Ethyl acetate extract from Angelica Dahuricae Radix inhibits lipopolysaccharide-induced production of nitric oxide, prostaglandin E2 and tumor necrosis factor-alphavia mitogen-activated protein kinases and nuclear factor-kappaB in macrophages.

Angelica dahurica (Umbelliferae) has been used to treat headache of common cold, supraorbital neuralgia, painful swelling on the body, nasal stuffiness, leukorrhea and arthralgia due to wind-dampness in Korean traditional medicine. It is also claimed to be effective in the treatment of acne, erythema, headache, toothache, sinusitis, colds and flu. The present study focused whether the ethyl acetate extract from Angelica Dahuricae Radix (EAAD) inhibits production of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and tumor necrosis factor (TNF)-alpha, as well as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPKs) in lipopolysaccharide (LPS)-stimulated macrophages. EAAD inhibited LPS-induced NO, PGE(2) and TNF-alpha production as well as expression of iNOS and COX-2 in RAW 264.7 cells. EAAD inhibited LPS-induced TNF-alpha production in THP-1 cells. Furthermore, EAAD suppressed LPS-induced phosphorylation of p38 MAPK and extracellular-signal regulated kinases 1/2 (ERK1/2), I-kappaBalpha degradation, and NF-kappaB activation in RAW 264.7 cells. These results suggest that EAAD has the inhibitory effects on LPS-induced TNF-alpha, NO and PGE(2) production, and expression of iNOS and COX-2 in macrophage through blockade in the phosphorylation of MAPKs, following I-kappaBalpha degradation and NF-kappaB activation.     

Sauchinone suppresses lipopolysaccharide-induced inflammatory responses through Akt signaling in BV2 cells

Activated microglial cells play an important role in inflammatory responses in the central nervous system (CNS) that are involved in neurodegenerative diseases. Sauchinone has been shown to modulate the expression of inflammatory factors through nuclear factor-kappa B (NF-κB) signaling pathway. Here, we examined the effect of sauchinone on the inflammatory responses of microglia cells induced by lipopolysaccharide (LPS) and explored the mechanism underlying action of sauchinone. BV2 cells treated with LPS showed an up-regulation of nitric oxide (NO) and prostaglandin (PGE(2)) release, whereas sauchinone suppressed this up-regulation. Sauchinone inhibited both mRNA and protein expression of COX-2, iNOS, TNF-α and IL-1β. In addition, sauchinone blocked the activation of NF-κB through its inhibition of I-κB phosphorylation. Interestingly, sauchinone had no effect on the LPS-induced phosphorylation of mitogen activated protein kinases (MAP kinases; ERK1/2, p38, JNK), but it did inhibit Akt phosphorylation. These results suggest that the inhibitory effect of sauchinone on the LPS-induced production of inflammatory mediator in BV2 cells is associated with the suppression of the NF-κB and Akt signaling pathways. Therefore, sauchinone may be a useful treatment for neurodegenerative disease by inhibiting inflammatory responses in activated microglia.     

Novel selective COX-1 inhibitors suppress neuroinflammatory mediators in LPS-stimulated N13 microglial cells

COX-1 plays a previously unrecognized part in the neuroinflammation. Genetic ablation or pharmacological inhibition of COX-1 activity attenuates the inflammatory response and neuronal loss. In this context, the effects of selective COX-1 inhibitors (P6, P10, SC-560, aspirin) and coxibs (celecoxib and etoricoxib) on LPS-stimulated microglial cell function (a worldwide accepted neuroinflammation model) were investigated, and the effects on COX-1/COX-2, cPGES mRNA and iNOS expression, PGE(2) and NO production and NF-κB activation by IκBα phosphorylation were evaluated. The total suppression of the expression of both COX-1 and COX-2 by their respective selective inhibitors occurred. NF-κB remained almost completely inactive in the presence of coxibs, as expected, and totally inactive in the presence of P6. P6 also markedly counteracted LPS enhancing cPGES mRNA expression and PGE(2) production. Since COX-1 is predominantly localized in microglia, its high selective inhibition rather than COX-2 (by coxibs) is more likely to reduce neuroinflammation and has been further investigated as a potential therapeutic approach and prevention in neurodegenerative diseases with a marked inflammatory component.     

Rengyolone inhibits inducible nitric oxide synthase expression and nitric oxide production by down-regulation of NF-kappaB and p38 MAP kinase activity in LPS-stimulated RAW 264.7 cells

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. Rengyolone, a cyclohexylethanoid isolated from the fruits of Forsythia koreana, exhibits anti-inflammatory activity with unknown mechanism. In this study, we found that rengyolone has a strong inhibitory effect on the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). Rengyolone also inhibited inducible nitric oxide synthase (iNOS) gene expression and cyclooxygenase 2 (COX-2) by lipopolysaccharide (LPS). In order to explore the mechanism responsible for the inhibition of iNOS gene expression by rengyolone, we investigated its effect on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by rengyolone, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaBalpha and phosphorylation of p38 MAP kinase. Furthermore, rengyolone suppressed the expression of ICE protein in IL-1beta-treated D10S cells. Taken together, these results suggest that rengyolone attenuates the inflammation through inhibition of NO production and iNOS expression by blockade of NF-kappaB and p38 MAPK activation in LPS-stimulated RAW 264.7 cells.     

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide and cyclooxygenase-2 by chiisanoside via suppression of nuclear factor-kappaB activation in RAW 264.7 macrophage cells

In the present study, the effects of several triterpenes isolated from the leaves of Acanthopanax chiisanensis (Araliaceae), namely, chiisanoside, isochiisanoside, 22-hydroxychiisanoside and chiisanogenin (the aglycone of chiisanoside) were evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production by the RAW 264.7 macrophage cell line. Of the triterpenes tested, chiisanoside was found to most potently inhibit NO and PGE2 production. In addition, chiisanoside significantly reduced the release of inflammatory cytokines like TNF-alpha and IL-1beta. Consistent with these observations, the protein and mRNA expression levels of iNOS and COX-2 enzyme were found to be inhibited by chiisanoside in a concentration-dependent manner. Furthermore, chiisanoside inhibited the nuclear factor-kappaB (NF-kappaB) activation induced by LPS and this was associated with a reduction in p65 protein in the nucleus and with the phosphorylations of ERK1/2 and JNK MAP kinases. Taken together, our data indicate that the anti-inflammatory properties of chiisanoside might be the result from the inhibition of iNOS, COX-2, TNF-alpha and IL-1beta expression through the down-regulation of NF-kappaB binding activity.     

Anti-inflammatory potential of Antrodia Camphorata through inhibition of iNOS, COX-2 and cytokines via the NF-kappaB pathway

Antrodia camphorata (A. camphorata), well known in Taiwan as a traditional Chinese medicine, has been shown to exhibit antioxidant and anticancer effects. In the present study, therefore, we have examined the effects of the fermented culture broth of A. camphorata (25-100 microg/ml) in terms of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in RAW 264.7 macrophages. Our results indicate concentration-dependent A. camphorata inhibition of LPS-induced NO and PGE2 production, without appreciable cytotoxicity on the RAW 264.7 cells. A. camphorata also attenuates the production of LPS-induced tumor necrosis factor (TNF-alpha) and interleukin (IL)-1beta. Furthermore, A. camphorata blocks the IkappaB-alpha degradation induced by LPS. These results indicate that A. camphorata inhibits LPS induction of cytokine, iNOS and COX-2 expression by blocking NF-kappaB activation. Therefore, we report the first confirmation of the anti-inflammatory potential of this traditionally employed herbal medicine in vitro.     

Inhibition of methanol extract from the aerial parts of Saururus chinensis on lipopolysaccharide-induced nitric oxide and prostagladin E2 production from murine macrophage RAW 264.7 cells

As an attempt to search for bioactive natural products exerting antiinflammatory activity, we have evaluated the effects of the methanol extract from the aerial parts of Saururus chinensis (LOUR.) BAILL (Saururaceae) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) release by the macrophage cell line RAW 264.7. Our data indicate that this extract is a potent inhibitor of NO production and it also significantly decreased PGE(2) release. Consistent with these observations, the protein and mRNA expression level of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 was inhibited by MeOH extracts of the aerial part of S. chinensis (SCM) in a dose-dependent manner. Furthermore, SCM inhibited the LPS-induced DNA binding activity of nuclear factor-kappaB (NF-kappaB), which was associated with decreased p65 protein levels in the nucleus. These results suggest that SCM inhibits LPS-induced iNOS and COX-2 expression by blocking NF-kappaB activation.     

Inhibitory action of minocycline on lipopolysaccharide-lnduced release of nitric oxide and prostaglandin E2 in BV2 microglial cells

Microglia are the major inflammatory cells in the central nervous system and become activated in response to brain injuries such as ischemia, trauma, and neurodegenerative diseases including Alzheimer's disease (AD). Moreover, activated microglia are known to release a variety of proinflammatory cytokines and oxidants such as nitric oxide (NO). Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory effects that are completely distinct form its antimicrobial action. In this study, the inhibitory effects of minocycline on NO and prostaglandin E2 (PGE2) release was examined in lipopolysaccharides (LPS)-challenged BV2 murine microglial cells. Further, effects of minocycline on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression levels were also determined. The results showed that minocycline significantly inhibited NO and PGE2 production and iNOS and COX-2 expression in BV2 microglial cells. These findings suggest that minocycline should be evaluated as potential therapeutic agent for various pathological conditions due to the excessive activation of microglia.     

Styraxoside A isolated from the stem bark of Styrax japonica inhibits lipopolysaccharide-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells by suppressing nuclear factor-kappa B activation

In the present study, the effects of terpenes (styraxosides A and B) and lignans (egonol, masutakeside I, and styraxlignolide A) isolated from the stem bark of Styrax japonica Sieb. et Zucc. (styracaceae) were evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production by the RAW 264.7 macrophage cell line. Of the tested compounds, styraxoside A was found to most potently inhibit the productions of NO and PGE2, and also significantly reduced the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Consistent with these observations, the protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the mRNA expression levels of iNOS, COX-2, TNF-alpha and IL-1beta were found to be inhibited by styraxoside A in a concentration-dependent manner. Furthermore, styraxoside A inhibited the LPS-induced DNA binding activity of nuclear factor-kappaB (NF-kappaB). Taken together, our data indicate that styraxoside A inhibits LPS-induced iNOS, COX-2, TNF-alpha, and IL-1beta expressions through the down-regulation of NF-kappaB-DNA binding activity.     

Down-regulation of microglial cyclo-oxygenase-2 and inducible nitric oxide synthase expression by lipocortin 1

1. Activated microglial cells are believed to play an active role in most brain pathologies, during which they can contribute to host defence and repair but also to the establishment of tissue damage. These actions are largely mediated by microglial secretory products, among which are prostaglandins (PGs) and nitric oxide (NO). 2. The anti-inflammatory protein, lipocortin 1 (LC1) was reported to have neuroprotective action and to be induced by glucocorticoids in several brain structures, with a preferential expression in microglia. In this paper we tested whether the neuroprotective effect of LC1 could be explained by an inhibitory effect on microglial activation. 3. We have previously shown that bacterial endotoxin (LPS) strongly stimulates PGE2 and NO production in rat primary microglial cultures, by inducing the expression of the key enzymes cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively. 4. Dexamethasone (DEX, 1-100 nM) and LC1-derived N-terminus peptide (peptide Ac2-26, 1-100 microg ml(-1)) dose-dependently inhibited the production of both PGE2 and NO from LPS-stimulated microglia. The inhibitory effects of DEX on NO and of the peptide on NO and PGE2 synthesis were partially abrogated by a specific antiserum, raised against the N-terminus of human LC1. The peptide Ac2-26 did not affect arachidonic acid release from control and LPS-stimulated microglial cultures. 5. Western blot experiments showed that the LPS-induced expression of COX-2 and iNOS was effectively down-regulated by DEX (100 nM) and peptide Ac2-26 (100 microg ml(-1)). 6. In conclusion, our findings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms.     

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.     

Ampelopsin attenuates lipopolysaccharide-induced inflammatory response through the inhibition of the NF-κB and JAK2/STAT3 signaling pathways in microglia

Increasing evidence suggests that microglia are a major cellular contributor to neuroinflammation. The present study investigated whether Ampelopsin (Amp), a type of flavanonol derivative from Ampelopsis grossedentata, may exert an anti-inflammatory effect on lipopolysaccharide (LPS)-induced BV2 and primary microglia cells. We found that pre-treatment of microglia cells with Amp before LPS with a non-cytotoxic concentration range decreased the production of nitric oxide (NO) and prostaglandin E2 (PGE2). Amp also suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the mRNA and protein levels. In addition, LPS-induced production of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) was obviously reduced by Amp. Our mechanistic study indicated that Amp suppressed LPS-induced activation of the IκB/NF-κB inflammation pathway without affecting changes in the phosphorylation levels of mitogen-activated protein kinases (MAPKs) in BV2 cells. Further studies revealed that Amp markedly reduced the phosphorylation levels of JAK2-STAT3 and STAT3 nuclear translocation. Overall, our data suggest that Amp can suppress the LPS-induced inflammatory response of microglial cells, indicating that Amp has potential for the treatment of inflammation-mediated neurodegenerative diseases.