Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1

Little is known about whether trans-isoferulic acid (TIA) regulates the production of lipopolysaccharide (LPS)-induced proinflammatory mediators. Therefore, we examined the effect of TIA isolated from Clematis mandshurica on LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in BV2 microglial cells. We found that TIA inhibited the production of LPS-induced NO and PGE₂ without accompanying cytotoxicity in BV2 microglial cells. TIA also downregulated the expression levels of specific regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing LPS-induced NF-κB activity via dephosphorylation of PI3K/Akt. In addition, we demonstrated that a specific NF-κB inhibitor PDTC and a selective PI3K/Akt inhibitor, LY294002 effectively attenuated the expression of LPS-stimulated iNOS and COX-2 mRNA, while LY294002 suppressed LPS-induced NF-κB activity, suggesting that TIA attenuates the expression of these proinflammatory genes by suppressing PI3K/Akt-mediated NF-κB activity. Our results showed that TIA suppressed NO and PGE₂ production through the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). Taken together, our data indicate that TIA suppresses the production of proinflammatory mediators such as NO and PGE₂, as well as their regulatory genes, in LPS-stimulated BV2 microglial cells, by inhibiting PI3K/Akt-dependent NF-κB activity and enhancing Nrf2-mediated HO-1 expression.     

Psoralidin inhibits LPS-induced iNOS expression via repressing Syk-mediated activation of PI3K-IKK-IκB signaling pathways

Psoralidin has been reported to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production, but the mechanisms of the action remain unclear. Thus, the impact of psoralidin on signaling pathways known to be implicated in NO synthesis was explored in LPS-activated RAW264.7 macrophages by using RT-PCR and Western blotting. Consistent with NO inhibition, psoralidin suppressed LPS-induced expression of inducible NO synthase (iNOS) by abolishing IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation without effecting mitogen-activated protein kinases (MAPKs) phosphorylation. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathway might also be phosphoinositide-3-kinase (PI3K) dependent. By using Src inhibitor PP2, Janus kinase 2 (JAK-2) inhibitor AG490, Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 and spleen tyrosine kinase (Syk) inhibitor piceatannol, the results showed that piceatannol clearly repressed NO production more potently than the other inhibitors. Furthermore, piceatannol significantly repressed LPS-induced PI3K/Akt phosphorylation and the downstream IKK/IκB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/Akt-mediated signaling. In fact, transfection with siRNA targeting Syk obviously reduced iNOS expression. Interestingly, LPS-induced phosphorylations of Syk and PI3K-p85 were both significantly blunted by psoralidin treatment. The present results show that interfering with Syk-mediated PI3K phosphorylation might contribute to the NO inhibitory effect of psoralidin via blocking IKK/IκB signaling propagation in LPS-stimulated RAW 264.7 macrophages.     

Ampelopsin reduces endotoxic inflammation via repressing ROS-mediated activation of PI3K/Akt/NF-κB signaling pathways

Ampelopsin (AMP), a plant flavonoid, has potent anti-inflammatory properties in vitro and in vivo. The molecular mechanisms of ampelopsin on pharmacological and biochemical actions of RAW264.7 macrophages in inflammation have not been clearly elucidated yet. In the present study, non-cytotoxic level of ampelopsin significantly inhibited the release of nitric oxide (NO) and pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in a dose-dependent manner. Consistent with NO inhibition, ampelopsin suppressed lipopolysaccharide (LPS)-induced expression of inducible NO synthase (iNOS) by inhibiting nuclear factor κB (NF-κB) activation, which highly correlated with its inhibitory effect on IκB kinase (IKK) phosphorylation, IκB phosphorylation and NF-κB nuclear translocation. Further study demonstrated that ampelopsin suppressed LPS-induced activation of Akt without effecting mitogen-activated protein kinases (MAPKs) phosphorylation. A pharmacological inhibitor of the phosphoinositide 3-kinase (PI3K)-Akt pathway, LY294002, abrogated IKK/IκB/NF-κB-mediated iNOS gene expression. Finally, we certificated that ampelopsin reduced reactive oxygen species (ROS) accumulation and an anti-oxidant N-acetyl-L-cysteine (NAC) significantly repressed LPS-induced PI3K/Akt phosphorylation and the downstream IKK/IκB activation. NAC thereby inhibited LPS-induced iNOS expression and NO production. The present results suggest that the anti-inflammatory effect of ampelopsin is due to inhibiting the interconnected ROS/Akt/IKK/NF-κB signaling pathways.     

Reduced scytonemin isolated from Nostoc commune suppresses LPS/IFNγ-induced NO production in murine macrophage RAW264 cells by inducing hemeoxygenase-1 expression via the Nrf2/ARE pathway

Reduced scytonemin (R-scy) and scytonemin (Scy) isolated from Nostoc commune exhibit anti-tumor and ultraviolet-absorbing properties. In this study, we examined the effects of R-scy and Scy on the induction of nitric oxide (NO) production by lipopolysaccharide (LPS) and interferon-γ (IFNγ) in murine macrophage RAW264 cells. While both R-scy and Scy suppressed LPS/IFNγ-induced NO production, R-scy exhibited a stronger inhibitory effect compared with Scy. To further elucidate the mechanisms underlying the anti-inflammatory effects of R-scy, we examined the changes in the intracellular signaling cascade after LPS/IFNγ stimulation in cells. In addition to the attenuation of LPS/IFNγ-induced upregulation of the inducible isoform of NO synthase, R-scy decreased the activity of nuclear factor-κB, phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinases (MAPKs) after LPS/IFNγ stimulation. R-scy treatment increased heme oxygenase-1 (HO-1) expression by increasing the intracellular levels of reactive oxygen species and thereby activating nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant response element signaling. The induction of HO-1 by R-scy was inhibited by pretreatment with an antioxidant, N-acetyl-cysteine (NAC), as well as SB203580 and LY294002, inhibitors for p38 MAPK and PI3K/Akt, respectively. Our findings suggest that the anti-inflammatory effects of R-scy could involve both the ROS/PI3K/Akt and the p38 MAPK/Nrf2 signaling pathways.     

Hyaluronan inhibits Akt, leading to nuclear factor-κB down-regulation in lipopolysaccharide-stimulated U937 macrophages

Hyaluronan (HA) of high molecular weight is used in the treatment of osteoarthritis and rheumatoid arthritis by intra-articular injection. While HA has been shown to suppress nuclear factor (NF)-κB activation by proinflammatory cytokines and lipopolysaccharide (LPS), intracellular upstream events that cause NF-κB down-regulation in response to HA remain unclear. Thus, this study was performed to investigate the involvement of phosphoinositide-3-OH kinase (PI3K)/Akt in the inhibition of the LPS-activated NF-κB pathway by HA in U937 macrophages. In adherent U937 macrophage cultures, pretreatment with HA of 2700 kDa (1 mg/ml, 1 h) significantly inhibited interleukin-6 (IL-6) production by LPS (200 ng/ml, 24 h)-stimulated U937 cells. LPS (200 ng/ml) activated Akt and NF-κB, whereas HA (1 mg/ml) down-regulated LPS-stimulated phosphorylation of Akt and NF-κB. Inhibition studies using LY294002 (20 μM) revealed the requirement of the PI3K/Akt pathway for LPS-stimulated IL-6 production and NF-κB activation. Pretreatment with anti-intercellular adhesion molecule-1 (ICAM-1) antibody (20 μg/ml) reversed the inhibitory effects of HA on LPS-induced production of IL-6 and activation of Akt and NF-κB. Herein, we provided the first evidence that HA suppresses the LPS-activated PI3K/Akt pathway, leading to down-regulation of NF-κB with diminished IL-6 production through interaction with ICAM-1.     

Opposite effects of wortmannin and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride on toll-like receptor-mediated nitric oxide production: negative regulation of nuclear factor-{kappa}B by phosphoinositide 3-kinase

A number of previous studies have suggested the involvement of phosphoinositide 3-kinase (PI3K) in Toll-like receptor (TLR) signaling. However, there have also been a number of conflicting reports. The PI3K inhibitor wortmannin greatly enhanced TLR-mediated inducible nitric-oxide synthase (iNOS) expression and cytokine production in the mouse macrophage cell line Raw264.7. The effect of wortmannin was common to TLR2, -3, -4, and -9 and was accompanied by activation of nuclear factor-kappaB and up-regulation of cytokine mRNA production. We were surprised to find that another PI3K inhibitor, LY294002, strongly suppressed the production of iNOS and cytokines. This effect of 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) was based on its inhibitory effect on mRNA synthesis. Expression of dominant-negative mutants of PI3K in macrophages augmented the lipopolysaccharideinduced expression of iNOS. Introduction of a pH1 vector producing short hairpin RNA that targets a catalytic subunit of PI3K (p110beta) also enhanced the TLR-mediated responses. Thus, the augmentation of TLR signals by wortmannin was mediated through the inhibition of PI3K, whereas the effect of LY294002 was not explained by its effect on PI3K. These discrepancies in the effects of pharmacological inhibitors in TLR-signaling may have caused confusion regarding the role of PI3K in innate immunity.     

Ent-pimara-8(14), 15-dien-19-oic acid isolated from the roots of Aralia cordata inhibits induction of inflammatory mediators by blocking NF-kappaB activation and mitogen-activated protein kinase pathways

Macrophages play central roles in the innate immune system. The roots of Aralia cordata are widely used in Oriental medicine as a remedy for arthritis. During our program to screen medicinal plants for potential anti-inflammatory compounds, ent-pimara-8(14), 15-dien-19-oic acid (pimaradienoic acid; PA) was isolated from the roots of A. cordata. We examined the effect of PA on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. PA was found to significantly inhibit the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and interleukin-6 (IL-6), as well as the expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and IL-6. Furthermore, we examined whether mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are involved in LPS-induced RAW 264.7 cells. We found that a p38 inhibitor (SB203580) and an ERK 1/2 inhibitor (PD98059) significantly affected LPS-induced IL-6 production. In contrast, a JNK 1/2 inhibitor (SP600125) and PI3K inhibitor (wortmannin or LY294002) did not block the induction of IL-6 production by LPS. The LPS-induced phosphorylation of p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2) was inhibited by PA, but not the phosphorylation of JNK 1/2 and AKT (Ser473). Moreover, PA suppressed I kappaB alpha degradation, NF-kappaB activation and luciferase activity. These results suggest that PA isolated from A. cordata has a potential regulatory effect on inflammatory iNOS, COX-2 and IL-6 expression through blockade of the phosphorylation of MAPKs following I kappaB alpha degradation and NF-kappaB activation.     

Inhibition of PI3K and calcineurin suppresses chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)-dependent responses of Th2 lymphocytes to prostaglandin D(2)

Interaction of prostaglandin D2 (PGD2) with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) triggers chemotaxis and pro-inflammatory cytokine production by Th2 lymphocytes. We have investigated the role of inhibitors of various cell-signalling pathways on the responses of human CRTH2+ CD4+ Th2 cells to PGD2. Phosphatidylinositol 3-kinase (PI3K) and Ca2+/calcineurin/nuclear factor of activated T cells (NFAT) pathways were activated by PGD2 in Th2 cells in a CRTH2-dependent manner. Inhibition of the PI3K pathway with LY294002 significantly reduced both PGD2-induced cell migration and cytokine (interleukin-4, interleukin-5 and interleukin-13) production. The inhibitory effect of LY294002 on cell migration is likely to be related to cytoskeleton reorganization as it showed a similar potency on PGD2-induced actin polymerization. The calcineurin inhibitors, tacrolimus (FK506) and cyclosporin A, had no effect on cell migration but completely blocked both cytokine production and the nuclear translocation of NFATc1 suggesting that Ca2+/calcineurin/NFAT is involved in CRTH2-dependent cytokine production but not chemotaxis. The promotion of NFAT nuclear location by PI3K activation may be mediated by negative regulation of glycogen synthase kinase-3beta (GSK3beta), since the PGD2-stimulated increase in phospho-GSK3beta was down-regulated by LY294002, and inhibition of GSK3beta by SB216763 enhanced PGD2-induced Th2 cytokine production and reversed the inhibitory effect of LY294002. These data suggest that PI3K and Ca2+/calcineurin/NFAT signalling pathways are critically involved in pro-inflammatory responses of Th2 cells to PGD2.     

The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor κB inhibition, in a PI3K-independent mechanism

The Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 (LY2), has been previously reported to inhibit nuclear factor κB (NFκB) activity, in a PI3K-independent mechanism. The goals of the current research were to determine the specificity of LY2 regarding NFκB subunits, and to identify relevant modulation of cytokine expression in LPS-stimulated macrophages. We found that LY2 specifically diminished the level of p50, but not p65, NFκB in the nucleus of LPS-stimulated mouse RAW264.7 macrophages and human THP-1 monocytes. This activity of LY2 was mimicked by its PI3K-inert analog LY303511 (LY3), but not by another PI3K inhibitor - wortmannin. We further show that LY2 inhibited LPS-induced IL-10 expression by RAW264.7 macrophages, in a PI3K-independent mechanism. Moreover, using a deletion mutant of an IL-10 promoter reporter gene we demonstrate that the activity of the NFκB enhancer site at the IL-10 promoter is regulated by LY2 in a PI3K-independent manner. Finally, both LY2 and LY3 elevated TNFα production in the LPS tolerant state which is regulated by p50 NFκB homodimers, but not before tolerance development. The effects of LY2 and LY3 on p50 translocation and on cytokine production in LPS-stimulated macrophages are thus consistent with specific PI3K-independent inhibition of p50 NFκB homodimer activity by LY2.     

Amlodipine inhibits TNF-α production and attenuates cardiac dysfunction induced by lipopolysaccharide involving PI3K/Akt pathway

Calcium channel blockers (CCBs) are widely used in the therapy of cardiovascular diseases. Recent studies have shown that several CCBs exerted distinct anti-inflammatory effect in myocardial dysfunction models. The purpose of the present study was to evaluate therapeutic effect and possible mechanism of action of amlodipine, one of the widely used CCBs, on rat cardiac dysfunction during sepsis induced by lipopolysaccharide (LPS). Pretreatment of the rats with amlodipine (10 or 30 mg/kg, i.v.) delayed the fall of mean arterial blood pressure caused by LPS. Amlodipine also significantly inhibited the elevation of plasma tumor necrosis factor α (TNF-α) and decreased levels of inducible nitric oxide synthase (iNOS) in response to LPS challenge. To investigate the mechanism of the action of amlodipine, neonatal rat cardiomyocytes were used as a model. Amlodipine concentration-dependently decreased the release of TNF-α and iNOS protein expression, and suppressed the degradation and phosphorylation of inhibitor of κB-α (IκB-α) in LPS-activated neonatal rat cardiomyocytes. Further studies revealed that amlodipine markedly activated phosphatidylinositiol 3-kinase (PI3K) and Akt, downstream of the PI3K signal cascade. Application of PI3K inhibitors, wortmannin and LY294002 attenuated the depression of TNF-α and iNOS expression by amlodipine in LPS-induced cardiomyocytes. These findings may explain some cardioprotective effects of amlodipine in LPS-mediated sepsis and suggest that the inhibition of TNF-α and iNOS expression by amlodipine is, at least in part, dependent on PI3K/Akt signaling pathway.     

Pifithrin-α, a pharmacological inhibitor of p53, downregulates lipopolysaccharide-induced nitric oxide production via impairment of the MyD88-independent pathway

The effect of pifithrin (PFT)-α, a pharmacological inhibitor of p53, on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage-like cells was examined. PFT-α inhibited the production of NO but not tumor necrosis factor (TNF)-α in response to LPS. PFT-α inhibited LPS-induced NO production via reduced expression of an inducible NO synthase (iNOS). Moreover, PFT-α inhibited LPS-induced iNOS expression in p53-silenced cells. PFT-α inhibited the production of interferon (IFN)-β, characteristic of the MyD88-independent pathway of LPS signaling, whereas it did not affect the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. PFT-α inhibited poly I:C-induced NO production whereas it did not inhibit IFN-β-induced NO production. Further, PFT-α reduced the expression of IFN regulatory factor 3 that leads to the IFN-β production in the MyD88-independent pathway. The most upstream event impaired by PFT-α was the reduced expression of TNF receptor-associated factor (TRAF) 3 in the MyD88-independent pathway. PFT-α also reduced the in vivo expression of iNOS in the livers of mice injected with LPS. Taken together, PFT-α was suggested to inhibit LPS-induced NO production via impairment of the MyD88-independent pathway and attenuated LPS-mediated inflammatory response.     

18β-Glycyrrhetinic acid suppresses TNF-α induced matrix metalloproteinase-9 and vascular endothelial growth factor by suppressing the Akt-dependent NF-κB pathway

Little is known about the molecular mechanism through which 18β-glycyrrhetinic acid (GA) inhibits metastasis and invasion of cancer cells. Therefore, this study aimed to investigate the effects of GA on the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in various types of cancer cells. We found that treatment with GA reduces tumor necrosis factor-α (TNF-α)-induced Matrigel invasion with few cytotoxic effects. Our findings also showed that MMP-9 and VEGF expression increases in response to TNF-α; however, GA reverses their expression. In addition, GA inhibited inhibitory factor kappa B degradation, sustained nuclear factor-kappa B (NF-κB) subunits, p65 and p50, in the cytosol compartments, and consequently suppressed the TNF-α-induced DNA-binding activity and luciferase activity of NF-κB. Specific NF-κB inhibitors, pyrrolidine dithiocarbamate, MG132, and PS-1145, also attenuated TNF-α-mediated MMP-9 and VEGF expression as well as activity by suppressing their regulatory genes. Furthermore, phosphorylation of TNF-α-induced phosphatidyl-inositol 3 kinase (PI3K)/Akt was significantly downregulated in the presence of GA accompanying with the inhibition of NF-κB activity, and as presumed, the specific PI3K/Akt inhibitor LY294002 significantly decreased MMP-9 and VEGF expression as well as activity. These results suggest that GA operates as a potential anti-invasive agent by downregulating MMP-9 and VEGF via inhibition of PI3K/Akt-dependent NF-κB activity. Taken together, GA might be an effective anti-invasive agent by suppressing PI3K/Akt-mediated NF-κB activity.     

Saucerneol F, a new lignan, inhibits iNOS expression via MAPKs, NF-κB and AP-1 inactivation in LPS-induced RAW264.7 cells

Saucerneol F (SF), a new tetrahydrofuran-type sesquilignan isolated from Saururus chinensis, dose-dependently inhibited nitric oxide (NO) production, with concomitant reduction of inducible nitric oxide synthase (iNOS) protein and mRNA expression in lipopolysaccharide (LPS)-stimulated murine macrophage RAW264.7 cells. To elucidate the molecular mechanism underlying the inhibition of iNOS expression by SF, we assessed the effects of SF on nuclear factor-κB (NF-κB) DNA-binding activity, NF-κB-dependent reporter gene activity, inhibitory factor-κB (IκB) phosphorylation and degradation, and p65 nuclear translocation. Treatment with SF decreased the luciferase activities of NF-κB reporter promoters in a dose-dependent manner and translocation of NF-κB p65. In addition, pretreatment of SF reduced LPS-stimulated activation of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and c-Jun NH(2)-terminal kinase (JNK). Furthermore, SF attenuated the luciferase activities of AP-1 reporter promoters and the DNA-binding capacity of AP-1. Taken together, the present results indicate that SF attenuates NO production and iNOS expression by blocking LPS-induced activation of NF-κB, MAPKs, and AP-1, suggesting that SF is potentially applicable as an anti-inflammatory drug.     

Differential modulation of immunostimulant-triggered NO production by endoplasmic reticulum stress inducers in vascular smooth muscle cells

We investigated the effects of endoplasmic reticulum (ER) stress inducers thapsigargin (TG) and tunicamycin (Tm) on immunostimulant lipopolysaccharide/interferon (LPS/IFN)-induced expression of isoform of nitric oxide synthase (iNOS) and nitric oxide (NO) production in vascular smooth muscle cells. LPS/IFN-induced iNOS mRNA expression was markedly enhanced by TG, whereas iNOS mRNA expression was strongly attenuated by Tm. Similarly, production of iNOS protein was markedly upregulated by TG but virtually eliminated by Tm. LPS/IFN-induced guanosine triphosphate cyclohydrolase I mRNA expression was slightly reduced by TG and markedly inhibited by Tm. Similarly, LPS/IFN-mediated induction of cellular biopterin was modestly reduced by TG and markedly inhibited by Tm. TG modestly enhanced LPS/IFN-induced activation of NF-κB, whereas Tm had no effect on it. Cellular respiration was reduced by TG and Tm in a concentration-dependent manner, which was confirmed by apoptosis assay. Thus, TG and Tm-induced ER stress and differently modulated NO production through alterations in iNOS expression and activity independently of NF-κB activation and caused a similar degree of ER stress-induced apoptosis.