Disseminated intravascular coagulation phenotype is regulated by the TRPM7 channel during sepsis.

BACKGROUND: Sepsis is an uncontrolled inflammatory response against a systemic infection that results in elevated mortality, mainly induced by bacterial products known as endotoxins, producing endotoxemia. Disseminated intravascular coagulation (DIC) is frequently observed in septic patients and is associated with organ failure and death. Sepsis activates endothelial cells (ECs), promoting a prothrombotic phenotype contributing to DIC. Ion channel-mediated calcium permeability participates in coagulation. The transient reception potential melastatin 7 (TRPM7) non-selective divalent cation channel that also contains an α-kinase domain, which is permeable to divalent cations including Ca2+, regulates endotoxin-stimulated calcium permeability in ECs and is associated with increased mortality in septic patients. However, whether endothelial TRPM7 mediates endotoxemia-induced coagulation is not known. Therefore, our aim was to examine if TRPM7 mediates coagulation during endotoxemia. RESULTS: The results showed that TRPM7 regulated endotoxin-induced platelet and neutrophil adhesion to ECs, dependent on the TRPM7 ion channel activity and by the α-kinase function. Endotoxic animals showed that TRPM7 mediated neutrophil rolling on blood vessels and intravascular coagulation. TRPM7 mediated the increased expression of the adhesion proteins, von Willebrand factor (vWF), intercellular adhesion molecule 1 (ICAM-1), and P-selectin, which were also mediated by the TRPM7 α-kinase function. Notably, endotoxin-induced expression of vWF, ICAM-1 and P-selectin were required for endotoxin-induced platelet and neutrophil adhesion to ECs. Endotoxemic rats showed increased endothelial TRPM7 expression associated with a procoagulant phenotype, liver and kidney dysfunction, increased death events and an increased relative risk of death. Interestingly, circulating ECs (CECs) from septic shock patients (SSPs) showed increased TRPM7 expression associated with increased DIC scores and decreased survival times. Additionally, SSPs with a high expression of TRPM7 in CECs showed increased mortality and relative risk of death. Notably, CECs from SSPs showed significant results from the AUROC analyses for predicting mortality in SSPs that were better than the Acute Physiology and Chronic Health Evaluation II (APACHE II) and the Sequential Organ Failure Assessment (SOFA) scores. CONCLUSIONS: Our study demonstrates that sepsis-induced DIC is mediated by TRPM7 in ECs. TRPM7 ion channel activity and α-kinase function are required by DIC-mediated sepsis-induced organ dysfunction and its expression are associated with increased mortality during sepsis. TRPM7 appears as a new prognostic biomarker to predict mortality associated to DIC in SSPs, and as a novel target for drug development against DIC during infectious inflammatory diseases.

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.

L-Carnitine and synbiotic co-supplementation: beneficial effects on metabolic-endotoxemia, meta-inflammation, and oxidative-stress biomarkers in obese patients: a double blind, randomized, controlled clinical trial.

Obesity, a chronic pandemic disease, is characterized by low-grade chronic inflammation, accompanied by over-expression of pro-inflammatory cytokines, thereby contributing to metabolic disorders pathogenesis. Oxidative-stress, an adverse cellular response to adipocyte hypertrophy, promotes inflammation. Furthermore, gut-microbiota dysbiosis may induce oxidative-stress, low-grade inflammation, and metabolic-endotoxemia as major drivers of obesity. Functional-foods/nutraceuticals have attracted extensive attention due to their plausible anti-inflammatory/anti-oxidative properties; evidence supports the superiority of the nutraceutical combined-supplementation approach versus conventional mono-therapies. Current data suggest the anti-oxidative/anti-inflammatory properties of either L-carnitine or pre/pro/synbiotics. This trial compared the effects of co-supplementing L-carnitine and multi-species/multi-strain synbiotic versusL-carnitine mono-therapy on inflammatory/anti-inflammatory, oxidative-stress, and metabolic-endotoxemia biomarkers in 46 female obese patients, receiving either co-supplementation (L-carnitine-tartrate (2 × 500 mg d-1) + multi-species/multi-strain synbiotic (1 capsule per day)) or mono-therapy (L-carnitine-tartrate (2 × 500 mg d-1) + maltodextrin (1 capsule per day)) for eight weeks. L-Carnitine + synbiotic co-supplementation significantly decreased interleukin-6 (IL-6, -33.98%), high-sensitivity-C-reactive-protein (hs-CRP, -10%), tumor-necrosis-factor-alpha (TNF-α, -18.73%), malondialdehyde (MDA, -21.73%), and lipopolysaccharide (LPS, -10.14%), whereas the increase in interleukin-10 (IL-10, 7.69%) and total-antioxidant-capacity (TAC, 4.13%) levels was not significant. No significant changes were observed for the above-mentioned parameters in the L-carnitine + placebo group, except for a significant reduction in IL-10 (-17.59%) and TNF-α (-14.78%); however, between-group differences did not reach the significant threshold. Co-supplementing L-carnitine + multi-strain synbiotic led to significant amelioration of inflammatory, oxidative, and metabolic-endotoxemia responses in female obese patients; nevertheless, no improving effects were observed in patients receiving single-supplementation, suggesting that L-carnitine + synbiotic co-supplementation might represent an adjuvant approach to improve oxidative-stress/pro-inflammatory indicators in women with obesity, possibly through beneficial effects of the synbiotic alone. Further longer duration studies with higher doses of L-carnitine in a three-group setting are warranted to elucidate the possibility of synergistic or complementary mechanisms.

Intestinal edema induced by LPS-induced endotoxemia is associated with an inflammasome adaptor ASC.

The apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/caspase-1/interleukin(IL)-1ß axis, also known as the inflammasome pathway, is indispensable for IL-1ß activation in response to various pathogens or own damages. Previously, we developed an NLRP3-inflammasome using a cell-free system and identified ASC targeting drugs; thus, examination of ASC-related histopathology in various diseases could help to provide indications for these drugs. Here, we generated mice deficient only in ASC-protein (ASC-deficient (AD) mice) using CRISPR/Cas9 technology, studied which tissues were most affected, and obtained histopathological images of lipopolysaccharide (LPS)-induced endotoxemia. C57BL/6 wild-type (WT) and (AD) mice were injected intraperitoneally with a lethal dose (50 µg/g) of LPS. Statistical analysis of the survival of C57BL/6 mice and AD mice was performed using the Kaplan-Meier method and the log-rank test. The histopathological findings of multiple tissues from these mice were compared. Acute inflammation (e.g., catarrhal inflammation), along with congestion was observed in the colon of WT mice but not in that of AD mice. Adhesion of neutrophils to capillaries, along with interstitial infiltration, were observed in multiple tissues from WT mice. In AD mice, neutrophil infiltration was less severe but remained evident in the stomach, small intestine, heart, liver, kidney, spleen, and brain. Notably, there was no difference between WT and AD mice with respect to alveolar neutrophil infiltration and interstitial edema. These findings suggest that even though ASC contributes to systemic inflammation, it is dependent on the tissue involved. Intestinal congestion and edema might be good candidates for anti-ASC-targeted therapy.

TRPV6 deficiency attenuates stress and corticosterone-mediated exacerbation of alcohol-induced gut barrier dysfunction and systemic inflammation.

Introduction: Chronic stress is co-morbid with alcohol use disorder that feedback on one another, thus impeding recovery from both disorders. Stress and the stress hormone corticosterone aggravate alcohol-induced intestinal permeability and liver damage. However, the mechanisms involved in compounding tissue injury by stress/corticosterone and alcohol are poorly defined. Here we explored the involvement of the TRPV6 channel in stress (or corticosterone) 3and alcohol-induced intestinal epithelial permeability, microbiota dysbiosis, and systemic inflammation. Methods: Chronic alcohol feeding was performed on adult wild-type and Trpv6-/- mice with or without corticosterone treatment or chronic restraint stress (CRS). The barrier function was determined by evaluating inulin permeability in vivo and assessing tight junction (TJ) and adherens junction (AJ) integrity by immunofluorescence microscopy. The gut microbiota composition was evaluated by 16S rRNA sequencing and metagenomic analyses. Systemic responses were assessed by evaluating endotoxemia, systemic inflammation, and liver damage. Results: Corticosterone and CRS disrupted TJ and AJ, increased intestinal mucosal permeability, and caused endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. Corticosterone and CRS synergistically potentiated the alcohol-induced breakdown of intestinal epithelial junctions, mucosal barrier impairment, endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. TRPV6 deficiency also blocked the effects of CRS and CRS-mediated potentiation of alcohol-induced dysbiosis of gut microbiota. Conclusions: These findings indicate an essential role of TRPV6 in stress, corticosterone, and alcohol-induced intestinal permeability, microbiota dysbiosis, endotoxemia, systemic inflammation, and liver injury. This study identifies TRPV6 as a potential therapeutic target for developing treatment strategies for stress and alcohol-associated comorbidity.

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.

RATIONALE: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: Our results showed that inflammation occurred in the brain 24 h 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. CONCLUSIONS: 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.

Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

Can increased intestinal permeability and low-grade endotoxemia be the triggering pathogenesis in isolated coronary artery ectasia?

PURPOSE: Isolated coronary artery ectasia (ICAE) is a rare coronary artery disease (CAD) encountered during coronary angiography. Although many mechanisms have been suggested today that may be associated with ICAE, the underlying pathogenesis has not been fully understood. In this study, we aimed to reveal the possible relationship between intestinal permeability and ICAE. METHODS: Of the 12 850 patients who underwent coronary angiography, 138 consecutive patients with ICAE and 140 age- and sex-matched subjects with normal coronary arteries as the control group and 140 subjects with stenotic CAD were included in the study. RESULTS: Serum zonulin and lipopolysaccharide levels were significantly higher in patients with ICAE than in the control group and CAD group. Additionally, zonulin and lipopolysaccharide levels were significantly higher in the CAD group than in the ICAE group. In the correlation analysis, serum zonulin levels were correlated with the mean diameter and length of the ecstatic segment. In multivariate analysis, zonulin and lipopolysaccharide were identified as independent predictors for ICAE. CONCLUSION: These results suggest that there may be a pathophysiological relationship between increased intestinal permeability and ICAE.

Novel transdermal curcumin therapeutic preserves endothelial barrier function in a high-dose LPS rat model.

Sepsis is a devastating complication of infection and injury that, through widespread endothelial dysfunction, can cause perfusion deficits and multi-organ failure. To address the recognised need for therapeutics targetting the endothelial barrier, a topical formulation (CUR; VASCEPTOR™; Vascarta Inc, Summit, NJ) was developed to transdermally deliver bio-active concentrations of curcumin-an anti-inflammatory and nitric oxide promoter. Male, Sprague Dawley rats were treated daily with lipopolysaccharide (LPS, 10 mg/kg, IP) to induce endotoxemia, and topical applications of Vehicle Control (LPS + VC; N = 7) or Curcumin (LPS + CUR; N = 7). A third group received neither LPS nor treatment (No-LPS; N = 8). After 72 h, animals were surgically prepared for measurements of physiology and endothelial dysfunction in the exteriorised spinotrapezius muscle through the extravasation of 67 kDa TRITC-BSA (albumin) and 500 kDa FITC-dextran (dextran). At 72 h, LPS + VC saw weight loss, and increases to pulse pressure, lactate, pCO2, CXCL5 (vs No-LPS) and IL-6 (vs 0 h; p < 0.05). LPS + CUR was similar to No-LPS, but with hypotension. Phenylephrine response was increased in LPS + CUR. Regarding endothelial function, LPS + CUR albumin and dextran extravasation were significantly reduced versus LPS + VC suggesting that Curcumin mitigated endotoxemic endothelial dysfunction. The speculated mechanisms are nitric oxide modulation of the endothelium and/or an indirect anti-inflammatory effect.

Protective Effect of Electroacupuncture on the Barrier Function of Intestinal Injury in Endotoxemia through HO-1/PINK1 Pathway-Mediated Mitochondrial Dynamics Regulation.

Background and Aims: Endotoxemia (ET) is a common critical illness in patients receiving intensive care and is associated with high mortality and prolonged hospital stay. The intestinal epithelial cell dysfunction is regarded as the "engine" of deteriorated ET. Although electroacupuncture (EA) can mitigate endotoxin-induced intestinal epithelial cell dysfunction in ET, the mechanism through which EA improves endotoxin-induced intestinal injury for preventing ET deterioration needs further investigation. Methods: An in vivo ET model was developed by injecting lipopolysaccharide (LPS) in wild-type and PINK1-knockout mice. An in vitro model was also established by incubating epithelial cells in the serum samples obtained from both groups of mice. Hemin and zinc protoporphyrin IX (ZnPP) were applied to activate or inhibit heme oxygenase 1 (HO-1) production. EA treatment was performed for 30 min consecutively for 5 days before LPS injection, and on the day of the experiment, EA was performed throughout the process. Samples were harvested at 6 h after LPS induction for analyzing tissue injury, oxidative stress, ATP production, activity of diamine oxidase (DAO), and changes in the levels of HO-1, PTEN-induced putative kinase 1 (PINK1), mitochondrial fusion and fission marker gene, caspase-1, and interleukin 1 beta (IL-1ß). Results: In the wild-type models (both in vivo and vitro), EA alleviated LPS-induced intestinal injury and mitochondrial dysfunction, as indicated by decreased reactive oxygen species (ROS) production and oxygen consumption rate (OCR) and reduced levels of mitochondrial fission proteins. EA treatment also boosted histopathological morphology, ATP levels, DAO activity, and levels of mitochondrial fusion proteins in vivo and vitro. The effect of EA was enhanced by hemin but suppressed by Znpp. However, EA + AP, Znpp, or hemin had no effects on the LPS-induced, PINK1-knocked out mouse models. Conclusion: EA may improve the HO-1/PINK1 pathway-mediated mitochondrial dynamic balance to protect the intestinal barrier in patients with ET.

Central Venous Waveform Analysis and Cardiac Output in a Porcine Model of Endotoxemic Hypotension and Resuscitation.

BACKGROUND: Cardiac output (CO) is a valuable proxy for perfusion, and governs volume responsiveness during resuscitation from distributive shock. The underappreciated venous system has nuanced physiology that confers valuable hemodynamic information. In this investigation, deconvolution of the central venous waveform by the fast Fourier transformation (FFT) algorithm is performed to assess its ability to constitute a CO surrogate in a porcine model of endotoxemia-induced distributive hypotension and resuscitation. STUDY DESIGN: Ten pigs were anesthetized, catheterized, and intubated. A lipopolysaccharides infusion protocol was used to precipitate low systemic vascular resistance hypotension. Four crystalloid boluses (10 cc/kg) were then given in succession, after which heart rate, mean arterial pressure, thermodilution-derived CO, central venous pressure (CVP), and the central venous waveform were collected, the last undergoing fast Fourier transformation analysis. The amplitude of the fundamental frequency of the central venous waveform's cardiac wave (f0-CVP) was obtained. Heart rate, mean arterial pressure, CVP, f0-CVP, and CO were plotted over the course of the boluses to determine whether f0-CVP tracked with CO better than the vital signs, or than CVP itself. RESULTS: Distributive hypotension to a 25% mean arterial pressure decrement was achieved, with decreased systemic vascular resistance (mean 918 ± 227 [SD] dyne/s/cm-5 vs 685 ± 180 dyne/s/cm-5; p = 0.038). Full hemodynamic parameters characterizing this model were reported. Slopes of linear regression lines of heart rate, mean arterial pressure, CVP, f0-CVP, and CO were -2.8, 1.7, 1.8, 0.40, and 0.35, respectively, demonstrating that f0-CVP values closely track with CO over the 4-bolus range. CONCLUSIONS: Fast Fourier transformation analysis of the central venous waveform may allow real-time assessment of CO during resuscitation from distributive hypotension, possibly offering a venous-based approach to clinical estimation of volume responsiveness.

Using heparan sulfate octadecasaccharide (18-mer) as a multi-target agent to protect against sepsis.

Sepsis is a lethal syndrome manifested by an unregulated, overwhelming inflammation from the host in response to infection. Here, we exploit the use of a synthetic heparan sulfate octadecasaccharide (18-mer) to protect against sepsis. The 18-mer not only inhibits the pro-inflammatory activity of extracellular histone H3 and high mobility group box 1 (HMGB1), but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). We demonstrate that the 18-mer protects against sepsis-related injury and improves survival in cecal ligation and puncture mice and reduces inflammation in an endotoxemia mouse model. The 18-mer neutralizes the cytotoxic histone-3 (H3) through direct interaction with the protein. Furthermore, the 18-mer enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide, a toxic complex contributing to cell death and tissue damage in sepsis. Our study provides strong evidence that the 18-mer mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.

The mechanistic and structural role of von Willebrand factor in endotoxemia-enhanced deep vein thrombosis in mice.

BACKGROUND: Although the concept of immunothrombosis has established a link between inflammation and thrombosis, the role of inflammation in the pathogenesis of deep vein thrombosis remains to be fully elucidated. Further, although various constituents of venous thrombi have been identified, their localizations and cellular and molecular interactions are yet to be combined in a single, multiplexed analysis. OBJECTIVES: The objective of this study was to investigate the role of the von Willebrand factor (VWF) in inflammation-associated venous thrombosis. We also performed a proof-of-concept study of imaging mass cytometry to quantitatively and simultaneously analyze the localizations and interactions of 10 venous thrombus constituents. METHODS: We combined the murine inferior vena cava stenosis model of deep vein thrombosis with the lipopolysaccharide model of endotoxemia. We also performed a proof-of-concept study of imaging mass cytometry to assess the feasibility of this approach in analyzing the structural composition of thrombi. RESULTS: We found that lipopolysaccharide-treated mice had significantly higher incidences of venous thrombosis, an effect that was mitigated when VWF was inhibited using inhibitory αVWF antibodies. Our detailed structural analysis also showed that most thrombus components are localized in the white thrombus regardless of endotoxemia. Moreover, although endotoxemia modulated the relative representation and interactions of VWF with other thrombus constituents, the scaffolding network, comprised VWF, fibrin, and neutrophil extracellular traps, remained largely unaffected. CONCLUSIONS: We observe a key role for VWF in the pathogenesis of inflammation-associated venous thrombosis while providing a more comprehensive insight into the molecular interactions that constitute the architecture of venous thrombi.

ACETAMINOPHEN ATTENUATES PULMONARY VASCULAR RESISTANCE AND PULMONARY ARTERIAL PRESSURE AND INHIBITS CARDIOVASCULAR COLLAPSE IN A PORCINE MODEL OF ENDOTOXEMIA.

ABSTRACT: Acetaminophen (paracetamol) is often used in critically ill patients with fever and pain; however, little is known about the effects of acetaminophen on cardiovascular function during systemic inflammation. Here, we investigated the effect of acetaminophen on changes in the systemic and pulmonary circulation induced by endotoxin (0.5 µg/kg per hour) in anesthetized pigs. Endotoxin infusion led to a rapid increase in pulmonary artery pressure and pulmonary vascular resistance index. Acetaminophen delayed and attenuated this increase. Furthermore, acetaminophen reduced tachycardia and decreased stroke volume, accompanied by systemic inflammation, without affecting inflammatory parameters such as white blood cell count and TNF-α in blood. As a proof of concept, we injected a high dose of endotoxin (100 µg), which induced rapid cardiovascular collapse in pigs. Pigs treated with acetaminophen survived with no obvious hemodynamic instability during the 50-min observation period. In conclusion, acetaminophen attenuates the effects of endotoxin on pulmonary circulation in anesthetized pigs. This may play a role in severe systemic inflammation.

HC067047 as a potent TRPV4 inhibitor repairs endotoxemia colonic injury.

Colonic injury causes severe inflammation during systemic infections in patients with endotoxemia. The prevention of colonic injury could effectively reduce the progression of endotoxemia. We investigated the protective effects and detailed mechanisms of the TRPV4 inhibitor HC067047 in the treatment of colonic injury caused by endotoxemia. An LPS-induced endotoxemia colonic injury model was used to assess the in vivo effects of HC067047. Colon slices were detected by hematoxylin and eosin (HE) staining and immunofluorescence assays. Spectrophotometry was used to determine the levels of MDA, calcium, GSH, and GSSG. Alterations in oxidative stress/mitophagy/inflammatory pyroptosis-related markers were evaluated by Q-PCR and western blot assays. HC067047 reduced the body weight loss and spleen weight index of endotoxemic mice and partly recovered the normal morphology of the colonic mucous layer. As an inhibitor of the calcium permeant cation channel, HC067047 suppressed the phosphorylation of the CAMKIIɑ protein and levels of MDA and calcium, upregulated the ratio of GSH/GSSG, shortened the expression of oxidative stress-related proteins, and enhanced the expression of the anti-oxidative protein CAT in damaged colon tissues. Additionally, HC067047 maintained normal mitochondrial functions in endotoxemia colons by promoting mitochondrial fusion and biosynthesis and suppressing mitochondrial fission and the PINK/Parkin/mitophagy pathway. HC067047 potently blocked inflammatory pyroptosis and protected the colonic tight junction barrier. HC067047 restores endotoxemia colons against oxidative stress, mitophagy, inflammatory pyroptosis, and colonic barrier dysfunction. Hence, HC067047 therapy may be potentially useful in the treatment of colonic injury in endotoxemia.

Sesamolin Attenuates Kidney Injury, Intestinal Barrier Dysfunction, and Gut Microbiota Imbalance in High-Fat and High-Fructose Diet-Fed Mice.

This study investigated the effects of sesamolin on kidney injury, intestinal barrier dysfunction, and gut microbiota imbalance in high-fat and high-fructose (HF-HF) diet-fed mice and explored the underlying correlations among them. The results indicated that sesamolin suppressed metabolic disorders and increased renal function parameters. Histological evaluation showed that sesamolin mitigated renal epithelial cell degeneration and brush border damage. Meanwhile, sesamolin inhibited the endotoxin-mediated induction of the Toll-like receptor 4-related IKKα/NF-κB p65 pathway activation. Additionally, sesamolin mitigated intestinal barrier dysfunction and improved the composition of gut microbiota. The correlation results further indicated that changes in the dominant phyla, including Firmicutes, Deferribacterota, Desulfobacterota, and Bacteroidota, were more highly correlated with a reduction in endotoxemia and metabolic disorders, as well as decreases in intestinal proinflammatory response and related renal risk biomarkers. The results of this study suggest that sesamolin attenuates kidney injuries, which might be associated with its effects on the reduction of endotoxemia and related metabolic disorders through the restoration of the intestinal barrier and the modulation of gut microbiota. Thus, sesamolin may be a potential dietary supplement for protection against obesity-associated kidney injury.

The renin-angiotensin system modulates endotoxic postconditioning of exacerbated renal vasoconstriction in preeclamptic offspring.

We recently reported exacerbated endotoxic signs of neuroinflammation and autonomic defects in offspring of preeclamptic (PE) dams. Here, we investigated whether PE programming similarly modifies hemodynamic and renal vasoconstrictor responsiveness to endotoxemia in PE offspring and whether this interaction is modulated by gestational angiotensin 1-7 (Ang1-7). Preeclampsia was induced by gestational treatment with L-NAME. Adult offspring was challenged with lipopolysaccharides (LPS, 5 mg/kg) and systolic blood pressure (SBP) and renal vasoconstrictions were assessed 4 h later. Male, but not female, offspring of PE rats exhibited SBP elevations that were blunted by LPS. Renal vasoconstrictions induced by angiotensin II (Ang II), but not phenylephrine, were intensified in perfused kidneys of either sex. LPS blunted the heightened Ang II responses in male, but not female, kidneys. While renal expressions of AT1-receptors and angiotensin converting enzyme (ACE) were increased in PE offspring of both sexes, ACE2 was upregulated in female offspring only. These molecular effects were diminished by LPS in male offspring. Gestational Ang1-7 caused sex-unrelated attenuation of phenylephrine vasoconstrictions and preferentially downregulated Ang II responses and AT1-receptor and nuclear factor-kB (NFkB) expressions in females. Together, endotoxemia and Ang1-7 offset in sexually-related manners imbalances in renal vasoconstriction and AT1/ACE/ACE2 signaling in PE offspring.

Increased Endotoxin Activity in COVID-19 Patients Admitted to the Intensive Care Unit.

OBJECTIVE: Endotoxin is a component of Gram-negative bacteria and can be measured in blood using the endotoxin activity assay (EAA). Endotoxin exposure initiates an inflammatory cascade that may contribute to organ dysfunction. Endotoxemia has been reported in previous viral pandemics and we investigated the extent of endotoxemia and its relationship to outcomes in critically ill patients with COVID-19. MATERIALS AND METHODS: We conducted a Prospective Cohort Study of 96 critically-ill COVID-19 patients admitted to the George Washington University Hospital ICU from 25 Mar-6 Jun 2020. EAA and inflammatory markers (ferritin, d dimer, IL-6, CRP) were measured on ICU admission and at the discretion of the clinical team. Clinical outcomes (mortality, LOS, need for renal replacement therapy (RRT), intubation) were measured. Statistical analysis was conducted using descriptive statistics and effect estimates with 95% confidence intervals. Comparisons were made using chi-square tests for categorical variables, and T-tests for continuous variables. RESULTS: A majority of patients (68.8%) had high EAA [≥ 0.60], levels seen in septic shock. Only 3 patients had positive bacterial cultures. EAA levels did not correlate with mortality, higher levels were associated with greater organ failure (cardiovascular, renal) and longer ICU LOS. Among 14 patients receiving RRT for severe AKI, one had EAA < 0.6 (p = 0.043). EAA levels did not directly correlate with other inflammatory markers. CONCLUSIONS: High levels of endotoxin activity were found in a majority of critically-ill COVID-19 patients admitted to the ICU and were associated with greater risk for cardiovascular and renal failure. Further investigation is needed to determine if endotoxin reducing strategies are useful in treating severe COVID-19 infection.

Oral Administration of Meloxicam and Flunixin Meglumine Have Similar Analgesic Effects After Lipopolysaccharide-Induced Inflammatory Response in Thoroughbred Horses.

Flunixin meglumine (FM), a nonselective cyclooxygenase (COX) inhibitor, is most frequently selected for the treatment of equine systemic inflammatory response syndrome (SIRS)/endotoxemia. However, FM has considerable adverse effects on gastrointestinal function. The aims of this study were to compare the effect of meloxicam (MX), a COX-2 selective inhibitor commonly used in equine clinical practice, with FM, and to investigate the potential for clinical application in horses with SIRS/endotoxemia. Fifteen horses were divided into three groups of five and orally administered MX (0.6 mg/kg), FM (1.1 mg/kg), or saline as placebo at 30 minutes after LPS challenge. Clinical parameters, including behavioral pain scores, were recorded and blood for clinical pathological data was collected at various times from 60 minutes before to 420 minutes after LPS infusion. The pain score were significantly lower in both the MX and FM groups than in the placebo group, with no significant difference between them. Body temperature was significantly lower in the MX and FM groups than in the placebo group. Heart rates and respiratory rates, hoof wall surface temperature, and leukocyte counts changed similarly between the MX and FM groups. TNF-α and cortisol were lower in the FM group than in the MX group. The results suggest that MX suppresses the inflammatory response after LPS infusion and has an analgesic effect similar to that of FM. Given the adverse effects of nonselective COX inhibitors, clinical application of MX may be beneficial in horses with SIRS/endotoxemia.

ALDH2 deficiency increases susceptibility to binge alcohol-induced gut leakiness, endotoxemia, and acute liver injury in mice through the gut-liver axis.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is the major enzyme responsible for metabolizing toxic acetaldehyde to acetate and acts as a protective or defensive protein against various disease states associated with alcohol use disorder (AUD), including alcohol-related liver disease (ARLD). We hypothesized that Aldh2-knockout (KO) mice are more susceptible to binge alcohol-mediated liver injury than wild-type (WT) mice through increased oxidative stress, gut leakiness and endotoxemia. Therefore, this study aimed to investigate the protective role of ALDH2 in binge alcohol-induced gut permeability, endotoxemia, and acute inflammatory liver injury by exposing Aldh2-KO or WT mice to a single oral dose of binge alcohol 3.5, 4.0, or 5.0 g/kg. Our findings showed for the first time that ALDH2 deficiency in Aldh2-KO mice increases their sensitivity to binge alcohol-induced oxidative and nitrative stress, enterocyte apoptosis, and nitration of gut tight junction (TJ) and adherent junction (AJ) proteins, leading to their degradation. These resulted in gut leakiness and endotoxemia in Aldh2-KO mice after exposure to a single dose of ethanol even at 3.5 g/kg, while no changes were observed in the corresponding WT mice. The elevated serum endotoxin (lipopolysaccharide, LPS) and bacterial translocation contributed to systemic inflammation, hepatocyte apoptosis, and subsequently acute liver injury through the gut-liver axis. Treatment with Daidzin, an ALDH2 inhibitor, exacerbated ethanol-induced cell permeability and reduced TJ/AJ proteins in T84 human colon cells. These changes were reversed by Alda-1, an ALDH2 activator. Furthermore, CRISPR/Cas9-mediated knockout of ALDH2 in T84 cells increased alcohol-mediated cell damage and paracellular permeability. All these findings demonstrate the critical role of ALDH2 in alcohol-induced epithelial barrier dysfunction and suggest that ALDH2 deficiency or gene mutation in humans is a risk factor for alcohol-mediated gut and liver injury, and that ALDH2 could be an important therapeutic target against alcohol-associated tissue or organ damage.