Caffeine suppresses lipopolysaccharide-stimulated BV2 microglial cells by suppressing Akt-mediated NF-κB activation and ERK phosphorylation

Since the anti-inflammatory effect of caffeine is unclear in microglial cells, we performed whether caffeine attenuates the expression of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Caffeine substantially suppressed the LPS-induced pro-inflammatory mediators nitric oxide (NO), prostaglandin E₂ (PGE₂) and tumor necrosis factor-α (TNF-α) in BV2 microglial cells. These effects resulted from the inhibition of their regulatory genes inducible NO synthase (iNOS), cycloxygenase-2 (COX-2) and TNF-α. In addition, caffeine significantly decreased LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) by suppressing the nuclear translocation of p50 and p65 subunits. A specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated the LPS-induced expression of iNOS, COX-2 and TNF-α genes. In addition, we elucidated that inhibition of Akt phosphorylation plays a crucial role in caffeine-mediated NF-κB regulation in LPS-stimulated BV2 microglial cells. Caffeine also attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) and a specific inhibitor of ERK, PD98059, subsequently downregulated the expression of the pro-inflammatory genes iNOS, COX-2 and TNF-α. Taken together, our data indicate that caffeine suppresses the generation of pro-inflammatory mediators, such as NO, PGE₂ and TNF-α as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting Akt-dependent NF-κB activation and the ERK signaling pathway.     

Pro-inflammatory effects of commercial alpha-lactalbumin on RAW 264.7 macrophages is due to endotoxin contamination

This study investigated the effects of alpha-lactalbumin (α-LA) on cellular signaling molecules associated with inflammatory responses in RAW 264.7 macrophages. The results indicated that commercial α-LA could increase prostaglandin E₂ (PGE₂) and the expression of COX-2 via increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and jun N-terminal kinase (JNK), and increase nitric oxide (NO) and the expression of iNOS via the activation of ERK1/2 and JNK. Furthermore, commercial α-LA could increase nuclear translocation of p65 nuclear factor-kappa B (p65 NF-κB) through stimulation on inhibitor kappa B-alpha (IκB-α) degradation. Since endotoxin also has these effects, we assayed the content of endotoxin in the commercial α-LA. We found to our surprise that endotoxin was there and that α-LA-induced NO and PGE₂ production could be suppressed by polymyxin B, a specific inhibitor of endotoxin. Thus, the pro-inflammatory effects of commercial α-LA might be caused by endotoxin contamination through activation and expression of iNOS and COX-2 which were upregulated by MAPKs or nuclear translocation of p65 NF-κB in RAW 264.7 cells. It is therefore crucial to assess the possibility of endotoxin contamination within any biological product being studied for immune augmenting activities before a meaning result can be obtained.     

Protective effect of Chrysanthemum indicum Linne against 1-methyl-4-phenylpridinium ion and lipopolysaccharide-induced cytotoxicity in cellular model of Parkinson’s disease

Chrysanthemum indicum Linn. (CI) has been used in Oriental medicine for several centuries. In the present study, the effect of CI extract was evaluated against 1-methyl-4-phenylpridinium ion (MPP⁺)-induced damage in SH-SY5Y cells and lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Cell viability, oxidative damage, reactive oxygen species, expression of Bcl-2/Bax, and poly (ADP-ribose) polymerase (PARP) proteolysis were evaluated using SH-SY5Y cells. Production of iNOS, prostaglandin E₂, and pro-inflammatory cytokines like tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interleukin (IL)-6, expression of cyclooxygenase type-2 (COX-2) and type-1 (COX-1) were examined in activated BV-2 microglia. At 1, 10 and 100 μg, CI inhibited cell loss, decreased the reactive oxygen species production, regulated the Bax/Bcl-2 ratio and inhibited PARP proteolysis in MPP⁺-induced SH-SY5Y cells. Furthermore, CI suppressed the production of prostaglandin E_2, expression of cyclooxygenase type-2 (COX-2), blocked IκB-α degradation and activation of NF-κB p65 in BV-2 cells in a dose-dependent manner. The molecular mechanisms involved by CI might involve its inhibitory actions both on neuronal apoptosis and neuroinflammatory NF-κB/IκB-α signaling pathway. The present investigation scientifically supports the long history and safe usage of CI as an important functional food with potential benefits in ameliorating deleterious conditions seen in PD.     

1,3,5-trihydroxy-4-prenylxanthone represses lipopolysaccharide-induced iNOS expression via impeding posttranslational modification of IRAK-1

Both high level of nitric oxide (NO) and its generating enzyme, inducible NO synthase (iNOS), play important roles in pathophysiological conditions such as inflammatory processes. We previously found that 1,3,5-trihydroxy-4-prenylxanthone (TH-4-PX) isolated from Cudrania cochinchinensis repressed lipopolysaccharide (LPS)-induced NO production in RAW264.7 macrophages. Here we further examined the underlying mechanisms using RT-PCR and Western blot analyses. Consistent with NO inhibition, suppression of LPS-induced iNOS expression by TH-4-PX through abolishing IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor-κB (NF-κB) nuclear translocation was observed. After LPS stimulation, the increased nuclear level of c-Fos and c-Jun (major components of activator protein-1, AP-1) and the phosphorylated level of upstream signal molecules, such as c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase, (ERK) were all significantly suppressed by TH-4-PX, while p38 remained unaffected. A further experiment revealed that TH-4-PX inhibited the phosphorylation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), an upstream signaling molecule required for IKK and mitogen-activated protein kinases (MAPKs) activation. Stimulation with LPS also triggered the modification (phosphorylation and ubiquitination) and eventually the proteasomal degradation of membrane-associated interleukin (IL)-1 receptor-associated serine/threonine kinase 1 (IRAK-1), an essential signaling component to toll-like receptor (TLR)-mediated TAK-1 activation. Interestingly, the modified pattern of IRAK-1 in the presence LPS was significantly attenuated by TH-4-PX treatment. In conclusion, TH-4-PX inhibited LPS-induced NF-κB and AP-1 activations by interfering with the posttranslational modification (phosphorylation and/or ubiquitinylation) of IRAK-1 in the cell membrane to impede TAK1-mediated activation of IKK and MAPKs signal transduction.     

Artemisia fukudo essential oil attenuates LPS-induced inflammation by suppressing NF-κB and MAPK activation in RAW 264.7 macrophages

In the present study, the chemical constituents of Artemisia fukudo essential oil (AFE) were investigated using GC–MS. The major constituents were α-thujone (48.28%), β-thujone (12.69%), camphor (6.95%) and caryophyllene (6.01%). We also examined the effects of AFE on the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Western blotting and RT-PCR tests indicated that AFE has potent dose-dependent inhibitory effects on pro-inflammatory cytokines and mediators. We investigated the mechanism by which AFE inhibits NO and PGE₂ by examining the level of nuclear factor-κB (NF-κB) activation within the mitogen-activated protein kinase (MAPK) pathway, which is an inflammation-induced signal pathway in RAW 264.7 cells. AFE inhibited LPS-induced ERK, JNK, and p38 phosphorylation. Furthermore, AFE inhibited the LPS-induced phosphorylation and degradation of Iκ-B-α, which is required for the nuclear translocations of the p50 and p65 NF-κB subunits in RAW 264.7 cells. Our results suggest that AFE might exert an anti-inflammatory effect by inhibiting the expression of pro-inflammatory cytokines. Such an effect is mediated by a blocking of NF-κB activation which consequently inhibits the generation of inflammatory mediators in RAW264.7 cells. AFE may be useful for treating inflammatory diseases.     

Anti-inflammatory activity of bark of Dioscorea batatas DECNE through the inhibition of iNOS and COX-2 expressions in RAW264.7 cells via NF-κB and ERK1/2 inactivation

We identified a bioactive herbal medicine with anti-inflammatory activity from an ethanol extract derived from the bark of Dioscorea batatas DECNE (BDB) in RAW264.7 cells. We examined the effects of BDB on nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in LPS-induced RAW264.7 cells. BDB consistently inhibited both NO and PGE₂ production in a dose-dependent manner, with an IC₅₀ of 87–71 μg/ml, respectively. The reduction of NO and PGE₂ production were accompanied by a reduction in iNOS and COX-2 protein expression, as evaluated by Western blotting. To evaluate the action mode of BDB and its ability to inhibit iNOS and COX-2 protein expression, we assessed the effects of BDB 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. BDB suppressed DNA-binding activity and reporter gene activity as well as translocation of the NF-κB p65 subunit. BDB also down-regulated IκB kinase (IKK), thus inhibiting LPS-induced both phosphorylation and the degradation of IκBα. In addition, BDB also inhibited the LPS-induced activation of ERK1/2.     

Evaluation of the new anti-inflammatory compound ethyl salicylate 2-O-β-d-glucoside and its possible mechanism of action

Ethyl salicylate 2-O-β-d-glucoside (ESG) is a derivative of natural salicylate isolated from Gaultheria yunnanensis (Franch.) Rehder, it has been used for the treatments of rheumatoid arthritis, swelling and pain. The aim of this study was to evaluate the anti-inflammatory effects of ESG and explore the anti-inflammatory mechanisms. We found that ESG had potent anti-inflammatory effects on the lipopolysaccharide (LPS)-activated murine macrophages RAW264.7. ESG exerted a dose-dependent inhibition of the LPS-stimulated release of the pro-inflammatory cytokines TNF-α and IL-1β. Moreover, it significantly inhibited LPS-stimulated the production of NO and PGE₂ by repressing the expression of iNOS and COX protein respectively. Western blot analysis showed that ESG prominently inhibited LPS-induced activation of NF-κB in RAW264.7 cells by blocking phosphorylation of inhibitor IκBα and p65. Consistent with these results, we found that ESG prevented the nuclear translocation of NF-κB induced by LPS. Our study suggests that ESG may be effective in the treatment of inflammatory diseases by inhibiting the pro-inflammatory cytokine production and regulating the NF-κB signal pathway.     

Water extract of processed Hydrangea macrophylla (Thunb.) Ser. leaf attenuates the expression of pro-inflammatory mediators by suppressing Akt-mediated NF-κB activation

Although Hydrangea macrophylla is native to Northeast Asia and widely cultivated in many parts of the world, no studies on its anti-inflammatory effects have been reported. In this study, we evaluated the anti-inflammatory effect of a water extract of processed H. macrophylla leaf (WH) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. WH inhibited the expression of LPS-stimulated pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α), as well as their regulatory genes inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α without any accompanying cytotoxicity. Moreover, WH significantly suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB), as well as the nuclear translocation of the NF-κB subunits, p65 and p50 by suppressing of IκBα phosphorylation and degradation. WH also increased Akt dephosphorylation, leading to the suppression of the DNA-binding activity of NF-κB in LPS-stimulated RAW264.7 macrophage cells. Our results indicate that WH downregulates the expression of pro-inflammatory mediators such as NO, PGE₂, and TNF-α by suppressing the Akt-mediated NF-κB activity in LPS-stimulated RAW264.7 macrophage cells.     

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by phenolic (3E)-4-(2-hydroxyphenyl)but-3-en-2-one in RAW 264.7 macrophages

The large amount of nitric oxide (NO) produced by inducible NO synthase (iNOS) contributes to cellular injury in inflammatory disease. In the present study, a novel synthetic compound (3E)-4-(2-hydroxyphenyl)but-3-en-2-one (HPB) was found to inhibit lipopolysaccharide (LPS)-induced NO generation, but not through the inhibition of iNOS activity, in RAW 264.7 macrophages. Administration of HPB into mice also inhibited the LPS-induced increase in serum nitrite/nitrate levels. To evaluate the underlying mechanisms of HPB inhibition of NO generation, the expression of the iNOS gene in RAW 264.7 macrophages was examined. HPB abolished the LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in a similar concentration-dependent manner. LPS-induced nuclear factor-κB (NF-κB) DNA binding and NF-κB-dependent reporter gene activity were both significantly inhibited by HPB. This effect was mediated through the inhibition of inhibitory factor-κBα (IκBα) phosphorylation and degradation, and of p65 nuclear translocation. HPB had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinases (MAPK), and c-Jun NH₂-terminal kinase (JNK). However, HPB suppressed the LPS-induced intracellular reactive oxygen species (ROS) production. These results indicate that HPB down-regulates iNOS gene expression probably through the inhibition of LPS-induced intracellular ROS production, which has been implicated in the activation of NF-κB.     

Narirutin fraction from citrus peels attenuates LPS-stimulated inflammatory response through inhibition of NF-κB and MAPKs activation

In this study, we examined the regulatory activity of narirutin fraction from citrus peels on the production of inflammatory mediators managing acute or chronic inflammatory diseases in macrophages. Narirutin fraction inhibited the release, by lipopolysaccharide (LPS)-stimulated macrophages, of nitric oxide (NO) and prostaglandin E₂ (PGE₂) through suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. The release, by LPS stimulated macrophages, of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was also reduced by narirutin fraction in a dose-dependent manner. Furthermore, narirutin fraction inhibited the LPS-mediated activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs), which are signaling molecules involved in production of pro-inflammatory factors. As a result of these properties, narirutin fraction has the potential to be used as a functional dietary supplement and effective anti-inflammatory agent.     

β-Ionone attenuates LPS-induced pro-inflammatory mediators such as NO,PGE2 and TNF-α in BV2 microglial cells via suppression of the NF-κB and MAPK pathway

β-Ionone, a precursor of carotenoids, possesses a variety of biological properties such as anti-cancerous, anti-mutagenic and anti-microbial activity. Nevertheless, anti-inflammatory effects of β-ionone remain unknown. In this study, we investigated whether ION attenuates the expression of lipopolysaccharide (LPS)-induced pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E₂ (PGE₂) and tumor necrosis factor-α (TNF-α) in BV2 microglia cells. Our data showed that β-ionone significantly inhibits secretion of NO, PGE₂ and TNF-α. β-Ionone also inhibits the expression of inducible NO synthesis (iNOS), cyclooxygenase-2 (COX-2) and TNF-α protein and their mRNA in LPS-stimulated BV2 microglia cells. In addition, β-ionone significantly reduced DNA-binding activity of nuclear factor-κB (NF-κB) through suppression of nuclear translocation of p50 and p65. We showed that NF-κB inhibitor N-acetyl-L-cysteine (NAC) effectively attenuates the expression of LPS-stimulated iNOS, COX-2 and TNF-α. We also found that LPS-induced NF-κB activation is significantly regulated through inhibition of Akt phosphorylation in the presence of β-ionone. Finally, we showed that β-ionone substantially inhibits the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, p38 and JNK, which are closely related to regulation of pro-inflammatory mediator secretion. Taken together, these data imply that β-ionone regulates LPS-induced NF-κB-dependent inflammatory pathways through suppression of Akt and MAPK activation.     

Ethylacetate extract from DraconisResina inhibits LPS-induced inflammatory responses in vascular smooth muscle cells and macrophages via suppression of ROS production

DraconisResina (DR) is a type of dragon’s blood resin obtained from Daemomoropsdraco BL. (Palmae). DR has long been used as a traditional Korean herbal medicine, and is currently used in traditional clinics to treat wounds, tumors, diarrhea, and rheumatism, insect bites and other conditions. In this study, we evaluated fractionated extracts of DR to determine if they inhibited the production of interleukin-1β (IL-1β) and the expression of cyclooxygenase (COX)-2. The results of this analysis revealed that the ethylacetate extract of DraconisResina (DREA) was more potent than that of other extracts. Moreover, DREA inhibited the production of nitric oxide (NO), reactive oxygen species (ROS), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), IL-8 and IL-6 in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMC) and RAW 264.7 macrophages. Furthermore, treatment with an NADPH oxidase assembly inhibitor, AEBSF, efficiently blocked LPS-induced mitogen-activated protein kinases (MAPKs) activation, as did DREA. These findings indicate that DREA inhibits the production of NO, PGE₂, TNF-α, IL-8, and IL-6 by LPS via the inhibition of ROS production, which demonstrates that DREA inhibits LPS-induced inflammatory responses via the suppression of ROS production. Taken together, these results indicate that DREA has the potential for use as an anti-atherosclerosis agent.     

Anti-inflammatory effect of corymbocoumarin from Seseli gummiferum subsp. corymbosum through suppression of NF-κB signaling pathway and induction of HO-1 expression in LPS-stimulated RAW 264.7 cells

This study investigated the anti-inflammatory activity of corymbocoumarin, an angular-type pyranocoumarin isolated from Seseli gummiferum subsp. corymbosum in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Corymbocoumarin not only inhibited the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂), but also inhibited the protein and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Corymbocoumarin also attenuated pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Investigation of the effect on nuclear factor κB (NF-κB) signaling pathway showed that corymbocoumarin inhibited the phosphorylation of Akt and inhibitory κB (IκB)-α and decreased the subsequent translocation of the p65 and p50 NF-κB subunits to the nucleus. A further study revealed that corymbocoumarin exerted anti-inflammatory activity through induction of heme oxygenase (HO)-1 expression. The in vivo study showed that corymbocoumarin (20 mg/kg, i.p.) reduced paw swelling in carrageenan-induced acute inflammation model. Taken together, these results suggest that corymbocoumarin exerts its anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation and inducing HO-1 expression. Corymbocoumarin may provide a useful therapeutic approach for inflammation-associated diseases.     

Exopolysaccharide of Laetiporus sulphureus var. miniatus downregulates LPS-induced production of NO, PGE2, and TNF-α in BV2 microglia cells via suppression of the NF-κB pathway

Our previous study showed that the exopolysaccharide (EPS) of Laetiporus sulphureus var. miniatus was well characterized and prevented cell damage in streptozotocin-induced apoptosis. However, little is known about the molecular mechanisms underlying its anti-inflammatory effects. Therefore, we attempted in this study to determine whether EPS induces a significant inhibition of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia cells. Our results showed that EPS significantly inhibited LPS-induced pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α), without any significant cytotoxicity. EPS also downregulated mRNA and protein expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-induced BV2 microglia cells. Our data also revealed that EPS treatment significantly reduced translocation of nuclear factor-κB (NF-κB) subunit p65 and its DNA-binding activity in LPS-stimulated BV2 microglia cells. Furthermore, we confirmed by using proteasome inhibitor N-acetyl-l-cysteine (NAC), that the inhibition of NF-κB activity influenced the expression of pro-inflammatory genes in LPS-induced BV2 microglia cells. As expected, NAC suppressed the expression of iNOS, COX-2, and TNF-α by blocking proteasome-mediated degradation. Taken together, our data indicate that EPS inhibits the expression of pro-inflammatory mediators by suppressing NF-κB activity.     

Incorporation of sciadonic acid into cellular phospholipids reduces pro-inflammatory mediators in murine macrophages through NF-κB and MAPK signaling pathways

Sciadonic acid (SCA; Δ5,11,14–20:3), a non-methylene-interrupted polyunsaturated fatty acid (NMIFA), can substitute for arachidonic acid (AA) and reduce prostaglandin E₂ (PGE₂) synthesis in macrophages. However, little is known about how SCA exerts its anti-inflammatory effects. The objectives of this study were to purify SCA from seeds of Podocarpus nagi and investigate mechanisms underlying the modulatory effects of SCA on inflammatory responses in murine RAW264.7 macrophages. We describe how high-purity SCA (>98%) can be obtained using argentated column chromatography. SCA was dose-dependently incorporated into cellular phospholipids, and increasing SCA incorporation correlated with decreases in the proportions of AA, total monounsaturated fatty acids (MUFA) and total saturated fatty acids (SFA). SCA decreased production of PGE₂ (29%), nitric oxide (NO) (31%), interleukin-6 (IL-6) (34%) and tumor necrosis factor-α (TNF-α) (14%). The suppression of pro-inflammatory mediators was due, in part, to decreased expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). SCA incorporation suppressed nuclear factor-kappa B (NF-κB) translocation and phosphorylation of mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK). These findings indicate that by altering the cellular fatty acid composition SCA can modulate the responsiveness of macrophages to LPS through inactivation of NF-κB and MAPK pathways.