Endotoxemia Model Research Articles

Inhaled molecular hydrogen reduces hippocampal neuroinflammation, glial reactivity and ameliorates memory impairment during systemic inflammation

Sepsis is associated with numerous physiological and biochemical abnormalities that result in a life-threatening condition. The involvement of the Central Nervous System (CNS) during sepsis has received considerable attention, especially the hippocampus which plays a key role in the learning and memory processes. The increased interest in this limbic region during systemic inflammation (SI) is related to the number of sepsis survivor patients who have cognitive impairments. A single injection of lipopolysaccharide (LPS)-induced systemic inflammation is the most commonly used murine endotoxemia model because it replicates several pathophysiological changes observed in severe sepsis. Molecular hydrogen (H2) has been used as an anti-inflammatory therapeutic strategy to prevent neuroinflammation. However, the mechanisms by which inhaled H2 mitigate memory loss during SI remains unknown. To understand how H2 acts in the hippocampus, the current study focused on specific mechanisms that may be involved in reducing neuroinflammation in rats during SI. We hypothesized that inhaled H2 decreases LPS-induced hippocampal pro-inflammatory cytokines surges and this effect is associated with reduced memory loss. Using different and integrative approaches, i.e., from hippocampal cells electrophysiology to animal behavior, we report that inhaled H2 decreased LPS-induced peripheral and hippocampal inflammation, decreased microglial and astrocytic activation, lessen memory loss without affecting long-term potentiation (LTP). To our knowledge, this is the first evidence showing that inhaled H2 reduces hippocampal microglial and glial cells inflammation, which may be associated with a reduced memory impairment induced by SI.

Delta 9-tetrahydrocannabinol conserves cardiovascular functions in a rat model of endotoxemia: Involvement of endothelial molecular mechanisms and oxidative-nitrative stress.

In endotoxemic models, the inflammatory parameters are altered to a favorable direction as a response to activation of cannabinoid receptors 1 and 2. The phytocannabinoid Δ9-tetrahydrocannabinol (THC) is an agonist/partial antagonist of both cannabinoid receptors. This report targets the effects of THC on the cardiovascular system of endotoxemic rats. In our 24-hour endotoxemic rat model (E. coli derived lipopolysaccharide, LPS i.v. 5mg/kg) with THC treatment (LPS+THC 10 mg/kg i.p.), we investigated cardiac function by echocariography and endothelium-dependent relaxation of the thoracic aorta by isometric force measurement compared to vehicle controls. To evaluate the molecular mechanism, we measured endothelial NOS and COX-2 density by immunohistochemistry; and determined the levels of cGMP, the oxidative stress marker 4-hydroxynonenal, the nitrative stress marker 3-nitrotyrosine, and poly(ADP-ribose) polymers. A decrease in end-systolic and end-diastolic ventricular volumes in the LPS group was observed, which was absent in LPS+THC animals. Endothelium-dependent relaxation was worsened by LPS but not in the LPS+THC group. LPS administration decreased the abundance of cannabinoid receptors. Oxidative-nitrative stress markers showed an increment, and cGMP, eNOS staining showed a decrement in response to LPS. THC only decreased the oxidative-nitrative stress but had no effect on cGMP and eNOS density. COX-2 staining was reduced by THC. We hypothesize that the reduced diastolic filling in the LPS group is a consequence of vascular dysfunction, preventable by THC. The mechanism of action of THC is not based on its local effect on aortic NO homeostasis. The reduced oxidative-nitrative stress and the COX-2 suggest the activation of an anti-inflammatory pathway.

DHCR7 Expression Predicts Poor Outcomes and Mortality From Sepsis.

Sepsis patients experience poor outcomes including chronic critical illness (CCI) or early death (within 14 d). We investigated lipid metabolic gene expression differences by outcome to discover therapeutic targets. Secondary analysis of samples from prospectively enrolled sepsis patients (first 24 hr) and a zebrafish endotoxemia model for drug discovery. Patients were enrolled from the emergency department or ICU at an urban teaching hospital. Enrollment samples from sepsis patients were analyzed. Clinical data and cholesterol levels were recorded. Leukocytes were processed for RNA sequencing and reverse transcriptase polymerase chain reaction. A lipopolysaccharide zebrafish endotoxemia model was used for confirmation of human transcriptomic findings and drug discovery. The derivation cohort included 96 patients and controls (12 early death, 13 CCI, 51 rapid recovery, and 20 controls) and the validation cohort had 52 patients (6 early death, 8 CCI, and 38 rapid recovery). The cholesterol metabolism gene 7-dehydrocholesterol reductase (DHCR7) was significantly up-regulated in both derivation and validation cohorts in poor outcome sepsis compared with rapid recovery patients and in 90-day nonsurvivors (validation only) and validated using RT-qPCR analysis. Our zebrafish sepsis model showed up-regulation of dhcr7 and several of the same lipid genes up-regulated in poor outcome human sepsis (dhcr24, sqlea, cyp51, msmo1, and ldlra) compared with controls. We then tested six lipid-based drugs in the zebrafish endotoxemia model. Of these, only the Dhcr7 inhibitor AY9944 completely rescued zebrafish from lipopolysaccharide death in a model with 100% lethality. DHCR7, an important cholesterol metabolism gene, was up-regulated in poor outcome sepsis patients warranting external validation. This pathway may serve as a potential therapeutic target to improve sepsis outcomes.

In an endotoxaemic model, antibiotic clearance can be affected by different central venous catheter positions, during renal replacement therapy

BackgroundIn intensive care, different central venous catheters (CVC) are often used for infusion of drugs. If a patient is treated with continuous renal replacement therapy (CRRT) a second catheter, a central venous dialysis catheter (CVDC), is needed. Placing the catheters close together might pose a risk that a drug infused in a CVC could be directly aspirated into a CRRT machine and cleared from the blood without giving the effect intended. The purpose of this study was to elucidate if drug clearance is affected by different catheter placement, during CRRT. In this endotoxaemic animal model, an infusion of antibiotics was administered in a CVC placed in the external jugular vein (EJV). Antibiotic clearance was compared, whether CRRT was through a CVDC placed in the same EJV, or in a femoral vein (FV). To reach a target mean arterial pressure (MAP), noradrenaline was infused through the CVC and the dose was compared between the CDVDs.ResultsThe main finding in this study was that clearance of antibiotics was higher when both catheter tips were in the EJV, close together, compared to in different vessels, during CRRT. The clearance of gentamicin was 21.0 ± 7.3 vs 15.5 ± 4.2 mL/min (p 0.006) and vancomycin 19.3 ± 4.9 vs 15.8 ± 7.1 mL/min (p 0.021). The noradrenaline dose to maintain a target MAP also showed greater variance with both catheters in the EJV, compared to when catheters were placed in different vessels.ConclusionThe results in this study indicate that close placement of central venous catheter tips could lead to unreliable drug concentration, due to direct aspiration, during CRRT.

Prophylactic simvastatin increased survival during endotoxemia and inhibited granulocyte trafficking in a cell-intrinsic manner

Cross sectional studies have shown that statin-users have improved odds of surviving severe sepsis. Meanwhile controlled clinical trials failed to demonstrate improved sepsis survival with acute statin administration following hospitalization. Here, a lethal murine peritoneal lipopolysaccharide (LPS) endotoxemia model was used to assess the efficacy of chronic versus acute simvastatin on survival. Mirroring clinical observations, chronic but not acute treatment with simvastatin significantly increased survival. At a pre-mortality time point in LPS-treated mice, chronic simvastatin suppressed granulocyte trafficking in to the lungs and peritoneum without otherwise suppressing emergency myelopoiesis, myeloid cells in circulation, or inflammatory cytokines. Chronic simvastatin treatment significantly downregulated inflammatory chemokine gene signature in the lungs of LPS-treated mice. Thus, it was unclear if simvastatin was inhibiting granulocyte chemotaxis in a cell intrinsic or extrinsic manner. Adoptive transfer of fluorescently labeled granulocytes from statin and vehicle treated mice into LPS-treated mice showed that simvastatin inhibited lung-granulocyte trafficking in a cell intrinsic manner. Congruent with this, chemotaxis experiments using in vitro macrophages and ex vivo granulocytes demonstrated that simvastatin inhibited chemotaxis in a cell-intrinsic manner. Collectively, chronic but not acute simvastatin treatment improved survival in murine endotoxemia, and this was associated with cell-intrinsic inhibition of granulocyte chemotaxis.

Bupleurum chinense exerts a mild antipyretic effect on LPS-induced pyrexia rats involving inhibition of peripheral TNF-α production.

Bupleuri Radix, the dried roots of Bupleurum chinense DC. (BC) or Bupleurum scorzonerifolium Willd., is one of the most frequently used traditional Chinese medicines. As the species in Xiao-Chai-Hu decoction, BC has been used as an antipyretic medicine with a long history. However, its antipyretic characteristics and underlying mechanism(s) remain unclear. To elucidate the antipyretic characteristics and mechanism(s) of BC used in its traditional way. The water extract of BC (BCE) was prepared according to the traditional decocting mode. Murine fever and endotoxemia models were induced by intravenous injection of lipopolysaccharide (LPS). In vitro complement activation assay and the levels of TNF-α, IL-6, IL-1β, and C5a were determined by ELISA. BCE exerted a confirmed but mild antipyretic effect on LPS-induced fever of rat. In vitro, it significantly lowered LPS-elevated TNF-α in the supernatant of rat complete blood cells and THP-1cells, but failed to decrease IL-6 and IL-1β. In murine endotoxemia models, BCE markedly decreased serum TNF-α, but had no impact on IL-6 and IL-1β. BCE also restricted complement activation in vitro and in vivo. Nevertheless, the mixture of saikosaponin A and D could not suppress supernatant TNF-α of monocytes and serum TNF-α of endotoxemia mice. The present study dissects the peripheral mechanism for the antipyretic effect of BC used in the traditional way. Our findings indicate that BCE directly suppresses monocyte-produced TNF-α, thus decreasing circulating TNF-α, which may be responsible for its mild but confirmed antipyretic action.

Mitochondrial pyruvate carrier-mediated metabolism is dispensable for the classical activation of macrophages.

Glycolysis is essential for the classical activation of macrophages (M1), but how glycolytic pathway metabolites engage in this process remains to be elucidated. Glycolysis leads to production of pyruvate, which can be transported into the mitochondria by the mitochondrial pyruvate carrier (MPC) followed by utilization in the tricarboxylic acid cycle. Based on studies that used the MPC inhibitor UK5099, the mitochondrial route has been considered to be of significance for M1 activation. Using genetic approaches, here we show that the MPC is dispensable for metabolic reprogramming and activation of M1 macrophages. In addition, MPC depletion in myeloid cells has no impact on inflammatory responses and macrophage polarization toward the M1 phenotype in a mouse model of endotoxemia. While UK5099 reaches maximal MPC inhibitory capacity at approximately 2-5 μM, higher concentrations are required to inhibit inflammatory cytokine production in M1 and this is independent of MPC expression. Taken together, MPC-mediated metabolism is dispensable for the classical activation of macrophages and UK5099 inhibits inflammatory responses in M1 macrophages due to effects other than MPC inhibition.

Effect of mitoTEMPO on Redox Reactions in Different Body Compartments upon Endotoxemia in Rats.

Mitochondrial ROS (mitoROS) control many reactions in cells. Biological effects of mitoROS in vivo can be investigated by modulation via mitochondria-targeted antioxidants (mtAOX, mitoTEMPO). The aim of this study was to determine how mitoROS influence redox reactions in different body compartments in a rat model of endotoxemia. We induced inflammatory response by lipopolysaccharide (LPS) injection and analyzed effects of mitoTEMPO in blood, abdominal cavity, bronchoalveolar space, and liver tissue. MitoTEMPO decreased the liver damage marker aspartate aminotransferase; however, it neither influenced the release of cytokines (e.g., tumor necrosis factor, IL-4) nor decreased ROS generation by immune cells in the compartments examined. In contrast, ex vivo mitoTEMPO treatment substantially reduced ROS generation. Examination of liver tissue revealed several redox paramagnetic centers sensitive to in vivo LPS and mitoTEMPO treatment and high levels of nitric oxide (NO) in response to LPS. NO levels in blood were lower than in liver, and were decreased by in vivo mitoTEMPO treatment. Our data suggest that (i) inflammatory mediators are not likely to directly contribute to ROS-mediated liver damage and (ii) mitoTEMPO is more likely to affect the redox status of liver cells reflected in a redox change of paramagnetic molecules. Further studies are necessary to understand these mechanisms.

Clusterin Neutralizes the Inflammatory and Cytotoxic Properties of Extracellular Histones in Sepsis.

Rationale: Extracellular histones, released into the surrounding environment during extensive cell death, promote inflammation and cell death, and these deleterious roles have been well documented in sepsis. Clusterin (CLU) is a ubiquitous extracellular protein that chaperones misfolded proteins and promotes their removal. Objectives: We investigated whether CLU could protect against the deleterious properties of histones. Methods: We assessed CLU and histone expression in patients with sepsis and evaluated the protective role of CLU against histones in invitro assays and invivo models of experimental sepsis. Measurements and Main Results: We show that CLU binds to circulating histones and reduces their inflammatory, thrombotic, and cytotoxic properties. We observed that plasma CLU levels decreased in patients with sepsis and that the decrease was greater and more durable in nonsurvivors than in survivors. Accordingly, CLU deficiency was associated with increased mortality in mouse models of sepsis and endotoxemia. Finally, CLU supplementation improved mouse survival in a sepsis model. Conclusions: This study identifies CLU as a central endogenous histone-neutralizing molecule and suggests that, in pathologies with extensive cell death, CLU supplementation may improve disease tolerance and host survival.

Linking platelet lipidome and inflammatory response in septic patients

Sepsis is characterized by major endothelial dysfunction, microvascular alterations, and coagulopathy. Platelets are important players in sepsis as they promote thrombo-inflammation and generate lipidic mediators of inflammation. Previously, we observed that platelet acetyl-CoA carboxylase 1 (ACC1), responsible for de novo lipid synthesis, is phosphorylated and thus inhibited by AMP-activated protein kinase (AMPK) upon thrombin stimulation, leading to platelet lipidome changes. The role of the AMPK-ACC signaling in the regulation of platelet lipidome during sepsis, and its potential impact on inflammation, has never been investigated. To investigate whether changes in ACC phosphorylation can influence the platelet lipidome and the inflammatory response of patients during sepsis. Platelets and plasmas were isolated from 48 septic and 48 control patients. In parallel, WT and DKI mice injected intraperitoneally with lipopolysaccharides were used as a model of endotoxemia. Platelets ACC1 phosphorylation/expression levels were measured by western blot. Lipidomics analysis was carried out by untargeted liquid chromatography–mass spectrometry or on the commercial Lipidyzer platform. Plasma factors were measured by Enzyme-linked immunosorbent assays (ELISA). Platelet ACC1 expression and phosphorylation were increased both in septic patients and in septic mice. Lipidomics analysis highlighted significant changes of lipids in human and murine platelets during sepsis, with a substantial reduction in phosphatidylcholines and phosphatidylethanolamines containing long polyunsaturated fatty acid chains (ω3 and ω6), which are key phospholipids for the generation of pro- and anti-inflammatory lipid mediators. Our preliminary data obtained in humans and mice, suggest that elevated ACC phosphorylation in sepsis is associated with a higher content of these phospholipids as well as a plasma reduction in myeloid inflammatory parameters. Our data reveal that critical changes in the platelet lipidome occur during sepsis and may contribute to the pathophysiology of the disease. Our results also suggest that ACC can influence the platelet lipid content and/or composition.

Ghrelin prevents lethality in a rat endotoxemic model through central effects on the vagal pathway and adenosine A2B signaling : Brain ghrelin and anti-septic action.

Accumulating evidence suggest that ghrelin plays a role as an antiseptic peptide. The present study aimed to clarify whether the brain may be implicated ghrelin's antiseptic action. We examined the effect of brain ghrelin on survival in a novel endotoxemic model achieved by treating rats with lipopolysaccharide (LPS) and colchicine. The observation of survival stopped three days after chemicals' injection or at death. Intracisternal ghrelin dose-dependently reduced lethality in the endotoxemic model; meanwhile, neither intraperitoneal injection of ghrelin nor intracisternal des-acyl-ghrelin injection affected the mortality rate. The brain ghrelin-induced lethality reduction was significantly blocked by surgical vagotomy. Moreover, intracisternal injection of a ghrelin receptor antagonist blocked the improved survival achieved by intracisternal ghrelin injection or intravenous 2-deoxy-d-glucose administration. Intracisternal injection of an adenosine A2B receptor agonist reduced the lethality and the ghrelin-induced improvement of survival was blocked by adenosine A2B receptor antagonist. I addition, intracisternal ghrelin significantly blocked the colonic hyperpermeability produced by LPS and colchicine. These results suggest that ghrelin acts centrally to reduce endotoxemic lethality. Accordingly, activation of the vagal pathway and adenosine A2B receptors in the brain may be implicated in the ghrelin-induced increased survival. Since the efferent vagus nerve mediates anti-inflammatory mechanisms, we speculate that the vagal cholinergic anti-inflammatory pathway is implicated in the decreased septic lethality caused by brain ghrelin.

Soluble TREM-like Transcript-1 Acts as a Damage-Associated Molecular Pattern through the TLR4/MD2 Pathway Contributing to Immune Dysregulation during Sepsis.

Studies have shown that elevated plasma levels of platelet-derived soluble TREM-like transcript-1 (sTLT-1) are associated with an unfavorable outcome in patients with septic shock. However, the underlying molecular mechanisms are not well defined. This research aimed to study the role of sTLT-1 in mediating immune dysfunction during the development of sepsis. Our study demonstrated that patients with septic shock have significantly higher plasma concentrations of sTLT-1, whereas sTLT-1 is not detectable in healthy subjects. Plasma concentrations of sTLT-1 were correlated with the degree of immunosuppressive parameters in monocytes from patients with septic shock. sTLT-1 can first activate monocytes by binding to the TLR4/MD2 complex but subsequently induce immunosuppressive phenotypes in monocytes. Blocking Abs against TLR4 and MD2 led to a significant decrease in sTLT-1-induced activation. Treatment with an anti-TLT-1 Ab also significantly reduces sTLT-1 binding to monocytes and proinflammatory cytokine secretion in a mouse model of endotoxemia. sTLT-1 acts as an endogenous damage-associated molecular pattern molecule, triggering the activation of monocytes through the TLR4/MD2 complex followed by sustained immune suppression. This process plays a crucial role in the development of sepsis-associated pathophysiology. Our findings outline, to our knowledge, a novel pathway whereby platelets counteract immune dynamics against infection through sTLT-1.

Modulation of the Acute Inflammatory Response Induced by the Escherichia coli Lipopolysaccharide through the Interaction of Pentoxifylline and Florfenicol in a Rabbit Model.

Experimental reports have demonstrated that florfenicol (FFC) exerts potent anti-inflammatory effects, improving survival in a murine endotoxemia model. Considering the anti-inflammatory and immunomodulatory properties of pentoxifylline (PTX) as an adjuvant to enhance the efficacy of antibiotics, the anti-inflammatory effects of the interaction FFC/PTX over the E. coli Lipopolysaccharide (LPS)-induced acute inflammatory response was evaluated in rabbits. Twenty-five clinically healthy New Zealand rabbits (3.8 ± 0.2 kg body weight: bw), were distributed into five experimental groups. Group 1 (control): treated with 1 mL/4 kg bw of 0.9% saline solution (SS) intravenously (IV). Group 2 (LPS): treated with an IV dose of 5 µg/kg of LPS. Group 3 (pentoxifylline (PTX) + LPS): treated with an oral dose of 30 mg/kg PTX, followed by an IV dose of 5 µg/kg of LPS 45 min after PTX. Group 4 (Florfenicol (FFC) + LPS): treated with an IM dose of 20 mg/kg of FFC, followed by an IV dose of 5 µg/kg of LPS 45 min after FFC administration. Group 5 (PTX + FFC + LPS): treated with an oral dose of 30 mg/kg of PTX, followed by an IM dose of 20 mg/kg of FFC, and, 45 min after an IV dose of 5 µg/kg of LPS was administered. The anti-inflammatory response was evaluated through changes in plasma levels of interleukins (TNF-α, IL-1β and IL-6), C-reactive protein (CRP), and body temperature. It has been shown that each drug produced a partial inhibition over the LPS-induced increase in TNF-α, IL-1β, and CRP. When both drugs were co-administered, a synergistic inhibitory effect on the IL-1β and CRP plasma concentrations was observed, associated with a synergic antipyretic effect. However, the co-administration of PTX/FFC failed to modify the LPS-induced increase in the TNF-α plasma concentrations. We concluded that the combination of FFC and PTX in our LPS sepsis models demonstrates immunomodulatory effects. An apparent synergistic effect was observed for the IL-1β inhibition, which peaks at three hours and then decreases. At the same time, each drug alone was superior in reducing TNF-α levels, while the combination was inferior. However, the peak of TNF-α in this sepsis model was at 12 h. Therefore, in rabbits plasma IL-1β and TNF-α could be regulated independently, thus, further research is needed to explore the effects of this combination over a more prolonged period.

CytoSorb hemoperfusion markedly attenuates circulating cytokine concentrations during systemic inflammation in humans in vivo

BackgroundThe CytoSorb hemoadsorption device has been demonstrated to be capable of clearing inflammatory cytokines, but has not yet been shown to attenuate plasma cytokine concentrations. We investigated the effects of CytoSorb hemoperfusion on plasma levels of various cytokines using the repeated human experimental endotoxemia model, a highly standardized and reproducible human in vivo model of systemic inflammation and immunological tolerance induced by administration of bacterial lipopolysaccharide (LPS).MethodsTwenty-four healthy male volunteers (age 18–35) were intravenously challenged with LPS (a bolus of 1 ng/kg followed by continuous infusion of 0.5 ng/kg/hr for three hours) twice: on day 0 to quantify the initial cytokine response and on day 7 to quantify the degree of endotoxin tolerance. Subjects either received CytoSorb hemoperfusion during the first LPS challenge (CytoSorb group), or no intervention (control group). Plasma cytokine concentrations and clearance rates were determined serially. This study was registered at ClinicalTrials.gov (NCT04643639, date of registration November 24th 2020).ResultsLPS administration led to a profound increase in plasma cytokine concentrations during both LPS challenge days. Compared to the control group, significantly lower plasma levels of tumor necrosis factor (TNF, − 58%, p < 0.0001), interleukin (IL)-6 ( − 71%, p = 0.003), IL-8 ( − 48%, p = 0.02) and IL-10 ( − 26%, p = 0.03) were observed in the CytoSorb group during the first LPS challenge. No differences in cytokine responses were observed during the second LPS challenge.ConclusionsCytoSorb hemoperfusion effectively attenuates circulating cytokine concentrations during systemic inflammation in humans in vivo, whereas it does not affect long-term immune function. Therefore, CytoSorb therapy may be of benefit in conditions characterized by excessive cytokine release.

Interferon regulatory factor 1 (IRF1) inhibits lung endothelial regeneration following inflammation-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is a respiratory condition caused by severe endothelial barrier dysfunction within the lung. In ARDS, excessive inflammation, tissue edema, and immune cell influx prevents endothelial cell regeneration that is crucial in repairing the endothelial barrier. However, little is known about the molecular mechanism that underpin endothelial cell regeneration in ARDS. R-based bioinformatics tools were used to analyze microarray-derived transcription profiles in human lung microvascular endothelial cells (HLMVECs) subjected to non-treatment or lipopolysaccharide (LPS) exposure. We generated endothelial cell-specific interferon regulatory factor 1 (Irf1) knockout (Irf1EC-/-) and Irf1fl/fl control mice for use in an endotoxemic murine model of acute lung injury (ALI). In vitro studies (qPCR, immunoblotting, and ChIP-qPCR) were conducted in mouse lung endothelial cells (MLECs) and HLMVECs. Dual-luciferase promoter reporter assays were performed in HLMVECs. Bioinformatics analyses identified IRF1 as a key up-regulated gene in HLMVECs post-LPS exposure. Endothelial-specific knockout of Irf1 in ALI mice resulted in enhanced regeneration of lung endothelium, while liposomal delivery of endothelial-specific Irf1 to wild-type ALI mice inhibited lung endothelial regeneration in a leukemia inhibitory factor (Lif)-dependent manner. Mechanistically, we demonstrated that LPS-induced Stat1Ser727 phosphorylation promotes Irf1 transactivation, resulting in downstream up-regulation of Lif that inhibits endothelial cell proliferation. These results demonstrate the existence of a p-Stat1Ser727-Irf1-Lif axis that inhibits lung endothelial cell regeneration post-LPS injury. Thus, direct inhibition of IRF1 or LIF may be a promising strategy for enhancing endothelial cell regeneration and improving clinical outcomes in ARDS patients.

A Dietary Nucleoside-Nucleotide Mixture Inhibits Endotoxin-Induced Bacterial Translocation in Mice Fed Protein-Free Diet

Nucleosides and nucleotides are important substrates utilized by the intestinal mucosa. To determine the relative effect of dietary nucleosides and nucleotides on the gut, we investigated the effects of these compounds on endotoxin-induced bacterial translocation, cecal bacterial populations and ileal histology in protein-malnourished mice. There was an inhibition of gram-negative enteric bacteria in the mesenteric lymph node and spleen of the surviving mice fed the protein-free diet supplemented with a nucleoside-nucleotide mixture compared with the nonsupplemented group. Histologically, the damage to the gut mucosal barrier was more pronounced in the nonsupplemented group than in the nucleoside-nucleotide supplemented group. However, the cecal bacterial populations in the groups were not different. The villous height, crypt depth and total wall thickness were more developed in the supplemented group compared with the nonsupplemented group, indicating that the nucleoside-nucleotide mixture blocked bacterial translocation by preventing endotoxin-induced mucosal or epithelial damage. These results suggest that the nucleoside-nucleotide mixture could be used to inhibit or reduce the incidence of bacterial translocation, decrease intestinal injury and improve survival in a lethal model of protein deficiency and endotoxemia.

Lipopolysaccharide-induced endotoxaemia during adolescence promotes stress vulnerability in adult mice via deregulation of nuclear factor erythroid 2-related factor 2 in the medial prefrontal cortex.

Sepsis is a severe inflammatory response to infection that leads to long-lasting cognitive impairment and depression after resolution. The lipopolysaccharide (LPS)-induced endotoxaemia model is a well-established model of gram-negative bacterial infection and recapitulates the clinical characteristics of sepsis. However, whether LPS-induced endotoxaemia during adolescence can modulate depressive and anxiety-like behaviours in adulthood remains unclear. To determine whether LPS-induced endotoxaemia in adolescence can modulate the stress vulnerability to depressive and anxiety-like behaviours in adulthood and explore the underlying molecular mechanisms. Quantitative real-time PCR was used to measure inflammatory cytokine expression in the brain. A stress vulnerability model was established by exposure to subthreshold social defeat stress (SSDS), and depressive- and anxiety-like behaviours were evaluated by the social interaction test (SIT), sucrose preference test (SPT), tail suspension test (TST), force swimming test (FST), elevated plus-maze (EPM) test, and open field test (OFT). Western blotting was used to measure Nrf2 and BDNF expression levels in the brain. Our results showed that inflammation occurred in the brain 24h after the induction of LPS-induced endotoxaemia at P21 but resolved in adulthood. Furthermore, LPS-induced endotoxaemia during adolescence promoted the inflammatory response and the stress vulnerability after SSDS during adulthood. Notably, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and BDNF in the mPFC were decreased after SSDS exposure in mice treated with LPS during adolescence. Activation of the Nrf2-BDNF signalling pathway by sulforaphane (SFN), an Nrf2 activator, ameliorated the effect of LPS-induced endotoxaemia during adolescence on stress vulnerability after SSDS during adulthood. Our study identified adolescence as a critical period during which LPS-induced endotoxaemia can promote stress vulnerability during adulthood and showed that this effect is mediated by impairment of Nrf2-BDNF signalling in the mPFC.

Short-Term Intake of Theobroma grandiflorum Juice Fermented with Lacticaseibacillus rhamnosus ATCC 9595 Amended the Outcome of Endotoxemia Induced by Lipopolysaccharide.

Endotoxemia is a condition caused by increasing levels of lipopolysaccharide (LPS) characterized by an impaired systemic response that causes multiple organ dysfunction. Lacticaseibacillus rhamnosus ATCC 9595 is a strain with probiotic potential which shows immunomodulatory properties. The incorporation of this bacterium in food rich in bioactive compounds, such as cupuaçu juice (Theobroma grandiflorum), could result in a product with interesting health properties. This work evaluated the effects of the oral administration of cupuaçu juice fermented with L. rhamnosus on the outcome of LPS-induced endotoxemia in mice. C57BL/6 mice (12/group) received oral doses (100 µL) of saline solution and unfermented or fermented cupuaçu juice (108 CFU/mL). After 5 days, the endotoxemia was induced by an intraperitoneal injection of LPS (10 mg/kg). The endotoxemia severity was evaluated daily using a score based on grooming behavior, mobility, presence of piloerection, and weeping eyes. After 6 h and 120 h, the mice (6/group) were euthanized for analysis of cell counts (in peritoneal lavage and serum) and organ weight. L. rhamnosus grew in cupuaçu juice and produced organic acids without the need for supplementation. The bacteria counts were stable in the juice during storage at 4 °C for 28 days. The fermentation with L. rhamnosus ATCC 9595 changed the metabolites profile of cupuaçu juice due to the biotransformation and enhancement of some compounds. In general, the administration of L. rhamnosus-fermented juice allowed a significant improvement in several characteristics of endotoxemic status (weight loss, hypothermia, severity index, cell migration). In addition, treatment with fermented juice significantly reduced the weight of the spleen, liver, intestine, and kidneys compared to the saline-treated endotoxemic group. Taken together, our data show that short-term intake therapy of cupuaçu juice fermented with L. rhamnosus ATCC 9595 can reduce systemic inflammation in an experimental model of LPS-induced endotoxemia in mice.

The Use of Diffusion Weighted Imaging and Intravoxel Incoherent Motion to Assess Edema and Perfusion in Abdominal Organs during Endotoxemia

Diffusion weighted magnetic resonance imaging (DW-MRI) technique probing the microscopic movement of water molecules in tissue, allows for the quantification of water diffusivity and the contribution of perfusion to the total amount of water. In the last decades, its use in organs other than the brain has increased and it has successfully been applied to abdominal organs; otherwise the use of DWI for the assessment of perfusion (by measuring perfusion fraction and edema (by measuring the apparent diffusion coefficient—ADC) of entire organs is not a clinical standard. The aim of this paper is to assess whether DWI, specifically IVIM, can accurately assess edema (the amount of water contained in a tissue) and perfusion in abdominal organs (liver, spleen, intestine and kidneys) in an endotoxemic porcine model. Endotoxemia was induced in 20 piglets and they were divided into three groups: HM group (HighMAP, mean arterial pressure was maintained &gt;65 mmHg by noradrenaline infusion), LM group (LowMAP, MAP was maintained between 50 and 60 mmHg) and LTD (the thoracic duct was ligated to increase abdominal edema). In addition, a fourth group of healthy controls (four animals) underwent MRI. Edema was also assessed by wet–dry weight in liver, spleen and intestine; and perfusion was assessed by hemodynamics, lactate concentration and SvO2. ADC was found to be higher in the intestine of the LTD group compared with the other groups, in accordance with wet–dry weight. In addition, ADC in kidneys was found to be correlated to glomerular filtration rate. f was correlated with hemodynamics in kidneys and liver. The presented data show that there is a potential for clinical use of the technique in septic patients.

Anti-Inflammatory and T-Cell Immunomodulatory Effects of Banana Peel Extracts and Selected Bioactive Components in LPS-Challenged In Vitro and In Vivo Models

Banana peel (BP) has potent antioxidative properties; however, the anti-inflammatory potential of BP and its related bioactive components remain unclear. This study used solvent extraction and gas chromatography–mass spectrometry (GC–MS) to isolate and identify the active fractions and compounds in BP. BP was extracted with 95% ethanol (BP-95E) and partitioned with an ethyl acetate (EA) and water mixture to obtain the BP-95E-EA and BP-95E-H2O fractions. The BP-95E-EA fractions were further partitioned with n-hexane (Hex) and methanol (MeOH) mixtures to obtain BP-95E-EA-Hex and BP-95E-EA-MeOH subfractions, and the BP-95E-H2O fractions were partitioned with n-butanol (BuOH) to obtain BP-95E-H2O-BuOH subfractions and the H2O residual. The results show that the BP-95E-H2O-BuOH subfractions exhibited the most potent inhibition of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion while down-regulating inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. In this active subfraction, five non-polyphenol compounds were identified, namely, 5-hydroxyethyl furfural (5-HMF), guaiol, oleic acid, linoleic acid, and oleamide. 5-HMF, guaiol, and oleamide were the most effective at suppressing IL-6 and TNF-α secretion. The in vivo immunomodulatory action of BP was evaluated in an LPS-induced endotoxemia model of BALB/c mice. Oral administration of BP-95E-H2O-BuOH extracts (42 and 166 mg/kg b.w.) for two weeks lowered the serum levels of IL-6 and TNF-α and normalized the activated T-cell population, as evidenced by an increase in CD3CD69 and decrease in IFN-γ/IL-4 (Th1/Th2) in mice with systemic inflammation. Our findings reveal that BP exhibits anti-inflammatory and T-cell immunomodulatory effects that may contribute to delaying endotoxemia-associated disorders.