measurements were performed to reveal their purinergic intercellular interaction. KEY FINDINGS: In heparinized blood, glibenclamide reduced LPS-induced release of IL-1 beta and TNF-alpha, tissue factor and PAI-2 mRNA in a concentration-dependent manner. When samples were subjected to strong hypoxemia using 95% N(2)/5% CO(2), these parameters became even more sensitive to the drug. No drug effect was observable in citrated blood or in isolated monocytes. IL-1 beta mRNA inhibition by glibenclamide appeared to be dependent on P2X7-receptor activation of monocytes by ATP-releasing erythrocytes during hypoxia. Cytosolic calcium values as well as the duration of calcium transients elicited by P2X7-receptor stimulation in isolated monocytes were strongly increased during hypoxia, both of which could be abolished by glibenclamide. SIGNIFICANCE: We conclude that the anti-inflammatory effect of glibenclamide is mainly based on the reduction of calcium entry by drug-induced depolarization of hypoxic monocytes. Thus, glibenclamide possesses a potentially beneficial shock-specific anti-inflammatory action.

Novel complementary peptides to target molecules.

We generated an evolutionary computer program that generates complementary peptide (C-pep) sequences, with the potential to interact with a target peptide, by comparing several physico-chemical parameters of each pair of the complementary peptides being analyzed. We generated C-peps to target several molecules. About 30% of synthesized C-peps interfered with the function of their targets. C5a stimulates generation of TNFα and other inflammatory cytokines. Inhibition of C5a should be effective against sepsis, which impairs the status of cancer-bearing patients. One of the inhibitory C-peps of C5a, termed AcPepA, was effective in Cynomolgus monkeys intravenously infused with a lethal dose of bacterial LPS (4 mg/kg) destined to die. The monkeys were rescued by intravenous administration of 2 mg/kg/h of AcPepA. The excellent therapeutic effect of AcPepA is likely to be due to restriction of high mobility group box 1 (HMGB1) surge induced by the effect of C5a on C5L2, which is the second C5a receptor, since the released HMGB1 has the capacity to stimulate TLR4 as an endogeneous ligand resulting in further activation of inflammatory cells to release inflammatory cytokines forming a positive feedback circuit of inflammation.

Baicalein, an active component of Scutellaria baicalensis Georgi, improves cardiac contractile function in endotoxaemic rats via induction of heme oxygenase-1 and suppression of inflammatory responses.

AIM OF THE STUDY: To evaluate the protective effect of baicalein on myocardial dysfunction caused by endotoxaemia in rats and to explore the possible mechanisms. MATERIALS AND METHODS: Baicalein (10mg/kg, intravenous) was administered to conscious Wistar rats 30min after lipopolysaccharide (LPS; 10mg/kg, intravenous) challenge. Six hours after LPS administration, the contractile function of the isolated heart was examined using the Langendorff technique. Cardiac protein expression related to inflammatory responses, superoxide anion production and caspase-3 activity were measured. RESULTS: Post-treatment with baicalein significantly attenuated the LPS-induced hypotension with accompanying tachycardia. The contractile function of isolated heart was significantly preserved 6h after LPS administration, following treatment with baicalein. Furthermore, baicalein induced the expression of heme oxygenase-1 protein and reduced superoxide anion formation in the myocardium of LPS-treated rats. Cardiac levels of inducible nitric oxide synthase, monocyte chemoattractant protein-1, phospho-IκBα and phospho-p65 protein and caspase-3 activity significantly increased 6h after LPS challenge but baicalein significantly attenuated these LPS-induced changes. CONCLUSIONS: Baicalein improves myocardial contractility in LPS-induced sepsis, which may be related to reductions in oxidative stress, myocardial inflammatory responses and apoptosis.

Changes of plasma glutamine concentration and hepatocyte membrane system N transporters expression in early endotoxemia.

BACKGROUND: Glutamine plays important roles in health and critical illness. During endotoxemia, glutamine metabolism, including its plasma level and transport, changes markedly. Previous studies have demonstrated that system N transporters in hepatocytes play a major role in hepatic glutamine transport. However, little is known about the changes of mRNA and protein expression of system N transporters in hepatocyte plasma membrane. Furthermore, the alteration of plasma glutamine concentration during endotoxemia is still controversial. In this study, we investigated the changes in early endotoxemic rats by intraperitoneal injection of lipopolysaccharide (LPS). MATERIALS AND METHODS: Three, 6, 12 mg/kg body weight doses of LPS were injected intraperitoneally to establish the endotoxemic rat model; equal volume of 0.9% saline was used as the control. Before and 2, 4, 6, 12, 24h after injections, plasma glutamine concentration, mRNA, and protein expression of SNAT3 and SNAT5 transporters in hepatocyte plasma membrane were detected by high performance liquid chromatography, real-time PCR, and Western blot, respectively. RESULTS: LPS injection resulted in a marked increase of the plasma glutamine concentration from 4 to 12h (3mg/kg) and 2 to 6h (6 mg/kg, 12 mg/kg) after the injection compared with its physiologic level, and a significant decrease in 6, 12 mg/kg groups at 24h. Both the mRNA and protein expression of SNAT3 and SNAT5 were enhanced by LPS in a time- and dose-dependent manner. CONCLUSIONS: The plasma glutamine concentration in endotoxemic rat increased transiently during early endotoxemia but subsequently decreased over time. The effect of LPS on system N expression occurs not only at the protein level, but also at the mRNA level. It is reasonable to supplement glutamine for patients with sepsis or endotoxemia begin at 6 to 12h after the development of disease.

Thioredoxin binding protein-2 mediates metabolic adaptation in response to lipopolysaccharide in vivo.

OBJECTIVES: Endotoxin triggers a reorganization of the energy metabolic pathway, including the promotion of fatty acid utilization to adapt to a high energy demand during endotoxemia. However, the factors responsible for the metabolic adaptation and characteristic pathologies resulting from defective utilization fatty acids during endotoxin response have not been fully clarified. The thioredoxin binding protein-2 (TBP-2) knockout (TBP-2) mouse is an animal model of fatty acid oxidation disorder. The aim of this study was to determine whether and how TBP-2 is involved in metabolic regulation in a lipopolysaccharide (LPS)-induced endotoxemia model in mice. DESIGN: Prospective animal trial. SETTING: Research laboratory. SUBJECTS: TBP-2 and wild control mice. INTERVENTION: TBP-2 and wild control mice were intraperitoneally injected with LPS. Mortality, serum levels of markers of hepatorenal injuries, cytokines, insulin, glucose and lipid derivatives, and the hepatic signaling pathway regulating gluconeogenesis were investigated. MEASUREMENTS AND MAIN RESULTS: Following the administration of LPS, TBP-2 mice showed a predisposition for death without any significant elevation of inflammatory cytokines, compared to the wild mice. LPS-challenged TBP-2 mice showed fat deposition in the liver and kidney, organ injuries, glycogen depletion, and elevation of serum lipid derivatives such as free fatty acids, triglyceride and cholesterol. Hyperinsulinemia and hypoglycemia were observed in TBP-2 mice after LPS injection. Death due to the LPS administration was prevented by supplementation of glucose. Phosphorylation of Akt and FoxO1, an inhibitory pathway of gluconeogenesis in the liver of LPS-challenged TBP-2 mice was demonstrated, suggesting the enhancement of insulin signaling. CONCLUSIONS: TBP-2 is involved in metabolic control during LPS-induced endotoxemia. After the LPS challenge, TBP-2 mice showed several characteristic aspects, such as hepatorenal injuries, and dysregulation of the lipid and glucose metabolisms. Furthermore, hypoglycemia promoted by hyperinsulinemia may be a critical risk factor for mortality in circumstances in which fatty acid utilization is impaired during endotoxemia.