LPS-induced Secretion Of Pro-inflammatory Cytokines Research Articles

Cepharanthine Alleviates DSS-Induced Ulcerative Colitis via Regulating Aconitate Decarboxylase 1 Expression and Macrophage Infiltration.

Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid from tubers of Stephania, protects against some inflammatory diseases. Aconitate decarboxylase 1 (ACOD1) is also known as immune-responsive gene 1 (IRG1), which plays an important immunometabolism role in inflammatory diseases by mediating the production of itaconic acid. ACOD1 exhibits abnormal expression in ulcerative colitis (UC). However, whether CEP can combat UC by affecting ACOD1 expression remains unanswered. This study was designed to explore the protective effects and mechanisms of CEP in treating colitis through in vitro and in vivo experiments. In vitro assays indicated that CEP inhibited LPS-induced secretion of pro-inflammatory cytokines and ACOD1 expression in RAW264.7 macrophages. Additionally, in the mouse model of DSS-induced colitis, CEP decreased macrophage infiltration and ACOD1 expression in colon tissue. After treatment with antibiotics (Abx), the expression of ACOD1 changed with the composition of gut microbiota. Correlation analysis also revealed that Family-XIII-AD3011-group and Rumini-clostridium-6 were positively correlated with ACOD1 expression level. Additionally, data of the integrative Human Microbiome Project (iHMP) showed that ACOD1 was highly expressed in the colon tissue of UC patients and this expression was positively correlated with the severity of intestinal inflammation. Collectively, CEP can counter UC by modulating gut microbiota and inhibiting the expression of ACOD1. CEP may serve as a potential pharmaceutical candidate in the treatment of UC.

PLCE1 alleviates lipopolysaccharide-induced acute lung injury by inhibiting PKC and NF-κB signaling pathways.

Acute lung injury (ALI) is a clinical syndrome characterized by hyperosmotic pulmonary edema and increased alveolar fluid. Phospholipase C epsilon-1 (PLCE1), identified as a member of phospholipase family, and the relationship between PLCE1 and lung injury is not clear. To assess the possible role of Phospholipase C Epsilon 1 (PLCE1) in Acute lung injury (ALI) progression and related mechanisms. The effects of LPS and PLCE1 on cell viability and apoptosis were examined by MTT and flow cytometry. Also, the level of PLCE1 was controlled by transfection of its plasmid and shRNA. The inflammatory response in response to PLCE1 overexpression or ablation was analyzed by quantitative PCR and ELISA assay. And the involvement of PKC and NF-κB signal pathway were detected by Immunoblot. In this study, we developed a LPS-induced ALI cell model. We found PLCE1 was upregulated in LPS-induced pneumonia cells and affected cell viability. Also, knockdown of PLCE1 reduced LPS-induced apoptosis of pneumonia cells. In addition, depletion of PLCE1 suppressed LPS-induced secretion of proinflammatory cytokines in pneumonia cells. Mechanically, we found depletion of PLCE1 inhibited PKC and NF-κB signal pathway, and therefore alleviated LPS-induced ALI. We therefore thought PLCE1 could serve as a promising drug for ALI.

Translation of IRF-1 Restricts Hepatic Interleukin-7 Production to Types I and II Interferons: Implications for Hepatic Immunity.

Interleukin-7 (IL-7) is an important cytokine with pivotal pro-survival functions in the adaptive immune system. However, the role of IL-7 in innate immunity is not fully understood. In the present study, the impact of hepatic IL-7 on innate immune cells was assessed by functional experiments as well as in patients with different stages of liver cirrhosis or acute-on-chronic liver failure (ACLF). Human hepatocytes and liver sinusoidal endothelial cells secreted IL-7 in response to stimulation with interferons (IFNs) of type I and II, yet not type III. De novo translation of interferon-response factor-1 (IRF-1) restricted IL-7 production to stimulation with type I and II IFNs. LPS-primed human macrophages were identified as innate immune target cells responding to IL-7 signaling by inactivation of Glycogen synthase kinase-3 (GSK3). IL-7-mediated GSK3 inactivation augmented LPS-induced secretion of pro-inflammatory cytokines and blunted LPS tolerance of macrophages. The IFN-IRF-1-IL-7 axis was present in liver cirrhosis patients. However, liver cirrhosis patients with or without ACLF had significantly lower concentrations of IL-7 in serum compared to healthy controls, which might contribute to LPS-tolerance in these patients. In conclusion, we propose the presence of an inflammatory cascade where IFNs of type I/II induce hepatocellular IL-7 in an IRF-1-restriced way. Beyond its role in adaptive immune responses, IL-7 appears to augment the response of macrophages to LPS and to ameliorate LPS tolerance, which may improve innate immune responses against invading pathogens.

The Mast Cell Is an Early Activator of Lipopolysaccharide-Induced Neuroinflammation and Blood-Brain Barrier Dysfunction in the Hippocampus.

Neuroinflammation contributes to or even causes central nervous system (CNS) diseases, and its regulation is thus crucial for brain disorders. Mast cells (MCs) and microglia, two resident immune cells in the brain, together with astrocytes, play critical roles in the progression of neuroinflammation-related diseases. MCs have been demonstrated as one of the fastest responders, and they release prestored and newly synthesized mediators including histamine, β-tryptase, and heparin. However, temporal changes in MC activation in this inflammation process remain unclear. This study demonstrated that MC activation began at 2 h and peaked at 4 h after lipopolysaccharide (LPS) administration. The number of activated MCs remained elevated until 24 h after LPS administration. In addition, the levels of histamine and β-tryptase in the hippocampus markedly and rapidly increased within 6 h and remained higher than the baseline level within 24 h after LPS challenge. Furthermore, mast cell-deficient KitW-sh/W-sh mice were used to investigate the effects of MCs on microglial and astrocytic activation and blood-brain barrier (BBB) permeability at 4 h after LPS stimulation. Notably, LPS-induced proinflammatory cytokine secretion, microglial activation, and BBB damage were inhibited in KitW-sh/W-sh mice. However, no detectable astrocytic changes were found in WT and KitW-sh/W-sh mice at 4 h after LPS stimulation. Our findings indicate that MC activation precedes CNS inflammation and suggest that MCs are among the earliest participants in the neuroinflammation-initiating events.

Tanshinone IIA protects against lipopolysaccharide-induced lung injury through targeting Sirt1.

This study was designed to investigate the effects and the mechanism of Tanshinone IIA (TIIA) on endotoxic shock-induced lung injury in a mouse model. Mice were administered intraperitoneally with TIIA (10mg/kg) 0.5h before lipopolysaccharide (LPS) challenge and then received additional injections every 24h during the 3-day experimental period. The physiological indexes, thesurvival rate and the parameters for lung injury were examined. The protein levels of Sirt1, and the acetylation and activation of NF-κB p65 were determined.The expression and secretion of pro-inflammatory factors were evaluated, respectively. Treatment with TIIA significantly improved physiological indexes and increased the survival rate of mice in response to LPS challenge. TIIA treatment displayed an obvious up-regulation of Sirt1 protein, in accompany with reduced acetylation and activation of NF-κB p65 following LPS stimulation. In addition, TIIA attenuated LPS-induced lung injury and prevented the expression and secretion of pro-inflammatory factors. However, the protective effects of TIIA were abolished by Sirt1 inhibitor. Tanshinone IIA prevents LPS-induced secretion of pro-inflammatory cytokines thus exerts protective effects against acute lung injury, probably via modulation of Sirt1/NF-κB signalling pathway.

Curative Anti-Inflammatory Properties of Chinese Optimized Yinxieling Formula in Models of Parkinson's Disease.

Parkinson's disease (PD) is marked by the progressive degeneration of dopaminergic neurons (DAN) accompanied by glial activation. Thus, inhibiting glial activation that occurs during this disease could be an effective method for treating PD. Optimized Yinxieling Formula (OYF), a Chinese medicinal formula, which is used to efficiently treat autoimmune disease psoriasis, has been proved to display potential immunomodulatory effects in inflammation-associated diseases. This study assessed the therapeutic benefits of OYF on glial-mediated neuroinflammation and neuroprotection in PD models in vitro and in vivo. First, the results showed that OYF significantly suppresses LPS-induced proinflammatory cytokine secretion and attenuates the overall inflammatory responses in BV-2 cells. Second, in vivo studies confirm that while the validity of our MPTP-induced PD mouse models possesses activated glia and significant neurobehavioral dysfunction, pretreatment with OYF prevents glial activation and ameliorates movement dysfunction in the MPTP-induced PD mouse models as evaluated by the pole and rotarod tests. Third, transcriptomic analyses were carried out to reveal the underlying molecular mechanism of the OYF treatment. Sixteen pathways were significantly upregulated in the OYF-treated PD model mice, including the cytokine-cytokine receptor interaction, cell adhesion molecules, coagulation, and complement cascades. Fifteen pathways were significantly downregulated in the OYF-treated PD model mice, such as the natural killer cell mediated cytotoxicity, hematopoietic cell lineage, phagosome, and others. These pathways share direct or indirect features of immunomodulation, suggesting that the physiological effects of OYF involve key roles of immune and inflammation regulations. Therefore, we prove that OYF is a useful immunomodulatory formula in developing prevention and treatment methods for neurodegenerative disease PD.

Immuno-suppressive function of nucleus-transducible BAF57-ΔPH in T cell activation via degradation of endogenous BAF57.

The BAF57 subunit, an indispensable member of the BAF complex, is functionally implicated in apoptosis, cell cycle, and T cell development through chromosomal remodeling. However, the precise roles of BAF57 in the T cell receptor (TcR)-mediated signaling pathway have not been elucidated. In this study, a nucleus-transducible form of BAF57, absent the proline-rich and HMG domains (ntBAF57-ΔPH), was generated to interfere with the interaction between BAF57 and its binding protein, BAF155. ntBAF57-ΔPH was effectively delivered into mouse CD4+ T cells in a dose- and time-dependent manner, without cellular toxicity. Inhibition of T cell activation by ntBAF57-ΔPH was mediated by its disruption of the interaction between BAF155 and BAF57, leading to the degradation of endogenous BAF57 and BAF155. This phenomenon led to alterations in gene expression similar to those associated with Ciclosporin A treatment. In vivo administration of ntBAF57-ΔPH enhanced survival rate of sepsis-induced mice and reduced the LPS-induced secretion of pro-inflammatory cytokines and the expression of endogenous BAF57. These results reveal a novel function of BAF57 as an essential regulator of T cell activation. ntBAF57-ΔPH represents a novel immune-suppressive drug candidate with potential uses in the treatment of autoimmunity and graft rejection.

TET-2 up-regulation is associated with the anti-inflammatory action of Vicenin-2

Vicenin-2, a C-glycoside flavone that is present in many plant sources, exerts potent anti-inflammatory effects in a number of cell and animal models of inflammation. Ten-eleven translocation (TET)-2 has recently gained considerable attention due to the role it plays in regulating the inflammasome. We studied the ability of Vicenin-2 (V-2) to regulate a range of lipopolysaccharide (LPS) stimulated inflammatory activities in PMA-differentiated THP-1 cells and human primary mononuclear cells. We also investigated the action of V-2 on the secretion of NLRP3 inflammasome regulated cytokines (IL-1β and IL-18) by ELISA, and determined if V-2 can regulate the expression of NLRP3, IL-10, IL-1Ra and TET-2. The effect of V-2 on NF-κB signalling was investigated by fluorescence microscopy and gene reporter assay. Additionally, the effect of V-2 on LPS-induced phosphorylation of IKB-α was also investigated by Western blot analysis.V-2 down-regulated LPS-induced secretion of proinflammatory cytokines (TNF-α and IL-1β), in both THP-1 and primary mononuclear cells. V-2 also decreased the LPS-stimulated secretion of IL-18 in THP-1 cells. V-2 significantly down-regulated TNF-α induced NF-κB reporter activity in HEK293T transfected cells and attenuated IKB-α phosphorylation in THP-1 cells. V-2 treatment also induced enhanced nuclear staining of the p50 subunit and reduced p65 subunit of NF-κB.V-2 treatment alone increased the expression of anti-inflammatory cytokine, IL-10, and the regulator of the inflammasome; IL-1Ra, in the presence of LPS. V-2 also significantly decreased LPS-induced NLRP3 expression while concomitantly increasing TET-2 expression.This study demonstrates that the anti-inflammatory actions of V-2 are associated not only with increased IL-10 and IL-1Ra expression, but also with TET-2 up-regulation. Further work is required to establish if the effects of V-2 can be definitively linked to TET-2 activity and that these actions are mirrored in a range of relevant cell types.

MicroRNA-146a-5p attenuates irradiation-induced and LPS-induced hepatic stellate cell activation and hepatocyte apoptosis through inhibition of TLR4 pathway

Elevated toll-like receptor 4 (TLR4) expression is associated with a high risk of radiation-induced liver disease (RILD). MicroRNA (miR)-146a-5p is a key regulator of lipopolysaccharide (LPS)/TLR4 signaling, but its role in modulation of RILD remains unclear. Here, we found that irradiation and LPS stimulation induced TLR4 and miR-146a-5p expression in the human hepatic stellate cell (HSC) line LX2. Ectopic expression of miR-146a-5p in LX2 inhibited irradiation-induced and LPS-induced pro-inflammatory cytokine secretion and cell proliferation, and promoted cell apoptosis by down-regulating the expression levels of TLR4, interleukin-1 receptor associated kinase 1 (IRAK1), tumor necrosis factor receptor associated factor 6 (TRAF6) and phosphorylation of nuclear factor-kappa B. In addition, the culture medium from the irradiated and LPS-stimulated HSCs transfected with miR-146a-5p significantly attenuated apoptosis in irradiated hepatocytes. Overexpression of miR-146a-5p reduced α-smooth muscle actin production in irradiated and LPS-stimulated LX2 cells, which was associated with inhibition of TRAF6-mediated JNK and Smad2 phosphorylation. Knockdown of TRAF6 or IRAK1 mimicked the effects of miR-146a-5p on HSC function. Furthermore, miR-146a-5p treatment alleviated irradiation-induced and endotoxin-induced hepatic inflammatory response and fibrogenesis in mice through inhibition of the TLR4 signaling pathway. Collectively, this study reveals the anti-pro-inflammatory and anti-fibrotic effects of miR-146a-5p on liver injury, and suggests a potential application of miR-146a-5p in the therapeutic prevention of RILD.

Dual requirement for STAT signaling in dendritic cell immunobiology

Dendritic cells (DC) represent an attractive target for therapeutic manipulation of the immune system and enhancement of insufficient immune response in cancer. STAT family members play key roles in the differentiation and activation of DC, a feature that is currently being exploited in DC-based therapies. We previously reported that the small-molecule Stattic, originally developed as a STAT3-specific inhibitor, also inhibits STAT1 and STAT2 phosphorylation in DC exposed to cytokines or LPS. Aim of this study was to investigate the functional consequences of in vitro treatment with Stattic on DC immunobiology. Interestingly, we observed an opposite effect of Stattic on DC immunophenotype depending on the activation state. While the expression of costimulatory, coinhibitory, MHC class II and CD83 molecules was enhanced in immature DC exposed to Stattic, the LPS induced up-modulation of these molecules was strongly repressed. An effective blockade of LPS-induced secretion of proinflammatory cytokines and capacity to stimulate a Th1 polarization was also observed in the presence of Stattic. Our results indicate that the immunological consequences of STAT inhibition in DC vary depending on the cell activation state. This knowledge is of relevance for anticipating potential effects of STAT-targeted therapeutics, and pursuing selective DC manipulation in clinical applications.

Schisandrin A suppresses lipopolysaccharide-induced inflammation and oxidative stress in RAW 264.7 macrophages by suppressing the NF-κB, MAPKs and PI3K/Akt pathways and activating Nrf2/HO-1 signaling.

Schisandrin A is a bioactive lignan occurring in the fruits of plants of the Schisandra genus that have traditionally been used in Korea for treating various inflammatory diseases. Although the anti-inflammatory and antioxidant effects of lignan analogues similar to schisandrin A have been reported, the underlying molecular mechanisms have remained elusive. In the present study, schisandrin A significantly suppressed the lipopolysaccharide (LPS)-induced production of the key pro-inflammatory mediators nitric oxide (NO) and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2 at the mRNA and protein levels in RAW 264.7 macrophages. Furthermore, schisandrin A was demonstrated to reduce the LPS-induced secretion of pro-inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β; this was accompanied by a simultaneous decrease in the respective mRNA and protein levels in the macrophages. In addition, the LPS- induced translocation of nuclear factor-κB (NF-κB), as well as activation of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3 kinase (PI3K)/Akt pathways were inhibited by schisandrin A. Furthermore, schisandrin A significantly diminished the LPS-stimulated accumulation of intracellular reactive oxygen species, and effectively enhanced the expression of NF erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). These results suggested that schisandrin A has a protective effect against LPS-induced inflammatory and oxidative responses in RAW 264.7 cells by inhibiting the NF-κB, MAPK and PI3K/Akt pathways; these effects are mediated, at least in part, by the activation of the Nrf2/HO-1 pathway. Based on these results, it is concluded that schisandrin A may have therapeutic potential for treating inflammatory and oxidative disorders caused by over-activation of macrophages.

Cordycepin negatively modulates lipopolysaccharide-induced cytokine production by up-regulation of heme oxygenase-1

AimsThe present study is to investigate the effect of cordycepin on the expression of heme oxygenase-1 (HO-1) in lipopolysaccharide (LPS)-activated microphages, as well as its mechanism of action. MethodsMouse RAW264.7 cells were treated with different concentrations of cordycepin for 0–16h. Western blotting was used to determine the expression of HO-1 and the phosphorylation of c-Src and the p47phox subunit of NADPH oxidase. Intracellular reactive oxygen species (ROS) level was determined using H2DCFDA as fluorescent probe. Laser-scanning confocal microscopy was used to visualize the nuclear translocation of NF-E2-related factor 2 (Nrf2). Enzyme-linked immunosorbent assay was performed to measure the inhibitory effect of cordycepin on LPS-induced secretion of tumor necrosis factor-α and interleukin-6. ResultsCordycepin induced the phosphorylation of c-Src and p47phox subunit of NADPH oxidase in RAW264.7 cells. Cordycepin increased the secretion of ROS by activating NADPH oxidase. In addition, cordycepin enhanced the expression of HO-1 in RAW264.7 cells in both dose- and time-dependent manners. Of note, elevated HO-1 expression induced by cordycepin treatment was regulated by c-Src/NADPH oxidase/ROS pathway. HO-1 expression induced by cordycepin was dependent on the activation of Nrf2, which was regulated by c-Src/NADPH oxidase/ROS. Cordycepin reduced LPS-induced secretion of proinflammatory cytokines through up-regulation of HO-1. ConclusionThe present study demonstrates that cordycepin induces the expression of HO-1 in RAW264.7 cells via c-Src/NADPH oxidase/ROS/Nrf2 pathway, and plays an anti-inflammatory role by inhibiting the secretion of cytokines from macrophages.

Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways.

Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H2O2-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expression of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.

Role of Notch signaling during lipopolysaccharide-induced preterm labor.

Notch signaling pathways exert effects throughout pregnancy and are activated in response to TLR ligands. To investigate the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligand Delta-like protein-1, transcriptional repressor hairy and enhancer of split-1, and Notch deregulator Numb were assessed. Preterm labor was initiated on gestation d 14.5 by 1 of 2 methods: 1) inflammation-induced preterm labor: intrauterine injection of LPS (a TLR4 agonist) and 2) hormonally induced preterm labor: subcutaneous injection of mifepristone. Delta-like protein-1, Notch1, and hairy and enhancer of split-1 were elevated significantly, and Numb was decreased in the uterus and placenta of inflammation-induced preterm labor mice but remained unchanged in hormonally induced preterm labor compared with their respective controls. F4/80(+) macrophage polarization was skewed in the uterus of inflammation-induced preterm labor toward M1-positive (CD11c(+)) and double-positive [CD11c(+) (M1) and CD206(+) (M2)] cells. This process is dependent on activation of Notch signaling, as shown by suppression of M1 and M2 macrophage-associated cytokines in decidual macrophages in response to γ-secretase inhibitor (an inhibitor of Notch receptor processing) treatment ex vivo. γ-Secretase inhibitor treatment also diminished the LPS-induced secretion of proinflammatory cytokines and chemokines in decidual and placental cells cultured ex vivo. Furthermore, treatment with recombinant Delta-like protein-1 ligand enhanced the LPS-induced proinflammatory response. Notch ligands (Jagged 1 and 2 and Delta-like protein-4) and vascular endothelial growth factor and its receptor involved in angiogenesis were reduced significantly in the uterus and placenta during inflammation-induced preterm labor. These results suggest that up-regulation of Notch-related inflammation and down-regulation of angiogenesis factors may be associated with inflammation-induced preterm labor but not with hormonally induced preterm labor.

Angiotensin-converting enzyme inhibition attenuates lipopolysaccharide-induced lung injury by regulating the balance between angiotensin-converting enzyme and angiotensin-converting enzyme 2 and inhibiting mitogen-activated protein kinase activation.

Activation of the renin-angiotensin system (angiotensin-converting enzyme [ACE]/angiotensin II [Ang II] and angiotensin-converting enzyme 2 [ACE2]/Ang-1-7) has been implicated in the pathophysiology of inflammatory response and acute lung injury (ALI). Previous studies have shown that the ACE inhibitor captopril (Cap) may be a potent therapeutic drug for ALI. However, the mechanisms of its protective effects on ALI are still largely unknown. In this study, we evaluated the effects of Cap on preventing lipopolysaccharide (LPS)-induced lung injury and further investigated the underlying mechanisms of these protective effects. Rats were intraperitoneally pretreated with Cap (50 mg/kg) 30 min prior to an intravenous administration of LPS (7.5 mg/kg). Furthermore, following a 30-min pretreatment with Cap (10 mol/mL) or combined with the ACE2 inhibitor MLN4760 (10 mol/mL), rat pulmonary microvascular endothelial cells (PMVECs) were stimulated with LPS (1 mg/mL). Captopril pretreatment significantly attenuated LPS-induced pathophysiological changes in the lung, inhibited secretion of tumor necrosis factor α and interleukin 6, reduced the ratio of Ang II to Ang-1-7, and reversed the increased ratio of ACE to ACE2, which was remarkably decreased from 7.07 (LPS only) to 1.71 (LPS + Cap). The protective effects of Cap on ALI were also confirmed by in vitro studies, in which Cap suppressed LPS-induced secretion of proinflammatory cytokines and modulated the expression levels of ACE and ACE2. After Cap pretreatment, the ratio of ACE to ACE2 expression was remarkably decreased from 5.18 (LPS alone) to 1.52 (LPS + Cap). Furthermore, Cap given before LPS administration led to inhibition of p38 mitogen-activated protein kinase (MAPK), ERK (extracellular signal-regulated kinase) 1/2, and JNK (c-Jun N-terminal kinase) phosphorylation in PMVECs, whereas MLN4760 abolished the protective effects of Cap on LPS-induced secretion of proinflammatory cytokines and abolished Cap-induced blockade of p38MAPK, ERK1/2, and JNK phosphorylation. Our findings reveal that Cap exerts protective effects on LPS-induced lung injury and the cytotoxicity of PMVECs, and these effects may, at least in part, regulate the balance of ACE and ACE2 expression and inhibit the activation of MAPKs.

Phosphorylation of Akt Mediates Anti-Inflammatory Activity of 1-p-Coumaroyl β-D-Glucoside Against Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells

Hydroxycinnamic acids have been reported to possess numerous pharmacological activities such as antioxidant, anti-inflammatory, and anti-tumor properties. However, the biological activity of 1-p-coumaroyl β-D-glucoside (CG), a glucose ester derivative of p-coumaric acid, has not been clearly examined. The objective of this study is to elucidate the anti-inflammatory action of CG in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. In the present study, CG significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2 and the protein expression of iNOS and COX-2. CG also inhibited LPS-induced secretion of pro-inflammatory cytokines, IL-1β and TNF-α. In addition, CG significantly suppressed LPS-induced degradation of IκB. To elucidate the underlying mechanism by which CG exerts its anti-inflammatory action, involvement of various signaling pathways were examined. CG exhibited significantly increased Akt phosphorylation in a concentration-dependent manner, although MAPKs such as Erk, JNK, and p38 appeared not to be involved. Furthermore, inhibition of Akt/PI3K signaling pathway with wortmannin significantly, albeit not completely, abolished CG-induced Akt phosphorylation and anti-inflammatory actions. Taken together, the present study demonstrates that Akt signaling pathway might play a major role in CG-mediated anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells.

Abstract 116: Slit-Robo Signaling Regulates Lipopolysaccharide-induced Endothelial Inflammation and Expression of Slit/Robo is Dysregulated During Endotoxemia

Abstract Introduction: Lipopolysaccharide (LPS) is one of the critical factors which induce endothelial inflammation during the pathogenesis of atherosclerosis, endocarditis and sepsis shock induced heart injury. The secretory Slit2 protein and its endothelial receptors Robo1 and Robo4 have been shown to regulate mobility and permeability of endothelial cells, which could be functional in regulating LPS induced endothelial inflammation. Hypothesis: We hypothesized that in addition to regulating permeability and migration of endothelial cells, Slit2-Robo1/4 signaling might regulate other LPS-induced endothelial inflammatory responses. Methods and Results: Using Human Umbilical Vein Endothelial Cells (HUVEC) culture, we observed that Slit2 treatment suppressed LPS-induced secretion of pro-inflammatory cytokines (including GM-CSF), cell adhesion molecule upregulation and monocyte (THP-1 cell) adhesion. With siRNA knock down techniques, we further confirmed that this anti-inflammatory effect is mediated by the interaction of Slit2 with its dominant receptor in endothelial cells, Robo4, though the much lesser expressed minor receptor Robo1 is pro-inflammatory. Our signaling studies showed that downstream of Robo4, Slit2 suppressed inflammatory gene expression by inhibiting the Pyk2 - NF-kB pathway following LPS-TLR4 interaction. In addition, Slit2 can induce a positive feedback to its expression and downregulate the pro-inflammatory Robo1 receptor via mediation of miR-218. Moreover, both in in vitro studies using HUVEC and in vivo mouse model studies indicated that LPS also causes endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 and upregulating the pro-inflammatory Robo1 during endotoxemia, especially in mouse arterial endothelial cells and whole heart. Conclusions: Slit2-Robo1/4 signaling is important in regulation of LPS induced endothelial inflammation, and LPS in turn causes inflammation by interfering with the expression of Slit2, Robo1 and Robo4. This implies that Slit2-Robo1/4 is a key regulator of endothelial inflammation and its dysregulation during endotoxemia is a novel mechanism for LPS induced cardiovascular pathogenesis.

An estrogen receptor dependent mechanism of Oroxylin A in the repression of inflammatory response.

Oroxylin A, a natural flavonoid, is one of the main bioactive compounds that underlie the anti-inflammatory effect of the medicinal herb Scutellaria baicalensis Georgi widely used in southeastern Asia; however, the molecular mechanisms for the therapeutic benefits remain largely unclear. In this study, we found that Oroxylin A induces estrogen-responsive gene expression and promoter activity. In macrophages, Oroxylin A treatment significantly attenuates lipopolysaccharide (LPS)-induced but not basal inflammatory response, including nitric oxide (NO) production and the expression of inflammatory mediators (i.e., iNOS and COX-2) and cytokines (i.e., TNF-α, IL-1β, and IL-6), in an estrogen receptor (ER)-dependent manner. Oroxylin A treatment also dramatically decreases LPS-induced secretion of pro-inflammatory cytokines. Furthermore, the downregulation of all these inflammatory parameters by Oroxylin A was abolished when cells were pretreated with specific ER antagonist. Thus, Oroxylin A is a novel phytoestrogen and exhibits anti-inflammatory effects that are mediated by ER activity.

Dietary Flavonoids as Therapeutics for Preterm Birth: Luteolin and Kaempferol Suppress Inflammation in Human Gestational TissuesIn Vitro

Infection/inflammation is commonly associated with preterm birth (PTB), initiating uterine contractions and rupture of fetal membranes. Proinflammatory cytokines induce matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) and prostaglandins which initiate uterine contractions. Nuclear factor-κB (NF-κB) and activator-protein- (AP-)1 have key roles in the formation of these prolabour mediators. In nongestational tissues, dietary flavonoids such as luteolin and kaempferol inhibit NF-κB, AP-1, and their downstream targets. The aim of this study was to determine if luteolin and kaempferol reduce infection-induced prolabour mediators in human gestational tissues. Fetal membranes were incubated with LPS, and primary amnion cells and myometrial cells were incubated with IL-1β in the absence or presence of luteolin or kaempferol. Luteolin and kaempferol significantly reduced LPS-induced secretion of proinflammatory cytokines (IL-6 and IL-8) and prostaglandins (PGE2 and PGF2α) in fetal membranes, IL-1β-induced COX-2 gene expression and prostaglandin production in myometrium, and IL-1β-induced MMP-9 activity in amnion and myometrial cells. Luteolin and kaempferol decreased IL-1β-induced NF-κB p65 DNA binding activity and nuclear c-Jun expression. In conclusion, luteolin and kaempferol inhibit prolabour mediators in human gestational tissues. Given the central role of inflammation in provoking preterm labour, phytophenols may be a therapeutic approach to reduce the incidence of PTB.

Somatostatin effect on TNFA receptors in Kupffer cells

<h3>Introduction</h3> Somatostatin is a mediator of immune functions and has been used as an antineoplastic agent in animal models and human neoplasias. We have demonstrated that octreotide-a somatostatin analogue- inhibits only LPS-induced secretion of pro-inflammatory cytokines including tumour necrosis factor (TNF) α by Kupffer cells.12 We therefore tested the hypothesis that somatostatin modulates the expression of TNFα receptors and apoptosis of Kupffer cells. <h3>Methods</h3> Rat Kupffer cells were isolated by centrifugal elutriation. TNFR1 and TNFR2 expression was studied by RT-PCR, quantitative PCR, Western Blot and immunofluorescence before and after octreotide pre-incubation. Apoptosis was assessed by quantitative measurement of cytoplasmic histone-associated DNA fragments (Roche). TNFα mRNa expression was assessed by a semiquantitative PCR and TNFα was measured in cell supernatants by ELISA. <h3>Results</h3> TNFR1 and TNFR2 m RNA are constitutively expressed in Kupffer cells. Octreotide incubation increased both receptors expression with a peak at 6 h and return to basal levels at 24 h. TNFR1 was mostly influenced. However only TNFR2 protein increase was identified by Western blot, while a band at 90 kD was present instead of a band at 55 kD as expected for TNFR1. TNFa mRNA expression and protein secretion was not influenced by octreotide at 24 h. However a significant inhibition was observed at 48 h. TNF had no effect on Kupffer cell apoptosis while octreotide significantly increased their apoptosis and this effect was not influenced by co-incubation with TNFα. <h3>Conclusion</h3> TNFR1 and TNFR2 are constitutively expressed in Kupffer cells and their expression is strongly increased by somatostatin. Moreover somatostatin increases Kupffer cell apoptosis. These findings may in part explain the antineoplasmatic effect of somatostatin.