LPS-induced Interleukin-6 Expression Research Articles

Origin recognition complex subunit 6 (ORC6) is a key mediator of LPS-induced NFκB activation and the pro-inflammatory response

Lipopolysaccharide (LPS)-activated pro-inflammatory responses play a critical role in sepsis, a life-threatening condition. This study investigates the role of origin recognition complex subunit 6 (ORC6) in LPS responses in macrophages and monocytes. Silencing ORC6 using targeted shRNA significantly reduced LPS-induced expression and production of IL-1β (interleukin-1 beta), TNF-α (tumor necrosis factor alpha), and IL-6 (interleukin-6) in THP-1 human macrophages, peripheral blood mononuclear cells (PBMCs), and bone marrow-derived macrophages (BMDMs). Additionally, ORC6 knockout (KO) via the CRISPR/Cas9 method in THP-1 macrophages inhibited LPS-induced pro-inflammatory responses, while ectopic overexpression of ORC6 enhanced LPS-induced expression and production of pro-inflammatory cytokines. ORC6 is crucial for the activation of the nuclear factor kappa B (NFκB) signaling cascade in macrophages and monocytes. LPS-induced NFκB activation was largely inhibited by ORC6 silencing or KO, but potentiated following ORC6 overexpression. Mechanistically, ORC6 associated with nuclear p65 after LPS stimulation, an interaction necessary for NFκB activation. Overexpression of ORC6 did not recover the reduced pro-inflammatory response to LPS in THP-1 macrophages with silenced p65. Furthermore, the NFκB inhibitor BMS-345,541 nearly eliminated the pro-inflammatory response enhanced by ORC6 overexpression in response to LPS. Further studies revealed that ORC6 depletion inhibited NFκB activation induced by double-stranded RNA (dsRNA) and high mobility group box 1 (HMGB1) in THP-1 macrophages. In vivo experiments demonstrated that macrophage-specific knockdown of ORC6 protected mice from LPS-induced septic shock and inhibited LPS-stimulated production of IL-1β, TNF-α, and IL-6 in mouse serum. ORC6 silencing also inhibited LPS-induced NFκB activation in ex vivo cultured PBMCs following macrophage-specific knockdown of ORC6. These findings highlight ORC6 as a pivotal mediator in LPS-induced NFκB activation and the pro-inflammatory response in sepsis, suggesting that targeting ORC6 could be a novel therapeutic strategy for managing sepsis and related inflammatory conditions.

Penehyclidine hydrochloride alleviates LPS-induced inflammatory responses and oxidative stress via ROS/Nrf2/HO-1 activation in RAW264.7 cells.

Inflammation is a biological response of the immune system to harmful stimuli. Penehyclidine hydrochloride (PCH) can alleviate inflammation and oxidative stress by activating reactive oxygen species (ROS), nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase 1 (HO-1) in animal models, but there is a lack of cellular evidence. This study investigated the effects of PHC on lipopolysaccharide (LPS)-induced inflammation response and oxidative stress in RAW264.7 cells. RAW264.7 cells were treated with 1 μg/mL or 5 μg/mL of PHC, with interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-1β, and prostaglandin E2 (PGE2) levels measured with enzyme-linked immunosorbent assay (ELISA) and nitric oxide (NO) measured using the Griess test. Reactive oxygen species were examined with flow cytometry and immunofluorescence, and b-related factor 2 (BRF-2) and NAD(P)H-quinone oxidoreductase 1 (NQO1) using western blot. Penehyclidine hydrochloride partly, but substantially, reversed LPS-related NO and PGE2 production by RAW264.7 cells in a dose-dependent manner and suppressed LPS-induced expression of IL-6, TNF-α and IL-1β messenger ribonucleic acid (mRNA), secretion of IL-6, TNF-α and IL-1β, and ROS production. Lipopolysaccharide stimulation did not affect Nrf2, heme oxygenase 1 (HO-1) or NQO1 protein expression in RWA264.7 cells not treated with PHC. However, PHC treatment significantly elevated Nrf2, HO-1 and NQO1 protein in LPS-treated RWA264.7 cells, an effect that was dose-dependent. The ROS scavenging using N-acetyl-L-cysteine abolished the PHC-induced upregulation of Nrf2 and HO-1. Penehyclidine hydrochloride may alleviate LPS-induced inflammation and oxidative stress by activating Nrf2 signaling in RAW264.7 macrophages. These findings suggest that PHC could alleviate inflammation by targeting activated macrophages.

Sebacic acid, a royal jelly-containing fatty acid, decreases LPS-induced IL-6 mRNA expression in differentiated human THP-1 macrophage-like cells.

Macrophages produce many inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in innate immune responses. However, excess production of these mediators by activated macrophages triggers deleterious effects, leading to disorders associated with inflammation. Royal jelly (RJ), a milky-white substance secreted by worker bees, contains unique fatty acids, including 10-hydroxy-2-decenoic acid (10H2DA) and sebacic acid (SA). 10H2DA has been reported to have various biological functions, such as anti-inflammation. However, the anti-inflammatory effect of SA is not fully understood. In this study, we investigated the effects of SA on lipopolysaccharide (LPS)-induced cytokine expression using differentiated human THP-1 macrophage-like cells. SA dose-dependently decreased LPS-induced mRNA expression of IL-6, but not TNF-α and IL-1β. SA suppressed the phosphorylation of signal transducers and activators of transcription 1 (STAT1) and STAT3, but hardly affected the activation of JNK, p38, or NF-κB. In addition, SA decreased LPS-induced interferon-β (IFN-β) expression, and the addition of IFN-β restored the inhibition by SA of LPS-induced STAT activation and IL-6 expression. Furthermore, SA suppressed LPS-induced nuclear translocation of interferon regulatory factor 3 (IRF3), a transcription factor responsible for IFN-β expression. Taken together, we conclude that SA selectively decreases LPS-induced expression of IL-6 mRNA through inhibition of the IRF3/IFN-β/STAT axis.

Human β-Defensin 3 Inhibits Porphyromonas Gingivalis Lipopolysaccharide-Induced Oxidative and Inflammatory Responses of Microglia by Suppression of Cathepsins B and L.

Recently, the effects of antibacterial peptides are suggested to have therapeutic potential in Alzheimer's disease. Furthermore, systemic treatment of Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) induced Alzheimer's disease-like neuropathological changes in middle-aged mice. Then, we examined whether human β-defensins (hBDs), antimicrobial peptides produced by the oral mucosa and salivary glands, can suppress Pg LPS-induced oxidative and inflammatory responses by microglia. hBD3 (1 μM) significantly suppressed Pg LPS-induced production of nitric oxide and interleukin-6 (IL-6) by MG6 cells, a mouse microglial cell line. hBD3 (1 μM) also significantly inhibited Pg LPS-induced expression of IL-6 by HMC3 cells, a human microglial cell line. In contrast, neither hBD1, hBD2 nor hBD4 failed to inhibit their productions. Furthermore, hBD3 suppressed Pg LPS-induced p65 nuclear translocation through the IκBα degradation. Pg LPS-induced expression of IL-6 was significantly suppressed by E64d, a cysteine protease inhibitor, and CA-074Me, a known specific inhibitor for cathepsin B, but not by pepstatin A, an aspartic protease inhibitor. Interestingly, hBD3 significantly inhibited enzymatic activities of recombinant human cathepsins B and L, lysosomal cysteine proteases, and their intracellular activities in MG6 cells. Therefore, hBD3 suppressed oxidative and inflammatory responses of microglia through the inhibition of cathepsins B and L, which enzymatic activities are necessary for the NF-κB activation.

Lactococcus lactis-fermented spinach juice suppresses LPS-induced expression of adhesion molecules and inflammatory cytokines through the NF-κB pathway in HUVECs.

Spinach (Spinacia oleracea L.), a green leafy vegetable, is widely regarded as a functional food due to its biological activities; however, to the best of our knowledge, there are no previous studies that have investigated the protective effects of fermented spinach against endothelial dysfunction and its underlying mechanisms. Therefore, this study investigated the effects and possible mechanisms of action of fresh spinach juice (S.juice) and fermented S.juice on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). The HUVECs were treated with S.juice and fermented S.juice for 18 h before LPS exposure, and the levels of cytokines and chemokines, such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6), were detected using enzyme-linked immunosorbent assays (ELISA). Furthermore, to examine the changes in inflammatory responses to the two treatments, immunofluorescence analysis was used to visualize the nuclear translocation of nuclear factor-κB (NF-κB). Western blot analysis was also performed to detect the differences in the expression of endothelial cell adhesion molecules, specifically vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Both S.juice and fermented S.juice inhibited the LPS-induced expression of MCP-1 and IL-6, and suppressed VCAM-1 and ICAM-1. Additionally, fermented S.juice inhibited the LPS-induced activation of NF-κB and degradation of the inhibitor of NF-κB (IκBα) in an LPS dose-dependent manner. These results suggest that the anti-inflammatory effect of vitamin K2-enriched fermented S.juice is mediated by the suppression of the NF-κB pathway, suggesting its potential as a novel therapeutic candidate for inflammatory cardiovascular disease.

Atorvastatin suppresses lipopolysaccharide-induced inflammation in human coronary artery endothelial cells

The present study was designed to examine the effects of atorvastatin on vascular inflammatory responses in human coronary artery endothelial cells(HCAECs), when challenged by lipopolysaccharide (LPS), a Toll-like receptor-4 (TLR4) ligand. HCAECs were pretreated with atorvastatin and induced by LPS. The expression of TLR4, interleukin -6(IL-6), monocyte chemoattractant protein 1(MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecular-1(ICAM-1), nuclear factor-κB (NF-κB) and p38 mitogen activated protein kinase(p38 MAPK) were evaluated using Real-time polymerase chain reaction, cytokine ELISA assay and Western blotting. The results showed that pretreatment with atorvastatin down-regulated the expression of TLR4 in LPS-activated HCAECs. Atorvastatin also attenuated the LPS-induced expression of interleukin IL-6 and MCP-1, at both the transcription and translation level in HCAECs. LPS-induced endothelial cell adhesion molecules, ICAM-1 and VCAM-1 expression were also reduced by pretreatment with atorvastatin. Furthermore, atorvastatin efficiently suppressed LPS-induced phosphorylation of NF-κB and p38 MAPK in HCAECs. These findings show that atorvastatin suppresses endothelial cell inflammation, suggesting that atorvastatin may be suitable for development as a therapeutic agent for inflammatory cardiovascular disease.

LncRNA CAIF suppresses LPS-induced inflammation and apoptosis of cardiomyocytes through regulating miR-16 demethylation.

BackgroundThe long noncoding RNA, cardiac autophagy inhibitory factor (CAIF), and microRNA (miR)‐16 are reported to be involved in lipopolysaccharide (LPS)‐induced inflammatory responses and cell apoptosis in many diseases. Herein, we investigated the interaction between CAIF and miR‐16 in sepsis‐induced chronic heart failure (CHF).MethodsThe expression of CAIF and miR‐16 in plasma samples from sepsis‐induced CHF patients (n = 60) and healthy controls (n = 60) were measured using quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR). The correlations between CAIF and miR‐16 across plasma samples from patients with sepsis‐induced CHF and healthy controls were analyzed using linear regression. The messenger RNA (mRNA) levels of inducible nitric oxide synthase, C‐C motif chemokine 2 (CCL2), growth‐regulated alpha protein (CXCL1), and interleukin‐6 (IL‐6) were evaluated using qRT‐PCR while nuclear factor κB activation was evaluated using luciferase assay.ResultsThe expression levels of CAIF and miR‐16 were downregulated in the plasma of sepsis‐induced CHF patients and were positively correlated in these patients. In cardiomyocytes, LPS treatment dose‐dependently decreased CAIF and miR‐16 levels. CAIF overexpression increased miR‐16 expression by demethylating miR‐16. CAIF and/or miR‐16 overexpression suppressed LPS‐induced CCL2, CXCL1, and IL‐6 expression at both the mRNA and protein levels. Analysis of cell apoptosis and western blot analysis showed that CAIF and/or miR‐16 overexpression inhibited LPS‐induced cardiomyocyte apoptosis by reducing Bax and cleaved caspase 3 levels and enhancing Bcl‐2 levels.ConclusionOur study is the first to report the abnormal expression of CAIF and miR‐16 in heart disease. CAIF plays a protective role in sepsis‐induced CHF by inhibiting cardiomyocyte apoptosis and inflammation, possibly by regulating miR‐16 demethylation.

Porphyromonas gingivalis lipopolysaccharide induces interleukin-6 and c-c motif chemokine ligand 2 expression in cultured hCMEC/D3 human brain microvascular endothelial cells.

This paper describes the effect of Porphyromonas gingivalis (Pgingivalis) lipopolysaccharide (LPS) on the expression of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in cultured hCMEC/D3 human brain microvascular endothelial cells. Pgingivalis is one of the important pathogens in periodontitis, and periodontitis is a risk factor for brain disorders including cerebrovascular diseases and Alzheimer's disease. However, the mechanisms underlying the pathogenesis of Pgingivalis-mediated brain diseases are incompletely understood. Effects of Pgingivalis LPS on brain endothelial cells are not known well. The hCMEC/D3 human brain microvascular endothelial cells were cultured and treated with Pgingivalis LPS. The expression of IL-6 and CCL2 mRNA and protein was examined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Effect of inhibitors of Toll-like receptor (TLR) 2, TLR4, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was also investigated. Phosphorylation of NF-κB p65, p38 MAPK and JNK was examined using Western blotting. Pgingivalis LPS-induced mRNA and protein expression of IL-6 and CCL2 in hCMEC/D3 cells in a concentration-dependent manner at the concentration of 0.5-50µg/mL. Maximal mRNA expression of IL-6 and CCL2 was found 2 and 4hours after stimulation, respectively. Induction of IL-6 and CCL2 by Pgingivalis LPS was almost completely inhibited by pretreatment of cells with TLR4 inhibitor but not by TLR2 inhibitor. Treatment of cells with Pgingivalis LPS for up to 2hours induced phosphorylation of NF-κB p65, p38 MAPK and JNK. IL-6 induction was decreased by pretreatment of cells with NF-κB inhibitor SN50 or p38 MAPK inhibitor SB203580, while CCL2 induction was reduced by SN50 or JNK inhibitor SP600125. IL-6 and CCL2 produced upon Pgingivalis LPS stimulation may contribute to the inflammatory reactions in brain endothelial cells and subsequent neurological disorders such as cerebrovascular and Alzheimer's diseases.

Senescent cells exacerbate chronic inflammation and contribute to periodontal disease progression in old mice.

Coinciding with other chronic comorbidities, the prevalence of periodontal disease increases with aging. Mounting evidence has established that senescent cells accumulate at sites of age-related pathologies, where they promote "non-microbial" inflammation. We hypothesized that alveolar bone osteocytes develop senescence characteristics in old age. Alveolar bone samples were obtained from young (6 months) and old (20 to 22 months) mice to evaluate the expression of senescence biomarkers by immunofluorescent staining. Osteocyte-enriched fractions were used to characterize the age-related senescence-associated secretory phenotype (SASP) gene expression profile. Primary alveolar bone cells were exposed to the SASP via in vitro senescent conditioned media (SCM) administration. A multiplex assay confirmed protein levels of specific cytokines. Interactions with bacterial components were evaluated by stimulating cells with lipopolysaccharide (LPS). Increased senescence-associated distension of satellites (SADS) and p16Ink4a mRNA expression were identified in alveolar bone osteocytes with aging. These findings were associated with increased levels of DNA damage, and activated p38 MAPK, both inducers of senescence. Furthermore, interleukin-6 (IL6), IL17, IGFBP4, and MMP13 were significantly upregulated with aging in osteocyte-enriched samples. Interestingly, SCM potentiated the LPS-induced expression of IL1α,IL1β,and IL6. Cell migration and differentiation were also impeded by SCM. These in vitro effects were ameliorated by the p38 MAPK inhibitor SB202190. Accumulation of senescent osteocytes contributes to deterioration of the periodontal environment by exacerbating chronic inflammation and reducing regeneration in old age. Cellular senescence is a cell-intrinsic response to DNA damage, and a host-related mechanism associated with aging that could potentiate inflammation induced by bacterial components.

Sodium Butyrate Alleviates Lipopolysaccharide-Induced Inflammatory Responses by Down-Regulation of NF-κB, NLRP3 Signaling Pathway, and Activating Histone Acetylation in Bovine Macrophages.

Sodium butyrate is the sodium salt of butyric acid, which possesses many biological functions including immune system regulation, anti-oxidant and anti-inflammatory ability. The present study was designed to elucidate the anti-inflammatory effects and mechanisms of sodium butyrate on lipopolysaccharide (LPS)-stimulated bovine macrophages. The effect of sodium butyrate on the cell viability of bovine macrophages was assayed by using the CCK-8 kit. Quantitative real-time PCR (qRT-PCR) was used to detect the gene expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), and inducible Nitric Oxide Synthase (iNOS). NF-κB, NLRP3 signaling pathway, and histone deacetylase were detected by western blotting. The results showed that sodium butyrate had no significant effect on cell viability at 0–1 mM, and inhibited LPS-induced IL-6, IL-1β, COX-2, and iNOS expression. Moreover, sodium butyrate suppressed LPS (5 μg/ml)-stimulated the phosphorylation of IκB and p65, inhibited the deacetylation of histone H3K9, and has also been found to inhibit protein expression in NLRP3 inflammasomes. Thus, our finding suggested that sodium butyrate relieved LPS-induced inflammatory responses in bovine macrophage by inhibiting the canonical NF-κB, NLRP3 signaling pathway, and histone decetylation, which might be helpful to prevent cow mastitis.

Involvement of Receptor for Advanced Glycation Endproducts in Hypertensive Disorders of Pregnancy.

Preeclampsia/hypertensive disorders of pregnancy (PE/HDP) is a serious and potentially life-threatening disease. Recently, PE/HDP has been considered to cause adipose tissue inflammation, but the detailed mechanism remains unknown. We exposed human primary cultured adipocytes with serum from PE/HDP and healthy controls for 24 h, and analyzed mRNA expression of several adipokines, cytokines, and ligands of the receptor for advanced glycation endproducts (RAGE). We found that the mRNA levels of interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), high mobility group box 1 (HMGB1), and RAGE were significantly increased by the addition of PE/HDP serum. Among RAGE ligands, advanced glycation endproducts (AGE) and HMGB1 increased mRNA levels of IL-6 and CCL2 in SW872 human adipocytes and mouse 3T3-L1 cells. The introduction of small interfering RNA for RAGE (siRAGE) into SW872 cells abolished the AGE- and HMGB1-induced up-regulation of IL-6 and CCL2. In addition, lipopolysaccharide (LPS), a ligand of RAGE, increased the expression of IL-6 and CCL2 and siRAGE attenuated the LPS-induced expression of IL-6 and CCL2. These results strongly suggest that the elevated AGE, HMGB1, and LPS in pregnant women up-regulate the expression of IL-6 and CCL2 via the RAGE system, leading to systemic inflammation such as PE/HDP.

Bioactive Constituents from the Roots of Eurycoma longifolia.

Four new phenolic components, eurylophenolosides A (1) and B (2), eurylolignanosides A (3) and B (4), along with twelve known compounds were isolated from the roots of Eurycoma longifolia Jack. The structure of these components was elucidated by using various spectral techniques and chemical reactions. Among the known isolates, syringaldehyde (12), 3-chloro-4-hydroxybenzoic acid (13), 3-chloro-4-hydroxyl benzoic acid-4-O-β-d-glucopyranoside (14), and isotachioside (15) were isolated from the Eurycoma genus for the first time. Further, the NMR data of 14 was reported here firstly. Meanwhile, the nitric oxide (NO) inhibitory activities of all compounds were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells at 40 μM. As results, piscidinol A (6), 24-epi-piscidinol A (7), bourjotinolone A (10), and scopoletin (16) were found to play important role in suppressing NO levels without cytotoxicity. Furthermore, the Western blot method was used to investigate the mechanism of compounds 6, 7, 10, and 16 by analysing the level of inflammation related proteins, such as inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in LPS-stimulated RAW264.7 cells. Consequently, compounds 6, 7, 10, and 16 were found to significantly inhibit LPS-induced protein expression of IL-6, NF-κB and iNOS in NF-κB signaling pathway. Moreover, it was found that the protein expression inhibitory effects of 6, 7, and 16 exhibited in a dose-dependent manner. The mechanism may be related to the inhibition of the iNOS expressions through suppressing the IL-6-induced NF-κB pathway.

Inhibition of the receptor for advanced glycation inhibits lipopolysaccharide-mediated High mobility group protein B1 and Interleukin-6 synthesis in human gingival fibroblasts through the NF-κB signaling pathway

AimsWe investigated the effect of a specific inhibitor of the receptor for advanced glycation (FPS-ZM1) against lipopolysaccharide (LPS)-induced increase in expressions of high mobility group protein B1 (HMGB1) and interleukin-6 (IL-6) in human gingival fibroblasts (HGFs). Furthermore, we explored the potential molecular mechanisms and assessed the involvement of the NF-κB pathway in mediating the changes in the expressions of HMGB1 and IL-6 expression in response to LPS and FPS-ZM1. MethodsHGFs were cultured with enzymatic digestion-tissue explants method. The proliferation of LPS-stimulated HGFs pretreated with FPS-ZM1 at 24, 48, and 72 h was determined by cell counting kit 8 assay. The expressions of HMGB1 and IL-6 were measured using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Western blot analysis was used to assess the expressions of receptor for advanced glycation end products (RAGE) and NF-κB. ResultsLPS enhanced the protein expression of RAGE in HGFs. At the same time, LPS stimulated mRNA and protein expressions of HMGB1 and IL-6 in HGFs. However, pretreatment with FPS-ZM1 attenuated these effects. Pretreatment with FPS-ZM1 (250, 500 nM) significantly inhibited the LPS-induced NF-κB activity. ConclusionFPS-ZM1 down-regulated the LPS-induced HMGB1 and IL-6 expression in HGFs through blocking NF-κB activation. FPS-ZM1 is a promising therapeutic agent for inflammatory diseases caused by oral bacteria.

Primary Cultures from Rat Dorsal Root Ganglia: Responses of Neurons and Glial Cells to Somatosensory or Inflammatory Stimulation

Primary cultures of rat dorsal root ganglia (DRG) consist of neurons, satellite glial cells and a moderate number of macrophages. Measurements of increased intracellular calcium [Ca2+]i induced by stimuli, have revealed that about 70% of DRG neurons are capsaicin-responsive nociceptors, while 10% responded to cooling and or menthol (putative cold sensors). Cultivation of DRG in the presence of a moderate dose of lipopolysaccharide (LPS, 1 µg/ml) enhanced capsaicin-induced Ca2+ signals. We therefore investigated further properties of DRG primary cultures stimulated with 10 µg/ml LPS for a short period. Exposure to LPS for 2 h resulted in pronounced release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) into the supernatants of DRG cultures, increased expression of both cytokines in the DRG cells and increased TNF immunoreactivity predominantly in macrophages. We further observed an accumulation of the inflammatory transcription factors NF-IL6 and STAT3 in the nuclei of LPS-exposed DRG neurons and macrophages. In the presence of the cytotoxic agent cisplatin (5 or 10 µg/ml), the number of macrophages was decreased significantly, the growth of satellite glial cells was markedly suppressed, but the vitality and stimulus-induced Ca2+ signals of DRG neurons were not impaired. Under these conditions the LPS-induced production and expression of TNF-α and IL-6 were blunted. Our data suggest a potential role for macrophages and satellite glial cells in the initiation of inflammatory processes that develop in sensory ganglia upon injury or exposure to pathogens.

LPS-induces IL-6 and IL-8 gene expression in bovine endometrial cells “through DNA methylation”

Uterine infection in dairy cows causes great economic loss. In bovine endometrial cells, lipopolysaccharide (LPS)-stimulated increase in interleukin 6 (IL-6) and interleukin 8 (IL-8) mRNA is crucial for the inflammatory response; however, the regulatory mechanisms remain unclear. Here, we investigated the role of DNA methylation in IL-6 and IL-8 mRNA expression following LPS-induction in bovine endometrial cells. IL-6 and IL-8 mRNA expression was evaluated under DNA methylation inhibition using 5-Aza-2′-deoxycytodine (5Aza) following LPS stimulation. Expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), methyl CpG-binding protein 2 (MeCP2) and DNA methylation at IL-6 and IL-8 regions, were analyzed using quantitative real-time PCR (qRT-PCR) and bisulfite sequencing PCR (BSP) following 24 h of LPS treatment. Inhibition of DNA methylation significantly enhanced LPS-induced IL-6 and IL-8 mRNA expression. LPS increased IL-6 and IL-8 mRNA expression, and decreased methylation levels of specific CpG sites at the IL-6 promoter (at −366 and −660) and the IL-8 promoter (at −120 and −48) after 24 h. Furthermore, LPS treatment for 24 h significantly increased DNMT1, DNMT3A, DNMT3B, and MeCP2 mRNA expression. Our results indicate that treating bovine endometrial cells with LPS induces the expression of IL-6 and IL-8 mRNA regulated by IL-6 and IL-8 promoter methylation.

Delayed treatment of propofol inhibits lipopolysaccharide-induced inflammation in microglia through the PI3K/PKB pathway.

Propofol is a short-acting intravenous anesthetic agent with potential neuroprotective effect. In this study, we aim to investigate whether delayed propofol treatment is protective against lipopolysaccharide (LPS)-stimulated inflammatory responses in microglial cells. Cultured BV2 microglial cells were exposed to propofol at various time points after initiation of LPS stimulation. Nitrite production and cell viability were assessed after stimulation with LPS for 24 h. The effect of propofol on mRNA levels of cyclooxygenase-2 (Cox-2), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) was analyzed using reverse transcription PCR (RT-PCR) 6 h after LPS stimulation. The production of TNF-α and reactive oxygen species was also studied. Propofol applied 0–4 h after the initiation of LPS dose-dependently inhibits nitric oxide production. Propofol application also decreased LPS-induced Cox-2, IL-6, iNOS, TNF-α, and IL-1β mRNA expression and induced significant protein kinase B (PKB) phosphorylation in BV2 cells. Treatment with phosphoinositide 3-kinase (PI3K)/PKB inhibitor wortmannin decreased PKB phosphorylation induced by propofol, and abolished the inhibitory effect of propofol on LPS-stimulated NO, reactive oxygen species and TNF-α production. Our results suggest that delayed propofol treatment can reduce LPS-induced activation of microglial cells. These effects may be mediated by activation of the PI3K/PKB pathway.

Pinus densiflora needle supercritical fluid extract suppresses the expression of pro-inflammatory mediators iNOS, IL-6 and IL-1\u03b2, and activation of inflammatory STAT1 and STAT3 signaling proteins in bacterial lipopolysaccharide-challenged murine macrophages

BackgroundRegulation of a persistently-activated inflammatory response in macrophages is an important target for treatment of various chronic diseases. Pine needle extracts are well known to have potent immunomodulatory effects. The current study was designed to evaluate the effects of Pinus densiflora needle supercritical fluid extract (PDN-SCFE) on bacterial lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 murine macrophages.MethodsCytotoxic effect of PDN-SCFE was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of nitric oxide (NO) and the corresponding enzyme, inducible nitric oxide synthase (iNOS), were quantified by Griess and immunoblotting methods, respectively. The levels of cytokines were quantified using commercial ELISA kits. Quantitative real-time PCR (qRT-PCR) analysis was performed to assess the mRNA expression of iNOS and cytokines. To elucidate the mechanism of action, the involvement of nuclear transcription factor-kappa B (NFκB), mitogen activated protein kinases (MAPKs) and Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathways were examined by an immunoblotting method. In addition, the cellular localization of NFκB was analyzed by immunofluorescence staining.ResultsMTT assay results indicated that PDN-SCFE is non-toxic to RAW 264.7 cells up to a maximum assayed concentration of 40 μg/mL. The PDN-SCFE exhibited a concentration-dependent inhibitory effect on LPS-induced NO production by down regulating the expression of iNOS. In addition, the extract suppressed the LPS-induced expression of interleukin-6 (IL-6) and interleukin-1β (IL-1β) but not tumour necrosis factor-α (TNFα). Mechanistic studies revealed that PDN-SCFE does not influence the NFκB and MAPK pathways. However, it showed a significant inhibitory effect on LPS-induced activation of STAT1 and STAT3 proteins in macrophages.ConclusionThe present findings revealed that the anti-inflammatory activity of PDN-SCFE in LPS-challenged RAW 264.7 macrophages is probably caused by the suppression of the JAK-STAT signaling pathway.Graphical

The membrane-type estrogen receptor G-protein-coupled estrogen receptor suppresses lipopolysaccharide-induced interleukin 6 via inhibition of nuclear factor-kappa B pathway in murine macrophage cells.

The female sex hormone estrogen exerts anti-inflammatory effects. The G-protein-coupled estrogen receptor (GPER) has been recently identified as a novel membrane-type estrogen receptor that can mediate non-genomic estrogenic effects on many cell types. We previously demonstrated that GPER inhibits tumor necrosis factor alpha-induced expression of interleukin 6 (IL-6) through repression of nuclear factor-kappa B (NF-κB) promoter activity using human breast cancer cells. Although several reports have indicated that GPER suppresses Toll-like receptor-induced inflammatory cytokine expression in macrophages, the molecular mechanisms of the inhibition of cytokine production via GPER remain poorly understood. In the present study, we examined GPER-mediated inhibition of IL-6 expression induced by lipopolysaccharide (LPS) stimulation in a mouse macrophage cell line. We found that the GPER agonist G-1 inhibited LPS-induced IL-6 expression in macrophage cells, and this inhibition was due to the repression of NF-κB promoter activity by GPER. G-1 treatment also decreased the phosphorylation of inhibitor of κB kinases. Among the mitogen-activated protein kinases, the phosphorylation of c-jun N-terminal kinase (JNK) was increased by G-1. These findings delineate the novel mechanism of the inhibition of LPS-induced IL-6 through GPER-activated JNK-mediated negative regulation of the NF-κB pathway in murine macrophage cells, which links anti-inflammatory effects to estrogen.

Recombinant CC16 protein inhibits the production of pro-inflammatory cytokines via NF-κB and p38 MAPK pathways in LPS-activated RAW264.7 macrophages.

Accumulating evidence indicates that Clara cell protein-16 (CC16) has anti-inflammatory functions, although the involved molecular pathways have not been completely elucidated. Here, we evaluated the effect of recombinant rat CC16 (rCC16) on the expression of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-8 in lipopolysaccharide (LPS)-stimulated mouse macrophages (RAW264.7 cells) and explored the underlying molecular mechanisms. It was found that rCC16 inhibited LPS-induced TNF-α, IL-6, and IL-8 expression at both the messenger ribonucleicacid (mRNA) level and protein level in a concentration-dependent manner, as demonstrated by real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. Such suppressive effects were accompanied by the inhibition of transcriptional activity and the deoxyribonucleic acid binding activity of nuclear factor (NF)-κB but not activator protein (AP)-1. Western blot analysis further revealed that rCC16 inhibited the increase of nuclear NF-κB and the reduction of cytosolic NF-κB, the phosphorylation and reduction of NF-κB inhibitory protein IκBα, and the p38 mitogen-activated protein kinase (MAPK)-dependent NF-κB activation by phosphorylation at Ser276 of its p65 subunit. Furthermore, rCC16 was found to have no effect on the phosphorylation of c-Jun N-terminal kinase, c-Jun, or the nuclear translocation of c-Jun. In addition, reduction of TNF-α, IL-6, and IL-8 were reversed when the level of endogenous uteroglobin-binding protein was reduced by RNA interference in rCC16- and LPS-treated RAW264.7 cells. Our data suggest that rCC16 suppresses LPS-mediated inflammatory mediator TNF-α, IL-6, and IL-8 production by inactivating NF-κB and p38 MAPK but not AP-1 in RAW264.7 cells.

Esculentoside A suppresses lipopolysaccharide-induced pro-inflammatory molecule production partially by casein kinase 2

Ethnopharmacological relevanceEsculentoside A (EsA) is a saponin isolated from the root of Phytolacca esculenta, an herb which has long been used in Traditional Chinese Medicine for various inflammatory diseases. EsA has been reported to have potent anti-inflammatory properties both in vitro and in vivo. Aim of the studyThe present study focused on the molecular mechanism of EsA for its anti-inflammatory effects in RAW264.7 cells stimulated with lipopolysaccharide (LPS). Methods and resultsEnzyme Linked Immunosorbent Assay (ELISA) showed EsA dose dependently inhibited the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and nitric oxide in RAW264.7 cells. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay further confirmed the suppression of LPS-induced TNF-α, IL-6 and iNOS gene expression by EsA on a transcriptional level. Moreover, EsA treatment markedly suppressed LPS-stimulated IκB phosphorylation and degradation as well as LPS-stimulated luciferase reporter construct driven by κB response elements in RAW264.7 cells. In addition, EsA significantly reduced LPS-induced stimulation of p38 and JNK, but not ERK1/2, phosphorylation. Furthermore, we used a computational method called “reverse docking” to search the possible binding proteins of EsA from the potential drug target database (PDTD), and focused on CK2 as the primary binding protein of EsA. Afterward, we further tested EsA directly interacts with recombinant CK2 using SPR assay. In CK2 kinase activity assay, EsA inhibited recombinant CK2 holoenzyme activity obviously in a dose-dependent manner. In addition, TBB (4, 5, 6, 7-tetrabromo-2-benzotriazole, a pharmacological inhibitor of CK2) blocked IL-6 release in a dose-dependent manner, whereas co-treatment of cells with EsA and TBB did not have an additive effect. ConclusionsTaken together, these results indicate that EsA blocks the LPS-induced pro-inflammatory molecules expression, at least in part, by impediment of LPS-triggered activation of NF-κB and p38/JNK MAPK pathways in macrophages. Furthermore, we discovered for the first time EsA as a ligand for CK2, which was involved in the inhibition of EsA to the expression of inflammatory cytokines. These findings extended our understanding on the cellular and molecular mechanisms responsible for the anti-inflammatory activity of EsA.