17-Hydroxy-jolkinolide B, a diterpenoid from Euphorbia fischeriana, inhibits inflammatory mediators but activates heme oxygenase-1 expression in lipopolysaccharide-stimulated murine macrophages

Jolkinolides are the main abietane-type diterpenoids isolated from the root of Euphorbia fischeriana Steud. In the present study, we investigated in vitro anti-inflammatory activity of four structural analogs of jolkinolide in lipopolysaccharide (LPS)-stimulated RAW264 macrophages. Among these jolkinolides, 17-hydroxy-jolkinolide B (HJB) exhibited the most potent inhibition of LPS-induced production of inflammatory mediators such as prostaglandin E(2) (PGE(2)), nitric oxide (NO), and pro-inflammatory cytokines [interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)]. HJB could decrease LPS-induced protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and the mRNA expressions of COX-2, iNOS, IL-6, and TNF-α in a concentration-dependent manner. These inhibitory effects were caused by suppression of MAPK phosphorylation and NF-κB activation. Furthermore, we demonstrated that HJB strongly induced heme oxygenase-1 (HO-1) protein and mRNA expressions. These findings suggest that HJB possesses anti-inflammatory actions in macrophages and may provide a potential therapeutic approach for inflammatory disorders.     

Sauchinone suppresses pro-inflammatory mediators by inducing heme oxygenase-1 in RAW264.7 macrophages

Sauchinone, a biologically active lignan isolated from the roots of Saururus chinensis (LOUR.) BAILL. (Saururaceae), is reported to exert a variety of biological activities, such as hepatoprotective, anti-inflammatory actions and inhibitory effects on bone resorption. In this study, we investigated the effect of sauchinone in suppressing cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, leading to a reduction in COX-2-derived prostaglandin E(2) (PGE(2)) and iNOS-derived nitric oxide (NO) production in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages. Present study also demonstrates the effects of sauchinone in inducing heme oxygenase-1 (HO-1) expression and an increase in heme oxygenase (HO) activity in RAW264.7 macrophages. The effects of sauchinone on LPS-induced PGE(2), NO, tumor necrosis factor-α (TNF-α) and interlukine-1β (IL-1β) production were partially reversed by the HO-1 inhibitor Tin protoporphyrin was also seen in this study. In addition, we found that treatment with extracellular signal-regulated kinase (ERK) inhibitor (PD98059) reduced sauchinone-induced HO-1 expression. Sauchinone also increased ERK phosphorylation. These results suggest that sauchinone inhibits pro-inflammatory mediators through expression of anti-inflammatory HO-1 via ERK pathway.     

Ethyl linoleate from garlic attenuates lipopolysaccharide-induced pro-inflammatory cytokine production by inducing heme oxygenase-1 in RAW264.7 cells

In the present study, an essential fatty acid, ethyl linoleate (ELA), was isolated from the cloves of Allium sativum, and its structure was elucidated by NMR and GC-MS analyses. In vitro systems were used to evaluate the anti-inflammatory activity of ELA. Our results indicate that ELA down-regulates inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and thereby reduces nitric oxide (NO) and prostaglandin E₂ production in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Immunofluorescent microscopy and western blot analyses revealed that these effects were mediated by impaired translocation of nuclear factor (NF)-κB and inhibition of phosphorylation of mitogen activated protein kinases. Furthermore, ELA exerted its anti-inflammatory activity by inducing heme oxygenase-1 (HO-1) expression, as determined by HO-1 small interfering (Si) RNA system. Si RNA-mediated knock-down of HO-1 abrogated the inhibitory effects of ELA on the production of NO, TNF-α, IL-1β, and IL-6 in LPS-induced macrophages. These findings indicate the potential therapeutic use of ELA as an anti-inflammatory agent.     

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.     

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.     

Salidroside attenuates inflammatory response via suppressing JAK2-STAT3 pathway activation and preventing STAT3 transfer into nucleus

Salidroside (SAL) is an active ingredient isolated from the Rhodiola rosea, has potent anti-inflammatory effect, but the mechanism is still elusive. The purpose of this study is to verify the effects of SAL on LPS-induced inflammatory response and investigate the possible underlying molecular mechanism. RAW264.7 cells were pre-incubated with SAL for 2 h, then stimulated with or without LPS for another 16 h. The levels of TNF-α, MCP-1, IL-6, and PGE2 were detected by ELISA, and the production of NO was determined by nitrite analysis. The expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by Western blotting. In RAW264.7 cells and murine peritoneal macrophages, the activation of signal molecules was also measured by Western blot. The nuclear translocation of STAT3 was determined by Laser confocal and nucleocytoplasmic separation experiments. Our results showed that SAL attenuated the productions of TNF-α, IL-6, MCP-1, PGE2 and NO dose dependently. SAL also suppressed LPS-induced expressions of iNOS and COX-2 significantly. Further studies revealed that SAL down-regulated the phosphorylation of JAK2-STAT3 signaling pathway and reduced the nuclear translocation of STAT3 induced by LPS in RAW264.7 cells and primary peritoneal macrophages. In addition, consistent with the results in vitro, in the model of mice acute lung injury (ALI) induced by LPS, SAL reduced the infiltration of inflammatory cells and decreased the levels of serum TNF-α and IL-6 obviously. Taken together, these data indicated that SAL exerted anti-inflammatory action via down-regulating LPS-induced activation of JAK2-STAT3 pathway and suppressing STAT3 transfer into the nucleus at least in part.     

Anti-inflammatory effects of glycyrol isolated from Glycyrrhiza uralensis in LPS-stimulated RAW264.7 macrophages

The anti-inflammatory effects of glycyrol, a benzofuran coumarin isolated from Glycyrrhizae Radix, were studied. Glycyrol of 5, 25 and 50 microM dose-dependently inhibited nitric oxide (NO) production by down-regulating inducible nitric oxide synthase (iNOS), and alleviated cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW264.7 macrophages, in both the mRNA and the protein. Furthermore, glycyrol dose-dependently decreased the mRNA of the pro-inflammatory cytokines IL-1beta and IL-6. LPS-induced NF-kappaB activation was prevented in RAW264.7 macrophages by inhibition of I-kappaBalpha phosphorylation. In addition, administration of glycyrol (30 and 100 mg/kg, i.p) reduced the thickness of carrageenan-induced mouse-paw edema swelling. Taken together, our results indicate that glycyrol is an important anti-inflammatory constituent of Glycyrrhizae Radix, and that its anti-inflammatory effect is attributed to the inhibition I-kappaBalpha phosphorylation.     

Anti-inflammatory activity of Angelica dahurica ethanolic extract on RAW264.7 cells via upregulation of heme oxygenase-1

In this study, we analyzed the anti-inflammatory effects of Angelica dahurica Bentham et Hooker ethanolic extract (ADEE) on RAW264.7 cells, to understand the mechanism underlying its observed effects. ADEE inhibited cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, leading to the suppression of COX-2-derived prostaglandin E₂ and iNOS-derived production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. These inhibitory effects of ADEE were accompanied by the reduced production of tumor necrosis factor α and interleukin (IL)-6. ADEE also inhibited nuclear factor κB (NFκB) translocation to the nucleus by interrupting inhibitor kappa Bα (IκBα) degradation. ADEE upregulated heme oxygenase 1 expression, and treatment with tin protoporphyrin IX (SnPP), a selective inhibitor of HO-1, reversed the LPS-induced generation of proinflammatory cytokines. ADEE also induced IL-4 and IL-5 expression in concanavalin-A-stimulated splenocytes. These results suggest that ADEE has anti-inflammatory activity, which acts via the suppression of the NF-κB 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.     

4,7-Dimethoxy-5-methyl-1,3-benzodioxole from Antrodia camphorata inhibits LPS-induced inflammation via suppression of NF-κB and induction HO-1 in RAW264.7 cells

Several benzenoid compounds have been isolated from Antrodia camphorata are known to have excellent anti-inflammatory activity. In this study, we investigated the anti-inflammatory potential of 4,7-dimethoxy-5-methyl-1,3-benzodioxole (DMB), one of the major benzenoid compounds isolated from the mycelia of A. camphorata. DMB significantly decreased the LPS-induced production of pro-inflammatory molecules, such as nitric oxide (NO), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in RAW264.7 cells. In addition, DMB suppressed the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose dependent manner. Moreover, DMB significantly suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB), and this inhibition was found to be associated with decreases in the phosphorylation and degradation of its inhibitor, inhibitory κB-α (IκB-α). Moreover, we found that DMB markedly inhibited the protein expression level of Toll-like receptor 4 (TLR4). Furthermore, treatment with DMB significantly increased hemoxygenase-1 (HO-1) expression in RAW264.7 cells, which is further confirmed by hemin, a HO-1 enhancer, significantly attenuated the LPS-induced pro-inflammatory molecules and iNOS and TLR4 protein levels. Taken together, the present study suggests that DMB may have therapeutic potential for the treatment of inflammatory diseases.     

Cornuside suppresses lipopolysaccharide-induced inflammatory mediators by inhibiting nuclear factor-kappa B activation in RAW 264.7 macrophages

Cornuside, a secoiridoid glucoside compound, was isolated from the fruit of Cornus officinalis SIEB. et ZUCC. Cornuside has been reported to possess immunomodulatory and anti-inflammatory activities. However, the effects and mechanism of action of cornuside in inflammation have not been fully characterized. The present study was therefore designed to examine whether cornuside suppresses inflammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Cornuside significantly inhibited the LPS-induced production of nitric oxide, prostaglandin E(2), tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-1beta. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were also decreased by cornuside. Furthermore, cornuside significantly attenuated the LPS-stimulated phosphorylation and degradation of inhibitory kappa B-alpha and the subsequent translocation of the p65 subunit of nuclear factor-kappa B (NF-κB) to the nucleus. Cornuside also reduced the phosphorylations of extracellular-signal-related kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2). These results suggest that the anti-inflammatory property of cornuside is related to the downregulations of iNOS and COX-2 due to NF-κB inhibition as well as the negative regulation of ERK1/2, p38, and JNK1/2 phosphorylations in RAW 264.7 cells.     

Anti-inflammatory effects of diaporisoindole B in LPS-stimulated RAW 264.7 macrophage cells via MyD88 activated NF-κB and MAPKs pathways

Diaporisoindole B (DPB), an isoprenylisoindole alkaloid isolated from the mangrove endophytic fungus Diaporthe sp. SYSU-HQ3, has been proved to inhibit the production of nitric oxide (NO) in lipopolysaccharide (LPS)-challenged RAW 264.7 mouse macrophages, showing potent anti-inflammatory effects. In this study, we further investigated the anti-inflammatory effects of DPB and explored the possible mechanisms in LPS-challenged RAW 264.7 mouse macrophages. The results showed that DPB (3.125, 6.2, 12.5 and 25 μM) could significantly reduce LPS-induced levels of PGE2, and inhibit the expressions of iNOS and COX-2 in a dose-dependent manner. In addition, DPB also inhibited LPS-induced production of inflammatory cytokines, including TNF-α, IL-1β, IL-6. Moreover, we further investigated signal transduction mechanisms by which DPB exerted anti-inflammatory effects. DPB could affect LPS-mediated nuclear factor kappa B (NF-κB) signaling pathway activation via down-regulating the upstream myeloid differentiation protein 88 (MyD88) at the protein level. Additionally, DPB also strongly inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38. Therefore, DPB might exert anti-inflammatory effects by suppressing NF-κB activation and MAPKs pathways via down-regulating MyD88 in RAW 264.7 cells.     

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.     

Polygonum cuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2 gene expressions in RAW 264.7 macrophages

Polygonum cuspidatum water extract (PCWE) was shown to be a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). PCWE was compared to baicalin isolated from Scutellaria baicalensis Georgi and berberine of Coptidis rhizoma and Phellodendri cortex, for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. Both PCWE and the compounds inhibited LPS-induced NO production in a concentration-dependent manner without a cytotoxicity. The decrease in NO production was in parallel with the inhibition of LPS-induced iNOS gene expression by PCWE and the compounds. In contrast, iNOS enzyme activity was not inhibited by PCWE and two agents. In addition, only PCWE inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity, while baicalin or berberine did not. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS protein expression, which was inhibited by these PCWE and two agents, although LPS-induced COX-2 protein expression was not affected by NLA and L-NAME. PCWE inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cell, however, baicalin or berberine did not. From the results, it was concluded that co-treatment with NOS inhibitors and PCWE effectively blocks acute production of NO and inhibits expression of iNOS and COX-2 genes.