Pyranocoumarins Isolated from Peucedanum praeruptorum Dunn Suppress Lipopolysaccharide-Induced Inflammatory Response in Murine Macrophages Through Inhibition of NF-κB and STAT3 Activation

Praeruptorin C, D, and E (PC, PD, and PE) are three pyranocoumarins isolated from the dried root of Peucedanum praeruptorum Dunn of Umbelliferae. In the present study, we investigated the anti-inflammatory effect of these compounds in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Pyranocoumarins significantly inhibited LPS-induced production of nitric oxide, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The mRNA and protein expressions of inducible nitric oxide synthase, IL-6, and TNF-α were also suppressed by these compounds. Both PD and PE exhibited greater anti-inflammatory activities than PC. Further study showed that pyranocoumarins suppressed the cytoplasmic loss of inhibitor κB-α protein and inhibited the translocation of NF-κB from cytoplasm to nucleus. In addition, pyranocoumarins suppressed LPS-induced STAT3 tyrosine phosphorylation. Taken together, the results suggest that pyranocoumarins may exert anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages through the inhibition of NF-κB and STAT3 activation.     

Upregulation of heme oxygenase-1 via PI3K/Akt and Nrf-2 signaling pathways mediates the anti-inflammatory activity of Schisandrin in Porphyromonas gingivalis LPS-stimulated macrophages

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is thought to induce periodontitis. In this study, we isolated Schisandrin from the dried fruits of Schisandra chinensis and examined the anti-inflammatory effect of Schisandrin in macrophages stimulated with LPS from P. gingivalis. First, Schisandrin inhibited LPS-induced pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. And Schisandrin suppressed the nuclear translocation and activity of NF-κB and phosphorylation of IκBα in LPS-stimulated RAW 264.7 cells. Next, the presence of a selective inhibitor of HO-1 (SnPP) and a siRNA specific for HO-1 inhibited Schisandrin-mediated anti-inflammatory activity. Furthermore, Schisandrin induced HO-1 expression of RAW 264.7 cells through Nrf-2, PI3K/Akt, and ERK activation. Therefore, these results suggest that the anti-inflammatory effects of Schisandrin on P. gingivalis LPS-stimulated RAW 264.7 cells may be due to a reduction of NF-κB activity and induction of the expression of HO-1, leading to TNF-α, IL-1β, and IL-6 down-regulation.     

Inhibition of LPS-induced inflammatory biomarkers by ethyl acetate fraction of Patrinia scabiosaefolia through suppression of NF-κB activation in RAW 264.7 cells

Patrinia scabiosaefolia (PS) has been used for curing various types of inflammatory-related disorders. However, the precise mechanism of the anti-inflammatory activity of PS remains unclear. Here, we investigated the anti-inflammatory effects of several fractions isolated from the PS in RAW 264.7 macrophages. The results indicated that the ethyl acetate fraction of PS (EAPS) concentration highly suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) and IL-6 productions without a cytotoxic effect on RAW 264.7 cells. EAPS inhibited the expressions of LPS-induced iNOS and COX-2 protein and their mRNA in a dose-dependent manner. Particularly, EAPS suppressed the level of nuclear factor-κB (NF-κB) activity, which was linked with the suppression of LPS-induced phosphorylation of p65 at serine 276 and p65 translocation into nuclei, but not MAPK signaling. In addition, treatment with EAPS inhibited the production of TNF-α in LPS-injected mice and suppressed the production of IL-6 and TNF-α in LPS-stimulated splenocytes from BALB/c mice. Therefore, we demonstrate here that Patrinia scabiosaefolia potentially inhibits the biomarkers related to inflammation through the blocking of NF-κB p65 activation, and it may be a potential therapeutic candidate for the treatment of inflammatory diseases.     

Inhibition of lipopolysaccharide-induced I-kappaB degradation and tumor necrosis factor-alpha expression by acriflavine, an antimicrobial agent

Acriflavine neutral (ACF) has been used for treatment of microbial infections for humans and fishes. Effects of ACF on the nuclear factor-kappaB (NF-kappaB) activation and tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS), an endotoxin, were examined in rat and RAW264.7 cells. Gel retardation analysis revealed that LPS (1 microg/kg) activated NF-kappaB in the liver, whereas pretreatment of rats with ACF (10 mg/kg) completely prevented the NF-kappaB activation. Selectivity of the NF-kappaB DNA binding was confirmed by immunodepletion with anti-p65 and anti-p50 antibodies. Translocation of NF-kappaB to the nucleus is preceded by phosphorylation and proteolytic degradation of inhibitor-kappaBalpha (I-kappaBalpha) subunit. Whereas the level of I-kappaBalpha protein was rapidly decreased after treatment of rats with LPS (1 microg/kg), ACF treatment prior to LPS attenuated the decrease in I-kappaBalpha protein level. LPS-induced increase in the production of TNF-alpha, the principal inflammatory mediator, was prevented by ACF pretreatment by 80%. Stimulation of RAW264.7 cells with 1 microg/ml of LPS caused an increase in DNA binding activity of NF-kappaB, which was 80% inhibited by 1 microg/ml of ACF. LPS reduced I-kappaBalpha level in RAW264.7 cells by 77%. ACF attenuated LPS-induced decrease in I-kappaBalpha protein in a concentration-dependent manner. Production of TNF-alpha by LPS from RAW264.7 cells was decreased by 84% in the presence of ACF. Data showed that ACF inhibited LPS-induced NF-kappaB activation through inhibition of I-kappaBalpha degradation and TNF-alpha production in both rat and RAW264.7 cells. Inhibition of NF-kappaB activation and TNF-alpha production may be associated with the anti-inflammatory activity of ACF.     

Glucocorticoids prevent NF-kappaB activation by inhibiting the early release of platelet-activating factor in response to lipopolysaccharide

Lipopolysaccharide (LPS) is a known inducer of numerous pro-inflammatory events including the production of platelet-activating factor (PAF). PAF released in response to LPS is a major contributor to the pathological events associated with endotoxemia. The present study demonstrates that dexmethasone (DEX) inhibited the LPS-induced early plasma PAF raise in a dose- and time-dependent manner. In addition, DEX prevented the subsequent PAF-mediated pathological phenomena such as anaphylactic shock-like symptoms, symptoms of disseminated intravascular coagulation and hemorrhage in renal medullae. DEX or the PAF antagonist BN 50739 significantly inhibited LPS-induced NF-kappaB activation. The inhibition of NF-kappaB activation by DEX was overcome by the injection of exogenous PAF. Administration of PAF or LPS resulted in a rapid loss of IkappaBalpha protein. The LPS-induced degradation of IkappaBalpha was prevented by pretreatment with BN 50739, suggesting that PAF is a critical intermediate in the LPS-triggered degradation of IkappaBalpha protein. DEX prevented the LPS-induced IkappaBalpha degradation, which was also reversed by exogenous PAF. Administration of DEX or BN 50739 caused an increase in cytoplasmic IkappaBalpha level. Our results indicate that DEX inhibits IkappaBalpha degradation and subsequent NF-kappaB activation through blocking the initial release of PAF.     

Beta2-adrenergic receptor regulates Toll-like receptor-4-induced nuclear factor-kappaB activation through beta-arrestin 2

Toll-like receptors (TLRs) play an important role in innate immunity while, beta(2)-adrenergic receptors (beta(2)AR) provide the key linkages for the sympathetic nervous system to regulate the immune system. However, their role in macrophages remains uncertain. Here, we demonstrate the cross-talk between beta(2)AR and TLR signalling pathways. Expression of beta(2)AR was down-regulated by TLR4 ligand lipopolysaccharide (LPS) stimulation. To investigate the physiological consequence of this down-regulation RAW264 cells, a macrophage cell line, were transfected with a beta(2)AR expression vector (RAWar). Both LPS-stimulated inducible nitric oxide synthase (NOS II) expression and NO production were markedly suppressed in the RAWar cells. The activation of nuclear factor-kappaB (NF-kappaB) and degradation of the inhibitor of NF-kappaB (IkappaBalpha) in response to LPS were markedly decreased in these cells. The level of beta-arrestin 2, which regulates beta(2)AR signalling, was also reduced in RAW264 cells after stimulation with LPS, but not in RAWar cells. Overexpression of beta-arrestin 2 (RAWarr2) also inhibited NO production and NOS II expression. Furthermore, we demonstrated that beta-arrestin 2 interacted with cytosolic IkappaBalpha and that the level of IkappaBalpha coimmunoprecipitated by anti-beta-arrestin 2 antibodies was decreased in the RAW264 cells but not in RAWar or RAWarr2 cells. These findings suggest that LPS-stimulated signals suppress beta(2)AR expression, leading to down-regulation of beta-arrestin 2 expression, which stabilizes cytosolic IkappaBalpha and inhibits the NF-kappaB activation essential for NOS II expression, probably to ensure rapid and sufficient production of NO in response to microbial attack.     

Inhibition of LPS-induced inflammatory biomarkers by ethyl acetate fraction of Patrinia scabiosaefolia through suppression of NF-κB activation in RAW 264.7 cells

Patrinia scabiosaefolia (PS) has been used for curing various types of inflammatory-related disorders. However, the precise mechanism of the anti-inflammatory activity of PS remains unclear. Here, we investigated the anti-inflammatory effects of several fractions isolated from the PS in RAW 264.7 macrophages. The results indicated that the ethyl acetate fraction of PS (EAPS) concentration highly suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) and IL-6 productions without a cytotoxic effect on RAW 264.7 cells. EAPS inhibited the expressions of LPS-induced iNOS and COX-2 protein and their mRNA in a dose-dependent manner. Particularly, EAPS suppressed the level of nuclear factor-κB (NF-κB) activity, which was linked with the suppression of LPS-induced phosphorylation of p65 at serine 276 and p65 translocation into nuclei, but not MAPK signaling. In addition, treatment with EAPS inhibited the production of TNF-α in LPS-injected mice and suppressed the production of IL-6 and TNF-α in LPS-stimulated splenocytes from BALB/c mice. Therefore, we demonstrate here that Patrinia scabiosaefolia potentially inhibits the biomarkers related to inflammation through the blocking of NF-κB p65 activation, and it may be a potential therapeutic candidate for the treatment of inflammatory diseases.     

Effects of Citral on Lipopolysaccharide-Induced Inflammation in Human Umbilical Vein Endothelial Cells

Citral is an active compound of lemongrass oil which has been reported to have anti-inflammatory effects. In this study, we investigated the effects of citral on lipopolysaccharide (LPS)-induced inflammatory response in a rat model of peritonitis and human umbilical vein endothelial cells (HUVECs). LPS was intraperitoneally injected into rats to establish a peritonitis model. The HUVECs were treated with citral for 12 h before exposure to LPS. The levels of TNF-α and IL-8 were measured using ELISA. Western blotting was used to detect the expression of VCAM-1, ICAM-1, NF-κB, and PPAR-γ. The results showed that citral had a protective effect against LPS-induced peritonitis. Citral decreased the levels of WBCs and inflammatory cytokines TNF-α and IL-6. Citral also inhibited LPS-induced myeloperoxidase (MPO) activity in the peritoneal tissue. Treatment of HUVECs with citral significantly inhibited TNF-α and IL-8 expression induced by LPS. LPS-induced VCAM-1 and ICAM-1 expression were also suppressed by citral. Meanwhile, we found that citral inhibited LPS-induced NF-κB activation in HUVECs. Furthermore, we found that citral activated PPAR-γ and the anti-inflammatory effects of citral can be reversed by PPAR-γ antagonist GW9662. In conclusion, citral inhibits LPS-induced inflammatory response via activating PPAR-γ which attenuates NF-κB activation and inflammatory mediator production.     

Effect of cross-tolerance between endotoxin and TNF-alpha or IL-1beta on cellular signaling and mediator production.

Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory-mediator production. This phenomenon also confers cross-tolerance to other stimuli including tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta. Post-receptor convergence of signal transduction pathways might occur after LPS, IL-1beta, and TNF-alpha stimulation. Therefore, it was hypothesized that down-regulation of common signaling molecules induces cross-tolerance among these stimuli. LPS tolerance and cross-tolerance were examined in THP-1 cells. Phosphorylation of MAP kinases and degradation of inhibitor kappaBalpha (IkappaBalpha) DNA binding of nuclear factor-kappaB (NF-kappaB), and mediator production were examined. In naive cells, LPS, TNF-alpha, and IL-1beta induced IkappaBalpha degradation, kinase phosphorylation, and NF-kappaB DNA binding. LPS stimulation induced production of TNF-alpha or TxB2 and degradation of IRAK. However, neither TNF-alpha nor IL-1beta induced IRAK degradation or stimulated TNF-alpha or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS-induced TxB2 and TNF-alpha production. LPS pretreatment induced cross-tolerance to TNF-alpha or IL-1beta stimulation. Pretreatment with TNF-alpha induced cross-tolerance to LPS-induced signaling events and TxB2 production. Although pretreatment with IL-1beta did not induce cross-tolerance to LPS-induced signaling events, it strongly inhibited LPS TNF-alpha and TxB2 production. These data demonstrate that IL-1beta induces cross-tolerance to LPS-induced mediator production without suppressing LPS-induced signaling to MAP kinases or NF-kappaB activation.     

ML365 inhibits lipopolysaccharide-induced inflammatory responses via the NF-κB signaling pathway

ML365 is a selective inhibitor of the twik-related acid-sensitive potassium channel 1/two-pore domain channel subfamily k member 3 two-pore domain potassium channel. There are no functional studies of the relationship between ML365 and inhibition of inflammation. In this study, we evaluated the anti-inflammatory effect of ML365 on lipopolysaccharide (LPS)-induced inflammation and elucidated the possible mechanism. ML365 showed no cytotoxicity and did not induce apoptosis on RAW264.7 cells and inhibited nitric oxide production. ML365 suppressed the release of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1β measured using enzyme-linked immunosorbent assay and quantitative polymerase chain reaction assays. LPS-induced activation and co-localization of NF-κB was inhibited by ML365 pre-treatment. ML365 inhibited the protein expression of Erk, p38 and Jnk. In vivo, ML365 appeared to prevent pathological damages in the LPS-induced endotoxin shock model. These findings suggest that ML365 inhibits LPS-induced inflammatory responses by regulating the NF-κB signaling pathway.     

Suppressing LPS-induced early signal transduction in macrophages by a polyphenol degradation product: a critical role of MKP-1

Macrophages represent the first defense line against bacterial infection and therefore, play a crucial role in early inflammatory response. In this study, we investigated the role of MAPKs and MKP-1 activation in regulation of an early inflammatory response in RAW 264.7 macrophage cells. We induced the inflammatory response by treating the macrophages with LPS and inhibited an early inflammatory response by using ferulaldehyde, a water-soluble end-product of dietary polyphenol degradation that we found previously to exert its beneficial anti-inflammatory effects during the early phase of in vivo inflammation. We found that LPS-induced ROS and nitrogen species formations were reduced by ferulaldehyde in a concentration-dependent manner, and ferulaldehyde protected mitochondria against LPS-induced rapid and massive membrane depolarization. LPS induced early suppression of MKP-1, which was accompanied by activation of JNK, ERK, and p38 MAPK. By reversing LPS-induced early suppression of MKP-1, ferulaldehyde diminished MAPK activation, thereby inhibiting NF-κB activation, mitochondrial depolarization, and ROS production. Taken together, our data suggest that ferulaldehyde exerts its early anti-inflammatory effect by preserving the mitochondrial membrane integrity and shifting the expression of MKP-1 forward in time in macrophages.     

Stevioside Suppressed Inflammatory Cytokine Secretion by Downregulation of NF-κB and MAPK Signaling Pathways in LPS-Stimulated RAW264.7 Cells

Stevioside, a diterpene glycoside isolated from Stevia rebaudiana, has been reported to have anti-inflammatory properties. However, the underlying molecular mechanisms are not well understood. The objective of this study was to investigate the molecular mechanism of stevioside in modifying lipopolysaccharide (LPS)-induced signal pathways in RAW264.7 cells. RAW264.7 cells were stimulated with LPS in the presence or absence of stevioside. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction. Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) were determined by western blot. The results showed that stevioside dose-dependently inhibited the expression of tumor necrosis factor-α, interleukin-6, and interleukin-1β in LPS-stimulated RAW264.7 cells. Western blot analysis showed that stevioside suppressed LPS-induced NF-κB activation, IκBa degradation, phosphorylation of ERK, JNK, and P38. Our results suggest that stevioside exerts an anti-inflammatory property by inhibiting the activation of NF-κB and mitogen-activated protein kinase signaling and the release of proinflammatory cytokines. These findings suggest that stevioside may be a therapeutic agent against inflammatory diseases.     

重组腺病毒△N IkBα的构建及其对A549细胞中NF-kB活性的抑制

目的：探讨△N IkBα基因对NF—kB活性的调节作用。方法：构建去除Ser^32和Ser^36磷酸化位点的IkBα重组腺病毒——Ad—△N IkBα。A549细胞分为3组：即LPS组、Ad—LacZ LPS组和Ad—△N IkBα LPS组．LPS组单纯用内毒素(LPS)激活NF—kB；Ad—LacZ LPS组及Ad—△N IkBα LPS组在用LPS前2d，分别感染Ad—LacZ和Ad—△N IkBα。用western blot、电泳迁移率变动分析(EMSA)和ELISA法分别检测LPS刺激后5h，细胞总蛋白中NF—kB的活性和培养上清中TNF—α及IL-6的含量。结果：Ad—△N IkBα LPS组NF—kB的活性，TNF—α和IL-6的含量，均显著低于LPS组及Ad—LacZ LPS组。结论：突变后的IkBα可明显抑制NF—kB活化，减少TNF—α和IL-6的释放，有望成为一种强有力的抗炎治疗制剂。     

Plumbagin,a Vitamin K3 Analogue,abrogates Lipopolysaccharide-Induced Oxidative Stress,Inflammation and Endotoxic Shock via NF-κB Suppression

Plumbagin has been reported to modulate cellular redox status and suppress NF-κB. In the present study, we investigated the effect of plumbagin on lipopolysaccharide (LPS)-induced endotoxic shock, oxidative stress and inflammatory parameters in vitro and in vivo. Plumbagin inhibited LPS-induced nitric oxide, TNF-α, IL-6 and prostaglandin-E2 production in a concentration-dependent manner in RAW 264.7 cells without inducing any cell death. Plumbagin modulated cellular redox status in RAW cells. Plumbagin treatment significantly reduced MAPkinase and NF-κB activation in macrophages. Plumbagin prevented mice from endotoxic shock-associated mortality and decreased serum levels of pro-inflammatory markers. Plumbagin administration ameliorated LPS-induced oxidative stress in peritoneal macrophages and splenocytes. Plumbagin also attenuated endotoxic shock-associated changes in liver and lung histopathology and decreased the activation of ERK and NF-κB in liver. These findings demonstrate the efficacy of plumbagin in preventing LPS-induced endotoxemia and also provide mechanistic insights into the anti-inflammatory effects of plumbagin.     

Inhibition of caspase 3 abrogates lipopolysaccharide-induced nitric oxide production by preventing activation of NF-kappaB and c-Jun NH2-terminal kinase/stress-activated protein kinase in RAW 264.7 murine macrophage cells

The effect of caspase inhibitors on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 267.4 murine macrophage cells was investigated. Pretreatment of RAW cells with a broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), resulted in a striking reduction in LPS-induced NO production. Z-VAD-FMK inhibited LPS-induced NF-kappaB activation. Furthermore, it blocked phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not that of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases. Similarly, a caspase 3-specific inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone, inhibited NO production, NF-kappaB activation, and JNK/SAPK phosphorylation in LPS-stimulated RAW cells. The attenuated NO production was due to inhibition of the expression of an inducible-type NO synthase (iNOS). The overexpression of the dominant negative mutant of JNK/SAPK and the addition of a JNK/SAPK inhibitor blocked iNOS expression but did not block LPS-induced caspase 3 activation. It was therefore suggested that the inhibition of caspase 3 might abrogate LPS-induced NO production by preventing the activation of NF-kappaB and JNK/SAPK. The caspase family, especially caspase 3, is likely to play an important role in the signal transduction for iNOS-mediated NO production in LPS-stimulated mouse macrophages.