Lipopolysaccharide Activation Research Articles

Bactericidal/Permeability-Increasing Protein Improves Cognitive Impairment in Diabetic Mice via Blockade of the LPS-LBP-TLR4 Signaling Pathway.

Emerging evidence suggests that the bactericidal/permeability-increasing protein (BPI) is involved in the process of cognitive impairment in diabetes. However, its underlying mechanism remains elusive. In this study, we found that BPI affects cognitive impairment due to diabetes through the lipopolysaccharide (LPS)-lipopolysacharide-binding protein (LBP)-toll-like receptor 4 (TLR4) signaling pathway. We examined the expression of BPI, LPS, LBP, CD14, and TLR4 in established mouse models of diabetes induced by high-fat diet (HFD) in combination with streptozotocin (STZ). Diabetic mice were then injected with adeno-associated-virus carrying BPI overexpression vectors and LPS. Fasting blood glucose, plasma insulin, and serum levels of inflammatory factors were examined. Then, glucose tolerance and, insulin resistance tests were used to measure systemic insulin sensitivity. Next, hippocampal tissue injury and cell apoptosis were examined by hematoxylin-eosin (HE) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Diabetic mice displayed increased LPS expression and activation of the LPS-CD14-TLR4 signaling pathway. HFD mice following LPS treatment showed significantly increased serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and expressions of Bcl-2-associated X protein (Bax) and Aβ but decreased expression of Bcl-2 in hippocampal tissues, as well as enhanced fasting blood glucose, plasma insulin, glucose tolerance, insulin tolerance, cell apoptosis, aggravated hippocampal tissue injury and, ultimately, cognitive impairment. However, overexpression of BPI was able to rescue the aforementioned phenotypes driven by LPS treatment. Taken together, BPI could potentially provide relief from cognitive impairment in diabetic mice by disrupting the LPS-LBP-TLR4 signaling pathway, underscoring a possible alternative therapeutic strategy against the cognitive impairment associated with diabetes.

Identification of tetragocarbone C and sideroxylin as the most potent anti-inflammatory components of Syncarpia glomulifera

In contrast to ancient Western and Asian cultures, medicinal plants of the Aboriginal and Torres Strait Islanders in Australia have not been as intensively studied for their molecular composition and molecular bioactivity. Syncarpia glomulifera subsp. glomulifera is a species in the plant family Myrtaceae. The resin of the plant has been traditionally used by the D'harawal people of Western Sydney to heal inflamed sores and ulcers. Hence, the anti-inflammatory activity of its leaf extract was investigated in RAW 264.7 macrophage and N11 microglia cell lines to isolate and identify the most active compounds. One new compound, tetragocarbone C, and three known compounds, tetragocarbone B, sideroxylin, and lumaflavanone A showed potent anti-inflammatory activity by downregulating nitric oxide and TNF-α production in LPS and IFN-γ stimulated cells. Except for the less potent tetragocarbone B, all compounds had an IC50 value (for nitric oxide downregulation) of <10 μg/mL and moderate cytotoxicity in both cell lines. The molecular targets along pro-inflammatory signaling pathways were further investigated in RAW 264.7 cells. All four compounds suppressed phosphorylation of ERK, c-Jun, and limited the phosphorylation of STAT-1 and STAT-3 in response to LPS and IFN-γ activation. The four compounds also suppressed NF-κB activation by preventing the translocation of the p65 subunit into the nucleus. Collectively, these findings suggest that the compounds isolated from Syncarpia glomulifera, especially tetragocarbone C and sideroxylin are promising anti-inflammatory agents, and could be further investigated for the treatment of diseases characterized by chronic inflammation.

Serum Amyloid A Aggravates Lipopolysaccharide-Induced Injury of BEAS-2B Cells by Activating Toll-Like Receptor 2/Activator Protein-1 Signaling

The serum amyloid A (sAA) is a common sensitive indicator for the diagnosis of infectious diseases, and sAA levels are increased in pneumonia. However, the detailed molecular mechanism is unknown. Previous studies have demonstrated the participation of Toll-like receptor (TLR) 2 and its downstream protein activator protein-1 (AP-1) in inflammatory lung injury. This study aimed to investigate the effect of sAA on LPS-induced BEAS-2B cells injury and uncover the possible mechanism. The human bronchial epithelial cell line BEAS-2B was exposed to sAA with or without lipopolysaccharide (LPS) treatment, then cell viability, inflammation and apoptosis were evaluated. The effects of TLR2 knockout on sAA + LPS-treated BEAS-2B cells were also determined. Results revealed that sAA treatment reduced cell viability in a concentration-dependent manner and the effect of 500 nM sAA on cell viability was approximately equivalent to LPS. The levels of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, monocyte chemotactic protein (MCP)-1 and IL-6 as well as cell apoptosis and expression of proteins related to apoptosis were significantly increased upon sAA or LPS stimulation. The expression of TLR2 and AP-1 was also elevated in cells challenged with sAA or LPS. Besides, sAA and LPS co-treatment further enhanced the actions of LPS. However, the knockdown of TLR2 obviously blunted the effects of LPS and sAA co-treatment on cell viability, inflammation and apoptosis. Taken together, our results revealed that sAA could exert an enhanced effect on LPS-induced BEAS-2B cells injury via promoting TLR2/AP-1 expression.

Diphenyl diselenide protects motor neurons through inhibition of microglia-mediated inflammatory injury in amyotrophic lateral sclerosis

Microglia-mediated neuroinflammatory response and neuron damage are considered as a self-propelling progressive cycle, being strongly implicated in the progression of neurodegeneration in amyotrophic lateral sclerosis (ALS). Diphenyl diselenide (DPDS), a simple organoselenium compound, has been known to possess multiple pharmacological properties. The purpose of this study was to explore the neuroprotective effects of DPDS against microglia-mediated neuroinflammatory injury in ALS models. We found that DPDS pretreatment inhibited LPS-induced activation of IκB/NF-κB pathway and subsequent release of proinflammatory factors from activated primary hSOD1G93A microglia. Moreover, DPDS suppressed NLRP3 inflammasome activation by decreasing protein nitration via reduction in NO and ROS levels, whose low levels are related to NF-κB inhibition responsible for iNOS and NOX2 down-regulations, respectively. Notably, DPDS-mediated ROS attenuation was not linked to Nrf2 activation in this cellular model. Furthermore, in the absence of activated microglia, DPDS has no significant effect on the individual hSOD1G93A-NSC34 cells; however, in in vitro neuron-microglia conditional culture and co-culture experiments, DPDS protected motor neurons from neurotoxic damage caused by LPS or BzATP-stimulated microglia activation. Above observations suggest that DPDS-afforded neuroprotection is linked to inhibition of microglia-mediated neuroinflammation in ALS, which was further verified in vivo as shown by improvements of motor deficits, prolonged survival, and reduction of motor neuron loss and reactive microgliosis in hSOD1G93A transgenic mouse. Altogether, our results show that DPDS elicited neuroprotection in ALS models through inactivation of microglia by inhibiting IκB/NF-κB pathway and NLRP3 inflammasome activation, suggesting that DPDS may be a promising candidate for potential therapy for ALS.

LPS-induced epithelial barrier disruption via hyperactivation of CACC and ENaC.

Gram-negative bacterial lipopolysaccharide (LPS) increases the susceptibility of cells to pathogenic diseases, including inflammatory diseases and septic syndrome. In our experiments, we examined whether LPS induces epithelial barrier disruption in secretory epithelia and further investigated its underlying mechanism. The activities of Ca2+-activated Cl- channels (CACC) and epithelial Na+ channels (ENaC) were monitored with a short-circuit current using an Ussing chamber. Epithelial membrane integrity was estimated via transepithelial electrical resistance and paracellular permeability assays. We found that the apical application of LPS evoked short-circuit current (Isc) through the activation of CACC and ENaC. Although LPS disrupted epithelial barrier integrity, this was restored with the inhibition of CACC and ENaC, indicating the role of CACC and ENaC in the regulation of paracellular pathways. We confirmed that LPS, CACC, or ENaC activation evoked apical membrane depolarization. The exposure to a high-K+ buffer increased paracellular permeability. LPS induced the rapid redistribution of zonula occludens-1 (ZO-1) and reduced the expression levels of ZO-1 in tight junctions through apical membrane depolarization and tyrosine phosphorylation. However, the LPS-induced epithelial barrier disruption and degradation of ZO-1 were largely recovered by blocking CACC and ENaC. Furthermore, although LPS-impaired epithelial barrier became vulnerable to secondary bacterial infections, this vulnerability was prevented by inhibiting CACC and ENaC. We concluded that LPS induces the disruption of epithelial barrier integrity through the activation of CACC and ENaC, resulting in apical membrane depolarization and the subsequent tyrosine phosphorylation of ZO-1.

Treatment with an Angiopoietin-1 mimetic peptide promotes neurological recovery after stroke in diabetic rats.

AimVasculotide (VT), an angiopoietin‐1 mimetic peptide, exerts neuroprotective effects in type one diabetic (T1DM) rats subjected to ischemic stroke. In this study, we investigated whether delayed VT treatment improves long‐term neurological outcome after stroke in T1DM rats.MethodsMale Wistar rats were induced with T1DM, subjected to middle cerebral artery occlusion (MCAo) model of stroke, and treated with PBS (control), 2 µg/kg VT, 3 µg/kg VT, or 5.5 µg/kg VT. VT treatment was initiated at 24 h after stroke and administered daily (i.p) for 14 days. We evaluated neurological function, lesion volume, vascular and white matter remodeling, and inflammation in the ischemic brain. In vitro, we evaluated the effects of VT on endothelial cell capillary tube formation and inflammatory responses of primary cortical neurons (PCN) and macrophages.ResultsTreatment of T1DM‐stroke with 3 µg/kg VT but not 2 µg/kg or 5.5 µg/kg significantly improves neurological function and decreases infarct volume and cell death compared to control T1DM‐stroke rats. Thus, 3 µg/kg VT dose was employed in all subsequent in vivo analysis. VT treatment significantly increases axon and myelin density, decreases demyelination, decreases white matter injury, increases number of oligodendrocytes, and increases vascular density in the ischemic border zone of T1DM stroke rats. VT treatment significantly decreases MMP9 expression and decreases the number of M1 macrophages in the ischemic brain of T1DM‐stroke rats. In vitro, VT treatment significantly decreases endothelial cell death and decreases MCP‐1, endothelin‐1, and VEGF expression under high glucose (HG) and ischemic conditions and significantly increases capillary tube formation under HG conditions when compared to non‐treated control group. VT treatment significantly decreases inflammatory factor expression such as MMP9 and MCP‐1 in macrophages subjected to LPS activation and significantly decreases IL‐1β and MMP9 expression in PCN subjected to ischemia under HG conditions.ConclusionDelayed VT treatment (24 h after stroke) significantly improves neurological function, promotes vascular and white matter remodeling, and decreases inflammation in the ischemic brain after stroke in T1DM rats.

Chemical Characterization and Biological Activity of Escherichia coli Lipopolysaccharides

Lipopolysaccharides (LPS) are specific components of the cell envelope of gram-negative bacteria, located at the external surface of their outer membrane and performing a number of important physicochemical and biological functions. The widespread in nature are representatives of Enterobacteriaceae family. Among them there are saprotrophic, useful human symbionts, as well as causative agents of acute intestinal infections. The role of saprophytic intestinal microbiota is not limited only to its participation in the digestion process. The endotoxin released as a result of self-renewal of the cell pool of Escherichia coli partially enters the portal blood and performs antigenic stimulation of the macroorganism. In addition, a small amount of endotoxin can also be released by live gram-negative bacteria, which, given the large population of E. coli in the intestine, can create a sufficiently high concentration of endotoxin. Aim. The study of composition and biological activity of lipopolysaccharides of new E. coli strains, found in the human body. Methods. The objects of investigation were strains of Escherichia coli, isolated from healthy patients at the epidemiological center in Kharkiv. Lipopolysaccharides were extracted from dried cells by 45% phenol water solution at 65–68°С by Westphal and Jann method. The amount of carbohydrates was determined by phenol-sulfuric method. Carbohydrate content was determined in accordance to the calibration curve, which was built using glucose as a standard. The content of nucleic acids was determined by Spirin method, protein − by Lowry method. Serological activity of LPS was investigated by double immunodiffusion in agar using the method of Ouchterlony. Results. In all studied E. coli LPS (2884, 2890, 2892), glucose was dominant monosaccharide (40.5, 41.1, 67.3%, respectively). LPS also contained rhamnose (1.8, 22.9, 1.6%, respectively), ribose (3.5, 6.1, 3.6%, respectively) and galactose (4.1, 20.2, 18.3%, respectively). E. coli 2884 LPS also contained arabinose (1.0%) and mannose (44.8%), while E. coli strains 2890 and 2892 LPS contained heptose (9.7 and 7.8%, respectively). Lipid A composition was presented by fatty acids with a carbon chain length from C12 to C18. As the predominant components were 3-hydroxytetradecanoic (39.2–51.3%) as well as tetradecanoic (23.1–28.5%), dodecanoic (8.9–10.9%), hexadecanoic (4.3–7.2%) and octadecanoic (1.8–2.4%) acids. Unsaturated fatty acids: hexadecenoic (2.0–17.9%) and octadecenoic (3.4–4.2%) have been also identified. It was found that octadecanoic and octadecenoic acids were absent in the LPS of 2884 and 2892 strains, respectively. In SDS-PAAG electrophoresis, a bimodal distribution typical for S-forms of LPS was observed. The studied LPS were toxic and pyrogenic. Double immunodiffusion in agar by Ouchterlony revealed that the tested LPS exhibited an antigenic activity in the homologous system. In heterologous system E. coli 2892 LPS had cross reactivity with LPS of E. coli 2890 and М-17. Since the structure of the O-specific polysaccharide (OPS) of E. coli M-17 was established by us earlier, the results of serological reactions make it possible to suggest an analogy of the E. coli 2892 and 2890 OPS structures with that of E. coli М-17 and their belonging to the same serogroup. Conclusions. The study of the composition and biological activity of LPS of new strains of Escherichia coli 2884, 2890 and 2892, isolated from the body of almost healthy patients, expands our knowledge about the biological characteristics of the species.

Characterisation of EFV12 a bio-active small peptide produced by the human intestinal isolate Lactobacillus gasseri SF1109.

EFV12 is a small bioactive peptide produced by Lactobacillus gasseri SF1109, a human intestinal isolate with probiotic features. In this study, EFV12 antimicrobial and anti-inflammatory properties are characterised. In particular, we propose a possible mechanism of action for EFV12 involving bacterial membranes targeting. Moreover, we show that this small peptide is able to bind lipopolysaccharides (LPS) and to counteract its inflammatory insult preventing LPS action on Toll-like receptor 4, thus interfering with extracellular signal-regulated kinase, p38 and Jun N-terminal kinase, mitogen-activated protein kinases signalling pathways. Altogether these observations suggest that the bioactive peptide EFV12 is a good candidate to promote L. gasseri induced gut homeostasis and counteracting intestinal pathogens.

Effect of lipopolysaccharide structure on functional response of whole blood cells

Lipopolysaccharides (LPSs) induce a wide spectrum of functional activities after interaction with blood cells. Effect of structure of toxic LPS from S- and Re-chemotypes of E. coli and/or non-toxic LPS of Rhodobacter capsulatus PG (R. caps.) on activation of neutrophils and monocytes of human whole blood were studied, particularly, expression of TLR4, CD14 and CD11b receptors, phagocytosis of BioParticles Alexa Fluor 488, synthesis of cytokines and chemokines. A leading role of CD11b receptor in phagocytic activity of neutrophils primed by LPS from various E. coli chemotypes was shown. The non-toxic LPS of R. caps. does not affect the efficiency of phagocytosis activity of the neutrophils. The LPS of R. caps. was shown to induce production of TRIF-dependent cytokine IFN-β in human whole blood leukocytes selectively, without activating MyD88-dependent pathway of pro-inflammatory cytokine synthesis, displaying properties of patrial agonist of TLR4. Structure and biological activity of LPS R. caps. allows considering it as a promising immunity stimulating pharmacological agent.

Photosensitizers attenuate LPS-induced inflammation: implications in dentistry and general health.

Antimicrobial photodynamic therapy (aPDT) is a complementary therapeutic modality for periodontal and endodontic diseases, in which Gram-negative bacteria are directly involved. Currently, there are few evidences regarding the effects of aPDT on bacterial components such as lipopolysaccharide (LPS) and it would represent a major step forward in the clinical use of this therapy. In this context, this study aimed to evaluate the efficacy of different photosensitizers (PSs) used in aPDT in LPS inhibition. Four PSs were used in this study: methylene blue (MB), toluidine blue (TBO), new methylene blue (NMB), and curcumin (CUR). Different approaches to evaluate LPS interaction with PSs were used, such as spectrophotometry, Limulus amebocyte lysate (LAL) test, functional assays using mouse macrophages, and an in vivo model of LPS injection. Spectrophotometry showed that LPS decreased the absorbance of all PSs used, indicating interactions between the two species. LAL assay revealed significant differences in LPS concentrations upon pre-incubation with the different PSs. Interestingly, the inflammatory potential of LPS decreased after previous treatment with the four PSs, resulting in decreased secretion of inflammatory cytokines by macrophages. In vivo, pre-incubating curcumin with LPS prevented animals from undergoing septic shock within the established time. Using relevant models to study the inflammatory activity of LPS, we found that all PSs used in this work decreased LPS-induced inflammation, with a more striking effect observed for NMB and curcumin. These data advance the understanding of the mechanisms of LPS inhibition by PSs.

Rosuvastatin protects against coronary microembolization-induced cardiac injury via inhibiting NLRP3 inflammasome-dependent pyroptosis

Abstract Background Coronary microembolization (CME), a common reason for periprocedural myocardial infarction (PMI) after coronary interventions, bears very important prognostic implications. However, the molecular mechanisms related to CME remain largely elusive. Statins have been shown to prevent PMI, but the underlying mechanism has not been identified. Purpose To determine whether NLRP3 inflammasome contributes to CME-induced cardiac injury and to investigate the effect of statin therapy on CME. Methods In vivo study, CME models were established in C57BL/6J mice by injecting 500,000 polyethylene microspheres (diameter 9 μm) into the left ventricle chamber with the occlusion of the ascending aorta. Mice with CME were treated with 40 mg/kg/d rosuvastatin orally or a selective NLRP3 inflammasome inhibitor MCC950 intraperitoneally (20 mg/kg/d). Cardiac function was evaluated by echocardiography. Cardiac histological analysis was performed. The NLRP3 inflammasome and pyroptosis-related genes and proteins were detected. Cardiac injury was evaluated by Heidenhain's iron hematoxylin stain and serum lactate dehydrogenase (LDH) measurement. Cardiac reactive oxygen species (ROS) production and mitochondrial morphology were also determined. In vitro study, H9c2 myoblasts, stimulated with the classic NLRP3 inflammasome activators lipopolysaccharide (LPS) plus nigericin (Nig), were used to reveal underlying mechanisms of the rosuvastatin's effects. Results The expression of NLRP3, caspase-1 and interleukin-1β (IL-1β) were all increased and peaking at 3 days after CME. Mice treated with MCC950 and rosuvastatin showed improved cardiac contractile function and morphological changes, diminished fibrosis and microinfarct size, and reduced serum LDH level. Mechanistically, rosuvastatin decreased the expression of NLRP3, caspase-1, IL-1β and the pyroptosis executor Gasdermin D. Furthermore, reduced ROS level and alleviated mitochondrial damage were observed in rosuvastatin-treated mice. In vitro study, rosuvastatin effectively inhibited the activation of NLRP3 inflammasome induced by LPS and Nig in H9c2 cells, as evidenced by decreased expression of NLRP3, caspase-1, IL-1β and Gasdermin D. Flow cytometry revealed that rosuvastatin rescued LPS and Nig-induced caspase-1 activation from 22.2±1.6% to 4.09±0.85%. Meanwhile, the pyroptosis was also suppressed by rosuvastatin, indicated by the increased cell viability, decreased LDH and propidium iodide uptake. Similarly, rosuvastatin also reduced the level of ROS generation in vitro. Conclusions NLRP3 inflammasome-dependent cardiac pyroptosis plays an important role in CME-induced cardiac injury and its inhibitor exerts cardioprotective effect following CME. We also uncover the anti-pyroptosis role of rosuvastatin in CME, which is associated with regulating mitochondrial ROS. Funding Acknowledgement Type of funding source: None

Temperate Propolis Has Anti-Inflammatory Effects and Is a Potent Inhibitor of Nitric Oxide Formation in Macrophages.

Previous research has shown that propolis has immunomodulatory activity. Extracts from two UK propolis samples were assessed for their anti-inflammatory activities by investigating their ability to alter the production of the cytokines: tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-10 from mouse bone marrow-derived macrophages co-stimulated with lipopolysaccharide (LPS). The propolis extracts suppressed the secretion of IL-1β and IL-6 with less effect on TNFα. In addition, propolis reduced the levels of nitric oxide formed by LPS-stimulated macrophages. Metabolomic profiling was carried out by liquid chromatography (LC) coupled with mass spectrometry (MS) on a ZIC-pHILIC column. LPS increased the levels of intermediates involved in nitric oxide biosynthesis; propolis lowered many of these. In addition, LPS produced an increase in itaconate and citrate, and propolis treatment increased itaconate still further while greatly reducing citrate levels. Moreover, LPS treatment increased levels of glutathione (GSH) and intermediates in its biosynthesis, while propolis treatment boosted these still further. In addition, propolis treatment greatly increased levels of uridine diphosphate (UDP)–sugar conjugates. Overall, the results showed that propolis extracts exert an anti-inflammatory effect by the inhibition of pro-inflammatory cytokines and by the metabolic reprogramming of LPS activity in macrophages.

Pathogen-Associated Molecules from Gut Translocation Enhance Severity of Cecal Ligation and Puncture Sepsis in Iron-Overload β-Thalassemia Mice.

IntroductionSystemic inflammation induced by gut translocation of lipopolysaccharide (LPS), a major component of Gram-negative bacteria, in thalassemia with iron-overload worsens sepsis. However, the impact of (1→3)-β-D-glucan (BG), a major fungal molecule, in iron-overload thalassemia is still unclear. Hence, the influence of BG was explored in 1) iron-overload mice with sepsis induced by cecal ligation and puncture (CLP) surgery; and 2) in bone marrow-derived macrophages (BMMs).MethodsThe heterozygous β-globin-deficient mice, Hbbth3/+ mice, were used as representative thalassemia (TH) mice. Iron overload was generated by 6 months of oral iron administration before CLP surgery- induced sepsis in TH mice and wild-type (WT) mice. Additionally, BMMs from both mouse strains were used to explore the impact of BG.ResultsWithout sepsis, iron-overload TH mice demonstrated more severe intestinal mucosal injury (gut leakage) with higher LPS and BG in serum, from gut translocation, when compared with WT mice. With CLP in iron-overload mice, sepsis severity in TH mice was more severe than WT as determined by survival analysis, organ injury (kidney and liver), bacteremia, endotoxemia, gut leakage (FITC-dextran) and serum BG. Activation by LPS plus BG (LPS+BG) in BMMs and in peripheral blood-derived neutrophils (both WT and TH cells) demonstrated more prominent cytokine production when compared with LPS activation alone. In parallel, LPS+BG also prominently induced genes expression of M1 macrophage polarization (iNOS, TNF-α and IL-1β) in both WT and TH cells in comparison with LPS activation alone. In addition, LPS+BG activated macrophage cytokine production was enhanced by a high dose of ferric ion (800 mM), more predominantly in TH macrophages compared with WT cells. Moreover, LPS+BG induced higher glycolysis activity with similar respiratory capacity in RAW264.7 (a macrophage cell line) compared with LPS activation alone. These data support an additive pro-inflammatory effect of BG upon LPS.ConclusionThe enhanced-severity of sepsis in iron-overload TH mice was due to 1) increased LPS and BG in serum from iron-induced gut-mucosal injury; and 2) the pro-inflammatory amplification by ferric ion on LPS+BG activation.

Nitrotyrosine formation, iNOS and the Na /K -ATPase activities in sepsis: The possible effects of CAPE

Sepsis is a response to infection characterized by the formation of highly reactive oxygen and nitrogen substances. The rat kidney was chosen for this purpose because many important inflammatory mediators, including inducible nitric oxide synthase (iNOS) and nitrotyrosine (nTyr) production, are expressed by kidney cells following either lipopolysaccharide (LPS) or bacterial challenge. The present study was aimed at investigating the relationship between nTyr formation with iNOS and Na+,K+-ATPase activities. We were also aimed at investigating the possible role of caffeic acid phenethyl ester (CAPE) on endogenous nTyr production, Na+,K+-ATPase and iNOS activities in the kidney. In this study, kidney Na+,K+-ATPase activity were maximally inhibited 6h after LPS injection and LPS treatment significantly increased iNOS activity of kidney. The regression analysis negative correlation between Na+,K+-ATPase activity and nTyr levels of LPS treated animals. Na+,K+-ATPase activity were also negatively correlated with iNOS activity in LPS-treated rats. These data suggest that nitric oxide (NO.) and peroxynitrite (ONOO-) contribute to the development of oxidant injury. Furthermore, the source of NO. may be iNOS. iNOS are expressed by the kindey, and their activity may increase following LPS administration. In addition, NO. and ONOO- formation inhibited Na+,K+-ATPase activity. This results also have strongly suggested that bacterial LPS disturbs activity of membran Na+,K+-ATPase that may be an important component leading to the pathological consequences such as renal dysfunction in which the production of reactive nitrogen substance (RNS) are increased as in the case of LPS challenge. CAPE treatment was decreased nTyr production and iNOS activites, was increased Na+,K+-ATPase activity. These data suggest that CAPE treatment contribute to the decrease of oxidant injury.

The role of maternal–foetal interface inflammation mediated by NLRP3 inflammasome in the pathogenesis of recurrent spontaneous abortion

IntroductionApproximately half of the recurrent spontaneous abortions (RSAs) that remain unidentified to date may be closely related to inflammation. Our previous study found excessive NLRP3 inflammasomes in RSA patients. Here, we investigated further the role of inflammasomes in the maternal–foetal interface of RSA patients. MethodsVillous and decidual tissues were collected during uterine curettage. The trophoblast cell line TEV-1 was cultured with lipopolysaccharide (LPS) or low molecular weight heparin (LMWH), and then the macrophage cell line RAW264.7 was treated with trophoblast media. The expression and localisation of inflammasomes in tissues and cells were detected, and the migration and proliferation of cells were analysed. ResultsA significantly increased expression of inflammasomes was observed in RSA tissues compared with those in the normal group, and it was more obvious in villous tissues than in decidual tissues. In TEV-1 cells, after LPS stimulation, the expression of inflammasomes was increased, but the cell activity was decreased, whereas in RAW264.7, both expression of inflammasomes and cell activity were increased in the LPS group. In addition, LMWH could inhibit the action of LPS in above cells. DiscussionIn patients experiencing RSA, abnormal inflammatory response might be mediated by NLRP3 inflammasomes on the maternal–foetal interface, which may reduce trophoblast activity and promote macrophage activity, leading to early embryo implantation failure. LMWH is expected to treat RSA patients by blocking this process.

Bacterial endotoxin lipopolysaccharide enhances synaptic transmission at low-output glutamatergic synapses

The endotoxin lipopolysaccharides (LPS), secreted from gram-negative bacteria, has direct effects on synaptic transmission independent of systemic secondary cytokine responses. High concentration of LPS (500 μg/mL) from Serratia marcescens increased synaptic efficacy at glutamatergic low-output synapses more than for high-output synapses. Over an hour of exposure was not toxic to the preparation and continued to enhance synaptic transmission. A small but significant rapid hyperpolarization of the post-synaptic cells occurred, in addition to a slower enhancement of in the amplitude of evoked excitatory junction potentials. LPS may promote reserve pool vesicles to the readily releasable pool for low-output synapses. The action of LPS at the glutamatergic synapses of the crayfish neuromuscular junction is unique in promoting synaptic transmission as compared to other glutamatergic synapses in Drosophila and mammals, where synaptic transmission is depressed.

Bacterial infections as novel risk factors of severe diabetic retinopathy in individuals with type 1 diabetes

Background/AimsDiabetic retinopathy (DR) is associated and shares many risk factors with other diabetic complications, including inflammation. Bacterial infections, potent inducers of inflammation have been associated with the development of diabetic...

Advanced glycation end products (AGEs) synergistically potentiated the proinflammatory action of lipopolysaccharide (LPS) and high mobility group box-1 (HMGB1) through their direct interactions.

Previously, we found that advanced glycation endproducts (AGEs) directly interact with tumor necrosis factor (TNF)-like weak inducer of apoptosis, a cytokine that controls inflammation, and that this interaction inhibited its action. This finding raised the novel possibility that AGEs alter the function of other cytokines through direct interaction. To investigate this possibility, we performed comprehensive screening for candidates that interacted with AGEs using protein array analysis.The array analysis revealed that high mobility group box-1 (HMGB1) had a markedly high affinity for AGEs. HMGB1 is a representative proinflammatory damage-associated molecular pattern molecule, and is reported to interact with lipopolysaccharide (LPS) directly to exert its inflammatory function. When LPS, HMGB1, and AGEs were mixed, the mobility of HMGB1 had shifted significantly in native PAGE, suggesting that these three molecules formed a triplet complex. The addition of AGEs to the LPS-HMGB1 mixture synergistically potentiated LPS-HMGB1-stimulated TNF-α mRNA expression in macrophage-like RAW264.7 cells. In addition, using receptor knockout clones, the increased proinflammatory response by LPS-HMGB1-AGEs complex was demonstrated to be mediated via Toll-like receptor 4 and receptor for AGEs.Taken together, this study suggested that AGEs carry out their pathophysiological roles by potentiating the LPS-HMGB1-stimulated proinflammatory response through direct interactions.

Expression of proteins linked to Alzheimer’s disease in C6 rat glioma cells under the action of lipopolysaccharide (LPS), nimesulide, resveratrol and citalopram

Abstract Objectives Alzheimer’s disease is complex and involves several proteins. Most affected are Tau protein and amyloid precursor protein (APP) which, when cleaved by the enzymes β-secretase (BACE1) and γ-secretase (Nicastrin), yield the amyloid peptide. Although these processes take place mainly in neurons, it is not exclusive of them, as glia cells also contribute to these processes. The objective of this study was to evaluate the effect of nimesulide, resveratrol and citalopram on C6 (glioma) cells when exposed to Lipopolysaccharide (LPS). Methods Expression levels of the proteins APP, BACE1, COX-2, Nicastrin and Tau-p were evaluated by Western-blot and ELISA in C6 cells by effect of LPS, and the drugs citalopram, nimesulide and resveratrol. Results It was found that LPS is able to hyperphosphorylate Tau in this cell model and the drugs decrease hyperphosphorylation. We also found that the drugs increase the expression of APP, decrease BACE1 and promote the expression of Nicastrin. COX-2 decreases its expression when nimesulide is used. Conclusions Our results suggest that C6 cell line is useful to analyze the effect of pro-inflammatory molecules on tau phosphorylation and APP expression in vitro. The beneficial effect on the reduction of tau hyperphosphorylation shown by citalopram, nimesulide and resveratrol should be taken with caution due to the limitations of the present study and further research on these compounds is needed to determine their therapeutic use in neurodegenerative diseases such as Alzheimer’s disease. Amaç Alzheimer hastalığı karmaşıktır ve birkaç protein içerir. En çok etkilenenler, u-sekretaz (BACE1) ve γ-sekretaz (Nicastrin) enzimleri tarafından parçalandığında amiloid peptidi veren Tau proteini ve amiloid öncü proteinidir (APP). Bu süreçler esas olarak nöronlarda gerçekleşmesine rağmen, glia hücreleri de bu süreçlere katkıda bulunduğundan, bunlardan ayrı değildir. Amaç: Bu çalışmanın amacı, LPS’ye maruz kaldığında nimesulid, resveratrol ve sitalopramın C6 (glioma) hücreleri üzerindeki etkisini değerlendirmektir. Gereç ve Yöntem APP, BACE1, COX-2, Nicastrin ve Tau-p proteinlerinin ekspresyon seviyeleri, LPS’nin etkisi ile C6 hücrelerinde Western-blot ve ELISA ve sitalopram, nimesulid ve resveratrol ilaçları ile değerlendirildi. Bulgular Bu hücre modelinde LPS’nin Tau’yu hiperfosforilat edebildiği ve ilaçların hiperfosforilasyonu azalttığı bulundu. Ayrıca ilaçların APP ekspresyonunu arttırdığını, BACE1’i azalttığını ve Nicastrin ekspresyonunu teşvik ettiğini bulduk. Nimesulid kullanıldığında COX-2 ekspresyonunu azaltır. Sonuçlar Sonuçlarımız, pro-enflamatuar moleküllerin tau fosforilasyonu ve in vitro APP ekspresyonu üzerindeki etkisini analiz etmek için C6 hücre hattının yararlı olduğunu göstermektedir. Sitalopram, nimesulid ve resveratrol tarafından gösterilen tau hiperfosforilasyonunun azaltılması üzerindeki yararlı etki, mevcut çalışmanın sınırlamaları nedeniyle dikkatle alınmalı ve bu bileşikler üzerinde, Alzheimer hastalığı gibi nörodejeneratif hastalıklarda terapötik kullanımlarının belirlenmesi için daha fazla araştırmaya ihtiyaç vardır.

Impact of lipopolysaccharide administration on luteinizing hormone/choriogonadotropin receptor (Lhcgr) expression in mouse ovaries

Lipopolysaccharide (LPS) is isolated from the genital tract of animals suffering from uterine damage and ovarian dysfunction. This study provides direct molecular evidence about the mechanism through which endotoxins cause reproductive disorders. Granulosa cells and ovaries were collected from immature mice treated with eCG or with eCG and LPS injection intraperitoneally. Normal large antral follicles were observed in ovaries obtained from eCG and LPS coinjected mice, and the morphology of the ovaries was similar to that observed in the control group. These antral follicles were not deemed atretic because few TUNEL-positive cells were observed. However, the granulosa cells of large antral follicles did not acquire the ability to respond to hCG stimulation. The number of ovulated oocytes was significantly lower in LPS-injected mice after superovulation compared to mice that were not exposed to LPS. The low reactivity was caused by the limited expression of the Lhcgr gene, which encodes the LH receptor in granulosa cells as well as an LPS-induced increase in the level of Dnmt1 expression. The methylation rate of the Lhcgr promoter region was significantly higher in granulosa cells obtained from the LPS treatment group compared with the control group. Together, these findings demonstrated that the decrease in the expression of Lhcgr due to LPS was a result of the epigenetic regulatory action of LPS. Our studies suggest that ovarian follicular cysts that is characterized by bacterial infection in humans and animals, is closely connected to the level of methylation of the Lhcgr promoter region.