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.     

2-(4-Hydroxyphenyl)-5-(3-Hydroxypropenyl)-7-Methoxybenzofuran,a Novel Ailanthoidol Derivative,Exerts Anti-Inflammatory Effect through Downregulation of Mitogen-Activated Protein Kinase in Lipopolysaccharide-Treated RAW 264.7 Cells

We reported that ailanthoidol, a neolignan from Zanthoxylum ailanthoides and Salvia miltiorrhiza Bunge, inhibited inflammatory reactions by macrophages and protected mice from endotoxin shock. We examined the anti-inflammatory activity of six synthetic ailanthoidol derivatives (compounds 1-6). Among them, compound 4, 2-(4-hydroxyphenyl)-5-(3-hydroxypropenyl)-7-methoxybenzofuran, had the lowest IC₅₀ value concerning nitric oxide (NO) release from lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Compound 4 suppressed the generation of prostaglandin (PG) E₂ and the expression of inducible NO synthase and cyclooxygenase (COX)-2 induced by LPS, and inhibited the release of LPS-induced pro-inflammatory cytokines from RAW264.7 cells. The underlying mechanism of compound 4 on anti-inflammatory action was correlated with the down-regulation of mitogen-activated protein kinase and activator protein-1 activation. Compound 4 is potentially an effective functional chemical candidate for the prevention of inflammatory diseases.     

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.     

Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1

The role of heme oxygenase-1 (HO-1) played in the inhibitory mechanism of flavonoids in lipopolysaccharide (LPS)-induced responses remained unresolved. In the present study, flavonoids, including 3-OH flavone, baicalein, kaempferol, and quercetin, induced HO-1 gene expression at the protein and mRNA levels in the presence or absence of LPS in RAW264.7 macrophages. This effect was associated with suppression of LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein expression. Hemin induced HO-1 protein expression and this was associated with the suppression of LPS-induced NO production and iNOS protein expression in a dose-dependent manner. In addition, an increase in bilirubin production was found in flavonoid- and hemin-treated cells. Hemin, at the doses of 10, 20, and 50 microM, dose-dependently stimulated the flavonoid (50 microM)-induced HO-1 protein expression, and enhanced their inhibitory effects on LPS-induced NO production and iNOS protein expression. Pretreatment of the HO-1 inhibitor, tin protoporphyrin (10 microM), attenuated the inhibitory activities of the indicated flavonoids on LPS-induced NO production. Morphologic analysis showed that 3-OH flavone, baicalein, kaempferol, quercetin, hemin, and tin protoporphyrin did not cause any change in cell viability in the presence or absence of LPS. In contrast, only 3-OH flavone showed a significant inhibition of cell growth using the MTT assay. Transfection of an HO-1 vector in macrophages (HO-1/RAW264.7) resulted in a 3-fold increase in HO-1 protein compared with that the parental RAW264.7 cells. NO production mediated by LPS in HO-1 over-expressed RAW264.7 cells (HO-1/RAW264.7) was significant less than that in parental RAW264.7 cells. 3-OH Flavone, baicalein, kaempferol, and quercetin showed a more significant inhibition on LPS-induced NO production in HO-1/RAW264.7 cells than in parental RAW264.7 cells. These results provide evidence on the role of HO-1 in the inhibition of LPS-induced NO production by flavonoids. A combination of HO-1 inducers (i.e. hemin) and flavonoids might be an effective strategy for the suppression of LPS-induced NO production.     

Anti-inflammatory effects of phlorofucofuroeckol B-rich ethyl acetate fraction obtained from Myagropsis myagroides on lipopolysaccharide-stimulated RAW 264.7 cells and mouse edema

Myagropsis myagroides has been used as a Chinese medicine and its extract has shown various biological activities, however, its anti-inflammatory mechanism remains unknown. In this study, we investigated the inhibitory effects of the ethyl acetate fraction of M. myagroides (EFM) on the production of inflammatory mediators and pro-inflammatory cytokines in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells. EFM significantly inhibited LPS-induced production of nitric oxide (NO), prostaglandin E₂, and pro-inflammatory cytokines in a dose-dependent manner and suppressed the production of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 in RAW 264.7 cells. Inhibitory effect of EFM on iNOS expression and NO production was further confirmed using LPS-activated mouse peritoneal macrophages. EFM treatment strongly suppressed the activation of nuclear factor-kappa B (NF-κB) by suppressing phosphorylation of Akt and extracellular signal-regulated kinases (ERKs). EFM as well as phlorofucofuroeckol B (PFF-B), a major compound isolated from EFM, reduced ear edema induced by phorbol 12-myristate 13-acetate in mice. These results indicate that the anti-inflammatory effect of EFM, rich in PFF-B, on LPS-stimulated macrophages is regulated by the inhibition of NF-κB pathway through the inhibition of ERKs and Akt phosphorylation in LPS-stimulated macrophage cells.     

Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-ß

RAW 264.7 macrophages induced with lipopolysaccharide and interferon-γ expressed nitric oxide (NO) synthase. Approximately two-thirds of the total induced NO synthase activity was found in the cytosolic fraction, whereas one-third was associated with the particulate fraction. Both enzymes formed L-citrulline in addition to NO-like material. NO and L-citrulline formation by both enzymes were calcium-independent and inhibited by N^G-nitro-L-arginine and N^G-methyl-L-arginine. Transforming growth factor-ß1 prevented the induction of both enzymes.     

1-苯甲酰基-4-(4-氯苄基)哌嗪衍生物的设计、合成以及抗炎活性研究

目的:以1-苯甲酰基-4-(4-氯苄基)哌嗪为先导化合物,经进一步的结构优化,期望获得一类抗炎活性更好的多靶点抑制剂。方法:以取代苯甲酸为起始原料,经取代、酰化反应合成18个目标化合物。测定所有化合物对脂多糖(lipopolysaccharide,LPS)诱导RAW264.7细胞分泌肿瘤坏死因子(tumor necrosis factor,TNF)-α和白细胞介素(interleukin,IL)-6的抑制作用,并对活性较好的化合物进行分子对接。结果:所有化合物均经¹H NMR,¹³C NMR,HRMS确定其结构,化合物3d,8k对TNF-α的分泌具有较好的抑制活性,化合物3b,3d对IL-6的分泌具有较好的抑制活性。结论:化合物3d对LPS诱导RAW264.7细胞分泌TNF-α和IL-6具有较好的抑制活性,有进一步研究的价值。     

Asperlin from the marine-derived fungus Aspergillus sp. SF-5044 exerts anti-inflammatory effects through heme oxygenase-1 expression in murine macrophages

Asperlin is a fungal metabolite isolated from Aspergillus sp. SF-5044. In the present study, we isolated asperlin from the marine-derived fungus Aspergillus sp. SF-5044 and demonstrated that it inhibited inducible nitric oxide synthase (iNOS) expression, reduced iNOS-derived NO, suppressed cyclooxygenase (COX)-2 expression, and reduced COX-derived prostaglandin (PG) E? production in lipopolysaccharide (LPS)-stimulated RAW264.7 and murine peritoneal macrophages. Similarly, asperlin reduced the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. In addition, asperlin inhibited the phosphorylation and degradation of IκB-α, as well as the nuclear translocation of p65 caused by the stimulation of LPS in RAW264.7 macrophages. Furthermore, asperlin induced heme oxygenase (HO)-1 expression through nuclear translocation of nuclear factor E2-related factor 2 and increased HO activity in RAW264.7 macrophages. The effects of asperlin on the LPS-induced expression of iNOS and COX-2 and production of NO, PGE?, TNF-α, and IL-1β were partially reversed by a HO-1 inhibitor, tin protoporphyrin. These findings suggest that asperlin-induced HO-1 expression plays a role in the anti-inflammatory effects of asperlin in macrophages.     

蟛蜞菊内酯对脂多糖诱导RAW264.7细胞环氧化酶2、NO及TNF-α的影响

目的:研究蟛蜞菊内酯对脂多糖(lipopo-lysaccharide,LPS)诱导RAW264.7巨噬细胞环氧化酶2(COX-2)、NO及TNF-α的作用。方法:ELISA方法检测0.2、2、20μmol/L不同浓度蟛蜞菊内酯对终浓度为10μg/mL LPS诱导RAW264.7细胞产生TNF-α、NO及前列腺素E2(PGE2)的影响,Western blot方法检测蟛蜞菊内酯对LPS诱导COX-2酶蛋白表达的影响。结果:LPS能够明显诱导小鼠RAW264.7细胞产生的COX-2酶蛋白,蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的COX-2酶蛋白表达。PGE2可以被LPS诱导增加,与空白组比有显著差异。蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的PGE2、NO和TNF-α,呈现剂量依赖性。结论:蟛蜞菊内酯抗炎的作用机制可能为抑制COX-2的蛋白表达,进而抑制PGE2的生成,也可能与抑制NO和TNF-α生成有关。     

Discovery of Novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole Derivatives as Potential Anti‐Inflammatory Agents

A novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 5 with good anti‐inflammatory activity was identified from our in‐house library. Based on hit compound 5 , two series of 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 6a – g and 7a – h were designed and synthesized as novel anti‐inflammatory agents. Most of synthesized compounds exhibited good inhibitory activity on NO and TNF‐α production in LPS‐stimulated RAW 264.7 macrophages, in which the compound 6e showed most potent inhibitory activity on NO (IC₅₀ = 0.86 μm ) and TNF‐α (IC₅₀ = 1.87 μm ) production. Further evaluation revealed that compound 6e displayed more potent in vivo anti‐inflammatory activity than ibuprofen did on xylene‐induced ear oedema in mice. Additionally, Western blot analysis revealed that compound 6e could restore phosphorylation level of IκBα and protein expression of p65 NF‐κB in LPS‐stimulated RAW 264.7 macrophages.     

Cudratricusxanthone A from Cudrania tricuspidata suppresses pro-inflammatory mediators through expression of anti-inflammatory heme oxygenase-1 in RAW264.7 macrophages

Cudratricusxanthone A (CTXA), isolated from the roots of Cudrania tricuspidata Bureau (Moraceae) has an isoprenylated xanthone skeleton that is known to exert a variety of biological activities. In the present study, we demonstrated that CTXA inhibited cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS) expression, and thereby reduced COX-2-derived prostaglandin E2 (PGE₂) and iNOS-derived NO production in lipopolysaccharide (LPS)-stimulated macrophages. Similarly, CTXA suppressed tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production. Moreover, CTXA inhibited the induced phosphorylation and degradation of IκB-α as well as the LPS-induced increase in p65 in the nuclear fraction of macrophages. CTXA also induced heme oxygenase-1 (HO-1) expression and increased heme oxygenase (HO) activity in RAW264.7 macrophages. We also demonstrated that the effects of CTXA on LPS-induced PGE₂, NO, TNF-α, and IL-1β production were partially reversed by the HO-1 inhibitor tin protoporphyrin, suggesting that CTXA-induced HO-1 expression was partly responsible for the resulting anti-inflammatory effects of the drug. Thus CTXA was shown to be an effective HO-1 inducer, capable of inhibiting macrophage-derived pro-inflammatory mediators.     

1-O-hexyl-2,3,5-trimethylhydroquinone inhibits IkappaB phosphorylation and degradation-linked inducible nitric oxide synthase expression: beyond antioxidant function

Inducible nitric oxide (NO) production in macrophages plays an important role in atherosclerosis, the protective effects of vitamin E and its derivatives perhaps being partly mediated by alteration in this parameter. We have investigated the influence of a novel synthesized vitamin E derivative, 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), on NO production in the RAW 264.7 mouse macrophage cell line. HTHQ dose-dependently inhibited lipopolysaccharide (LPS)-induced NO production through reducing LPS-triggered inducible nitric oxide synthase (iNOS) expression. The phosphorylation and subsequent degradation of IkappaB caused by LPS in RAW 264.7 cells was markedly blocked. The free radical scavenging activity of HTHQ was only 2-fold that of vitamin E, whereas its inhibition of NO production was found to be nearly 500-fold stronger. Our results indicated that HTHQ suppressed NO production in macrophages by blocking IkappaB degradation and thus inhibiting iNOS expression. The inhibitory activity of HTHQ on NO production did not parallel its free radical scavenging activity, implying a possible involvement of additional functions.     

Myrrh mediates haem oxygenase-1 expression to suppress the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages

Objectives To elucidate a novel anti‐inflammatory mechanism of myrrh against lipopolysaccharide (LPS)‐induced inflammation. Methods RAW264.7 macrophages were cultured in DMEM and then cells were treated with LPS or LPS plus a myrrh methanol extract (MME) for 24 h. The culture medium was collected for determination of nitric oxide (NO), prostaglandin (PG)E₂, interleukin (IL)‐1β, and tumour necrosis factor (TNF)‐α, and cells were harvested by lysis buffer for Western blot analysis. Key findings Our data showed that treatment with the MME (1～100 µg/ml) did not cause cytotoxicity or activate haem oxygenase‐1 (HO‐1) protein synthesis in RAW264.7 macrophages. Furthermore, the MME inhibited LPS‐stimulated NO, PGE₂, IL‐1β and TNF‐α release and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)‐2 protein expression. Zn(II) protoporphyrin IX, a specific inhibitor of HO‐1, blocked the inhibition of iNOS and COX‐2 expression by the MME. Conclusions These results suggest that among mechanisms of the anti‐inflammatory response, the MME inhibited the production of NO, PGE₂, IL‐1β and TNF‐α by downregulating iNOS and COX‐2 gene expression in macrophages and worked through the action of HO‐1.     

Bis‐(3‐hydroxyphenyl) diselenide inhibits LPS‐stimulated iNOS and COX‐2 expression in RAW 264.7 macrophage cells through the NF‐kB inactivation

Objectives Previously, we reported that diaryl diselenide compounds have strong inhibitory effects on lipopolysaccharide (LPS)‐induced nitric oxide (NO) production in macrophages. In this study, we investigated the molecular mechanisms underlying NO suppression and prostaglandin E₂ (PGE₂) production by diaryl diselenide compounds, bis‐(2‐hydroxyphenyl) diselenide (DSE‐A), bis‐(3‐hydroxyphenyl) diselenide (DSE‐B), bis‐(4‐hydroxyphenyl) diselenide (DSE‐C), dipyridyl diselenide (DSE‐D) and diphenyl diselenide (DSE‐E). Methods The effect of these compounds on NO suppression and PGE₂ production was investigated in RAW 264.7 macrophages. Key findings Our data indicate that of the above, DSE‐B most potently inhibits NO and PGE₂ production, and that it also significantly reduces the releases of tumour necrosis factor (TNF)‐α, interleukin(IL)‐1β and IL‐6. Consistent with these observations, DSE‐B also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase‐2 (COX‐2), and the mRNA levels of iNOS, COX‐2, TNF‐α, IL‐1β and IL‐6. Furthermore, DSE‐B inhibited LPS‐induced nuclear factor‐κB (NF‐κB) activation, which was associated with the prevention of the inhibitor κB‐α (IκB‐α) degradation and a subsequent reduction in nuclear p65 protein levels. Conclusions Taken together, our data suggest that the anti‐inflammatory properties of DSE‐B are due to reduction in the expression of iNOS, COX‐2, TNF‐α, IL‐1β and IL‐6 through the down‐regulation of NF‐κB binding activity.     

人参总皂苷在巨噬细胞RAW264.7中降低脂多糖诱导的炎症和氧化应激

目的:通过脂多糖诱导巨噬细胞RAW264.7建立炎症和氧化应激模型,探讨人参总皂苷的抗炎和抗氧化效果以及对脂多糖诱导的巨噬细胞RAW264.7自噬水平的影响。方法:培养RAW264.7细胞,给予脂多糖刺激并加入不同浓度人参总皂苷,使用硝酸还原酶法检测细胞内一氧化氮水平;ELISA法检测小鼠肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的分泌;DCFH-DA荧光探针法检测人参总皂苷处理后活性氧的变化;超氧化物阴离子荧光探针(dihydroethidium, DHE)检测细胞内超氧化物阴离子水平;吖啶橙染色法检测自噬体的形成;Western Blot法检测COX-2、NF-кB、Nrf2、HO-1、GCLC、GCLM、Beclin1、ATG7和LC3的蛋白表达。结果:与脂多糖模型组相比,随着TGS的浓度增加,细胞一氧化氮水平、TNF-α的分泌降低,细胞内活性氧和超氧阴离子的含量降低,巨噬细胞RAW264.7酸性自噬体的数量增加;Nrf2、HO-1、GCLC、GCLM、Beclin1、ATG7及LC3的蛋白表达提高,COX-2、NF-кB的蛋白表达降低。结论:在...     

Inhibitory effect of CDP, a polysaccharide extracted from the mushroom Collybia dryophila, on nitric oxide synthase expression and nitric oxide production in macrophages

The effect of Collybia dryophila polysaccharide (CDP), a (1-->3), (1-->4)-beta-D-glucan extracted from the mushroom C. dryophila, was evaluated on nitric oxide (NO) production induced by lipopolysaccharide (LPS) and gamma interferon (IFNgamma) or by LPS alone in RAW 264.7 cells. CDP significantly inhibited NO production in a dose-dependent manner without affecting cell viability. The inhibition of NO by CDP was consistent with decreases in both inducible nitric oxide synthase (iNOS) protein and mRNA expression suggesting that CDP exerts its effect by inhibiting iNOS gene expression. In addition, CDP at concentrations of 400 and 800 microg/ml was shown to significantly increase prostaglandin E2 (PGE2) production in LPS- and IFNgamma-induced macrophages when compared to the control.     

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.