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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Membrane insertion for the detection of lipopolysaccharides: Exploring the dynamics of amphiphile-in-lipid assays

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assays to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. In addition, we also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Leveraging microfluidic dielectrophoresis to distinguish compositional variations of lipopolysaccharide in E. coli

Lipopolysaccharide (LPS) is the unique feature that composes the outer leaflet of the Gram-negative bacterial cell envelope. Variations in LPS structures affect a number of physiological processes, including outer membrane permeability, antimicrobial resistance, recognition by the host immune system, biofilm formation, and interbacterial competition. Rapid characterization of LPS properties is crucial for studying the relationship between these LPS structural changes and bacterial physiology. However, current assessments of LPS structures require LPS extraction and purification followed by cumbersome proteomic analysis. This paper demonstrates one of the first high-throughput and non-invasive strategies to directly distinguish Escherichia coli with different LPS structures. Using a combination of three-dimensional insulator-based dielectrophoresis (3DiDEP) and cell tracking in a linear electrokinetics assay, we elucidate the effect of structural changes in E. coli LPS oligosaccharides on electrokinetic mobility and polarizability. We show that our platform is sufficiently sensitive to detect LPS structural variations at the molecular level. To correlate electrokinetic properties of LPS with the outer membrane permeability, we further examined effects of LPS structural variations on bacterial susceptibility to colistin, an antibiotic known to disrupt the outer membrane by targeting LPS. Our results suggest that microfluidic electrokinetic platforms employing 3DiDEP can be a useful tool for isolating and selecting bacteria based on their LPS glycoforms. Future iterations of these platforms could be leveraged for rapid profiling of pathogens based on their surface LPS structural identity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Diet-induced obesity reprograms the inflammatory response of the murine lung to inhaled endotoxin

The co-occurrence of environmental factors is common in complex human diseases and, as such, understanding the molecular responses involved is essential to determine risk and susceptibility to disease. We have investigated the key biological pathways that define susceptibility for pulmonary infection during obesity in diet-induced obese (DIO) and regular weight (RW) C57BL/6 mice exposed to inhaled lipopolysaccharide (LPS). LPS induced a strong inflammatory response in all mice as indicated by elevated cell counts of macrophages and neutrophils and levels of proinflammatory cytokines (MDC, MIP-1γ, IL-12, RANTES) in the bronchoalveolar lavage fluid. Additionally, DIO mice exhibited 50% greater macrophage cell counts, but decreased levels of the cytokines, IL-6, TARC, TNF-α, and VEGF relative to RW mice. Microarray analysis of lung tissue showed over half of the LPS-induced expression in DIO mice consisted of genes unique for obese mice, suggesting that obesity reprograms how the lung responds to subsequent insult. In particular, we found that obese animals exposed to LPS have gene signatures showing increased inflammatory and oxidative stress response and decreased antioxidant capacity compared with RW. Because signaling pathways for these responses can be common to various sources of environmentally induced lung damage, we further identified biomarkers that are indicative of specific toxicant exposure by comparing gene signatures after LPS exposure to those from a parallel study with cigarette smoke. These data show obesity may increase sensitivity to further insult and that co-occurrence of environmental stressors result in complex biosignatures that are not predicted from analysis of individual exposures. - Highlights: ► Obesity modulates inflammatory markers in BAL fluid after LPS exposure. ► Obese animals have a unique transcriptional signature in lung after LPS exposure. ► Obesity elevates inflammatory stress and reduces antioxidant capacity in the lung. ► Toxicant-specific biomarkers predict exposure independent of systemic inflammation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Long noncoding RNA HAGLROS regulates cell apoptosis and autophagy in lipopolysaccharides-induced WI-38 cells via modulating miR-100/NF-κB axis

Highlights: • LncRNA HAGLROS was higher expressed in patients with pneumonia. • LPS caused WI-38 cell injury and increased HAGLROS levels. • Downregulation of HAGLROS alleviated LPS-induced WI-38 cell injury by miR-100. • NFΚB3 was verified as a functional target of miR-100. • Knockdown of HAGLROS inhibited the activation of PI3K/AKT/NF-κB pathway. Pneumonia is a lower respiratory disease caused by pathogens or other factors. This study aimed to explore the roles and mechanism of long noncoding RNA HAGLROS in lipopolysaccharides (LPS)-induced inflammatory injury in pneumonia. The HAGLROS expression in serum of patients with acute stage pneumonia was detected. To induce pulmonary injury, WI-38 human lung fibroblasts were stimulated with lipopolysaccharides (LPS). The HAGLROS expressions in LPS-treated WI-38 cells and the effects of HAGLROS knockdown on the viability, apoptosis, and autophagy of LPS-induced cells were detected. Moreover, the regulatory relationship between HAGLROS and miR-100 was explored as well as the functional targets of miR-100 were identified. Furthermore, the regulatory relationship between miR-100 and PI3K/AKT/NF-κB pathway was elucidated. LncRNA HAGLROS was higher expressed in serum of patients with acute stage pneumonia compared with that in serum of healthy control. LPS caused WI-38 cell injury and increased HAGLROS levels. Downregulation of HAGLROS alleviated LPS-induced cell injury via increasing cell viability, and inhibiting apoptosis and autophagy. Moreover, there was a negative correlation between HAGLROS and miR-100, and the effects of HAGLROS downregulation on LPS-induced apoptosis and autophagy in WI-38 cells were by regulation of miR-100. Furthermore, NFΚB3 was verified as a functional target of miR-100 and effects of miR-100 inhibition on LPS-induced WI-38 cell injury were alleviated by knockdown of NFΚB3. Besides, Knockdown of HAGLROS inhibited the activation of PI3K/AKT/NF-κB pathway. Our findings reveal that downregulation of HAGLROS may alleviate LPS-induced inflammatory injury in WI-38 cells via modulating miR-100/NF-κB axis. HAGLROS/miR-100/NF-κB axis may provide a new strategy for treating acute stage of pneumonia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Multiple mechanisms involved in diabetes protection by lipopolysaccharide in non-obese diabetic mice

Toll-like receptor 4 (TLR4) activation has been proposed to be important for islet cell inflammation and eventually β cell loss in the course of type 1 diabetes (T1D) development. However, according to the “hygiene hypothesis”, bacterial endotoxin lipopolysaccharide (LPS), an agonist on TLR4, inhibits T1D progression. Here we investigated possible mechanisms for the protective effect of LPS on T1D development in non-obese diabetic (NOD) mice. We found that LPS administration to NOD mice during the prediabetic state neither prevented nor reversed insulitis, but delayed the onset and decreased the incidence of diabetes, and that a multiple-injection protocol is more effective than a single LPS intervention. Further, LPS administration suppressed spleen T lymphocyte proliferation, increased the generation of CD4{sup +}CD25{sup +}Foxp3{sup +} regulatory T cells (Tregs), reduced the synthesis of strong Th1 proinflammatory cytokines, and downregulated TLR4 and its downstream MyD88-dependent signaling pathway. Most importantly, multiple injections of LPS induced a potential tolerogenic dendritic cell (DC) subset with low TLR4 expression without influencing the DC phenotype. Explanting DCs from repeated LPS-treated NOD mice into NOD/SCID diabetic mice conferred sustained protective effects against the progression of diabetes in the recipients. Overall, these results suggest that multiple mechanisms are involved in the protective effects of LPS against the development of diabetes in NOD diabetic mice. These include Treg induction, down-regulation of TLR4 and its downstream MyD88-dependent signaling pathway, and the emergence of a potential tolerogenic DC subset. - Highlights: • Administration of lipopolysaccharide (LPS) prevented type 1 diabetes in NOD mice. • Downregulating TLR4 level and MyD88-dependent pathway contributed to protection of LPS. • LPS administration also hampered DC maturation and promoted Treg differentiation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Anti-inflammatory effects of methylthiouracil in vitro and in vivo

The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases. Here, methylthiouracil (MTU), an antithyroid drug, was examined for its effects on lipopolysaccharide (LPS)-mediated vascular inflammatory responses. The anti-inflammatory activities of MTU were determined by measuring permeability, human neutrophil adhesion and migration, and activation of pro-inflammatory proteins in LPS-activated human umbilical vein endothelial cells and mice. We found that post-treatment with MTU inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs), and adhesion/transendothelial migration of human neutrophils to human endothelial cells. MTU induced potent inhibition of LPS-induced endothelial cell protein C receptor (EPCR) shedding. It also suppressed LPS-induced hyperpermeability and neutrophil migration in vivo. Furthermore, MTU suppressed the production of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, and the activation of nuclear factor-κB (NF-κB) and extracellular regulated kinases (ERK) 1/2 by LPS. Moreover, post-treatment with MTU resulted in reduced LPS-induced lethal endotoxemia. These results suggest that MTU exerts anti-inflammatory effects by inhibiting hyperpermeability, expression of CAMs, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases. - Highlights: • MTU reduced LPS-mediated hyperpermeability in vitro and in vivo. • MTU inhibited LPS-mediated leukocyte adhesion and migration. • MTU inhibited LPS-mediated production of IL-6 and TNF-α. • MTU reduced LPS-mediated mortality and lung injury.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Edaravone attenuates lipopolysaccharide-induced acute respiratory distress syndrome associated early pulmonary fibrosis via amelioration of oxidative stress and transforming growth factor-β1/Smad3 signaling

Highlights: • The ARDS associated early pulmonary fibrosis in rat can be induced by injecting LPS. • Edaravone has anti-fibrosis activities against ARDS associated lung fibrosis. • The mechanism relies on amelioration of oxidative stress and TGF-β1/Smad3 signaling. Pulmonary fibrosis is responsible for the both short-term and long-term outcomes in patients with acute respiratory distress syndrome (ARDS). There is still no effective cure to improve prognosis. The purpose of this study was to investigate whether edaravone, a free radical scavenger, have anti-fibrosis effects in the rat model of ARDS associated early pulmonary fibrosis by lipopolysaccharide (LPS) administration. Rats were subjected to intravenous injection of LPS, and edaravone was given intraperitoneally after LPS administration daily for 7 consecutive days. LPS treatment rapidly increased lung histopathology abnormalities, coefficient of lung, hydroxyproline and collagen I levels, stimulated myofibroblast differentiation and induced expression of TGF-β1 and activation of TGF-β1/Smad3 signaling as early as day 7 after LPS injection. Moreover, LPS intoxication significantly increased the contents of malondialdehyde (MDA), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), whereas it dramatically decreased superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities from day 1 after LPS treatment. On the contrary, edaravone treatment ameliorated LPS-induced myofibroblast differentiation and pulmonary fibrosis, simultaneously, and attenuated LPS-stimulated oxidative stress and activation of TGF-β1/Smad3 signaling. Collectively, edaravone may attenuate ARDS associated early pulmonary fibrosis through amelioration of oxidative stress and TGF-β1/Smad3 signaling pathway. Edaravone may be a promising drug candidate for the treatment of ARDS-related pulmonary fibrosis in early period.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Disruption of IDH2 attenuates lipopolysaccharide-induced inflammation and lung injury in an α-ketoglutarate-dependent manner

Highlights: • IDH2 KO mice exhibit a decreased susceptibility to LPS-induced lung injury. • Depletion of IDH2 attenuates LPS-induced inflammatory response. • IDH2 disruption inhibits LPS-induced NF-κB signaling via modulation of α-KG levels. Acute lung injury (ALI) is an acute failure of the respiratory system with unacceptably high mortality, for which effective treatment is urgently necessary. Infiltrations by immune cells, such as leukocytes and macrophages, are responsible for the inflammatory response in ALI, which is characterized by excessive production of pro-inflammatory mediators in lung tissues exposed to various pathogen-associated molecules such as lipopolysaccharide (LPS) from microbial organisms. α-Ketoglutarate (α-KG) is a key metabolic intermediate and acts as a pro-inflammatory metabolite, which is responsible for LPS-induced proinflammatory cytokine production through NF-κB signaling pathway. Mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDH2) has been reported as an essential enzyme catalyzing the conversion of isocitrate to α-KG with concurrent production of NAPDH. Therefore, we evaluated the role of IDH2 in LPS-induced ALI using IDH2-deficient mice. We observed that LPS-induced inflammation and lung injury is attenuated in IDH2-deficient mice, leading to a lengthened life span of the mice. Our results also suggest that IDH2 disruption suppresses LPS-induced proinflammatory cytokine production, resulting from an inhibition of the NF-κB signaling axis in an α-KG-dependent manner. In conclusion, disruption of IDH2 leads to a decrease in α-KG levels, and the activation of NF-κB in response to LPS is attenuated by reduction of α-KG levels, which eventually reduces the inflammatory response in the lung during LPS-induced ALI. The present study supports the rationale for targeting IDH2 as an important therapeutic strategy for the treatment of systemic inflammatory response syndromes, particularly ALI.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Inflammation, mucous cell metaplasia, and Bcl-2 expression in response to inhaled lipopolysaccharide aerosol and effect of rolipram

Our previous studies have characterized the inflammatory response of intratracheally instilled lipopolysaccharides (LPS) in F344/N rats. To better reflect the environmentally relevant form of LPS exposure, the present study evaluated the inflammatory response of F344/N rats exposed to LPS by inhalation. Rats were exposed by nose-only inhalation to aerosolized LPS at a median particle diameter of 1 {mu}m and a dose range from 0.08 to 480 {mu}g. Animals were euthanized 72 h post exposure and the inflammatory cell counts and differentials, the cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF), and the changes in intraepithelial stored mucosubstances, mucous cells per mm basal lamina, and Bcl-2-positive mucous cells were quantified. We observed a dose-dependent increase reaching maximum values at the 75 {mu}g LPS dose for the numbers of neutrophils, macrophages and lymphocytes, for the levels of IL-6, IL-1{alpha}, IL-1{beta}, TNF{alpha}, MCP-1 and GRO-KC. In addition, mucous cell metaplasia and the percentage of Bcl-2-positive mucous cells were increased with an increasing deposited LPS dose. When rats were treated with the phosphodiesterase-4 (PDE4) inhibitor, rolipram (10 mg/kg), prior to exposure to aerosolized LPS neutrophil numbers in the BAL were reduced at 8 h but not at 24 or 72 h post LPS exposure. These results demonstrate that exposure to aerosolized LPS resulted in a more potent inflammatory response at lower doses and that inflammation was more uniformly distributed throughout the lung compared to inflammation caused by intratracheal LPS instillation. Therefore, this animal model will be useful for screening efficacy of anti-inflammatory drugs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Nilotinib ameliorates lipopolysaccharide-induced acute lung injury in rats

The present study aimed to investigate the effect of the new tyrosine kinase inhibitor, nilotinib on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and explore its possible mechanisms. Male Sprague-Dawley rats were given nilotinib (10 mg/kg) by oral gavage twice daily for 1 week prior to exposure to aerosolized LPS. At 24 h after LPS exposure, bronchoalveolar lavage fluid (BALF) samples and lung tissue were collected. The lung wet/dry weight (W/D) ratio, protein level and the number of inflammatory cells in the BALF were determined. Optical microscopy was performed to examine the pathological changes in lungs. Malondialdehyde (MDA) content, superoxidase dismutase (SOD) and reduced glutathione (GSH) activities as well as nitrite/nitrate (NO{sub 2}{sup -}/NO{sub 3}{sup -}) levels were measured in lung tissues. The expression of inflammatory cytokines, tumor necrosis factor-{alpha} (TNF-{alpha}), transforming growth factor-{beta}{sub 1} (TGF-{beta}{sub 1}) and inducible nitric oxide synthase (iNOS) were determined in lung tissues. Treatment with nilotinib prior to LPS exposure significantly attenuated the LPS-induced pulmonary edema, as it significantly decreased lung W/D ratio, protein concentration and the accumulation of the inflammatory cells in the BALF. This was supported by the histopathological examination which revealed marked attenuation of LPS-induced ALI in nilotinib treated rats. In addition, nilotinib significantly increased SOD and GSH activities with significant decrease in MDA content in the lung. Nilotinib also reduced LPS mediated overproduction of pulmonary NO{sub 2}{sup -}/NO{sub 3}{sup -} levels. Importantly, nilotinib caused down-regulation of the inflammatory cytokines TNF-{alpha}, TGF-{beta}{sub 1} and iNOS levels in the lung. Taken together, these results demonstrate the protective effects of nilotinib against the LPS-induced ALI. This effect can be attributed to nilotinib ability to counteract the inflammatory cells infiltration and hence ROS generation and regulate cytokine effects. - Research highlights: > The protective effects of nilotinib against LPS-induced ALI in rats were studied. > Nilotinib showed potent anti-inflammatory activity as it attenuated PMN infiltration and hence ROS generation. > In addition, nilotinib caused down-regulation of proinflammatory cytokine production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Mechanistic Insights into Surface Chemical Interactions between Lithium Polysulfides and Transition Metal Oxides

The design and development of materials for electrochemical energy storage and conversion devices requires fundamental understanding of chemical interactions at electrode/electrolyte interfaces. For Li–S batteries that hold the promise for outperforming the current generation of Li ion batteries, the interactions of lithium polysulfide (LPS) intermediates with the electrode surface strongly influence the efficiency and cycle life of the sulfur cathode. While metal oxides have been demonstrated to be useful in trapping LPS, the actual binding modes of LPS on 3d transition metal oxides and their dependence on the metal element identity across the periodic table remain poorly understood. Here, we investigate the chemical interactions between LPS and oxides of Mn, Fe, Co, and Cu by combining X-ray photoelectron spectroscopy and density functional theory calculations. We believe that Li–O interactions dominate LPS binding to the oxides (Mn 3 O 4 , Fe 2 O 3 , and Co 3 O 4 ), with increasing strength from Mn to Fe to Co. For Co 3 O 4 , LPS binding also involves metal–sulfur interactions. Moreover, we find that the metal oxides exhibit different binding preferences for different LPS, with Co 3 O 4 binding shorter-chain LPS more strongly than Mn 3 O 4 . In contrast to the other oxides, CuO undergoes intense reduction and dissolution reactions upon interaction with LPS. The reported findings are thus particularly relevant to the design of LPS/oxide interfaces for high-performance Li–S batteries.

25 ENERGY STORAGE↗
DOE OSTI2023

Influence of monsoon low pressure systems on South Asian disasters and implications for disaster prediction

Transient atmospheric vortices called monsoon low pressure systems (LPS) generate a large fraction of total rainfall over South Asia and often produce extreme precipitation. Here, we assess the influence of these storms on the occurrence of disasters, using information from the Emergency Events Database (EM-DAT) that we geocoded and then associated with LPS tracks. We show that more than half of hydro-meteorological disasters over South Asia during summer are associated with these LPS events. Weaker LPS (which are called monsoon lows) occur more frequently than stronger LPS (called monsoon depressions), but the stronger LPS produce a larger number of disasters. Furthermore, although many prior studies have shown that the peak rainfall in LPS falls southwest of the vortex centre, the disasters are concentrated on the northern edge of the LPS tracks, along the Himalayas and upper basins of the Ganga and Brahmaputra rivers. Observations show a sharp peak in rainfall on the day of disasters, confirming the physical link between LPS and these hydro-meteorological disasters. A similar peak in rainfall is found in weather forecasts made up to five days before the disaster, suggesting that short-term precipitation forecasts can be useful in disaster preparation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2013

Lipopolysaccharide-induced inhibition of transcription of tlr4 in vitro is reversed by dexamethasone and correlates with presence of conserved NFκB binding sites

Highlights: ► Chimpanzees, horses and humans have regions of similarity on TLR4 and MD2 promoters. ► Rodents have few regions of similarity on TLR4 promoter when compared to primates. ► Conserved NFkB binding sites were found in the promoters of TLR4 and MD2. ► LPS-induced inhibition of TLR4 transcription is reversed by dexamethasone. ► LPS-induced transcription of MD2 is inhibited by dexamethasone. -- Abstract: Engagement of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) is a master trigger of the deleterious effects of septic shock. Horses and humans are considered the most sensitive species to septic shock, but the mechanisms explaining these phenomena remain elusive. Analysis of tlr4 promoters revealed high similarity among LPS-sensitive species (human, chimpanzee, and horse) and low similarity with LPS-resistant species (mouse and rat). Four conserved nuclear factor kappa B (NFκB) binding sites were found in the tlr4 promoter and two in the md2 promoter sequences that are likely to be targets for dexamethasone regulation. In vitro treatment of equine peripheral blood mononuclear cells (eqPBMC) with LPS decreased transcripts of tlr4 and increased transcription of md2 (myeloid differentiation factor 2) and cd14 (cluster of differentiation 14). Treatment with dexamethasone rescued transcription of tlr4 after LPS inhibition. LPS-induced transcription of md2 was inhibited in the presence of dexamethasone. Dexamethasone alone did not affect transcription of tlr4 and md2.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

3-(Naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride attenuates NLRP3 inflammasome-mediated signaling pathway in lipopolysaccharide-stimulated BV2 microglial cells

Highlights: • KHG26792 attenuated LPS-induced TNF-α, IL-1β, and IL-6 level in BV2 cells. • KHG26792 attenuated LPS-induced ROS and nitrotyrosine level. • KHG26792 attenuated LPS-induced damage in mitochondrial potential. • KHG26792 attenuated LPS-induced expression of NLRP3, activated caspase-1, and ASC. • KHG26792 attenuated LPS-induced ATP level through P2X7 receptor. The nucleotide-binding and oligomerization domain-like receptor containing a pyrin domain 3 (NLRP3) inflammasome is a multiprotein complex with a role in innate immune responses. NLRP3 inflammasome dysfunction is a common feature of chronic inflammatory diseases. Microglia activation is also associated with neuroinflammatory pathologies. We previously reported that 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) reduced hypoxia-induced toxicity by modulating inflammation. However, no studies have elucidated the precise mechanisms for the anti-inflammatory action of KHG26792, in particular via inflammasome mediation. This study investigated the effects of KHG26792 on the inflammasome-mediated signaling pathway in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. KHG26792 significantly attenuated several inflammatory responses including tumor necrosis factor-α, interleukin-1β, interleukin-6, reactive oxygen species, and mitochondrial potential in these cells. KHG26792 also suppressed LPS-induced increase NLRP3, activated caspase-1, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) levels. Furthermore, KHG26792 successfully blocked LPS-activated adenosine triphosphate (ATP) level, likely through the purinergic receptor P2X ligand-gated ion channel 7 (P2X7) receptor. Our results suggest that the anti-inflammatory functions of KHG26792 may be, at least in part, due to regulation of the P2X7R/NLRP3-mediated signaling pathway during microglial activation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Interleukin-18 binding protein attenuates lipopolysaccharide-induced acute lung injury in mice via suppression NF-κB and activation Nrf2 pathway

Highlights: • IL-18BP protectes against LPS-induced ALI. • IL-18BP inhibits the activation of NF-κB in LPS-induced ALI mice. • IL-18BP enhances the activation of Nrf2 in LPS-induced ALI mice. Interleukin (IL)-18 belongs to a rather large IL-1 gene family and is a proinflammatory cytokine. IL-18 plays important roles in lung injury. IL-18 binding protein (IL-18BP), a natural antagonist of IL-18, binds IL-18 with high affinity. IL-18BP is able to neutralize IL-18 biological activity and has a protective effect against renal fibrosis. The aim of this study was to evaluate the potential protective effect of IL-18BP on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and to illuminate the underlying mechanisms. Results indicated that pretreatment with IL-18BP significantly attenuated LPS-induced pulmonary pathological injury. Meanwhile, IL-18BP pretreatment markedly inhibited infiltration of inflammatory cell and release of inflammatory factor in ALI mice in vivo and in primary macrophages after LPS insult in vitro. IL-18BP treatment dramatically reduced oxidative stress through increasing superoxide dismutase (SOD) and glutathione (GSH) contents, and decreasing the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in LPS-induced ALI mice and primary macrophages. Additionally, IL-18BP was also observed to markedly decreased the activation of nuclear factor-kappa B (NF-κB) and upregulated the nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, IL-18BP possessed protective effect against LPS-induced ALI, which might be associated with its regulation of NF-κB and Nrf2 activities. The results rendered IL-18BP worthy of further development into a pharmaceutical drug for the treatment of ALI.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Effects of acteoside on lipopolysaccharide-induced inflammation in acute lung injury via regulation of NF-κB pathway in vivo and in vitro

The purpose of the present study was to investigate the protective role of acteoside (AC) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). BalB/c mice intraperitoneally received AC (30, and 60 mg/kg) or dexamethasone (2 mg/kg) 2 h prior to or after intratracheal instillation of LPS. Treatment with AC significantly decreased lung wet-to-dry weight (W/D) ratio and lung myeloperoxidase (MPO) activity and ameliorated LPS-induced lung histopathological changes. In addition, AC increased super oxide dismutase (SOD) level and inhibited malondialdehyde (MDA) content, total cell and neutrophil infiltrations, and levels of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF) in LPS-stimulated mice. Furthermore, we demonstrated that AC inhibited the phosphorylation of IκBα, nuclear factor-κB (NF-κB) p65, inhibitor of nuclear factor kappa-B kinase-α (IKK-α) and inhibitor of nuclear factor kappa-B kinase-β (IKKβ) in LPS-induced inflammation in A549 cells. Our data suggested that LPS evoked the inflammatory response in lung epithelial cells A549. The experimental results indicated that the protective mechanism of AC might be attributed partly to the inhibition of proinflammatory cytokine production and NF-κB activation. - Highlights: • Acteoside inhibited inflammation in LPS-induced lung injury in mice. • Acteoside inhibited inflammation in lung epithelial cells A549. • Acteoside inhibited NF-kB activation in LPS-induced mice and lung epithelial cells A549.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Glycyrrhetinic acid attenuates lipopolysaccharide-induced fulminant hepatic failure in D-galactosamine-sensitized mice by up-regulating expression of interleukin-1 receptor-associated kinase-M

Glycyrrhetinic acid (GA), the main active ingredient of licorice, reportedly has anti-inflammatory and hepatoprotective properties, but its molecular mechanisms remain be elusive. In the present study, Balb/c mice were pretreated with GA (10, 30, or 100 mg/kg) 1 h before lipopolysaccharide (LPS)/D-galactosamine (D-GalN) administration. In other in vitro experiment, RAW264.7 macrophages were pretreated with GA before LPS exposure. The mortality, hepatic tissue histology, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed. Toll like receptor 4 (TLR4), interleukin-1 receptor-associated kinases (IRAKs), activation of mitogen-activated protein kinases (MAPKs) and NF-κB, and production of TNF-α were assessed by flow cytometry, western blotting, and enzyme-linked immunosorbent assay (ELISA), respectively. Our results showed that pretreatment with GA protected mice against LPS/D-GalN-induced fulminant hepatic failure (FHF), including a dose-dependent alleviation of mortality and ALT/AST elevation, ameliorating hepatic pathological damage, and decreasing TNF-α release. Moreover, GA inhibited LPS-induced activation of MAPKs and NF-κB in response to LPS, but the expression of TLR4 was not affected in vivo and in vitro. Notably, GA pretreatment in vivo suppressed IRAK-1 activity while inducing IRAK-M expression. Silencing of IRAK-M expression with siRNA blocked these beneficial effects of GA on the activation of MAPKs and NF-κB as well as TNF-α production in LPS-primed macrophages. Taken together, we conclude that GA could prevent LPS/D-GalN-induced FHF. The underlying mechanisms may be related to up-regulation of IRAK-M, which in turn caused deactivation of IRAK-1 and subsequent MAPKs and NF-κB, resulting in inhibiting TNF-α production. - Highlights: • Glycyrrhetinic acid protected from LPS/D-GalN-induced liver injury in mice. • Glycyrrhetinic acid inhibited LPS-induced TNF-α production in vivo and in vitro. • Glycyrrhetinic acid alleviated LPS-activated TLR4 signal pathway in vivo and in vitro. • Glycyrrhetinic acid upregulated the expression of IRAK-M in vivo and in vitro. • IRAK-M mediated the protective effect of Glycyrrhetinic acid on LPS-induced inflammation.

60 APPLIED LIFE SCIENCES↗