Effects of CDDO-EA in sepsis-induced acute lung injury: mouse model of endotoxaemia.

OBJECTIVE: Aim: The aim of this research is to clarify the potential effect of CDDO-EA against experimentally sepsis induced lung injury in mice. PATIENTS AND METHODS: Materials and Methods: Mice have divided into four groups: Sham group CLP group, Vehicle-treatment group, CDDO-EA-treated group: mice in this group received CDDO-EA 2mg/kg intraperitoneally, 1hr before CLP, then the animals were sacrificed 24hr after CLP. After exsAngpuinations, tissue samples of lung were collected, followed by markers measurement including, TNF-α, IL-1ß, VEGF, MPO, caspase11, Angp-1and Angp-2 by ELISA, gene expression of TIE2 and VE-cadherin by qRT-PCR, in addition to histopathological study. RESULTS: Results: A significant elevation (p<0.05) in TNF-α, IL-1ß, MPO, ANGP-2, VEGF, CASPASE 11 in CLP and vehicle groups when compared with sham group. CDDO-EA group showed significantly lower levels p<0.05, level of ANGP-1 was significantly lower p<0.05 in the CLP and vehicle groups as compared with the sham group. Quantitative real-time PCR demonstrated a significant decrement in mRNA expression of TIE2&ve-cadherin genes p<0.05 in sepsis & vehicle. CONCLUSION: Conclusions: CDDO-EA has lung protective effects due to its anti-inflammatory and antiAngpiogenic activity, additionally, CDDO-EA showes a lung protective effect as they affect tissue mRNA expression of TIE2 and cadherin gene. Furthermore, CDDO-EA attenuate the histopathological changes that occur during polymicrobial sepsis thereby lung protection effect.

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.

A novel blood-based bioassay to monitor adiponectin signaling.

The diverse beneficial effects of adiponectin-receptor signaling, including its impact on the regulation of inflammatory processes in vivo, have resulted in development of adiponectin receptor agonists as a treatment for metabolic disorders. However, there are no established non-invasive bioassays for detection of adiponectin target engagement in humans or animal models. Here, we designed an assay using small amounts of blood to assess adiponectin action. Specifically, we tested effects of the small 10-amino acid peptide adiponectin receptor agonist, ALY688, in a sublethal LPS endotoxemia model in mice. LPS-induced pro-inflammatory cytokine levels in serum were significantly reduced in mice treated with ALY688, assessed via multiplex ELISA in flow cytometry. Furthermore, ALY688 alone significantly induced TGF-ß release in serum 1 h after treatment and was elevated for up to 24 h. Additionally, using a flow-cytometry panel for detection of changes in circulating immune cell phenotypes, we observed a significant increase in absolute T cell counts in mice after ALY688 treatment. To assess changes in intracellular signaling effectors downstream of adiponectin, phospho-flow cytometry was conducted. There was a significant increase in phosphorylation of AMPK and p38-MAPK in mice after ALY688 treatment. We then used human donor immune cells (PBMCs) treated with ALY688 ex vivo and observed elevation of AMPK and p38-MAPK phosphorylation from baseline in response to ALY688. Together, these results indicate we can detect adiponectin action on immune cells in vivo by assessing adiponectin signaling pathway for AMPK and p38-MAPK, as well as pro-inflammatory cytokine levels. This new approach provides a blood-based bioassay for screening adiponectin action.

Lung single-cell RNA profiling reveals response of pulmonary capillary to sepsis-induced acute lung injury.

Background: Sepsis-induced acute lung injury (ALI) poses a significant threat to human health. Endothelial cells, especially pulmonary capillaries, are the primary barriers against sepsis in the lungs. Therefore, investigating endothelial cell function is essential to understand the pathophysiological processes of sepsis-induced ALI. Methods: We downloaded single-cell RNA-seq expression data from GEO with accession number GSE207651. The mice underwent cecal ligation and puncture (CLP) surgery, and lung tissue samples were collected at 0, 24, and 48 h. The cells were annotated using the CellMarker database and FindAllMarkers functions. GO enrichment analyses were performed using the Metascape software. Gene set enrichment Analysis (GSEA) and variation Analysis (GSVA) were performed to identify differential signaling pathways. Differential expression genes were collected with the "FindMarkers" function. The R package AUCell was used to score individual cells for pathway activities. The Cellchat package was used to explore intracellular communication. Results: Granulocytes increased significantly as the duration of endotoxemia increased. However, the number of T cells, NK cells, and B cells declined. Pulmonary capillary cells were grouped into three sub-clusters. Capillary-3 cells were enriched in the sham group, but declined sharply in the CLP.24 group. Capillary-1 cells peaked in the CLP.24 group, while Capillary-2 cells were enriched in the CLP.48 group. Furthermore, we found that Cd74+ Capillary-3 cells mainly participated in immune interactions. Plat+ Capillary-1 and Clec1a+ Capillary-2 are involved in various physiological processes. Regarding cell-cell interactions, Plat+ Capillary-1 plays the most critical role in granulocyte adherence to capillaries during ALI. Cd74+ Capillary cells expressing high levels of major histocompatibility complex (MHC) and mainly interacted with Cd8a+ T cells in the sham group. Conclusion: Plat+ capillaries are involved in the innate immune response through their interaction with neutrophils via ICAM-1 adhesion during endotoxemia, while Cd74+ capillaries epxressed high level of MHC proteins play a role in adaptive immune response through their interaction with T cells. However, it remains unclear whether the function of Cd74+ capillaries leans towards immunity or tolerance, and further studies are needed to confirm this.

Self-Cascade Redox Modulator Trilogically Renovates Intestinal Microenvironment for Mitigating Endotoxemia.

Endotoxemia is a life-threatening multiple organ failure disease caused by bacterial endotoxin infection. Unfortunately, current single-target therapy strategies have failed to prevent the progression of endotoxemia. Here, we reported that alanine fullerene redox modulator (AFRM) remodeled the intestinal microenvironment for multiple targets endotoxemia mitigation by suppressing inflammatory macrophages, inhibiting macrophage pyroptosis, and repairing epithelial cell barrier integrity. Specifically, AFRM exhibited broad-spectrum and self-cascade redox regulation properties with superoxide dismutase (SOD)-like enzyme, peroxidase (POD)-like enzyme activity, and hydroxyl radical (•OH) scavenging ability. Guided by proteomics, we demonstrated that AFRM regulated macrophage redox homeostasis and down-regulated LPS/TLR4/NF-κB and MAPK/ERK signaling pathways to suppress inflammatory hyperactivation. Of note, AFRM could attenuate inflammation-induced macrophage pyroptosis via inhibiting the activation of gasdermin D (GSDMD). In addition, our results revealed that AFRM could restore extracellular matrix and cell-tight junction proteins and protect the epithelial cell barrier integrity by regulating extracellular redox homeostasis. Consequently, AFRM inhibited systemic inflammation and potentiated intestinal epithelial barrier damage repair during endotoxemia in mice. Together, our work suggested that fullerene based self-cascade redox modulator has the potential in the management of endotoxemia through synergistically remodeling the inflammation and epithelial barriers in the intestinal microenvironment.

Cholinergic signaling via the α7 nicotinic acetylcholine receptor regulates the migration of monocyte-derived macrophages during acute inflammation.

BACKGROUND: The involvement of the autonomic nervous system in the regulation of inflammation is an emerging concept with significant potential for clinical applications. Recent studies demonstrate that stimulating the vagus nerve activates the cholinergic anti-inflammatory pathway that inhibits pro-inflammatory cytokines and controls inflammation. The α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages plays a key role in mediating cholinergic anti-inflammatory effects through a downstream intracellular mechanism involving inhibition of NF-κB signaling, which results in suppression of pro-inflammatory cytokine production. However, the role of the α7nAChR in the regulation of other aspects of the immune response, including the recruitment of monocytes/macrophages to the site of inflammation remained poorly understood. RESULTS: We observed an increased mortality in α7nAChR-deficient mice (compared with wild-type controls) in mice with endotoxemia, which was paralleled with a significant reduction in the number of monocyte-derived macrophages in the lungs. Corroborating these results, fluorescently labeled α7nAChR-deficient monocytes adoptively transferred to WT mice showed significantly diminished recruitment to the inflamed tissue. α7nAChR deficiency did not affect monocyte 2D transmigration across an endothelial monolayer, but it significantly decreased the migration of macrophages in a 3D fibrin matrix. In vitro analysis of major adhesive receptors (L-selectin, ß1 and ß2 integrins) and chemokine receptors (CCR2 and CCR5) revealed reduced expression of integrin αM and αX on α7nAChR-deficient macrophages. Decreased expression of αMß2 was confirmed on fluorescently labeled, adoptively transferred α7nAChR-deficient macrophages in the lungs of endotoxemic mice, indicating a potential mechanism for α7nAChR-mediated migration. CONCLUSIONS: We demonstrate a novel role for the α7nAChR in mediating macrophage recruitment to inflamed tissue, which indicates an important new aspect of the cholinergic regulation of immune responses and inflammation.

MALAT1 DEREPRESSES MIR-433-3P-MEDIATED RPTOR SUPPRESSION TO IMPAIR AUTOPHAGY AND DRIVE PYROPTOSIS IN ENDOTOXEMIA.

ABSTRACT: Objective: Autophagy elevation in endotoxemia plays a protective role by negatively regulating the pyroptosis of vascular endothelial cells, but the molecular mechanisms are still poorly understood. The present study aimed to identify the mechanism underlying autophagy and pyroptosis in endotoxemia. Methods: Bioinformatics analysis and whole-gene transcriptome sequencing prediction were used to identify the endotoxemia-related lncRNA-miRNA-mRNA axis of interest. Human umbilical vein endothelial cells (HUVECs) were activated by lipopolysaccharide (LPS) to mimic the inflammatory environment encountered in endotoxemia. Autophagy and pyroptosis of LPS-treated HUVECs were assessed in response to the knockdown of MALAT1 (metastasis-associated lung adenocarcinoma transcript 1)/miR-433-3p (miRNA-433-3p)/RPTOR (regulatory-associated protein of mTOR). The binding affinity of MALAT1, miR-433-3p, and RPTOR was detected by RNA pull-down and luciferase activity assays. The endothelial cell-specific RPTOR knockout mice were developed and rendered septic using LPS induction to verify the role of RPTOR in autophagy, pyroptosis, and inflammatory response in vivo . Results: The in vitro experiments indicated that LPS could stimulate HUVECs to highly express RPTOR, and its knockdown enhanced cellular autophagy and restricted pyroptosis to curb inflammatory responses. Mechanically, MALAT1 is competitively bound to miR-433-3p to release RPTOR expression, thereby promoting pyroptosis and aggravating endotoxemia. In vivo experiments further confirmed that the knockdown of RPTOR activated autophagy and curtailed pyroptosis in septic mice. Conclusion: MALAT1 is highly expressed in endotoxemia. MALAT1 promotes RPTOR expression by competitively absorbing miR-433-3p, inhibits LPS-activated HUVEC cell autophagy, promotes cell death, enhances LPS-induced inflammatory activation of vascular endothelial cells, and ultimately promotes the progression of endotoxemia.

MIR-155 PROMOTES ACUTE ORGAN INJURY IN LPS-INDUCED ENDOTOXEMIC MICE BY ENHANCING CCL-2 EXPRESSION IN MACROPHAGES.

ABSTRACT: Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Macrophages play important roles in the inflammatory process of sepsis by secreting chemokines. Chemokine (CC-motif) ligand 2 (CCL-2) is one of the main proinflammatory chemokines secreted by macrophages that plays a critical role in the recruitment of more monocytes and macrophages to the sites of injury in sepsis, but the mechanisms that regulate CCL-2 expression in macrophages during sepsis are still unknown. In the present study, by using the LPS-induced endotoxemia model, we found that LPS induced the expression of microRNA (miR)-155 and CCL-2 in endotoxemic mice and RAW264.7 cells. MiR-155 mimics or miR-155 inhibitor treatment experiment suggested that miR-155 was sufficient to increase LPS-induced CCL-2 expression in macrophages, but miR-155 was not the only factor promoting CCL-2 expression. We further demonstrated that miR-155-induced increase of CCL-2 promoted chemotaxis of additional macrophages, which subsequently enhanced lung injury in endotoxemic mice. Serum/glucocorticoid regulated kinase family member 3 (SGK3), a potential target of miR-155, was identified by RNA sequencing and predicted by TargetScan and miRDB. We further confirmed miR-155 regulated SGK3 to increase LPS-induced CCL-2 by using miR-155 mimics and SGK3 overexpression. Thus, our study demonstrates that miR-155 targets SGK3 to increase LPS-induced CCL-2 expression in macrophages, which promotes macrophage chemotaxis and enhances organs injury during endotoxemia. Our study contributed to a better understanding of the mechanisms underlying the inflammatory response during sepsis.

Role of brain-derived neurotrophic factor in endotoxaemia-induced acute lung injury.

Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), which is a pulmonary manifestation of a systemic reactive inflammatory syndrome, is a serious disease with high mortality, and sepsis is an important risk factor in the development of ALI. Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor family. It plays an essential role in the regulation of the modification of synaptic efficacy and brain metabolic activity and enhances neuronal survival. However, the role and underlying mechanism of BDNF in sepsis-induced ALI remain unclear. Here, we sought to observe the expression of BDNF in the lung tissues of mice. C57BL/6J mice were divided randomly into two groups: saline (n = 4) and lipopolysaccharide (LPS) (n = 4). We found that BDNF expression was elevated in the lung tissues of septic mice. Furthermore, we found that BDNF colocalized with aquaporin 5, a marker for type I alveolar epithelial cells, by immunofluorescence staining. In addition, we also found that tropomyosin-related kinase B, the specific receptor of BDNF, colocalized with surfactant protein C, a marker for type II alveolar epithelial cells, by immunofluorescence staining. Finally, the present study indicated that BDNF may alleviate excessive LPS-induced autophagy in alveolar epithelial cells. Overall, we hypothesize that BDNF expression increases in the lung tissues of septic mice as a compensatory mechanism to ameliorate sepsis-induced ALI by inhibiting excessive alveolar epithelial cell autophagy.

Macrophage-specific deletion of MIC26 (APOO) mitigates advanced atherosclerosis by increasing efferocytosis.

BACKGROUND AND AIMS: Recent studies have suggested that MIC26 (apolipoprotein O, APOO), a novel mitochondrial inner membrane protein, is involved in inflammation. Thus, the role of macrophage MIC26 in acute inflammation and chronic inflammatory disease atherosclerosis was investigated. METHODS: Macrophage-specific MIC26 knockout mice (MIC26LysM) were generated by crossing Apooflox/flox and LysMcre+/- mice. An endotoxemia mouse model was generated to explore the effects of macrophage MIC26 deficiency on acute inflammation, while an atherosclerosis mouse model was constructed by crossing MIC26LysM mice with Apoe-/- mice and challenged with a Western diet. Atherosclerotic plaques, primary macrophage function, and mitochondrial structure and function were analyzed. RESULTS: MIC26 knockout did not affect the median survival time and post-injection serum interleukin 1ß concentrations in mice with endotoxemia. Mice with MIC26 deficiency in an Apoe-/- background had smaller atherosclerotic lesions and necrotic core than the control group. In vitro studies found that the loss of MIC26 did not affect macrophage polarization, apoptosis, or lipid handling capacity, but increased efferocytosis (the ability to clear apoptotic cells). An in situ efferocytosis assay of plaques also showed that the ratio of macrophage-associated apoptotic cells to free apoptotic cells was higher in the MIC26-deficient group than in the control group, indicating increased efferocytosis. In addition, an in vivo thymus efferocytosis assay indicated that MIC26 deletion promoted efferocytosis. Mechanistically, the loss of MIC26 resulted in an abnormal mitochondrial inner membrane structure, increased mitochondrial fission, and decreased mitochondrial membrane potential. Loss of MIC26 reduced mitochondria optic atrophy type 1 (OPA1) protein, and OPA1 silencing in macrophages promoted efferocytosis. Overexpression of OPA1 abolished the increase in efferocytosis produced by MIC26 deficiency. CONCLUSIONS: Macrophage MIC26 deletion alleviated advanced atherosclerosis and necrotic core expansion by promoting efferocytosis. This mechanism may be related to the increased mitochondrial fission caused by reduced mitochondrial OPA1.

Raman Spectroscopy Profiling of Splenic T-Cells in Sepsis and Endotoxemia in Mice.

Sepsis is a life-threatening condition that results from an overwhelming and disproportionate host response to an infection. Currently, the quality and extent of the immune response are evaluated based on clinical symptoms and the concentration of inflammatory biomarkers released or expressed by the immune cells. However, the host response toward sepsis is heterogeneous, and the roles of the individual immune cell types have not been fully conceptualized. During sepsis, the spleen plays a vital role in pathogen clearance, such as bacteria by an antibody response, macrophage bactericidal capacity, and bacterial endotoxin detoxification. This study uses Raman spectroscopy to understand the splenic T-lymphocyte compartment profile changes during bona fide bacterial sepsis versus hyperinflammatory endotoxemia. The Raman spectral analysis showed marked changes in splenocytes of mice subjected to septic peritonitis principally in the DNA region, with minor changes in the amino acids and lipoprotein areas, indicating significant transcriptomic activity during sepsis. Furthermore, splenocytes from mice exposed to endotoxic shock by injection of a high dose of lipopolysaccharide showed significant changes in the protein and lipid profiles, albeit with interindividual variations in inflammation severity. In summary, this study provided experimental evidence for the applicability and informative value of Raman spectroscopy for profiling the immune response in a complex, systemic infection scenario. Importantly, changes within the acute phase of inflammation onset (24 h) were reliably detected, lending support to the concept of early treatment and severity control by extracorporeal Raman profiling of immunocyte signatures.

Palmitic Acid Modulates Microglial Cell Response to Metabolic Endotoxemia in an In Vitro Study.

Metabolic endotoxemia (ME) is characterized by a 2-3-fold increase in blood endotoxin levels and low-grade systemic inflammation without apparent infection. ME is usually accompanied by metabolic syndrome, characterized by central obesity and hyperlipidemia. According to numerous studies, ME may lead to functional brain disorders, including cognitive decline, depression, and dementia. In the current in vitro study, we aimed to determine the direct and indirect impact of endotoxin (LPS) and palmitic acid (PA), representing saturated fatty acids, on the inflammatory and oxidative stress response in the human microglial HMC3 cells unstimulated and stimulated with IFNγ. The study's results revealed that direct HMC3 cell exposition to endotoxin and PA increased inflammatory response measured as levels of IL-6 and MCP-1 released into the medium and PGE2 levels in cell lysates. Moreover, direct HMC3 cell treatment with PA and LPS induced oxidative stress, i.e., ROS and COX-2 production and lipid peroxidation. On the contrary, an indirect effect of LPS and PA on microglial cells, assessed as the impact of macrophage metabolites, was much lower regarding the inflammatory response, although still associated with oxidative stress. Interestingly, IFNγ had a protective effect on microglial cells, reducing the production of pro-inflammatory mediators and oxidative stress in HMC3 cells treated directly and indirectly with LPS and PA.

Reduced Tie2 in Microvascular Endothelial Cells Is Associated with Organ-Specific Adhesion Molecule Expression in Murine Health and Endotoxemia.

Endothelial cells (ECs) in the microvasculature in organs are active participants in the pathophysiology of sepsis. Tyrosine protein kinase receptor Tie2 (Tek; Tunica interna Endothelial cell Kinase) is thought to play a role in their inflammatory response, yet data are inconclusive. We investigated acute endotoxemia-induced changes in the expression of Tie2 and inflammation-associated endothelial adhesion molecules E-selectin and VCAM-1 (vascular cell adhesion molecule-1) in kidneys and lungs in inducible, EC-specific Tie2 knockout mice. The extent of Tie2 knockout in healthy mice differed between microvascular beds, with low to absent expression in arterioles in kidneys and in capillaries in lungs. In kidneys, Tie2 mRNA dropped more than 70% upon challenge with lipopolysaccharide (LPS) in both genotypes, with no change in protein. In renal arterioles, tamoxifen-induced Tie2 knockout was associated with higher VCAM-1 protein expression in healthy conditions. This did not increase further upon challenge of mice with LPS, in contrast to the increased expression occurring in control mice. Also, in lungs, Tie2 mRNA levels dropped within 4 h after LPS challenge in both genotypes, while Tie2 protein levels did not change. In alveolar capillaries, where tamoxifen-induced Tie2 knockout did not affect the basal expression of either adhesion molecule, a 4-fold higher E-selectin protein expression was observed after exposure to LPS compared to controls. The here-revealed heterogeneous effects of absence of Tie2 in ECs in kidney and lung microvasculature in health and in response to acute inflammatory activation calls for further in vivo investigations into the role of Tie2 in EC behavior.

The Role of Palmitic Acid in the Co-Toxicity of Bacterial Metabolites to Endothelial Cells.

Introduction: Metabolic endotoxemia most often results from obesity and is accompanied by an increase in the permeability of the intestinal epithelial barrier, allowing co-absorption of bacterial metabolites and diet-derived fatty acids into the bloodstream. A high-fat diet (HFD) leading to obesity is a significant extrinsic factor in developing vascular atherosclerosis. In this study, we evaluated the effects of palmitic acid (PA) as a representative of long-chain saturated fatty acids (LCSFA) commonly present in HFDs, along with endotoxin (LPS; lipopolysaccharide) and uremic toxin indoxyl sulfate (IS), on human vascular endothelial cells (HUVECs). Methods: HUVECs viability was measured based on tetrazolium salt metabolism, and cell morphology was assessed with fluorescein-phalloidin staining of cells' actin cytoskeleton. The effects of simultaneous treatment of endothelial cells with PA, LPS, and IS on nitro-oxidative stress in vascular cells were evaluated quantitatively with fluorescent probes. The expression of vascular cell adhesion molecule VCAM-1, E-selectin, and occludin, an essential tight junction protein, in HUVECs treated with these metabolites was evaluated in Western blot. Results: PA, combined with LPS and IS, did not influence HUVECs viability but induced stress on actin fibers and focal adhesion complexes. Moreover, PA combined with LPS significantly enhanced reactive oxygen species (ROS) production in HUVECs but decreased nitric oxide (NO) generation. PA also considerably increased the expression of VCAM-1 and E-selectin in HUVECs treated with LPS or IS but decreased occludin expression. Conclusion: Palmitic acid enhances the toxic effect of metabolic endotoxemia on the vascular endothelium.

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.

The impact of metabolic endotoxaemia on the browning process in human adipocytes.

BACKGROUND: Dysfunctional adipose tissue (AT) is known to contribute to the pathophysiology of metabolic disease, including type 2 diabetes mellitus (T2DM). This dysfunction may occur, in part, as a consequence of gut-derived endotoxaemia inducing changes in adipocyte mitochondrial function and reducing the proportion of BRITE (brown-in-white) adipocytes. Therefore, the present study investigated whether endotoxin (lipopolysaccharide; LPS) directly contributes to impaired human adipocyte mitochondrial function and browning in human adipocytes, and the relevant impact of obesity status pre and post bariatric surgery. METHODS: Human differentiated abdominal subcutaneous (AbdSc) adipocytes from participants with obesity and normal-weight participants were treated with endotoxin to assess in vitro changes in mitochondrial function and BRITE phenotype. Ex vivo human AbdSc AT from different groups of participants (normal-weight, obesity, pre- and 6 months post-bariatric surgery) were assessed for similar analyses including circulating endotoxin levels. RESULTS: Ex vivo AT analysis (lean & obese, weight loss post-bariatric surgery) identified that systemic endotoxin negatively correlated with BAT gene expression (p < 0.05). In vitro endotoxin treatment of AbdSc adipocytes (lean & obese) reduced mitochondrial dynamics (74.6% reduction; p < 0.0001), biogenesis (81.2% reduction; p < 0.0001) and the BRITE phenotype (93.8% reduction; p < 0.0001). Lean AbdSc adipocytes were more responsive to adrenergic signalling than obese AbdSc adipocytes; although endotoxin mitigated this response (92.6% reduction; p < 0.0001). CONCLUSIONS: Taken together, these data suggest that systemic gut-derived endotoxaemia contributes to both individual adipocyte dysfunction and reduced browning capacity of the adipocyte cell population, exacerbating metabolic consequences. As bariatric surgery reduces endotoxin levels and is associated with improving adipocyte functionality, this may provide further evidence regarding the metabolic benefits of such surgical interventions.

Are gut dysbiosis, barrier disruption, and endotoxemia related to adipose tissue dysfunction in metabolic disorders? Overview of the mechanisms involved.

Recently, compelling evidence points to dysbiosis and disruption of the epithelial intestinal barrier as major players in the pathophysiology of metabolic disorders, such as obesity. Upon the intestinal barrier disruption, components from bacterial metabolism and bacteria itself can reach peripheral tissues through circulation. This has been associated with the low-grade inflammation that characterizes obesity and other metabolic diseases. While circulating bacterial DNA has been postulated as a common feature of obesity and even type 2 diabetes, almost no focus has been given to the existence and effects of bacteria in peripheral tissues, namely the adipose tissue. As a symbiont population, it is expected that gut microbiota modulate the immunometabolism of the host, thus influencing energy balance mechanisms and inflammation. Gut inflammatory signals cause direct deleterious inflammatory responses in adipose tissue and may also affect key gut neuroendocrine mechanisms governing nutrient sensing and energy balance, like incretins and ghrelin, which play a role in the gut-brain-adipose tissue axis. Thus, it is of major importance to disclose how gut microbiota and derived signals modulate neuroendocrine and inflammatory pathways, which contribute to the dysfunction of adipose tissue and to the metabolic sequelae of obesity and related disorders. This review summarizes the current knowledge regarding these topics and identifies new perspectives in this field of research, highlighting new pathways toward the reduction of the inflammatory burden of metabolic diseases.

Animal board invited review: The contribution of adipose stores to milk fat: implications on optimal nutritional strategies to increase milk fat synthesis in dairy cows.

A wide range of nutritional and non-nutritional factors influence milk fat synthesis and explain the large variation observed in dairy herds. The capacity of the animal to synthesize milk fat will largely depend on the availability of substrates for lipid synthesis, some of which originate directly from the diet, ruminal fermentation or from adipose tissue stores. The mobilization of non-esterified fatty acids from adipose tissues is important to support the energy demands of milk synthesis and will therefore have an impact on the composition of milk lipids, especially during the early lactation period. Such mobilization is tightly controlled by insulin and catecholamines, and in turn, can be affected indirectly by factors that influence these signals, namely diet composition, lactation stage, genetics, endotoxemia, and inflammation. Environmental factors, such as heat stress, also impact adipose tissue mobilization and milk fat synthesis, mainly through endotoxemia and an immune response-related increase in concentrations of plasma insulin. Indeed, as proposed in the present review, the central role of insulin in the control of lipolysis is key to improving our understanding of how nutritional and non-nutritional factors impact milk fat synthesis. This is particularly the case during early lactation, as well as in situations where mammary lipid synthesis is more dependent on adipose-derived fatty acids.

P2X7 receptor antagonist A-438079 alleviates oxidative stress of lung in LPS-induced septic rats.

Sepsis is a deadly systemic inflammatory response of the body against infection resulting in immune response, cell differentiation and organ damage. Endotoxemia is one of the causes of sepsis-related acute respiratory distress and respiratory burst is an important generator of oxidants. Inflammation may be aggravated by overexpression of ATP-gated purinergic receptors (i.e., P2X7R) following cell damage. We aimed to evaluate the effects of P2X7R antagonist A-438079 on lung oxidative status and the receptor expression in endotoxemia of sepsis. Rats were subjected to sepsis by E. coli lipopolysaccharide (LPS) and treated with 15 mg/kg A-438079. The increase in circulatory IL-1ß and IL-8 concentrations in LPS group confirmed the systemic inflammatory response to endotoxemia compared with Control groups (p < 0.001). Besides, there was an increase in P2X7R expression in lung tissue after LPS administration. Compared with Control groups, there were significant increases in the values of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) (p < 0.001), and myeloperoxidase (MPO) (p < 0.05) in lung tissue of LPS group. P2X7R expression in lung and IL-1ß level in blood did not increase in LPS + A-438079 group. A-438079 decreased the lung levels of MDA, GSH, CAT and SOD (p < 0.001), and MPO (p < 0.01) in septic rats. As a result, administration of pathogen-associated LPS led to increased P2X7R expression into lung tissue and elevated lipid peroxidation product MDA with regard to oxidative damage. The P2X7R antagonist A-438079 alleviated the oxidative stress of lung with a balance of tissue oxidant/antioxidant factors in experimental sepsis in rats.

Nicaraven protects against endotoxemia-induced inflammation and organ injury through modulation of AMPK/Sirt1 signaling in macrophages.

Endotoxemia is a disease characterized by systemic inflammatory responses and organ injury caused by lipopolysaccharide (LPS) infection, with high mortality. Nicaraven (AVS), a potent hydroxyl radical scavenger, has been proven to regulate the inflammatory response in tumors. To investigate the protective effects and mechanisms of AVS in endotoxemia, mice were injected intraperitoneally with LPS to induce endotoxemia. AVS treatment significantly decreased the levels of pro-inflammatory cytokines in the serum, reduced neutrophil infiltration, attenuated multiple organ injury, and increased the survival rate in LPS-challenged mice. In the LPS-induced inflammatory model of macrophages, AVS inhibited macrophage activation, suppressed nitric oxide (NO) production, and inhibited the expression and secretion of pro-inflammatory cytokines. Mechanistically, AVS treatment up-regulated silence information regulator transcript-1 (Sirt1) expression in a time- and dose-dependent manner. AVS treatment activated the AMP-dependent protein kinase (AMPK)/Sirt1 signaling pathway and suppressed the activation of nuclear factor kappa B (NF-κB) in macrophages exposed to LPS. However, the anti-inflammatory effects of AVS could be reversed by the AMPK, the Sirt1 inhibitor, or the histone deacetylase inhibitor. We confirmed that the AMPK inhibitor inhibited AVS-mediated AMPK/Sirt1 activation and NF-κB p65 acetylation. These results suggested that AVS alleviated endotoxemia by activating the AMPK/Sirt1 signaling pathway in macrophages.