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.     

Rheosmin,a naturally occurring phenolic compound inhibits LPS-induced iNOS and COX-2 expression in RAW264.7 cells by blocking NF-κB activation pathway

Inflammation is part of the host defense mechanism against harmful matters and injury; however, aberrant inflammation is associated to the development of chronic disease such as cancer. Raspberry ketone is a natural phenolic compound. It is used in perfumery, in cosmetics, and as a food additive to impart a fruity odor. In this study, we evaluated whether rheosmin, a phenolic compound isolated from pine needles regulates the expression of iNOS and COX-2 protein in LPS-stimulated RAW264.7 cells. Rheosmin dose-dependently inhibited NO and PGE₂ production and also blocked LPS-induced iNOS and COX-2 expression. Rheosmin potently inhibited the translocation of NF-κB p65 into the nucleus by IκB degradation following IκB-α phosphorylation. This result shows that rheosmin inhibits NF-κB activation. In conclusion, our results suggest that rheosmin inhibits LPS-induced iNOS and COX-2 expression in RAW264.7 cells by blocking NF-κB activation pathway.     

Inhibitory effects of ivermectin on nitric oxide and prostaglandin E2 production in LPS-stimulated RAW 264.7 macrophages

We have previously shown that ivermectin inhibits LPS-induced production of inflammatory cytokines. In the present study, we investigated the effect of ivermectin on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in RAW 264.7 macrophages. Ivermectin inhibited LPS-induced NO and PGE₂ production. Consistent with these observations, the protein and mRNA expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes were inhibited by ivermectin in a concentration-dependent manner. Furthermore, the phosphorylation of p38, ERK1/2, and JNK in LPS-stimulated RAW 264.7 cells was suppressed by ivermectin in a dose-dependent manner. These results suggest that ivermectin suppresses NO and PGE₂ production, as well as iNOS and COX-2 expression, by inhibiting phosphorylation of mitogen-activated protein kinases (MAPK) (p38, ERK1/2, and JNK) in LPS-stimulated RAW 264.7 cells.     

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.     

穿琥宁抗炎作用的血红素加氧酶-1的信号转导机制

目的初步探讨穿琥宁抗炎作用的信号转导机制。方法用WST-8细胞计数试剂盒检测穿琥宁的细胞毒性;用IN Cell Analyzer 2000活细胞成像系统及组成型增强表达绿色荧光蛋白偶联NF-κBP65(EGFP-NF-κBP65)融合蛋白的CHO细胞和EGFP偶联促分裂原活化蛋白激酶APk2(EGFP-MAPK-APk2)融合蛋白的BHK细胞观察穿琥宁对白细胞介素1β(IL-1β)或肿瘤坏死因子α(TNF-α)诱导NF-κBP65核转位及脂多糖(LPS)诱导的P38MAPK下游分子MAPK-APk2核转位的影响;Western印迹法检测穿琥宁和血红素加氧酶-1(HO-1)特异性抑制剂锌原卟啉(ZnPP)对RAW264.7细胞HO-1、诱导型一氧化氮合酶(iNOS)和环氧化酶2(COX-2)表达的影响;Griess反应和ELISA法测定穿琥宁对RAW264.7细胞一氧化氮(NO)和前列腺素E2(PGE2)生成的影响。结果穿琥宁3～100μmol·L-1作用24 h对RAW264.7细胞无细胞毒性。穿琥宁不能抑制由IL-1β诱导的NF-κBP65核转位和LPS诱导的MAPK-APk2核转位,对TNF-α诱导的NF-κBP65核转位有较弱的抑制作用,抑制率为20%,无明显浓度效应关系。穿琥宁3,10,30和100μmol·L-1作用4 h能诱导RAW264.7细胞HO-1表达,穿琥宁100μmol· L-1作用6 h显著抑制IL-1β诱导的COX-2表达和PGE2产生,作用12 h抑制iNOS表达和NO产生。HO-1活性抑制剂ZnPP能部分逆转穿琥宁的上述作用。结论穿琥宁的抗炎作用可能主要通过HO-1信号转导,继而抑制iNOS和COX-2的表达及NO和PGE2的产生。对NF-κB信号传导系统的调节作用尚不清楚。     

Britanin suppresses LPS-induced nitric oxide,PGE2 and cytokine production via NF-κB and MAPK inactivation in RAW 264.7 cells

Little is known about the biological properties of britanin, which is isolated from the flowers of Inula japonica (Inulae Flos). Based on our previous studies that Inulae Flos had anti-inflammation and anti-asthmatic activities, we tried to find the bioactive compounds from it. In this study, the anti-inflammatory effects of britanin on the inflammatory mediators as well as on nuclear factor (NF)-кB and mitogen-activated protein (MAP) kinase activation were evaluated in RAW 264.7 cells. Britanin inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE₂) along with the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In addition, britanin reduced the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Furthermore, the phosphorylations of MAP kinases (p38 and JNK) in LPS-stimulated RAW 264.7 cells were suppressed by britanin. Moreover, britanin inhibited the NF-κB activation induced by LPS, which was associated with the abrogation of IκBα degradation and subsequent decreases in nuclear p65 levels. This study suggests that the anti-inflammatory activities of britanin might be attributed to the inhibition of iNOS and COX-2 and cytokine expression at least in part, through the attenuation of the phosphorylations of MAP kinases and NF-κB activation via IκBα degradation in macrophages. We conclude that britanin may have potential for the treatment of inflammatory diseases through the down-regulation of MAP kinases and NF-κB mediated activation of macrophages.     

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.     

3‑Bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol inhibits LPS-induced pro-inflammatory responses by preventing ROS production and downregulating NF-κB in vitro and in a zebrafish model

The anti-inflammatory effects of 3‑bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol (BEMB) from Polysiphonia morrowii were evaluated in lipopolysaccharide (LPS)-induced RAW264.7 cells and zebrafish embryo. BEMB showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO) and reactive oxygen species (ROS), and the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in the LPS-activated RAW264.7 cells and zebrafish embryo without cytotoxicity. Moreover, BEMB suppressed the protein and mRNA expression levels of nuclear factor (NF)-κB (p65 and inhibitor of NF-κB [IκB]-A) in RAW264.7 cells and zebrafish embryo, respectively. Collectively, the results of this study indicate that BEMB suppressed the production of pro-inflammatory mediators such as NO, iNOS, and COX-2 as well as their regulation in LPS-induced RAW264.7 cells and zebrafish embryos by inhibiting ROS production and NF-κB expression. Therefore, this study suggests that BEMB could be a potential anti-inflammatory agent for the treatment of inflammatory diseases.     

A new sulfonic acid derivative, (Z)-4-methylundeca-1,9-diene-6-sulfonic acid,isolated from the cold water sea urchin inhibits inflammatory responses through JNK/p38 MAPK and NF-κB inactivation in RAW 264.7

In this study, we isolated a new sulfonic acid derivative, (Z)-4-methylundeca-1,9-diene-6-sulfonic acid (1), from the sea urchin collected from the Sea of Okhotsk. We established the structure of this new compound by analysis of NMR and HRMS data, along with comparison of the data with those of the related compounds reported in the literature. In addition, we investigated its anti-inflammatory effects in LPS-stimulated RAW264.7 macrophages. Compound 1 inhibited the production of NO, iNOS, PGE₂, and COX-2, and it also suppressed the production of pro-inflammatory cytokines, such as TNF-α and IL-1β. It inhibited the translocation of the NF-κB subunit p65 into the nucleus by interrupting the phosphorylation and degradation of IκB-α. In addition, compound 1 significantly decreased the phosphorylation of JNK and p38 in LPS-stimulated RAW264.7 macrophages, suggesting that suppression of the inflammation process by compound 1 was mediated through the MAPK pathway. Taken together, this study showed that the anti-inflammatory effects of a new sulfonic acid derivative, (Z)-4-methylundeca-1,9-diene-6-sulfonic acid were mediated through the inhibition of NF-κB and JNK/p38 MAPK signaling pathways.     

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.     

Anti-inflammatory effect of 2-methoxy-4-vinylphenol via the suppression of NF-κB and MAPK activation,and acetylation of histone H3

Although inflammation acts as host defense mechanism against infection or injury and is primarily a self limiting process, inadequate resolution of inflammatory responses leads to various chronic disorders. This work aimed to elucidate the anti-inflammatory effects of 2-methoxy-4-vinylphenol (2M4VP) isolated from pine needles in LPS-stimulated RAW264.7 cells. Some key pro-inflammatory mediators including nitric oxide (NO), prostaglandins (PGE₂), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) were studied by sandwich ELISA and western blot. In addition, suppression of NF-κB and MAPK activation, and histone acetylation was studied by western blot analysis and immunostaining. 2M4VP dosedependently inhibited NO and PGE₂ production and also blocked LPS-induced iNOS and COX-2 expression. In addition, 2M4VP potently inhibited the translocation of NF-κB p65 into the nucleus by IκB degradation following IκB-α phosphorylation and the phosphorylation of MAPKs such as p38, ERK1/2, and JNK. Also, 2M4VP inhibited hyper-acetylation of histone H3 (Lys9/Lys14) induced by LPS. Taken together, our results suggest that 2M4VP, a naturally occurring phenolic compound, exert potent anti-inflammatory effects by inhibiting LPS-induced NO, PGE₂, iNOS, and COX-2 in RAW264.7 cells. These effects are mediated by suppression of NF-κB and MAPK activation and histone acetylation.     

Isotrifoliol inhibits pro-inflammatory mediators by suppression of TLR/NF-κB and TLR/MAPK signaling in LPS-induced RAW264.7 cells

Soybeans, produced by Glycine max (L.) Merr., contain high levels of isoflavones, such as genistein and daidzein. However, soy leaves contain more diverse and abundant flavonol glycosides and coumestans, as compared to the soybean. This study investigated the anti-inflammatory effects of the major coumestans present in soy leaf (coumestrol, isotrifoliol, and phaseol) in lipopolysaccharide (LPS)-induced RAW264.7 cells. Coumestans significantly reduced LPS-induced nitric oxide (NO), prostaglandin E2 (PGE₂), and reactive oxygen species (ROS) production; isotrifoliol had the most potent anti-inflammatory activity. Isotrifoliol reduced LPS-mediated induction of mRNA expression of inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-6, tumor necrosis factor alpha (TNFα), and chemokines, such as chemokine (C-C motif) ligand (CCL) 2, CCL3, and CCL4. Isotrifoliol prevented NF-κB p65 subunit activation by reducing the phosphorylation and degradation of the inhibitor of NF-κB. And isotrifoliol significantly suppressed phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Furthermore, isotrifoliol suppressed LPS-induced Toll-like Receptor (TLR) signaling pathway, including mRNA expression of TNF receptor associated factor 6, transforming growth factor beta-activated kinase 1 (TAK1), TAK1 binding protein 2 (TAB2), and TAB3. These results demonstrate that isotrifoliol exerts an anti-inflammatory effect by suppressing the expression of inflammatory mediators via inhibition of TLR/NF-κB and TLR/MAPK signaling in LPS-induced RAW264.7 macrophages. Therefore, isotrifoliol can be used as an anti-inflammatory agent, and coumestan-rich soy leaf extracts may provide a useful dietary supplement.     

Inhibitory effects of (−)-α-bisabolol on LPS-induced inflammatory response in RAW264.7 macrophages

Although (−)-α-bisabolol, a natural monocyclic sesquiterpene alcohol, is often used as a cosmetic soothing supplement, little is known about its mechanisms of anti-inflammatory effects. Therefore, this study was designed to investigate anti-inflammatory effects of (−)-α-bisabolol and its mechanisms of action. In this study, we found that (−)-α-bisabolol inhibited lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E₂ (PGE₂) in RAW264.7 cells. In addition, expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes was reduced, as evidenced by Western blot and luciferase reporter assays for COX-2 and iNOS. To assess the mechanism of the anti-inflammatory property of (−)-α-bisabolol, its effects on the activity of AP-1 and NF-κB promoters were examined. LPS-induced activation of AP-1 and NF-κB promoters was significantly reduced by (−)-α-bisabolol. Consistently, (−)-α-bisabolol reduced LPS-induced phosphorylation of IκBα. In addition, while LPS-induced phosphorylation of ERK and p38 was attenuated by (−)-α-bisabolol, significant changes in the level of phosphorylated JNK were not observed. Our results indicate that (−)-α-bisabolol exerts anti-inflammatory effects by downregulating expression of iNOS and COX-2 genes through inhibition of NF-κB and AP-1 (ERK and p38) signaling.     

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.     

8,8′-Bieckol,isolated from edible brown algae,exerts its anti-inflammatory effects through inhibition of NF-κB signaling and ROS production in LPS-stimulated macrophages

Ecklonia cava (E. cava) is an abundant brown alga that contains high levels of phlorotannins, which are unique marine polyphenolic compounds. It has been suggested that E. cava phlorotannins exert anti-inflammatory effects. However, the anti-inflammatory effects and underlying molecular mechanism exerted by 8,8′-bieckol isolated from E. cava have not been reported. Thus, in this study, we examined the anti-inflammatory effects of 8,8′-bieckol on lipopolysaccharide (LPS)-stimulated primary macrophages and RAW 264.7 macrophages. We found that 8,8′-bieckol suppressed key inflammatory mediator [i.e., nitric oxide (NO) and prostaglandin E₂ (PGE₂)] production in both primary and RAW 264.7 macrophages. 8,8′-Bieckol inhibited NO by suppressing LPS-induced expression of inducible nitric oxide synthase (iNOS) at the mRNA and protein levels in primary macrophages and RAW 264.7 cells. In addition, 8,8′-bieckol decreased the production and mRNA expression of the inflammatory cytokine interleukin-6 (IL-6), but not tumor necrosis factor (TNF)-α, in RAW 264.7 cells. Moreover, 8,8′-bieckol treatment diminished transactivation of nuclear factor-kappa B (NF-κB) and nuclear translocation of the NF-κB p65 subunit and suppressed LPS-induced intracellular reactive oxygen species (ROS) production in macrophages. Furthermore, 8,8′-bieckol markedly reduced mortality in LPS-induced septic mice. Taken together, these data indicate that the anti-inflammatory properties of 8,8′-bieckol are associated with the suppression of NO, PGE₂, and IL-6 via negative regulation of the NF-κB pathway and ROS production in LPS-stimulated RAW 264.7 cells. Moreover, 8,8′-bieckol protects mice from endotoxin shock.     

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.