Active CNS delivery of oxycodone in healthy and endotoxemic pigs.

BACKGROUND: The primary objective of this study was to advance our understanding of active drug uptake at brain barriers in higher species than rodents, by examining oxycodone brain concentrations in pigs. METHODS: This was investigated by a microdialysis study in healthy and endotoxemic conditions to increase the understanding of inter-species translation of putative proton-coupled organic cation (H+/OC) antiporter-mediated central nervous system (CNS) drug delivery in health and pathology, and facilitate the extrapolation to humans for improved CNS drug treatment in patients. Additionally, we sought to evaluate the efficacy of lumbar cerebrospinal fluid (CSF) exposure readout as a proxy for brain unbound interstitial fluid (ISF) concentrations. By simultaneously monitoring unbound concentrations in blood, the frontal cortical area, the lateral ventricle (LV), and the lumbar intrathecal space in healthy and lipopolysaccharide (LPS)-induced inflammation states within the same animal, we achieved exceptional spatiotemporal resolution in mapping oxycodone transport across CNS barriers. RESULTS: Our findings provide novel evidence of higher unbound oxycodone concentrations in brain ISF compared to blood, yielding an unbound brain-to-plasma concentration ratio (Kp,uu,brain) of 2.5. This supports the hypothesis of the presence of the H+/OC antiporter system at the blood-brain barrier (BBB) in pigs. Despite significant physiological changes, reflected in pig Sequential Organ Failure Assessment, pSOFA scores, oxycodone blood concentrations and its active net uptake across the BBB remained nearly unchanged during three hours of i.v. infusion of 4 µg/kg/h LPS from Escherichia coli (O111:B4). Mean Kp,uu,LV values indicated active uptake also at the blood-CSF barrier in healthy and endotoxemic pigs. Lumbar CSF concentrations showed minimal inter-individual variability during the experiment, with a mean Kp,uu,lumbarCSF of 1.5. LPS challenge caused a slight decrease in Kp,uu,LV, while Kp,uu,lumbarCSF remained unaffected. CONCLUSIONS: This study enhances our understanding of oxycodone pharmacokinetics and CNS drug delivery in both healthy and inflamed conditions, providing crucial insights for translating these findings to clinical settings.

Recombinant thrombomodulin and recombinant antithrombin attenuate pulmonary endothelial glycocalyx degradation and neutrophil extracellular trap formation in ventilator-induced lung injury in the context of endotoxemia.

BACKGROUND: Vascular endothelial damage is involved in the development and exacerbation of ventilator-induced lung injury (VILI). Pulmonary endothelial glycocalyx and neutrophil extracellular traps (NETs) are endothelial protective and damaging factors, respectively; however, their dynamics in VILI and the effects of recombinant thrombomodulin and antithrombin on these dynamics remain unclear. We hypothesized that glycocalyx degradation and NETs are induced by VILI and suppressed by recombinant thrombomodulin, recombinant antithrombin, or their combination. METHODS: VILI was induced in male C57BL/6J mice by intraperitoneal lipopolysaccharide injection (20 mg/kg) and high tidal volume ventilation (20 mL/kg). In the intervention groups, recombinant thrombomodulin, recombinant antithrombin, or their combination was administered at the start of mechanical ventilation. Glycocalyx degradation was quantified by measuring serum syndecan-1, fluorescence-labeled lectin intensity, and glycocalyx-occupied area in the pulmonary vascular lumen. Double-stranded DNA in the bronchoalveolar fluid and fluorescent areas of citrullinated histone H3 and myeloperoxidase were quantified as NET formation. RESULTS: Serum syndecan-1 increased, and lectin fluorescence intensity decreased in VILI. Electron microscopy revealed decreases in glycocalyx-occupied areas within pulmonary microvessels in VILI. Double-stranded DNA levels in the bronchoalveolar lavage fluid and the fluorescent area of citrullinated histone H3 and myeloperoxidase in lung tissues increased in VILI. Recombinant thrombomodulin, recombinant antithrombin, and their combination reduced glycocalyx injury and NET marker levels. There was little difference in glycocalyx injury and NET makers between the intervention groups. CONCLUSION: VILI induced glycocalyx degradation and NET formation. Recombinant thrombomodulin and recombinant antithrombin attenuated glycocalyx degradation and NETs in our VILI model. The effect of their combination did not differ from that of either drug alone. Recombinant thrombomodulin and antithrombin have the potential to be therapeutic agents for biotrauma in VILI.

A flavonoid-rich extract of bergamot juice improves high-fat diet-induced intestinal permeability and associated hepatic damage in mice.

Consumption of high-fat diets (HFDs) is a contributing factor to obesity, insulin resistance and non-alcoholic fatty liver disease (NAFLD). Several studies suggested the protective role of bioactives present in Citrus fruits against the above mentioned chronic metabolic conditions. In this study, we evaluated if a flavonoid-rich extract of Citrus bergamia (bergamot) juice (BJe) could inhibit HFD-induced intestinal permeability and endotoxemia and, through this mechanism, mitigate the associated hepatic damage in C57BL/6J mice. After 12 weeks of the treatment, HFD consumption caused high body weight (BW) gain, hyperinsulinemia, hyperglycemia, and dyslipidemia, which were mitigated by BJe (50 mg per kg BW) supplementation. Furthermore, supplementation with BJe prevented HFD-induced liver alterations, including increased plasma alanine aminotransferase (ALT) activity, increased hepatic lipid deposition, high NAS, and fibrosis. Mice fed a HFD for 12 weeks showed (i) a decrease in small intestine tight junction protein levels (ZO-1, occludin, and claudin-1), (ii) increased intestinal permeability, and (iii) endotoxemia. All these adverse events were mitigated by BJe supplementation. Linking the capacity of BJe to prevent HFD-associated endotoxemia, supplementation with this extract decreased the HFD-induced overexpression of hepatic TLR-4, downstream signaling pathways (MyD88, NF-κB and MAPK), and the associated inflammation, evidenced by increased MCP-1, TNF-α, IL-6, iNOS, and F4/80 levels. Overall, we suggest that BJe could mitigate the harmful consequences of western style diet consumption on liver physiology by protecting the gastrointestinal tract from permeabilization and associated metabolic endotoxemia.

Shenfu injection ameliorates endotoxemia-associated endothelial dysfunction and organ injury via inhibiting PI3K/Akt-mediated glycolysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Microcirculatory dysfunction is one of the main characteristics of sepsis. Shenfu Injection (SFI) as a traditional Chinese medicine is widely applied in clinical severe conditions. Recent studies have shown that SFI has the ability to ameliorate sepsis-induced inflammation and to improve microcirculation perfusion. AIM OF THE STUDY: This study aims to investigate the underlying mechanism of SFI for ameliorating sepsis-associated endothelial dysfunction and organ injury. MATERIALS AND METHODS: Side-stream dark-field (SDF) imaging was used to monitor the sublingual microcirculation of septic patients treated with or without SFI. Septic mouse model was used to evaluate the effects of SFI in vivo. Metabolomics and transcriptomics were performed on endothelial cells to identify the underlying mechanism for SFI-related protective effect on endothelial cells. RESULTS: SFI effectively abolished the disturbance and loss of sublingual microcirculation in septic patients. Twenty septic shock patients with or without SFI administration were enrolled and the data showed that SFI significantly improved the levels of total vessel density (TVD), perfused vessel density (PVD), microvascular flow index (MFI), and the proportion of perfused vessels (PPV). The administration of SFI significantly decreased the elevated plasma levels of Angiopoietin-2 (Ang2) and Syndecan-1, which are biomarkers indicative of endothelial damage in sepsis patients. In the mouse septic model in vivo, SFI inhibited the upregulation of endothelial adhesion molecules and Ly6G + neutrophil infiltration while restored the expression of VE-Cadherin in the vasculature of the lung, kidney, and liver tissue. Additionally, SFI reduced the plasma levels of Ang2, Monocyte Chemoattractant Protein-1(MCP1), and Interleukin-6 (IL6), and alleviated liver and kidney injury in septic mice. Moreover, SFI significantly inhibited the inflammatory activation and increased permeability of endothelial cells induced by endotoxins in vitro. By performing metabolomics and transcriptomics, we identified the activation of PI3K/Akt-mediated glycolysis as the underlying mechanism for SFI-related protective effect on endothelial cells. CONCLUSIONS: Our findings revealed that SFI may improve microcirculation perfusion and endothelial function in sepsis via inhibiting PI3K/Akt-mediated glycolysis, providing theoretical evidence for the clinical application of SFI.

The possible cardioprotective effect of ghrelin during experimental endotoxemia in mice.

This study aimed to evaluate the cardioprotective effects of ghrelin in septic mice, focusing on its anti-inflammatory and antioxidant properties. Thirty-five male Swiss mice (8-12 weeks old, 23-33g) were randomly assigned to five groups (n = 7 each): (1) Normal, fed usual diets, (2) Sham, subjected to anesthesia and laparotomy, (3) Sepsis, subjected to cecal ligation and puncture, (4) Vehicle, given an equivalent volume of intraperitoneal saline injections immediately after cecal ligation and puncture, and (5) Ghrelin-treated, administered 80 µg/kg ghrelin intraperitoneal injections immediately following cecal ligation and puncture. Serum levels of tumor necrosis factor-alpha (TNF-α), macrophage migration inhibitory factor (MIF), toll-like receptor 4 (TLR4), and 8-epi-prostaglandin F2 alpha (8-epi-PGF2α) were measured. The extent of cardiac damage was also evaluated histologically. The mean serum levels of TNF-α, MIF, TLR4, and 8-epi-PGF2α levels were significantly higher in the sepsis and vehicle groups than in the normal and sham groups. The levels were significantly lower in the ghrelin-treated group than in the vehicle and sepsis groups. Histological analysis revealed normal myocardial architecture in the normal and sham groups, whereas the sepsis and vehicle groups had severe myocardial injury. The ghrelin-treated group displayed histological features similar to the sham group, indicating reduced myocardial damage. Ghrelin ameliorated sepsis-induced cardiotoxicity in mice by exhibiting strong anti-inflammatory and antioxidant effects. These findings suggest that ghrelin may be a promising therapeutic candidate for the prevention of sepsis-induced cardiotoxicity.

Citrulline and ADI-PEG20 reduce inflammation in a juvenile porcine model of acute endotoxemia.

Background: Arginine is a conditionally essential amino acid that is depleted in critically ill or surgical patients. In pediatric and adult patients, sepsis results in an arginine-deficient state, and the depletion of plasma arginine is associated with greater mortality. However, direct supplementation of arginine can result in the excessive production of nitric oxide (NO), which can contribute to the hypotension and macrovascular hypo-reactivity observed in septic shock. Pegylated arginine deiminase (ADI-PEG20, pegargiminase) reduces plasma arginine and generates citrulline that can be transported intracellularly to generate local arginine and NO, without resulting in hypotension, while maintaining microvascular patency. The objective of this study was to assess the efficacy of ADI-PEG20 with and without supplemental intravenous citrulline in mitigating hypovolemic shock, maintaining tissue levels of arginine, and reducing systemic inflammation in an endotoxemic pediatric pig model. Methods: Twenty 3-week-old crossbred piglets were implanted with jugular and carotid catheters as well as telemetry devices in the femoral artery to measure blood pressure, body temperature, heart rate, and respiration rate. The piglets were assigned to one of three treatments before undergoing a 5 h lipopolysaccharide (LPS) infusion protocol. Twenty-four hours before LPS infusion, control pigs (LPS; n=6) received saline, ADI-PEG20 pigs (n=7) received an injection of ADI-PEG20, and seven pigs (ADI-PEG20 + CIT pigs [n=7]) received ADI-PEG20 and 250 mg/kg citrulline intravenously. Pigs were monitored throughout LPS infusion and tissue was harvested at the end of the protocol. Results: Plasma arginine levels decreased and remained low in ADI-PEG20 + CIT and ADI-PEG20 pigs compared with LPS pigs but tissue arginine levels in the liver and kidney were similar across all treatments. Mean arterial pressure in all groups decreased from 90 mmHg to 60 mmHg within 1 h of LPS infusion but there were no significant differences between treatment groups. ADI-PEG20 and ADI-PEG20 + CIT pigs had less CD45+ infiltrate in the liver and lung and lower levels of pro-inflammatory cytokines in the plasma. Conclusion: ADI-PEG20 and citrulline supplementation failed to ameliorate the hypotension associated with acute endotoxic sepsis in pigs but reduced systemic and local inflammation in the lung and liver.

Protective effects of nordalbergin against LPS-induced endotoxemia through inhibiting MAPK/NF-κB signaling pathway, NLRP3 inflammasome activation, and ROS production.

OBJECTIVE: Nordalbergin is a coumarin extracted from Dalbergia sissoo DC. To date, the biological effects of nordalbergin have not been well investigated. To investigate the anti-inflammatory responses and the anti-oxidant abilities of nordalbergin using lipopolysaccharide (LPS)-activated macrophages and LPS-induced sepsis mouse model. MATERIALS AND METHODS: Production of nitrite oxide (NO), prostaglandin E2 (PGE2), pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1ß), reactive oxygen species (ROS), tissue damage and serum inflammatory markers, and the activation of the NLRP3 inflammasome were examined. RESULTS: Our results indicated that nordalbergin reduced the production of NO and pro-inflammatory cytokines in vitro and ex vivo. Nordalbergin also suppressed iNOS and cyclooxygenase-2 expressions, decreased NF-κB activity, and attenuated MAPKs signaling pathway activation by decreasing JNK and p38 phosphorylation by LPS-activated J774A.1 macrophages. Notably, nordalbergin diminished NLRP3 inflammasome activation via repressing the maturation of IL-1ß and caspase-1 and suppressing ROS production by LPS/ATP- and LPS/nigericin-activated J774A.1 macrophages. Furthermore, nordalbergin exhibited protective effects against the infiltration of inflammatory cells and also inhibited the levels of organ damage markers (AST, ALT, BUN) by LPS-challenged mice. CONCLUSION: Nordalbergin possesses anti-inflammatory effects in macrophage-mediated innate immune responses, alleviates ROS production, decreases NLRP3 activation, and exhibits protective effects against LPS-induced tissue damage in mice.

1,25-Dihydroxyvitamin D3 reduces early mortality post severe burn injury via alleviating endotoxemia, oxidative stress and inflammation.

Severe burn patients frequently suffer from 1,25-Dihydroxyvitamin D3 (1,25-[OH]2-D3) deficiency. In this study, we investigated the effect of 1,25-[OH]2-D3 on early mortality post severe burn and potential underlying mechanisms. Our results indicate that 1,25-[OH]2-D3 significantly reduced early mortality in mice post severe burn injury. A decrease in serum lipopolysaccharide levels and an increase in serum superoxide dismutase activity were found after administration of 1,25-[OH]2-D3. Furthermore, 1,25-[OH]2-D3 demonstrated protective effects on both intestinal and lung histology and ameliorated lung inflammation. Its anti-inflammatory effect was further confirmed in airway epithelial cells. In conclusion, our study provides evidence that 1,25-[OH]2-D3 has a significant impact on the reduction of early mortality post severe burn injury, possibly through its ability to alleviate endotoxemia, oxidative stress, and inflammation. Our findings highlight the potential of 1,25-[OH]2-D3 to protect the intestinal mucosal barrier in the early stage following major burn injury and opens up new avenues for clinical application of 1,25-[OH]2-D3 in burn patients.

Ghrelin attenuates the inflammatory response induced by experimental endotoxemia in mice.

OBJECTIVE: Aim: The aim of this research is to assess the anti-inflammatory effect of ghrelin in mice models of polymicrobial sepsis. PATIENTS AND METHODS: Materials and Methods: 35 male albino Swiss mice, ages 8-12 weeks, weighing 23-33g, were randomly separated into five groups n = 7; normal group was fed their usual diets until time of sampling, the sham group subjected to Anaesthesia and laparotomy, sepsis group subjected to cecal ligation and puncture, vehicle group was given an equivalent volume of intraperitoneal saline injections immediately after cecal ligation and puncture, and the ghrelin group was treated with 80 µg/kg of ghrelin intraperitoneal injections immediately following cecal ligation and puncture. Twenty hours after cecal ligation and puncture, mice were sacrificed; myocardial tissue and serum samples were collected. Serum IL-1ß, NF-κB, and TLR4 levels were measured, and inflammatory response's effects on cardiac tissue were evaluated. RESULTS: Results: The mean serum IL-1ß, NF-κB, and TLR4 levels were markedly elevated in the sepsis and vehicle groups than in the normal and sham groups. The mean serum levels of IL-1ß, NF-κB, and TLR4 were considerably lower in the ghrelin-treated group than in the vehicle and sepsis groups. Myocardium tissue of the normal and sham groups showed normal architecture. The sepsis and vehicle groups had a severe myocardial injury. The histological characteristics of ghrelin-treated mice differed slightly from those of the normal and sham groups. CONCLUSION: Conclusions: Our study concluded that ghrelin exerts anti-inflammatory effects in polymicrobial sepsis, as indicated by a considerable decrease in the IL-1ß, NF-κB and TLR4 serum levels.

A synthetic bioactive peptide of the C-terminal fragment of adhesion protein Fibulin7 attenuates the inflammatory functions of innate immune cells in LPS-induced systemic inflammation.

OBJECTIVE: Systemic inflammation is associated with improper localization of hyperactive neutrophils and monocytes in visceral organs. Previously, a C-terminal fragment of adhesion protein Fibulin7 (Fbln7-C) was shown to regulate innate immune functionality during inflammation. Recently, a shorter bioactive peptide of Fbln7-C, FC-10, via integrin binding was shown to reduce ocular angiogenesis. However, the role of FC-10 in regulating the neutrophils and monocyte functionality during systemic inflammatory conditions is unknown. The study sought to explore the role of FC-10 peptide on the functionality of innate immune cells during inflammation and endotoxemic mice. METHODS: Neutrophils and monocytes were isolated from healthy donors and septic patient clinical samples and Cell adhesion assay was performed using a UV spectrophotometer. Gene expression studies were performed using qPCR. Protein level expression was measured using ELISA and flow cytometry. ROS assay, and activation markers analysis in vitro, and in vivo were done using flow cytometry. TREATMENT: Cells were stimulated with LPS (100 ng/mL) and studied in the presence of peptides (10 µg, and 20 µg/mL) in vitro. In an in vivo study, mice were administered with LPS (36.8 mg/kg bw) and peptide (20 µg). RESULTS: This study demonstrates that human neutrophils and monocytes adhere to FC-10 via integrin ß1, inhibit spreading, ROS, surface activation markers (CD44, CD69), phosphorylated Src kinase, pro-inflammatory genes, and protein expression, compared to scrambled peptide in cells isolated from healthy donors and clinical sample. In line with the in vitro data, FC-10 (20 µg) administration significantly decreases innate cell infiltration at inflammatory sites, improves survival in endotoxemia animals & reduces the inflammatory properties of neutrophils and monocytes isolated from septic patients. CONCLUSION: FC-10 peptide can regulate neutrophils and monocyte functions and has potential to be used as an immunomodulatory therapeutic in inflammatory diseases.

Cardioprotective properties of OMT-28, a synthetic analog of omega-3 epoxyeicosanoids.

OMT-28 is a metabolically robust small molecule developed to mimic the structure and function of omega-3 epoxyeicosanoids. However, it remained unknown to what extent OMT-28 also shares the cardioprotective and anti-inflammatory properties of its natural counterparts. To address this question, we analyzed the ability of OMT-28 to ameliorate hypoxia/reoxygenation (HR)-injury and lipopolysaccharide (LPS)-induced endotoxemia in cultured cardiomyocytes. Moreover, we investigated the potential of OMT-28 to limit functional damage and inflammasome activation in isolated perfused mouse hearts subjected to ischemia/reperfusion (IR) injury. In the HR model, OMT-28 (1 µM) treatment largely preserved cell viability (about 75 versus 40% with the vehicle) and mitochondrial function as indicated by the maintenance of NAD+/NADH-, ADP/ATP-, and respiratory control ratios. Moreover, OMT-28 blocked the HR-induced production of mitochondrial reactive oxygen species. Pharmacological inhibition experiments suggested that Gαi, PI3K, PPARα, and Sirt1 are essential components of the OMT-28-mediated pro-survival pathway. Counteracting inflammatory injury of cardiomyocytes, OMT-28 (1 µM) reduced LPS-induced increases in TNFα protein (by about 85% versus vehicle) and NF-κB DNA binding (by about 70% versus vehicle). In the ex vivo model, OMT-28 improved post-IR myocardial function recovery to reach about 40% of the baseline value compared to less than 20% with the vehicle. Furthermore, OMT-28 (1 µM) limited IR-induced NLRP3 inflammasome activation similarly to a direct NLRP3 inhibitor (MCC950). Overall, this study demonstrates that OMT-28 possesses potent cardio-protective and anti-inflammatory properties supporting the hypothesis that extending the bioavailability of omega-3 epoxyeicosanoids may improve their prospects as therapeutic agents.

Central angiotensin-(1-7) attenuates hypoglycemia in sepsis-like conditions via reducing systemic and hepatic inflammation.

Sepsis is understood as the result of initiating systemic inflammation derived from an inadequate host response against pathogens. In its acute phase, sepsis is marked by an exacerbated reaction to infection, tissue damage, organ failure, and metabolic dysfunction. Among these, hypoglycemia, characterized by disorders of the gluconeogenesis pathway, is related to one of the leading causes of mortality in septic patients. Recent research has investigated the involvement of sympathetic efferent neuroimmune pathways during systemic inflammation. These pathways can be stimulated by several centrally administered drugs, including Angiotensin-(1-7) (Ang-(1-7)). Therefore, the present study aims to evaluate the effects of central treatment with Ang-(1-7) on hypoglycemia during endotoxemia. For this, male Wistar Hannover rats underwent stereotaxic surgery for intracerebroventricular (i.c.v.) administration of Ang-(1-7) and cannulation of the jugular vein for lipopolysaccharide (LPS) injection. Our results demonstrate that LPS was capable of inducing hypoglycemia and that prior central treatment with Ang-(1-7) attenuated this effect. Our data also show that Ang-(1-7) reduced plasma concentrations of TNF-α, IL-1ß, IL-6, and nitric oxide, in addition to the decrease and increase of hepatic IL-6 and IL-10 respectively, in animals subjected to systemic inflammation by LPS, resulting in the reduction of systemic and hepatic inflammation, thus attenuating the deleterious effects of LPS on phosphoenolpyruvate carboxykinase protein content. In summary, the data suggest that central treatment with Ang-(1-7) attenuates hypoglycemia induced by endotoxemia, probably through anti-inflammatory action, leading to reestablishing hepatic gluconeogenesis.

Mogroside Alleviates Diabetes Mellitus and Modulates Intestinal Microflora in Type 2 Diabetic Mice.

Mogroside, the main component of Siraitia grosvenorii (Swingle) C. Jeffrey (Cucurbitaceae) is a natural product with hypoglycemic and intestinal microbiota regulating properties. However, whether the alteration of intestinal microbiota is associated with the antidiabetic effect of mogroside remains poorly understood. This study investigated the mechanism underlying the hypoglycemic effect of mogroside in regulating intestinal flora and attenuating metabolic endotoxemia. Kunming mice with type 2 diabetes mellitus (T2DM) induced by high-fat diet and intraperitoneal injection of streptozotocin were randomly divided into model, pioglitazone (2.57 mg/kg) and mogroside (200, 100, and 50 mg/kg) groups. After 28 d of administration, molecular changes related to glucose metabolism and metabolic endotoxemia in mice were evaluated. The levels of insulin receptor substrate-1 (IRS-1), cluster of differentiation 14 (CD14) and toll-like receptor 4 (TLR4) mRNAs were measured, and the composition of intestinal microflora was determined by 16s ribosomal DNA (rDNA) sequencing. The results showed that mogroside treatment significantly improved hepatic glucose metabolism in T2DM mice. More importantly, mogroside treatment considerably reduced plasma endotoxin (inhibition rate 65.93%, high-dose group) and inflammatory factor levels, with a concomitant decrease in CD14 and TLR4 mRNA levels. Moreover, mogroside treatment reduced the relative abundance of Firmicutes and Proteobacteria (the inhibition rate of Proteobacteria was 85.17% in the low-dose group) and increased the relative abundance of Bacteroidetes (growth rate up to 40.57%, high-dose group) in the intestines of diabetic mice. This study reveals that mogroside can relieve T2DM, regulating intestinal flora and improving intestinal mucosal barrier, indicating that mogroside can be a potential therapeutic agent or intestinal microbiota regulator in the treatment of T2DM.

An orthosteric/allosteric bivalent peptide agonist comprising covalently linked protease-activated receptor-derived peptides mimics in vitro and in vivo activities of activated protein C.

BACKGROUND: Activated protein C (APC) has anticoagulant and cytoprotective cell-signaling activities, which often require protease-activated receptor (PAR) 1 and PAR3 and PAR cleavages at noncanonical sites (R46-N47 and R41-G42, respectively). Some PAR1-derived (P1) peptides and PAR3-derived (P3) peptides, eg, P1-47-66 and P3-42-65, mimic APC's cell signaling. In anti-inflammatory assays, these 2 peptides at low concentrations synergistically attenuate cellular inflammation. OBJECTIVES: To determine whether a P1 peptide covalently linked to a P3 peptide mimics APC's anti-inflammatory and endothelial barrier stabilization activities. METHODS: Anti-inflammatory assays employed stimulated THP-1 cells and caspase-1 measurements. Cultured human EA.hy926 or murine aortic endothelial cells (ECs) exposed to thrombin were monitored for transendothelial electrical resistance. Bivalent covalently linked P1:P3 peptides were studied for APC-like activities. RESULTS: In anti-inflammatory assays, P1-47-55 was as active as P1-47-66 and some P3 peptides (eg, P3-44-54 and P3-51-65) were as active as P3-42-65. The bivalent P1:P3 peptide comprising P1-47-55-(Gly[10 residues])-P3-51-65 (designated "G10 peptide") was more potently anti-inflammatory than the P1 or P3 peptide alone. In transendothelial electrical resistance studies of thrombin-challenged ECs, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was more potent than the P1-47-55 peptide. In murine EC studies, the murine PAR-sequence-derived G10 peptide mimicked murine APC's activity. Anti-PAR1 and anti-PAR3 antibodies, but not anti-endothelial protein C receptor antibodies, abated G10's cytoprotection, showing that G10's actions involve PAR1:PAR3. G10 significantly increased survival in murine endotoxemia. CONCLUSION: The PAR-sequence-derived G10 peptide is a bivalent agonist that mimics APC's cytoprotective, anti-inflammatory, and endothelial barrier-stabilizing actions and APC's protection against endotoxemic mortality.

Propofol and Dexmedetomidine Ameliorate Endotoxemia-Associated Encephalopathy via Inhibiting Ferroptosis.

Background: Sepsis is recognized as a multiorgan and systemic damage caused by dysregulated host response to infection. Its acute systemic inflammatory response highly resembles that of lipopolysaccharide (LPS)-induced endotoxemia. Propofol and dexmedetomidine are two commonly used sedatives for mechanical ventilation in critically ill patients and have been reported to alleviate cognitive impairment in many diseases. In this study, we aimed to explore and compare the effects of propofol and dexmedetomidine on the encephalopathy induced by endotoxemia and to investigate whether ferroptosis is involved, finally providing experimental evidence for multi-drug combination in septic sedation. Methods: A total of 218 C57BL/6J male mice (20-25 g, 6-8 weeks) were used. Morris water maze (MWM) tests were performed to evaluate whether propofol and dexmedetomidine attenuated LPS-induced cognitive deficits. Brain injury was evaluated using Nissl and Fluoro-Jade C (FJC) staining. Neuroinflammation was assessed by dihydroethidium (DHE) and DCFH-DA staining and by measuring the levels of three cytokines. The number of Iba1+ and GFAP+ cells was used to detect the activation of microglia and astrocytes. To explore the involvement of ferroptosis, the levels of ptgs2 and chac1; the content of iron, malondialdehyde (MDA), and glutathione (GSH); and the expression of ferroptosis-related proteins were investigated. Conclusion: The single use of propofol and dexmedetomidine mitigated LPS-induced cognitive impairment, while the combination showed poor performance. In alleviating endotoxemic neural loss and degeneration, the united sedative group exhibited the most potent capability. Both propofol and dexmedetomidine inhibited neuroinflammation, while propofol's effect was slightly weaker. All sedative groups reduced the neural apoptosis, inhibited the activation of microglia and astrocytes, and relieved neurologic ferroptosis. The combined group was most prominent in combating genetic and biochemical alterations of ferroptosis. Fpn1 may be at the core of endotoxemia-related ferroptosis activation.

Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

OBJECTIVE: To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. METHODS: Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 ß, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 ß were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. RESULTS: SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 ß, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). CONCLUSIONS: SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

Thromboelastographic evaluation of the effectiveness of choline or CDP-choline treatment on endotoxin-induced hemostatic alterations in dogs.

Sepsis/endotoxemia associates with coagulation abnormalities. We showed previously that exogenous choline treatment reversed the changes in platelet count and function as well as prevented disseminated intravascular coagulation (DIC) in endotoxemic dogs. The aim of this follow-up study was to evaluate the effect of treatment with choline or cytidine-5'-diphosphocholine (CDP-choline), a choline donor, on endotoxin-induced hemostatic alterations using thromboelastography (TEG). Dogs were randomized to six groups and received intravenously (iv) saline, choline (20 mg/kg) or CDP-choline (70 mg/kg) in the control groups, whereas endotoxin (0.1 mg/kg, iv) was used alone or in combination with choline or CDP-choline at the same doses in the treatment groups. TEG variables including R- and K-time (clot formation), maximum amplitude (MA) and α-angle (clot stability), G value (clot elasticity), and EPL, A, and LY30 (fibrinolysis), as well as overall assessment of coagulation (coagulation index - CI), were measured before and at 0.5-48 h after the treatments. TEG parameters did not change significantly in the control groups, except for CI parameter after choline administration. Endotoxemia resulted in increased R-time and A value (P < 0.05), decreased K-time (P < 0.05), α-angle (P < 0.001) and CI values (P < 0.01) at different time points. Treatment with either choline or CDP-choline attenuated or prevented completely the alterations in TEG parameters in endotoxemic dogs with CDP-choline being more effective. These results confirm and extend the effectiveness of choline or CDP-choline in endotoxemia by further demonstrating their efficacy in attenuating or preventing the altered viscoelastic properties of blood clot measured by TEG.

A green tea extract confection decreases circulating endotoxin and fasting glucose by improving gut barrier function but without affecting systemic inflammation: A double-blind, placebo-controlled randomized trial in healthy adults and adults with metabolic syndrome.

Anti-inflammatory activities of catechin-rich green tea extract (GTE) in obese rodents protect against metabolic endotoxemia by decreasing intestinal permeability and absorption of gut-derived endotoxin. However, translation to human health has not been established. We hypothesized that GTE would reduce endotoxemia by decreasing gut permeability and intestinal and systemic inflammation in persons with metabolic syndrome (MetS) compared with healthy persons. A randomized, double-blind, placebo-controlled, crossover trial in healthy adults (n = 19, 34 ± 2 years) and adults with MetS (n = 21, 40 ± 3 years) examined 4-week administration of a decaffeinated GTE confection (890 mg/d total catechins) on serum endotoxin, intestinal permeability, gut and systemic inflammation, and cardiometabolic parameters. Compared with the placebo, the GTE confection decreased serum endotoxin (P = .023) in both healthy persons and those with MetS, while increasing concentrations of circulating catechins (P < .0001) and γ-valerolactones (P = .0001). Fecal calprotectin (P = .029) and myeloperoxidase (P = .048) concentrations were decreased by GTE regardless of health status. Following the ingestion of gut permeability probes, urinary lactose/mannitol (P = .043) but not sucralose/erythritol (P > .05) was decreased by GTE regardless of health status. No between-treatment differences (P > .05) were observed for plasma aminotransferases, blood pressure, plasma lipids, or body mass nor were plasma tumor necrosis factor-α, interleukin-6, or the ratio of lipopolysaccharide-binding protein/soluble cluster of differentiation-14 affected. However, fasting glucose in both study groups was decreased (P = .029) by the GTE confection compared with within-treatment arm baseline concentrations. These findings demonstrate that catechin-rich GTE is effective to decrease circulating endotoxin and improve glycemic control in healthy adults and those with MetS, likely by reducing gut inflammation and small intestinal permeability but without affecting systemic inflammation.

Potential Therapeutic Strategies and Drugs That Target Vascular Permeability in Severe Infectious Diseases.

Severe infection pathogenicity is induced by processes such as pathogen exposure, immune cell activation, inflammatory cytokine production, and vascular hyperpermeability. Highly effective drugs, such as antipathogenic agents, steroids, and antibodies that suppress cytokine function, have been developed to treat the first three processes. However, these drugs cannot completely suppress severe infectious diseases, such as coronavirus disease 2019 (COVID-19). Therefore, developing novel drugs that inhibit vascular hyperpermeability is crucial. This review summarizes the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced vascular hyperpermeability and identifies inhibitors that increase endothelial cell (EC) junction-related proteins and determines their efficacy in COVID-19 and endotoxemia models. Analyzing the effects of SARS-CoV-2 on vascular permeability revealed that SARS-CoV-2 suppresses Claudin-5 (CLDN5) expression, which is responsible for adhesion between ECs, thereby increasing vascular permeability. Inhibiting CLDN5 function in mice induced vascular hyperpermeability and pulmonary edema. In contrast, Enhancing CLDN5 expression suppressed SARS-CoV-2-induced endothelial hyperpermeability, suggesting that SARS-CoV-2-induced vascular hyperpermeability contributes to pathological progression, which can be suppressed by upregulating EC junction proteins. Based on these results, we focused on Roundabout4 (Robo4), another EC-specific protein that stabilizes EC junctions. EC-specific Robo4 overexpression suppressed vascular hyperpermeability and mortality in lipopolysaccharide-treated mice. An ALK1 inhibitor (a molecule that increases Robo4 expression), suppressed vascular hyperpermeability and mortality in lipopolysaccharide- and SARS-CoV-2-treated mice. These results indicate that Robo4 expression-increasing drugs suppress vascular permeability and pathological phenotype in COVID-19 and endotoxemia models.

The protective effects of naringenin, a citrus flavonoid, non-complexed or complexed with hydroxypropyl-ß-cyclodextrin against multiorgan damage caused by neonatal endotoxemia.

BACKGROUND: Endotoxemia is a severe and dangerous clinical syndrome that results in elevated morbidity, especially in intensive care units. Neonates are particularly susceptible to endotoxemia due to their immature immune systems. There are few effective treatments for neonatal endotoxemia. One group of compounds with potential in the treatment of neonatal inflammatory diseases such as endotoxemia is the flavonoids, mainly due to their antioxidant and anti-inflammatory properties. Among these, naringenin (NGN) is a citrus flavonoid which has already been reported to have anti-inflammatory, antioxidant, anti-nociceptive and anti-cancer effects. Unfortunately, its clinical application is limited by its low solubility and bioavailability. However, cyclodextrins (CDs) have been widely used to improve the solubility of nonpolar drugs and enhance the bioavailability of these natural products. OBJECTIVE: We, therefore, aimed to investigate the effects of NGN non-complexed and complexed with hydroxypropyl-ß-cyclodextrin (HPßCD) on neonatal endotoxemia injuries in a rodent model and describe the probable molecular mechanisms involved in NGN activities. METHOD: We used exposure to a bacterial lipopolysaccharide (LPS) to induce neonatal endotoxemia in the mice. RESULTS: It was found that NGN (100 mg/kg i.p.) exposure during the neonatal period reduced leukocyte migration and decreased pro-inflammatory cytokine (TNF-α, IL-1ß and IL-6) levels in the lungs, heart, kidneys or cerebral cortex. In addition, NGN upregulated IL-10 production in the lungs and kidneys of neonate mice. The administration of NGN also enhanced antioxidant enzyme catalase and SOD activity, reduced lipid peroxidation and protein carbonylation and increased the reduced sulfhydryl groups in an organ-dependent manner, attenuating the oxidative damage caused by LPS exposure. NGN decreased ERK1/2, p38MAPK and COX-2 activation in the lungs of neonate mice. Moreover, NGN complexed with HPßCD was able to increase the animal survival rate. CONCLUSION: NGN attenuated inflammatory and oxidative damage in the lungs, heart and kidneys caused by neonatal endotoxemia through the MAPK signaling pathways regulation. Our results show that NGN has beneficial effects against neonatal endotoxemia and could be useful in the treatment of neonatal inflammatory injuries.