Leakage Of Lipopolysaccharide Research Articles

LPS-LBP complex induced endothelial cell pyroptosis in aortic dissection is associated with gut dysbiosis

Acute aortic dissection (AAD) is the most severe traumatic disease affecting the aorta. Pyroptosis-mediated vascular wall inflammation is a crucial trigger for AAD, and the exact mechanism requires further investigation. In this study, our proteomic analysis showed that Lipopolysaccharide (LPS)-binding protein (LBP) was significantly upregulated in the plasma and aortic tissue of patients with AAD. Further, 16S rRNA sequencing of stool samples suggested that patients with AAD exhibit gut dysbiosis, which may lead to an impaired intestinal barrier and LPS leakage. By comparing with control mice, we found that LBP, including Pyrin Domain Containing Protein3 (NLRP3), the CARD-containing adapter apoptosis-associated speck-like protein (ASC), and Cleaved caspase-1, were upregulated in the AAD aorta, whereas gut intestinal barrier-related proteins were downregulated. Moreover, treated with LBPK95A (an LBP inhibitor) attenuated the incidence of AAD, the expression levels of pyroptosis-related factors, and the extent of vascular pathological changes compared to those in AAD mice. In addition, LPS and LBP treatment of human umbilical vein endothelial cells (HUVECs) activated TLR4 signaling and intracellular reactive oxygen species (ROS) production, which stimulated NLRP3 inflammasome formation and mediated pyroptosis in endothelial cells. Our findings showed that gut dysbiosis mediates pyroptosis by the LPS-LBP complex, thus providing new insights into developing AAD.

Lipopolysaccharide released from gut activates pyroptosis of macrophages via Caspase 11-Gasdermin D pathway in systemic lupus erythematosus.

Noncanonical pyroptosis is triggered by Caspase 4/5/11, which cleaves Gasdermin D (GSDMD), leading to cell lysis. While GSDMD has been studied previously in systemic lupus erythematosus (SLE), the role of pyroptosis in SLE pathogenesis remains unclear and contentious, with limited understanding of Caspase 11-mediated pyroptosis in this condition. In this study, we explored the level of Caspase 11-mediated pyroptosis in SLE, identifying both the upstream pathways and the interaction between pyroptosis and adaptive immune responses. We observed increased Caspase 5/11 and GSDMD-dependent pyroptosis in the macrophages/monocytes of both lupus patients and mice. We identified serum lipopolysaccharide (LPS), released from the gut due to a compromised gut barrier, as the signal that triggers Caspase 11 activation in MRL/lpr mice. We further discovered that pyroptotic macrophages promote the differentiation of mature B cells independently of T cells. Additionally, inhibiting Caspase 11 and preventing LPS leakage proved effective in improving lupus symptoms in MRL/lpr mice. These findings suggest that elevated serum LPS, resulting from a damaged gut barrier, induces Caspase 11/GSDMD-mediated pyroptosis, which in turn promotes B cell differentiation and enhances autoimmune responses in SLE. Thus, targeting Caspase 11 could be a viable therapeutic strategy for SLE.

Alpha-glycosyl isoquercitrin alleviates subchronic social defeat stress-induced depression symptoms by modulating the microbiota-gut-brain axis in mice

AimsIncreasing evidence suggests a link between gut microbial dysbiosis and the pathogenesis of depression. Alpha-glycosyl isoquercitrin (AGIQ), consisting of isoquercitrin and its glycosylated quercetin, has beneficial effects on the gut microbiome and brain function. Here, we detected the potential antidepressant impact of a four-week administration of AGIQ and its underlying mechanisms using a mouse model of depression. Main methodsMale C57BL/6 mice were orally administered AGIQ (0.05 % or 0.5 % in drinking water) for 28 days; subchronic social defeat stress was performed in the last 10 days. Behavior tests were conducted to assess anxiety and depressive-like behaviors. Additionally, evaluations encompassed 5-hydroxytryptamine (5-HT) levels, the gut microbiota composition, lipopolysaccharide (LPS) concentrations, short-chain fatty acids levels, and intestinal barrier integrity changes. Key findingsAGIQ significantly alleviated depression-like behaviors and increased hippocampal 5-HT levels. Further, AGIQ mitigated stress-induced gut microbial abnormalities and reduced the levels of LPS in the serum, which affected the relative gene expression levels of 5-HT biosynthesis enzymes in vitro. Furthermore, AGIQ reversed the reduced butyrate levels in cecal contents and improved the impaired intestinal barrier by increasing the expression of colonic zonula occluden-1 (ZO-1) and occludin, thereby decreasing LPS leakage. SignificanceOur results suggest that AGIQ could improve stress-induced depression by regulating the gut microbiome, which inhibits LPS production and maintains the gut barrier. This is the first report on the potential effect of AGIQ on depression via the gut microbiota-brain axis, shedding new light on treatment options.

Echinacea purpurea polysaccharide intervene in hepatocellular carcinoma via modulation of gut microbiota to inhibit TLR4/NF-κB pathway

Echinacea purpurea polysaccharide (EPP) exhibit various pharmacological activities, including immunomodulatory, anti-inflammatory, and anti-tumor effects. In this study, we investigated the potential mechanism of EPP intervention in hepatocellular carcinoma (HCC). The results demonstrated that EPP effectively mitigated liver injury caused by HCC, inhibited the proliferation of HCC, and induced apoptosis. Following EPP intervention, there was a significant increase in propionic acid and butyric acid-producing gut microbiota such as Coprococcus, Clostridium and Roseburia, leading to enhanced expression of intestinal tight junction proteins and the repair of the intestinal barrier. This controls lipopolysaccharide (LPS) leakage, which in turn inhibits the TLR4/NF-κB pathway and reduces the expression of inflammatory factors such as IL-6, as well as migration factors like MMP-2. Metabolomics revealed the downregulation of pyrimidine metabolism and nucleotide metabolism, along with the upregulation of butyrate metabolism in tumor cells. This study demonstrated that EPP effectively regulated LPS leakage by modulating gut microbes, and this modulation influenced the TLR4/NF-κB pathway, ultimately disrupting tumor cell survival induced by HCC in mice.

Wolfberry, Yam, and Chrysanthemum polysaccharides increased intestinal Akkermansia muciniphila abundance and hepatic YAP1 expression to alleviate DILI.

Drug-induced liver injury (DILI) is frequently induced by high dose of acetaminophen (APAP) and is concomitant with disturbances of gut flora. Akkermansia muciniphila is beneficial for the repair of liver injury. Lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide all have anti-inflammatory and antioxidation effects. The objective of this study is to investigate the potential of lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide (LYC) in improving DILI by increasing the abundance of A. muciniphila. Initially, screening for the optimal concentrations of wolfberry, yam, and chrysanthemum (WYC) or LYC to promote A. muciniphila proliferation invitro and validated in antibiotic (ATB)-treated KM mice. Subsequently, APAP-induced DILI model in BALB/c mice were constructed to examine the treatment effects of LYC. Our findings indicate that the optimal concentration ratio of WYC was 2:3:2, and LYC was 1:1:1. WYC increased A. muciniphila proliferation invitro and in ATB-treated mice under this ratio. Meanwhile, LYC increased A. muciniphila abundance invitro and the combination LYC with A. muciniphila promoted the proliferation of A. muciniphila in ATB-treated mice. The overdose of APAP resulted in the impairment of the intestinal barrier function and subsequent leakage of lipopolysaccharide (LPS). Moreover, LYC increased A. muciniphila abundance, reduced intestinal inflammation and permeability, and upregulated the expression of the tight junction protein zonula occludens protein 1 (ZO-1) and occludin contents in the gut. Lastly, LYC inhibited LPS leakage and upregulated hepatic YAP1 expression, ultimately leading to the repair of DILI.

The effects of astragaloside IV on gut microbiota and serum metabolism in a mice model of intracerebral hemorrhage

BackgroundAstragaloside IV (AS-IV) is the main active component of “Astragalus membranaceus (Fisch.) Bunge, a synonym of Astragalus propinquus Schischkin (Fabaceae)”, which demonstrated to be useful for the treatment of intracerebral hemorrhage (ICH). However, due to the low bioavailability and barrier permeability of AS-IV, the gut microbiota may be an important key regulator for AS-IV to work. ObjectiveTo explore the influences of gut microbiota on the effects of AS-IV on ICH. MethodsMice were randomly divided into five groups: sham, ICH, and AS-IV-treated groups (25 mg/kg, 50 mg/kg, and 100 mg/kg). Behavioral tests, brain histopathology, and immunohistochemistry analysis were used to evaluate the degree of brain injury. Western blot was employed to verify peri‑hematoma inflammation. The plasma lipopolysaccharide (LPS) leakage, the fluorescein isothiocyanate-dextran permeability, the colonic histopathology, and immunohistochemistry were detected to evaluate the barrier function of intestinal mucosal. Moreover, 16S rDNA sequencing and metabolomic analysis was applied to screen differential bacteria and metabolites, respectively. The correlation analysis was adopted to determine the potential relationship between differential bacteria and critical metabolites or neurological deficits. ResultsAS-IV alleviated neurological deficits, neuronal injury and apoptosis, and blood-brain barrier disruption. This compound reduced tumor necrosis factor (TNF)-α expression, increased arginase (Arg)-1 and interleukin (IL)-33 levels around the hematoma. Next, 16S rRNA sequencing indicated that AS-IV altered the gut microbiota, and inhibited the production of conditional pathogenic bacteria. Metabolomic analysis demonstrated that AS-IV regulated the serum metabolic profiles, especially the aminoacid metabolism and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Additionally, AS-IV mitigated intestinal barrier damage and LPS leakage. ConclusionThis study provides a new perspective on the use of AS-IV for the treatment of ICH. Among them, gut microbiota and its metabolites may be the key regulator of AS-IV in treating ICH.

Sealing Behavior of a New Material Combination in Two-Part Dental Implant Systems: An In Vitro Examination.

To test a newly introduced implant-abutment material combination against bacterial endotoxin leakage in a human whole blood assay. Two dental implant systems with internal connections and the following material combinations at the implant-abutment interface (IAI) were used (implant material/abutment material): yttrium-stabilized tetragonal zirconium dioxide (Y-TZP)/polyetherketoneketone (PEKK), and titanium (Ti/Ti). Test implants were inoculated with lipopolysaccharide (LPS) and sealed and submerged in human whole blood. Untreated implants served as the control groups. Changes in gene expression levels of inflammatory markers indicating LPS leakage were assessed after 1, 8, and 24 hours using quantitative real-time polymerase chain reaction. In the Y-TZP/PEKK test group, a significant influence of the implant system (P < .001) on increases in gene expression indicating leakage were detected after 8 hours for TLR-4 and after 24 hours for interleukin 1-β and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), indicating microleakage of LPS at the IAI. In the Ti/Ti test group, differences in gene expression were found only for NF-κB after 8 hours. The internal hexalobe IAI of two-piece dental implants fabricated from Y-TZP and PEKK do not prevent LPS molecular microleakage.

Fruits of Hippophaë rhamnoides in human leukocytes and Caco-2 cell monolayer models-A question about their preventive role in lipopolysaccharide leakage and cytokine secretion in endotoxemia.

Preparations from Hippophaë rhamnoides L. (sea buckthorn) have been traditionally used in the treatment of skin and digestive disorders, such as gastritis, gastric and duodenal ulcers, uterine erosions, as well as oral, rectal, and vaginal mucositis, in particular in the Himalayan and Eurasian regions. An influence of an aqueous extract from the fruits of H. rhamnoides (HR) on leakage of lipopolysaccharide (LPS) from Escherichia coli through gut epithelium developed from the human colorectal adenocarcinoma (Caco-2) monolayer in vitro and glucose transporter 2 (GLUT2) translocation were the principal objectives of the study. Additionally, the effect of HR on the production of pro- and anti-inflammatory cytokines (interleukins: IL-8, IL-1β, IL-10, IL-6; tumor necrosis factor: TNF-α) by the Caco-2 cell line, human neutrophils (PMN), and peripheral blood mononuclear cells (PBMC) was evaluated. The concentration of LPS on the apical and basolateral sides of the Caco-2 monolayer was evaluated with a Limulus Amebocyte Lysate (LAL) assay. GLUT2 translocation was evaluated using an immunostaining assay, whereas secretion of cytokines by cell cultures was established with an enzyme-linked immunosorbent (ELISA) assay. HR (500 μg/ml) significantly inhibited LPS leakage through epithelial monolayer in vitro in comparison with non-treated control. The treatment of Caco-2 cells with HR (50–100 μg/ml) showed GLUT2 expression similar to the non-treated control. HR decreased the secretion of most pro-inflammatory cytokines in all tested models. HR might prevent low-grade chronic inflammation caused by metabolic endotoxemia through the prevention of the absorption of LPS and decrease of chemotactic factors released by immune and epithelial cells, which support its use in metabolic disorders in traditional medicine.

A potential new pathway for heparin treatment of sepsis-induced lung injury: inhibition of pulmonary endothelial cell pyroptosis by blocking hMGB1-LPS-induced caspase-11 activation.

Sepsis is a significant cause of mortality in critically ill patients. Acute lung injury (ALI) is a leading cause of death in these patients. Endothelial cells exposed to the bacterial endotoxin lipopolysaccharide (LPS) can progress into pyroptosis, a programmed lysis of cell death triggered by inflammatory caspases. It is characterized by lytic cell death induced by the binding of intracellular LPS to caspases 4/5 in human cells and caspase-11 in mouse cells. In mice,caspase-11-dependent pyroptosis plays an important role in endotoxemia. HMGB1 released into the plasma binds to LPS and is internalized into lysosomes in endothelial cells via the advanced glycation end product receptor. In the acidic lysosomal environment, HMGB1 permeates the phospholipid bilayer, which is followed by the leakage of LPS into the cytoplasm and the activation of caspase-11. Heparin is an anticoagulant widely applied in the treatment of thrombotic disease. Previous studies have found that heparin could block caspase-11-dependent inflammatory reactions, decrease sepsis-related mortality, and reduce ALI, independent of its anticoagulant activity. Heparin or modified heparin with no anticoagulant property could inhibit the alarmin HMGB1-LPS interactions, minimize LPS entry into the cytoplasm, and thus blocking caspase-11 activation. Heparin has been studied in septic ALI, but the regulatory mechanism of pulmonary endothelial cell pyroptosis is still unclear. In this paper, we discuss the potential novel role of heparin in the treatment of septic ALI from the unique mechanism of pulmonary endothelial cell pyroptosis.

Andrographolide sodium bisulfite ameliorates dextran sulfate sodium-induced colitis and liver injury in mice via inhibiting macrophage proinflammatory polarization from the gut-liver axis

Ulcerative colitis (UC), an inflammatory disease, is widely thought to be associated with colonic barrier damage and inflammatory response. With the destruction of the colonic barrier, lipopolysaccharide (LPS) enters the liver through the portal vein and causes liver injury. Liver injury in turn exacerbates UC to form a vicious cycle, so the treatment of liver injury cannot be ignored. Andrographolide (Andro) has a protective effect against colitis and liver injury, but with low bioavailability. Andrographolide sodium bisulfite (ASB), a water-soluble sulfonate of Andro, has better bioavailability, whether it has a better curative effect against UC and liver injury is rarely reported. Hence, we investigated the protective effect and potential mechanism of ASB against dextran sulfate sodium (DSS)-induced UC and liver injury in mice. The results showed that treatment with ASB significantly relieved the clinical symptoms of UC and liver injury by reducing disease activity index, inhibiting gut-derived LPS leakage, and improving colonic and hepatic injury, and its curative effect was better than Andro. Moreover, ASB effectively decreased the YAP-mediated colonic inflammation and TLR4/MyD88/NF-κB-mediated pro-inflammatory factor release in the liver. Both colonic and hepatic inflammation were associated with macrophage proinflammatory polarization, but they were significantly inhibited by ASB. ASB showed good safety in the treatment of UC and liver injury and has no nephrotoxicity as previously described. In conclusion, ASB has an effective protective effect on DSS-induced UC and liver injury, mainly by suppressing macrophage proinflammatory polarization from the gut-liver axis.

The Mechanism of Lipopolysaccharide Escaping the Intestinal Barrier in Megalobrama amblycephala Fed a High-Fat Diet.

With the popularity of western food characterized by excessive fat and sugars, obesity has currently been a public health issue. Low-grade chronic inflammation accompanied by obesity increases the risk of multiple epidemics such as diabetes, cancer and cardiovascular diseases. Here, we show that feeding Megalobrama amblycephala with a high-fat diet (HFD) drives obesity-related chronic inflammation and the penetration of lipopolysaccharide (LPS). Interference with antibiotics inhibits the produce of LPS and this alleviates the sustained release of pro-inflammatory factors induced by HFD. LPS penetration is attributed to weakened intestinal mucus barrier after high-fat exposure. Mechanically, the consumption of HFD inhibits the secretion of mucin 2 (MUC2) due to the induction of endoplasmic reticulum stress mediated by the inositol-requiring enzyme 1 (IRE1) /X box-binding protein 1 (XBP1) pathway in goblet cells. Furthermore, excessive lipid exacerbates the leakage of LPS across the intestinal epithelial cell barrier via the transcellular pathway. Mechanically, lipid increases the internalization of LPS in intestinal epithelial cells depending on the activation of fatty acid translocase (FAT/CD36). These results demonstrate that HFD causes the penetration of LPS due to the weakened intestinal mucosal barrier and the assistance of CD36.

Nonalcoholic Steatohepatitis and HCC in a Hyperphagic Mouse Accelerated by Western Diet.

Background & AimsHow benign liver steatosis progresses to nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC) remains elusive. NASH progression entails diverse pathogenic mechanisms and relies on complex cross-talk between multiple tissues such as the gut, adipose tissues, liver, and the brain. Using a hyperphagic mouse fed with a Western diet (WD), we aimed to elucidate the cross-talk and kinetics of hepatic and extrahepatic alterations during NASH–HCC progression, as well as regression.MethodsHyperphagic mice lacking a functional Alms1 gene (Foz/Foz) and wild-type littermates were fed WD or standard chow for 12 weeks for NASH/fibrosis and for 24 weeks for HCC development. NASH regression was modeled by switching back to normal chow after NASH development.ResultsFoz+WD mice were steatotic within 1 to 2 weeks, developed NASH by 4 weeks, and grade 3 fibrosis by 12 weeks, accompanied by chronic kidney injury. Foz+WD mice that continued on WD progressed to cirrhosis and HCC within 24 weeks and had reduced survival as a result of cardiac dysfunction. However, NASH mice that were switched to normal chow showed NASH regression, improved survival, and did not develop HCC. Transcriptomic and histologic analyses of Foz/Foz NASH liver showed strong concordance with human NASH. NASH was preceded by an early disruption of gut barrier, microbial dysbiosis, lipopolysaccharide leakage, and intestinal inflammation. This led to acute-phase liver inflammation in Foz+WD mice, characterized by neutrophil infiltration and increased levels of several chemokines/cytokines. The liver cytokine/chemokine profile evolved as NASH progressed, with subsequent predominance by monocyte recruitment.ConclusionsThe Foz+WD model closely mimics the pathobiology and gene signature of human NASH with fibrosis and subsequent HCC. Foz+WD mice provide a robust and relevant preclinical model of NASH, NASH-associated HCC, chronic kidney injury, and heart failure.

Aging in Male Wistar Rats Associates With Changes in Intestinal Microbiota, Gut Structure, and Cholecystokinin-Mediated Gut-Brain Axis Function.

Aging in mammals is characterized by failure of the homeostatic mechanisms that regulate energy balance. Several mechanisms have been proposed such as the presence of a low-grade chronic inflammation in different tissues, as well as leptin and insulin resistance, but the primary alteration is not fully elucidated. The gut microbiota has recently emerged as a key player in a variety of metabolic and neurological disorders. A main concept in this context is the gut-brain axis that refers to alterations in the gut that mediate effects in the central nervous system, including those related with the control of energy balance. Using 16S rRNA analysis, we demonstrate that aged male Wistar rats have increased presence of mucin-degrading and lipopolysaccharide (LPS)-producing bacteria. In addition, old animals exhibit a lower number of neutral mucin secreting goblet cells, and a decrease of tight junctions and adherens junctions marker proteins, zonula occludens protein-1 (ZO-1) and β-catenin, respectively. These data are compatible with a thinner mucus layer and a weaker gut barrier in older animals that likely facilitate LPS leakage. Our data also show that cholecystokinin (CCK) satiating effect is impaired in aged rats, one of the expected effects of increased LPS leakage. In contrast, no overt signs of gut or systemic inflammation are observed. Changes in microbiota in old male Wistar rats present features of situations of increased adiposity, but different from those of obese animals. These could partly explain the increased adiposity and fat deposition in liver and heart as observed here.

Crocin-I alleviates the depression-like behaviors probably via modulating “microbiota-gut-brain” axis in mice exposed to chronic restraint stress

BackgroundDepressive disorder is rapidly advancing in the worldwide, and therapeutic strategy through “gut-brain” axis has been proved to be effective. Crocin, has been found to have antidepressant activity. However, there is no thorough research for the effects of crocin-I (a major active component of crocin) on depression and its underlying mechanism. MethodsWe investigated the antidepressant effect of six-week oral administration of crocin-I in a mice model of depression induced by four-week CRS. Based on the “microbiota-gut-brain” axis, we determined the effects of crocin-I administration on gut microbiota, intestinal barrier function, short chain fatty acids and neurochemical indicators. ResultsAdministration of crocin-I at a dose of 40 mg/kg for six weeks mitigated depression-like behaviors of depressed mice as evidenced by behaviors tests. In addition, crocin-I reduced the levels of lipopolysaccharide (LPS), Interleukin-6and tumor necrosis factor-α (TNF-α) in serum and TNF-α expression in the hippocampus, and increased the hippocampal brain-derived neurotrophic factor. Besides, 16 s rRNA sequencing revealed that crocin-I mitigated the gut microbiota dysbiosis in depressed mice as represented by the decreased abundance of Proteobacteria and Bacteroidetes, Sutterella spp. and Ruminococcus spp. and increased abundances of Firmicutes, Lactobacillus spp. and Bacteroides spp. Moreover, gas chromatography-mass spectrometry revealed that crocin-I reversed the decreased levels of short-chain fatty acids (SCFAs) in feces of depressed mice. Furthermore, crocin-I improved the impaired intestinal barrier by increasing expression of Occludin and Claudin-1, which contributed to the decreased LPS leakage. LimitationsOnly the male mice were used; the dose-effect relationship should be observed. ConclusionThese results suggested that crocin-I effectively alleviated depression-like behavior, likely depended on the gut microbiota and its modulation of intestinal barrier and SCFAs.

Characterization of an Adapted Murine Model of Intrauterine Inflammation–Induced Preterm Birth

Preterm birth (PTB) affects nearly 15 million infants each year. Of these PTBs, >25% are a result of inflammation or infection. Animal models have improved our understanding of the mechanisms leading to PTB. Prior work has described induction of intrauterine inflammation in mice with a single injection of lipopolysaccharide (LPS). Herein, we have improved the reproducibility and potency of LPS in the model using two injections distal to the cervix. An in vivo imaging system revealed more uniform distribution of Evans Blue Dye using a double distal injection (DDI) approach compared with a single proximal injection (SPI). Endotoxin concentrations in vaginal lavage fluid from SPI dams were significantly higher than from DDI dams. At equivalent LPS doses, DDI consistently induced more PTB than SPI, and DDI showed a linear dose-response, whereas SPI did not. Gene expression in myometrial tissue revealed increased levels of inflammatory markers in dams that received LPS DDI compared with LPS SPI. The SPI group showed more significant overexpression in cervical remodeling genes, likely due to the leakage of LPS from the uterine horns through the cervix. The more reliable PTB induction and uniform uterine exposure provided by this new model will be useful for further studying fetal outcomes and potential therapeutics for the prevention of inflammation-induced PTB.

Sodium butyrate ameliorates insulin resistance and renal failure in CKD rats by modulating intestinal permeability and mucin expression.

BackgroundThe associated increase in the lipopolysaccharide (LPS) levels and uremic toxins in chronic kidney disease (CKD) has shifted the way we focus on intestinal microbiota. This study shows that a disruption of the intestinal barrier in CKD promotes leakage of LPS from the gut, subsequently decreasing insulin sensitivity. Butyrate treatment improved the intestinal barrier function by increasing colonic mucin and tight junction (TJ) proteins. This modulation further ameliorated metabolic functions such as insulin intolerance and improved renal function.MethodsRenal failure was induced by 5/6th nephrectomy (Nx) in rats. A group of Nx and control rats received sodium butyrate in drinking water. The Nx groups were compared with sham-operated controls.ResultsThe Nx rats had significant increases in serum creatinine, urea and proteinuria. These animals had impaired glucose and insulin tolerance and increased gluconeogenesis, which corresponded with decreased glucagon-like peptide-1 (GLP-1) secretion. The Nx animals suffered significant loss of intestinal TJ proteins, colonic mucin and mucin 2 protein. This was associated with a significant increase in circulating LPS, suggesting a leaky gut phenomenon. 5′adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, known to modulate epithelial TJs and glucose metabolism, was significantly reduced in the intestine of the Nx group. Anti-inflammatory cytokine, interleukin 10, anti-bacterial peptide and cathelicidin-related antimicrobial peptide were also lowered in the Nx cohort. Butyrate treatment increased AMPK phosphorylation, improved renal function and controlled hyperglycemia.ConclusionsButyrate improves AMPK phosphorylation, increases GLP-1 secretion and promotes colonic mucin and TJ proteins, which strengthen the gut wall. This decreases LPS leakage and inflammation. Taken together, butyrate improves metabolic parameters such as insulin resistance and markers of renal failure in CKD animals.

Assessment of lipopolysaccharide microleakage at conical implant-abutment connections

The aim of this in vitro study was to assess lipopolysaccharide microleakage at conical implant-abutment connections of two-piece dental implants in terms of the expression levels of genes involved in lipopolysaccharide-mediated proinflammatory cytokine production. Two implant systems with conical implant-abutment connections were inoculated with lipopolysaccharide and submerged in human whole blood. Positive-control blood samples (without implants) were stimulated with 4μg/ml, 2μg/ml, 200ng/ml, and 20ng/ml lipopolysaccharide. Sampling was performed after 1, 8, and 24h of incubation. Changes of gene expression levels of Toll-like receptor 9, tumor necrosis factor-α, nuclear factor kappa light chain enhancer of activated B cells, interleukin-1β, and interferon-γ were assessed by real-time quantitative PCR. In addition, protein expression levels of interleukin-6, tumor necrosis factor-α, interleukin-1β, and interferon-γ were determined by immunoassay. Changes in cytokine expression at the genomic and proteomic levels indicated lipopolysaccharide leakage at the interfaces of both tested implant systems, although some implants showed no sign of microleakage. Any tested concentration of lipopolysaccharide stimulated similar gene expression. Conical implant-abutment connections of two-piece dental implants do not prevent microleakage on a molecular level. Changes in lipopolysaccharide-induced proinflammatory cytokine gene expression facilitate the detection of lipopolysaccharide microleakage at implant-abutment interfaces. Small amounts of lipopolysaccharide released from intra-implant cavities can stimulate a detectable immunological response in human whole blood and may induce alveolar bone resorption via the osteoclast-activating pathway.

Toll-like Receptor 4 Mediates LPS-Induced Release of Nitric Oxide and Tumor Necrosis Factor-&amp;#945; by Embryonal Cardiomyocytes: Biological Significance and Clinical Implications in Human Pathology

Lipopolysaccharide (LPS) the major structural component of the outer membrane of Gram-negative bacteria contributes to the cardiovascular collapse and death observed in septic patients, as well as in the immunocompromised host. LPS activates multiple cells to release proinflammatory cytokines, nitric oxide (NO) and other reactive molecules able to depress cardiac functions. It has been appreciated that the pattern recognition receptor, TLR4, is a signalling receptor for LPS, but its role in the embryonal cardiomyocytes is poorly understood. Here, we provide evidence for TLR4-dependent functional responses by LPS treated embryonal cardiomyocytes. It will be reported that LPS is able to induce TNF-alpha and NO release from cultured cardiomyocytes, while molecular and morphological evidence demonstrates the expression of TLR4 on surface membrane of embryonal cardiomyocytes. LPS-induced signalling was studied evaluating the expression of the extracellular signal-regulated kinase (ERK) and signal transducer and activators of transcription (STAT) protein families in response to LPS. The role of TLR4 was investigated with blocking assays using monoclonal antibody against this endotoxin receptor. Our results indicated that LPS-induced activation of signal transduction in embryonal cardiomyocytes occurs by a TLR4-dependent mechanism. In summary, chick embryonal cardiomyocytes may constitute a valid experimental model in order to study the LPS induced inflammatory responses by cardiomyocytes, useful not only to identify the signalling pathways evoked by endotoxin receptor, including TLR4, but also to suggest therapeutic targets for the control of myocardial dysfunction induced by infectious agents. In this respect, in elderly a continuous leakage of LPS from gut flora and/or external environment should be regarded as a possible cause of cardiac failure and, therefore, adequately prevented or treated.

Potential Therapeutic Intervention Suggested by Profound Deficiency of Neutrophil-Derived Bactericidal/Permeability-Increasing Protein (BPI) and Other Alterations in LPS-Directed Innate Immunity after Myeloablative Hematopoietic Stem Cell Transplantation (HSCT).

Myeloablative conditioning used for allogeneic HSCT damages the gut resulting in leakage of lipopolysaccharide (LPS, or endotoxin) into the circulation. LPS activates antigen-presenting cells to secrete inflammatory cytokines, including TNF, that enhance donor T cell responses to alloantigens. The relationship of TNF production and responsiveness to aGVHD and HSCT mortality has been demonstrated in animal models and with human correlative data. Use of either TNF or LPS antagonists in animal models or mice deficient in TNF or LPS responsiveness has led to profound amelioration of aGVHD. To determine whether LPS antagonists might have a therapeutic role in allogeneic HSCT, we measured plasma levels of 3 soluble LPS-modulating proteins in 30 sibling donor:recipient pairs undergoing myeloablative HSCT. Measurements included: 1) BPI, a constituent of neutrophil primary (azurophilic) granules with potent LPS-neutralizing activity at nanomolar concentrations in all body fluids, 2) LPS-binding protein (LBP) and 3) soluble CD14 (sCD14), which are both acute phase reactants that at basal levels enhance LPS activity by delivering LPS to its cell surface receptor but that at high levels detoxify LPS by delivering it to plasma lipoproteins. Spontaneous TNF production by whole blood and monocyte surface expression of TLR4, a key component of the LPS receptor, were also measured. Peripheral blood was obtained from donors (single time point) and patients pre-HSCT (baseline), Days 0, 7, 14, 21, 28 and on day of aGVHD. Plasma BPI levels fall dramatically from baseline, concurrent with severe neutropenia, while LBP (D7) and then sCD14 (D21–28) rise. Monocyte TLR4 expression was elevated at D7 and at onset of aGVHD. Patients with high spontaneous TNF production were at higher risk of aGVHD (p<.05) while those demonstrating greater concentrations of BPI at baseline appeared at lower risk for aGVHD (p<.01). Trends were observed suggesting lower aGVHD risk in patients with higher (i.e. potentially LPS inhibitory) levels of LBP and sCD14. Our results are consistent with a pathophysiologic pathway in which LPS, acting via TLR4, induces TNF production that contributes to aGVHD. The demonstration that patients are deficient in BPI and that deficiency of BPI and perhaps other LPS detoxifying proteins is associated with increased aGVHD risk suggest that replenishing BPI may represent a novel approach to shielding patients during the periHSCT period from LPS-induced pathophysiology, including cytokine driven events leading to aGVHD. Therefore, we are initiating a clinical trial of a recombinant N-terminal human BPI fragment (rBPI21 or Opebecan; XOMA (U.S.) L.L.C.), an investigational agent with an excellent safety profile and in vivo LPS-neutralizing activity, in myeloablative HSCT.

In vitro evidence that lipopolysaccharide of an oral pathogen leaks from root-end filled teeth.

The ability to achieve a complete apical seal of the root canal system is thought to be important in the success of non-surgical and surgical endodontics. The aim of this study was to establish whether or not root-end filled teeth allow leakage of lipopolysaccharide (LPS) from a known oral pathogen in vitro. Lipopolysaccharide (LPS) from a virulent strain of Prophyromonas gingivalis (P. gingivalis) (A7436 from patient with refractory periodontitis), was isolated by the Westphall and Jann technique, dialysed extensively, lyophilyzed, resuspended in distilled water and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Root canals from 10 teeth were instrumented endodontically and the apical 3 mm of resected roots were filled with gutta-percha. The teeth were mounted in 12 mL polypropylene vials by using sticky wax and root surfaces were covered with two layers of nail varnish. Teeth were filled with 3.3 mg mL-1 LPS and the vials filled with 11 mL of Tris Buffered Saline (TBS) containing 0.05% sodium azide. Both positive and negative controls were run in parallel with the experimental specimens. Aliquots were removed each day and subjected to slot blot analysis to quantitate the amount of LPS that had leaked into the bottom of the vials. The density of slots was analyzed using a laser densitometer and regression analysis was used to generate a standard curve, confidence intervals and experimental values. The data indicated that teeth obturated apically with gutta-percha leaked, whilst no LPS leakage was detected in teeth covered completely with nail varnish (P < 0.05). In vitro teeth with gutta-percha root-end fills can permit leakage of LPS from an identified oral pathogen.