LPS-mediated Inflammatory Responses Research Articles

Multifunctional injectable hydrogel with self-supplied H2S release and bacterial inhibition for the wound healing with enhanced macrophages polarization via interfering with PI3K/Akt pathway.

Hydrogen sulfide (H2S) gas therapy is beneficial for accelerating wound healing and alleviating the inflammatory process, but is seriously hindered by insufficient delivery and unsustainable release in vivo. This study presents a multifunctional injectable hydrogel, OC@ε-PL-SATO, composed of oxidized hyaluronic acid and N-acetylcysteine (NAC) as an initiator, carboxymethyl chitosan and S-aroylthiooxime modified ε-Poly-(l-lysine) (ε-PL-SATO). ε-PL-SATO is a NAC-responsive H2S donor. OC@ε-PL-SATO hydrogel is designed for the desired wound healing process, with rapid gelation (<30s) and a sustained H2S release. After mixing and gelling, H2S could be long-term released from the hydrogel and effectively drives macrophages toward M2 polarization, thereby ameliorating the inflammatory response. Revealed by transcriptome analysis, the underlying mechanism is that OC@ε-PL-SATO hydrogel releasing H2S inhibits LPS-mediated inflammatory responses in RAW264.7cells by interfering with phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling and NF-κB activation. Furthermore, the OC@ε-PL-SATO hydrogel effectively eliminates the bacterial burden and alleviates the accompanying inflammation in a rat model of cutaneous wound infection. Importantly, the sustained generation of H2S gas significantly promotes angiogenesis and collagen deposition, ultimately accelerating the wound repair. In conclusion, this study provides a multifunctional injectable hydrogel with rapid gelatinization and continuous H2S release for accelerating the infected wound healing.

Anti-rheumatic property and physiological safety of KMU-11342 in in vitro and in vivo models

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder characterized by joint destruction due to synovial hypertrophy and the infiltration of inflammatory cells. Despite substantial progress in RA treatment, challenges persist, including suboptimal treatment responses and adverse effects associated with current therapies. This study investigates the anti-rheumatic capabilities of the newly identified multi-protein kinase inhibitor, KMU-11342, aiming to develop innovative agents targeting RA. In this study, we synthesized the novel multi-protein kinase inhibitor KMU-11342, based on indolin-2-one. We assessed its cardiac electrophysiological safety using the Langendorff system in rat hearts and evaluated its toxicity in zebrafish in vivo. Additionally, we examined the anti-rheumatic effects of KMU-11342 on human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), THP-1 cells, and osteoclastogenesis in RAW264.7 cells. KMU-11342 demonstrated the ability to inhibit LPS-induced chemokine inhibition and the upregulation of pro-inflammatory cytokines, cyclooxygenase-2, inducible nitric oxide synthase, p-IKKα/β, p-NF-κB p65, and the nuclear translocation of NF-κB p65 in RA-FLS. It effectively suppressed the upregulation of NLR family pyrin domain containing 3 (NLRP3) and caspase-1 cleavage. Furthermore, KMU-11342 hindered the activation of osteoclast differentiation factors such as RANKL-induced TRAP, cathepsin K, NFATc-1, and c-Fos in RAW264.7 cells. KMU-11342 mitigates LPS-mediated inflammatory responses in THP-1 cells by inhibiting the activation of NLRP3 inflammasome. Notably, KMU-11342 exhibited minimal cytotoxicity in vivo and electrophysiological cardiotoxicity ex vivo. Consequently, KMU-11342 holds promise for development as a therapeutic agent in RA treatment.

Transient receptor potential vanilloid 1 interacts with Toll-like receptor 4 (TLR4)/cluster of differentiation 14 (CD14) signaling pathway in lipopolysaccharide-mediated inflammation in macrophages

Transient receptor potential vanilloid 1 (TRPV1), a ligand-gated cation channel, is a receptor for vanilloids on sensory neurons and is also activated by capsaicin, heat, protons, arachidonic acid metabolites, and inflammatory mediators on neuronal or non-neuronal cells. However, the role of the TRPV1 receptor in pro-inflammatory cytokine secretion and its potential regulatory mechanisms in lipopolysaccharide (LPS)-induced inflammation has yet to be entirely understood. To investigate the role and regulatory mechanism of the TRPV1 receptor in regulating LPS-induced inflammatory responses, bone marrow-derived macrophages (BMDMs) harvested from wild-type (WT) and TRPV1 deficient (Trpv1-/-) mice were used as the cell model. In WT BMDMs, LPS induced an increase in the levels of tumor necrosis factor-α, IL-1β, inducible nitric oxide synthase, and nitric oxide, which were attenuated in Trpv1-/- BMDMs. Additionally, the phosphorylation of inhibitor of nuclear factor kappa-Bα and mitogen-activated protein kinases, as well as the translocation of nuclear factor kappa-B and activator protein 1, were all decreased in LPS-treated Trpv1-/- BMDMs. Immunoprecipitation assay revealed that LPS treatment increased the formation of TRPV1-Toll-like receptor 4 (TLR4)-cluster of differentiation 14 (CD14) complex in WT BMDMs. Genetic deletion of TRPV1 in BMDMs impaired the LPS-triggered immune-complex formation of TLR4, myeloid differentiation protein 88, and interleukin-1 receptor-associated kinase, all of which are essential regulators in LPS-induced activation of the TLR4 signaling pathway. Moreover, genetic deletion of TRPV1 prevented the LPS-induced lethality and pro-inflammatory production in mice. In conclusion, the TRPV1 receptor may positively regulate the LPS-mediated inflammatory responses in macrophages by increasing the interaction with the TLR4-CD14 complex and activating the downstream signaling cascade.

Tirzepatide attenuates lipopolysaccharide-induced left ventricular remodeling and dysfunction by inhibiting the TLR4/NF-kB/NLRP3 pathway

BackgroundsSepsis-induced cardiac dysfunction is a leading cause of mortality in intensive care units. Tirzepatide, a dual glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor agonist, possess cardio-protective, their effects on sepsis-induced cardiomyopathy remain unknown. MethodsC57BL/6 mice received subcutaneous injections of tirzepatide once a day for 14 days before subjected to LPS challenge for 12 h. LPS-induced cardiac dysfunction and its potential mechanisms were estimated by pathological analysis, echocardiographic measurement, electrocardiography, langendorff-perfused heart and molecular analysis. ResultsPretreatment with tirzepatide attenuates LPS-induced cardiac dysfunction. tirzepatide remarkably reduces LPS-mediated inflammatory responses by inhibiting the cardiac protein levels of TNF-α, IL-6, and IL-1B in mice. Interestingly, tirzepatide administration also improves cardiomyocytes apoptosis caused by LPS treatment. Furthermore, the protective roles of irzepatide against LPS-mediated increased inflammatory responses and decreased cardiomyocytes apoptosis are partially blunted by inhibiting TLR4/NF-kB/NLRP3 inflammation signaling. In addition, tirzepatide reduce the susceptibility ventricular arrhythmia in LPS-treated mice. ConclusionIn brief, tirzepatide attenuates LPS-induced left ventricular remodeling and dysfunction by inhibiting the TLR4/NF-kB/NLRP3 pathway.

IL-21 impairs pro-inflammatory activity of M1-like macrophages exerting anti-inflammatory effects on rheumatoid arthritis

Objective：Macrophages are the main source of inflammatory mediators and play important roles in the pathogenesis of rheumatoid arthritis (RA). Interleukin-21 (IL-21) regulates both innate and adaptive immune responses and exerts major effects on inflammatory responses that promote the development of RA. However, its effect on macrophage polarisation remains unclear. Methods：CD14+ monocytes of the peripheral blood of Human healthy donors (HD) and RA, and macrophages of RA synovial fluid (RA-SF MΦs) were isolated. IL-21 receptor (IL-21R) was detected by flow cytometry. Cytokine production by MΦs from different sources pre-treated with IL-21 and/or LPS was measured by real-time polymerase chain reaction (RT-PCR) and ELISA. CD14+ monocytes were differentiated into M1-like and M2-like macrophages via stimulation with GM-CSF, interferon-γ (IFN-γ), and LPS or M-CSF, IL-4, and IL-13, respectively. To determine the effect of IL-21 on macrophage polarisation, macrophage phenotypes, gene expression, and cytokine secretion were detected by flow cytometry, RT-PCR, and ELISA. TLR4 and ERK1/2 were determined by western blotting. Results：IL-21 exerted different effects on LPS-mediated inflammatory responses in various derived MΦs, and inhibited macrophages polarisation to M1-like macrophages and promote their polarisation to M2-like macrophages in HD and RA. Moreover, IL-21 inhibited LPS-mediated secretion of inflammatory cytokines, probably by downregulating the ERK1/2, in RA-SF MΦs. Conclusion：For the first time, we indicated that IL-21 inhibits LPS-mediated cytokine production in RA-SF MΦs, and impairs pro-inflammatory activity of M1-like macrophages, hereby exerting anti-inflammatory effects on RA. Thus, IL-21 might not be an appropriate therapeutic target for RA.

Anti-neuroinflammatory activity of 6,7-dihydroxy-2,4-dimethoxy phenanthrene isolated from Dioscorea batatas Decne partly through suppressing the p38 MAPK/NF-κB pathway in BV2 microglial cells

Ethnopharmacological relevanceThe rhizome of Dioscorea batatas Decne (called Chinses yam) widely distributed in East Asian countries including China, Japan, Korea and Taiwan has long been used in oriental folk medicine owing to its tonic, antitussive, expectorant and anti-ulcerative effects. It has been reported to have anti-inflammatory, antioxidative, cholesterol-lowering, anticholinesterase, growth hormone-releasing, antifungal and immune cell-stimulating activities. Aim of the studyNeuroinflammation caused by activated microglia contributes to neuronal dysfunction and neurodegeneration. In the present study, the anti-neuroinflammatory activity of 6,7-dihydroxy-2,4-dimethoxy phenanthrene (DHDMP), a phenanthrene compound isolated from Dioscorea batatas Decne, was examined in microglial and neuronal cells. Materials and methodsA natural phenanthrene compound, DHDMP, was isolated from the peel of Dioscorea batatas Decne. The anti-neuroinflammatory capability of the compound was examined using the co-culture system of BV2 murine microglial and HT22 murine neuronal cell lines. The expression levels of inflammatory mediators and cytoprotective proteins in the cells were quantified by enzyme-linked immunosorbent assay and Western blot analysis. ResultsDHDMP at the concentrations of ≤1 μg/mL did not exhibit a cytotoxic effect for BV2 and HT22 cells. Rather DHDMP effectively restored the growth rate of HT22 cells, which was reduced by co-culture with lipopolysaccharide (LPS)-treated BV2 cells. DHDMP significantly decreased the production of proinflammatory mediators, such as nitric oxide, tumor necrosis factor-α, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2 in BV2 cells. Moreover, DHDMP strongly inhibited the nuclear translocation of nuclear factor κB (NF-κB) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in BV2 cells. The compound did not affect the levels and phosphorylation of ERK and JNK. Concurrently, DHDMP increased the expression of heme oxygenase-1 (HO-1), an inducible cytoprotective enzyme, in HT22 cells. ConclusionsOur findings indicate that DHDMP effectively dampened LPS-mediated inflammatory responses in BV2 microglial cells by suppressing transcriptional activity of NF-κB and its downstream mediators and contributed to HT22 neuronal cell survival. This study provides insight into the therapeutic potential of DHDMP for inflammation-related neurological diseases.

Protective Effects of Curcumin against Lipopolysaccharide-Induced Toxicity.

Lipopolysaccharide (LPS), a Gram-negative bacterial cell wall component, evokes intensive inflammatory responses in the human body. Naturally, inflammation is a part of the host immune response to an infection; nonetheless, an exaggerated response can lead to a series of pathophysiological consequences, collectively known as LPS toxicity or septic shock. This review will explore the cellular and experimental investigations that mainly focus on Curcumin's therapeutic effects on the LPS-mediated inflammatory responses. A literature review of all relevant studies was performed. Curcumin has been reported to exert anti-inflammatory properties by interfering with LPS-induced inflammatory pathways, including binding to cell surface receptors of LPS, NF-kB activation pathway, and inflammasome activation. Further clinical studies on the effect of Curcumin in reducing the pathophysiological consequences of LPS toxicity would substantiate the use of this molecule for future therapeutic approaches.

KMU-1170, a Novel Multi-Protein Kinase Inhibitor, Suppresses Inflammatory Signal Transduction in THP-1 Cells and Human Osteoarthritic Fibroblast-Like Synoviocytes by Suppressing Activation of NF-κB and NLRP3 Inflammasome Signaling Pathway.

Protein kinases regulate protein phosphorylation, which are involved in fundamental cellular processes such as inflammatory response. In this study, we discovered a novel multi-protein kinase inhibitor, KMU-1170, a derivative of indolin-2-one, and investigated the mechanisms of its inflammation-inhibiting signaling in both THP-1 cells and human osteoarthritic fibroblast-like synoviocytes (FLS). We demonstrated that in THP-1 cells, KMU-1170 inhibited lipopolysaccharide (LPS)-induced upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and, furthermore, suppressed LPS-induced phosphorylation of transforming growth factor-β-activated kinase 1, JNK, ERK, inhibitor of NF-κB kinase α/β (IKKα/β), and NF-κB p65 as well as nuclear translocation of NF-κB p65. Moreover, KMU-1170 suppressed LPS-induced upregulation of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, and, notably, inhibited LPS-induced upregulation of the NLRP3 inflammasome in THP-1 cells. Importantly, KMU-1170 attenuated LPS-mediated inflammatory responses in human osteoarthritic FLS, such as the upregulation of IL-1β, TNF-α, IL-6, iNOS, and COX-2 and the phosphorylation of IKKα/β and NF-κB p65. Collectively, these results suggest that KMU-1170 inhibits inflammatory signal transduction and could be developed as a potential anti-inflammatory agent.

MiR-599 regulates LPS-mediated apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1 in human umbilical vein endothelial cells.

MicroRNA plays an integral role in the development of atherosclerosis. Our study aimed to investigate the roles of miR-599 in lipopolysaccharide (LPS)-induced endothelial damage in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with a miR-599 mimic and negative control, and then exposed to LPS. The expression of miR-599 was detected by quantitative real time-polymerase chain reaction (RT-qPCR). Cell viability was analyzed by CCK-8 assay and trypan blue exclusion assay; the formation of DNA fragments was tested by Cell Death Detection ELISA Plus kit; the incidence of apoptosis was detected by flow cytometry; the expression of p53 and cleaved-caspase 3 (c-caspase 3) was evaluated by western blot. Moreover, the mRNA levels and concentrations of tumour necrosis factor (TNF)-α, interleukin (IL)-6, ICAM-1 and VCAM-1 were assayed by RT-qPCR and ELISA. The results showed that overexpression of miR-599 increased cell viability, reduced DNA fragments, the incidence of apoptosis, as well as the protein levels of p53 and c-caspase 3 in the presence of LPS. TNF-α, IL-6, ICAM-1 and VCAM-1 mRNA levels and concentrations were also decreased upon miR-599 upregulation. In addition, the dual luciferase reporter assay demonstrated that ROCK1 is a direct target of miR-599. MiR-599 overexpression inhibited ROCK1 expression. Induced expression of ROCK1 reversed the roles of miR-599 in apoptosis and inflammation. The gain function of miR-599 function inhibited activation of the JAK2/STAT3 signalling pathway, which was abrogated by overexpression of ROCK1. Taken together, our results indicate that miR-599 attenuates LPS-caused cell apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1.

Protective effect of triterpenes of Ganoderma lucidum on lipopolysaccharide-induced inflammatory responses and acute liver injury

Ganoderma lucidum is a popular medicinal mushroom, which has been used as therapeutic for centuries in traditional Chinese medicine. Although G. lucidum showed strong protective effects in prevention or treatment of a variety of inflammatory diseases, the mechanisms underlying the anti-inflammatory properties of triterpenes of G. lucidum remain undefined. In the current study, we demonstrated that ethanol extract and triterpenes of G. lucidum specifically suppressed LPS-mediated inflammatory responses. Notably, ganodermanontriol inhibited the expressions and interactions of TLR4 and MyD88, NF-κB translocation to nucleus and its DNA binding activity, phosphorylation of p38, ErK1/2 and JNK. In vivo, we showed that ganodermanontriol effectively prevented LPS/D-Galactosamine-induced liver injury by reducing TNF-α and IL-6 production, and decrease of ALT/AST release. Collectively, our results revealed a novel role in inhibition of inflammatory diseases for triterpenes that may act through potential inhibition of TLR4-MyD88-mediated NF-κB and MAPK signaling pathways.

1732-P: ERK1 and ERK2 Are Required for Lipopolysaccharide-Induced Production of Cytokines and Chemokines by Macrophages

Low-grade chronic inflammation may be a factor in the development of insulin resistance. Studies suggest that chronic inflammation may be due in part to changes in composition and function of gut microflora, which provide an intestinal barrier preventing bacterial lipopolysaccharide (LPS) release. Animal and human studies demonstrate that obesity increases gut permeability leading to elevated plasma LPS levels resulting in inflammation and metabolic dysfunction. We hypothesized that insulin resistance could be produced by inflammatory factors secreted by macrophages when exposed to gut-released LPS. We examined the extracellular signal-regulated kinase (ERK) signaling pathway, which may be responsible for the macrophage inflammatory response. We examined whether inhibition of ERK activity blocked LPS-mediated responses in bone marrow derived macrophages (BMDM). To determine which ERK isoform is involved in the regulation of inflammatory factor production, we used siRNA to knockdown ERK1, ERK2, or both. BMDM were treated with LPS (100 ng/ml, 6hr). LPS induced TNF-α, RANTES, MCP-1, and IFN-β production of 106371±18250 pg/ml, 11827 ± 1168 pg/ml, 851 ± 73 pg/ml, and 454 ± 46 pg/ml, respectively. Knockdown of ERK1 decreased the release of TNF-α and RANTES to 26534 ± 1471 pg/ml and 5938 ± 806 pg/ml, respectively. Knockdown of ERK2 decreased the release of TNF-α and RANTES to 11600±3610 pg/ml and 7289 ± 265 pg/ml, respectively. Knockdown of either ERK1 or ERK2 did not decrease the release of MCP-1 and IFN-β. However, double knockdown of ERK1 and ERK2 had the greatest inhibition of TNF-α, RANTES, MCP-1, and IFN-β release (not detected, 1540 ± 240 pg/ml, 549 ± 32 pg/ml, and 31 ± 1 pg/ml, respectively). In summary, knockdown of both ERK isoforms is necessary to completely abrogate the LPS effect in macrophages. We propose that ERK positively regulates LPS-mediated inflammatory responses and inhibition of ERK signaling may protect against development of insulin resistance. Disclosure P.F. Chang: None. S.M. Reyna: None. Funding National Institutes of Health; University of Texas Rio Grande Valley

Isoquercitrin Attenuated Cardiac Dysfunction Via AMPKα-Dependent Pathways in LPS-Treated Mice.

Isoquercitrin (IQC) has been reported to play a protective role in many pathological conditions. Here, the effects of IQC on lipopolysaccharide (LPS)-induced cardiac dysfunction are investigated, exploring its potential molecular mechanisms. C57BL/6 mice or H9c2 cardiomyoblasts are subjected to LPS challenge for 12 h. Pretreatment with IQC attenuates LPS-induced cardiac dysfunction. IQC remarkably reduces LPS-mediated inflammatory responses by inhibiting the mRNA levels of TNF-α, IL6, and MCP1 as well as the protein levels of p-IKKβ, p-IκBα, and p-p65 in vivo and in vitro. Interestingly, IQC administration also improves energy deficiencies caused by LPS, manifesting as significant increases in cardiac and cellular ATP levels. Furthermore, ATP levels increase due to the upregulation of PGC1β and PPAR-α, which enhances fatty acid oxidation in vivo and in vitro. However, the protective roles of IQC against LPS-mediated increased inflammatory responses and decreased acid fatty oxidation are partially blunted by inhibiting AMPKα in vitro, and suppressing AMPKα partially blocks the increased cardiac function elicited by IQC in LPS-treated mice. IQC attenuates LPS-induced cardiac dysfunction by inhibiting inflammatory responses and by enhancing fatty acid oxidation, partially by activating AMPKα. IQC might be a potential drug for sepsis-induced cardiac dysfunction.

Haem oxygenase-1 up-regulation by rosiglitazone via ROS-dependent Nrf2-antioxidant response elements axis or PPARγ attenuates LPS-mediated lung inflammation.

Haem oxygenase-1 (HO-1) is induced by thiazolidinediones including rosiglitazone and exerts anti-inflammatory effects in various models. However, the molecular mechanisms underlying rosiglitazone-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). HO-1 expression was determined by real time-PCR, Western blotting and promoter reporter analyses. Signalling pathways were investigated using pharmacological inhibitors or specific siRNAs. Interactions between nuclear factor erythroid-2-related factor (Nrf2) and antioxidant response elements (ARE) binding site of the HO-1 promoter were investigated with chromatin immunoprecipitation assays. Up-regulation of HO-1 in HPAEpiCs or in mice by rosiglitazone blunted ICAM-1 expression and monocyte adhesion to HPAEpiCs challenged with LPS. Rosiglitazone-induced HO-1 expression was significantly attenuated by NADPH oxidase (NOX) inhibitors (apocynin and diphenyleneiodonium) or ROS scavenger (N-acetyl cysteine). The involvement of NOX activity and ROS generation in rosiglitazone-induced HO-1 expression was confirmed by transfection with p47phox or NOX2 siRNA. Moreover, pretreatment with the inhibitors of c-Src (c-Srci II), proline-rich tyrosine kinase 2 (Pyk2) (PF431396), Akt (Akti VIII) or PPARγ (GW9662) and transfection with siRNA of c-Src, Pyk2, Akt or PPARγ abolished the rosiglitazone-induced HO-1 expression in HPAEpiCs. Subsequently, Nrf2 was activated by phosphorylation of c-Src, Pyk2 and Akt, which turned on transcription of HO-1 gene by binding to AREs binding site and enhancing ARE promoter activity. Rosiglitazone induces HO-1 expression via either NOX/ROS/c-Src/Pyk2/Akt-dependent Nrf2 activation or PPARγ in HPAEpiCs and suppresses LPS-mediated inflammatory responses, suggesting that PPARγ agonists may be useful for protection against pulmonary inflammation.

The CH&lt;SUB&gt;2&lt;/SUB&gt;Cl&lt;SUB&gt;2&lt;/SUB&gt;&lt;i&gt; &lt;/i&gt;Extract Fraction from&lt;i&gt; Ficus erecta&lt;/i&gt; var. &lt;i&gt;sieboldii&lt;/i&gt; (Miq.) King Suppresses Lipopolysaccharide-mediated Inflammatory Responses in Raw264.7 Cells

A phytochemical application of leaves from Ficus erecta var. sieboldii (Miq.) King has not been widely investigated. We determined an anti-inflammatory effect of F. erecta extracts on lipopolysaccharide (LPS)-mediated production through modulation of several pro-inflammatory mediators and prostaglandin E2 (PGE2). Among the F. erecta extracts, the CH2Cl2 fraction (CFE) most effectively suppressed the LPS-mediated production of nitric oxide (NO) in Raw264.7 cells. As determined by immunoblotting and PCR, CFE was shown to have an inhibitory effect on LPS-induced mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, CFE showed significant inhibitory effects on LPS-mediated production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and PGE2 (P2 production were syringaresinol (C1) and 6,7-furano-5-methoxy hydrocoumaric acid (C2). In conclusion, CFE showed an inhibitory effect on LPS-mediated inflammatory responses by suppressing iNOS, COX-2, TNF-α, IL-1β, and IL-6 production. The compounds C1 and C2 showed strong inhibitory effects on LPS-mediated production of PGE2 and may be applicable as starter compounds for developing anti-inflammatory and anti-nociceptive drugs.

γ-Oryzanol suppresses COX-2 expression by inhibiting reactive oxygen species-mediated Erk1/2 and Egr-1 signaling in LPS-stimulated RAW264.7 macrophages

Cyclooxygenase (COX)-2 produces prostanoids, which contribute to inflammatory responses. Nuclear factor (NF)-κB is a key transcription factor mediating COX-2 expression. γ-Oryzanol is an active component in rice bran oil, which inhibits lipopolysaccharide (LPS)-mediated COX-2 expression by inhibiting NF-κB. However, the inhibition of COX-2 expression by γ-oryzanol independently of NF-κB is poorly understood. We found that LPS upregulated Egr-1 expression at the transcriptional level. Forced expression of Egr-1 trans-activated the Cox-2 promoter independently of NF-κB. In contrast, silencing of Egr-1 abrogated LPS-mediated COX-2 expression. LPS produced reactive oxygen species (ROS), which, in turn, induced Egr-1 expression via the Erk1/2 MAPK pathway. ROS scavenging activity of γ-oryzanol suppressed Egr-1 expression by inhibiting the Erk1/2 MAPK pathway. Our results suggest that γ-oryzanol inhibits LPS-mediated COX-2 expression by suppressing Erk1/2-mediated Egr-1 expression. This study supports that γ-oryzanol may be useful for ameliorating LPS-mediated inflammatory responses.

Sex Differences in Estrogen Receptor α and β Levels and Activation Status in LPS-Stimulated Human Macrophages.

Immune function, inflammation, and atherosclerosis display sex differences and are influenced by 17β-estradiol through estrogen receptors subtypes ERα and ERβ. Male tissues express active ERs, but their possible involvement in inflammation in males has never been assessed. Macrophages express both ERα and ERβ and offer the opportunity to evaluate the role of ER levels and activation in inflammation. We assessed the ability of lipopolysaccharide (LPS) to modulate, in a sex-specific way, the expression and the activation status of ERα and ERβ in blood monocytes-derived macrophages (MDMs) from men and women. MDMs were incubated with 100 ng/ml LPS for 24 h and used to evaluate ERα, ERβ, P-ERα, p38, and P-p38 expression by Western Blotting. In basal conditions, ERα and ERβ were significantly higher in female MDMs than in male MDMs. LPS up-regulated ERα and ERα phosphorylation in both sexes, with a significantly higher effect observed in male MDMs, and down-regulated ERβ level only in female MDMs. p38 and P-p38 proteins, indicative of ERβ activity, did not show sex differences both in basal conditions and after LPS treatment. Finally, ERα/ERβ and P-ERα/ERα ratios were significantly higher in male MDMs than in female ones. Our data indicate, for the first time, that LPS affects ERα but not ERβ activation status. We identify a significant role of ERα in LPS-mediated inflammatory responses in MDMs, which represents an initial step in understanding the influence of sex in the relationship between LPS and ERα. J. Cell. Physiol. 232: 340-345, 2017. © 2016 Wiley Periodicals, Inc.

LFP-20, a porcine lactoferrin peptide, ameliorates LPS-induced inflammation via the MyD88/NF-κB and MyD88/MAPK signaling pathways

LFP-20 is one of the 20 amino acid anti-microbial peptides identified in the N terminus of porcine lactoferrin. Apart from its extensively studied direct anti-bacterial activity, its potential as an activator of immune-related cellular functions is unknown. Therefore, this study investigated its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated pig alveolar macrophages in vitro and systemic inflammation in an in vivo mouse model. We found that the inhibitory effects of LFP-20 on production of pro-inflammatory cytokines were independent of its LPS-binding activity. However, they were associated with NF-κB and MAPK-dependent signaling. Furthermore, LFP-20 might directly influence MyD88 levels to block its interaction with NF-κB and MAPK-dependent signaling molecules that might alter LPS-mediated inflammatory responses in activated macrophages. Taken together, our data indicated that LFP-20 prevents the LPS-induced inflammatory response by inhibiting MyD88/NF-κB and MyD88/MAPK signaling pathways, and sheds light on the potential use of LFP-20 in the therapy of LPS-mediated sepsis.

Inhibitory Effects of Portulaca Oleracea Ethanol Extract and Glechoma Hederacea Ethanol Extract on the Periodontitis

Both Portulaca oleracea (PO) and Glechoma hederacea (GH) have been used as traditional medicine due to the multiple pharmacological activities. However, the effects of PO and GH in the pathology of periodontitis is still elusive. In this study, we examined anti-microbial activity of PO ethanol extract (POEE) and GH ethanol extract (GHEE) in vitro, and physiological effects of POEE and GHEE on the cell inflammatory responses and the severity of periodontitis were determined using the rat periodontitis model. Our results indicate that POEE and GHEE had no effects on the proliferation of streptococcus mutans and on LPS-mediated inflammatory responses in gingival fibroblast cells. Notably, ingestion of POEE and GHEE resulted in attenuating the severity of periodontitis and population change of immune cells. These data suggests that PO and GH should be considered as candidates for relieving the severity of periondontitis.

Dipterocarpus tuberculatus ethanol extract strongly suppresses in vitro macrophage-mediated inflammatory responses and in vivo acute gastritis

Ethnopharmacological relevanceDipterocarpus tuberculatus Roxb. (Dipterocarpaceae) has been traditionally used to treat various inflammatory symptoms. However, no mechanistic studies on the anti-inflammatory actions of D. tuberculatus have been reported. This study is therefore aimed at exploring the anti-inflammatory effects of 95% ethanol extracts (Dt-EE) of this plant. Materials and methodsThe regulatory activity of Dt-EE and its molecular mechanism on the release of nitric oxide (NO) and prostaglandin (PG)E2 in lipopolysaccharide (LPS)-treated macrophage-like RAW264.7 cells were elucidated by evaluating the activation of transcription factors and their upstream signals and by analyzing the kinase activities of target enzymes. Furthermore, to confirm its availability for oral use, an EtOH/HCl-induced acute gastritis model was tested with this extract. ResultsDt-EE effectively suppressed LPS-mediated inflammatory responses such as the production of NO and PGE2 from macrophages in a dose-dependent manner. In particular, Dt-EE clearly blocked the activation of NF-κB by blocking the phosphorylation of its upstream enzymes IKK and Akt. Using a direct enzyme assay, Dt-EE was shown to block the enzyme activity of PDK1. Finally, this extract also remarkably ameliorated inflammatory lesions in the stomach induced by EtOH/HCl. ConclusionOur data strongly suggest that Dt-EE can be considered as a novel anti-inflammatory remedy with PDK1/NF-κB inhibitory properties and can also be used to treat gastritis symptoms. In addition, our findings can serve as a basis for further phytochemical and pharmacological studies in the future.

Human monocyte responses to lipopolysaccharide and 9-cis retinoic acid after laparoscopic surgery for colon cancer

Surgery, even modern minimal invasive laparoscopic surgery, induces an initial inflammatory and acute phase response which is followed by a period of immunosuppression rendering surgical patients more susceptible to infection. Here, we aimed to study changes in monocyte inflammatory responses and inflammatory modulation mechanisms following laparoscopic colorectal surgery for colon cancer. Blood samples were collected from 19 colon cancer patients before, directly after and daily for 3 days following surgery. Blood cells were exposed ex vivo to bacterial lipopolysaccharide (LPS) or the inflammatory modulator 9-cis retinoic acid (9cisRA). In blood samples taken prior to surgery, we found significant pro-inflammatory responses to LPS, indicating classical monocyte activation. Directly after surgery, LPS induced significantly less early pro-inflammatory cytokines and monocyte/granulocyte-attracting chemokines. The LPS-mediated release of interleukin (IL)-1β was still significantly attenuated 3 days after surgery. In patient monocytes collected after surgery, we found increased levels of suppressors of cytokine signaling (SOCS)1 and SOCS3 mRNA, reported to be associated with polarization towards resolving macrophages. The retinoic acid isomer 9cisRA, reported to attenuate LPS-mediated inflammatory responses and alter chemokine responses in cultured monocytes, had a similar effect in patient blood. Three days after surgery, 9cisRA still attenuated pro-inflammatory responses, but the induction of monocyte chemoattractive protein (MCP)-1/CCL2 mRNA in monocytes was reduced. This study indicates changes in monocyte responses that last for at least 3 days after laparoscopic surgery.