Ran Joo Choi

Alantolactone suppresses inducible nitric oxide synthase and cyclooxygenase-2 expression by down-regulating NF-κB, MAPK and AP-1 via the MyD88 signaling pathway in LPS-activated RAW 264.7 cells.

Several sesquiterpene lactones are the active components of several medicinal plants and have been demonstrated to perform various pharmacological functions. In this study, we investigated the anti-inflammatory effects of alantolactone, a sesquiterpene lactone isolated from the root of Aucklandia lappa, in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and peritoneal macrophages. Alantolactone inhibited inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein and mRNA transcription, as well as the downstream products, nitric oxide (NO), prostaglandin E(2) (PGE(2)) and tumor necrosis factor-α (TNF-α). Investigation of the effects on nuclear factor κB (NF-κB) signaling showed that alantolactone inhibits the phosphorylation of inhibitory κB (IκB)-α and IκB kinase (IKK) and the subsequent translocation of the p65 and p50 NF-κB subunits to the nucleus. Moreover, inhibition of mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 MAPK, and activator protein-1 (AP-1) was also observed. A further study indicated that alantolactone attenuated the phosphorylation of Akt and inhibited the expression of MyD88 and Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP), an upstream signaling molecule required for IKK and MAPKs activation. Taken together, these results suggest that alantolactone exerts its anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation and MAPKs phophorylation via downregulation of the MyD88 signaling pathway. Thus, alantolactone may provide a useful therapeutic approach for inflammation-associated diseases.

Anti-inflammatory Activity of Edible Brown Alga Saccharina japonica and its Constituents Pheophorbide a and Pheophytin a in LPS-stimulated RAW 264.7 Macrophage Cells

Anti-inflammatory activity of Saccharina japonica and its active components was evaluated via in vitro inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) expression in RAW 264.7 murine macrophage cells. Since the methanolic extract of S. japonica showed strong anti-inflammatory activity, it was fractionated with several solvents. Among the fractions, the ethyl acetate fraction demonstrated the highest inhibition of LPS-induced NO production (IC50=25.32μg/mL), followed by the CH2Cl2 fraction (IC50=75.86μg/mL). Considering the yield and anti-inflammatory potential together, the CH2Cl2 fraction was selected for chromatographic separation to yield two active porphyrin derivatives, pheophorbide a and pheophytin a, together with an inactive fucoxanthin. In contrast to fucoxanthin, pheophorbide a and pheophytin a showed dose-dependent inhibition against LPS-induced NO production at nontoxic concentrations in RAW 264.7 cells. Both compounds also suppressed the expression of iNOS proteins, while they did not inhibit the COX-2 expression in LPS-stimulated macrophages. These results indicate that pheophorbide a and pheophytin a are two important candidates of S. japonica as anti-inflammatory agents which can inhibit the production of NO via inhibition of iNOS protein expression. Thus, these compounds hold great promise for use in the treatment of various inflammatory diseases.

Suppression of LPS‐induced inflammatory and NF‐κB responses by anomalin in RAW 264.7 macrophages

The treatment of inflammatory diseases today is largely based on interrupting the synthesis or action of the mediators that drive the hosts response to injury. It is on the basis of this concept that most of the anti‐inflammatory drugs have been developed. In our continuous search for novel anti‐inflammatory agents from traditional medicinal plants, Saposhnikovia divaricata has been a focus of our investigations. Anomalin, a pyranocoumarin constituent of S. divaricata, exhibits potent anti‐inflammatory activity. To clarify the cellular signaling mechanisms underlying the anti‐inflammatory action of anomalin, we investigated the effect of anomalin on the production of inflammatory molecules in LPS‐stimulated murine macrophages. The anomalin dose‐dependently inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) mRNA and protein expression in LPS‐stimulated RAW 264.7 macrophage. Molecular analysis using quantitative real time polymerase chain reaction (qRT‐PCR) revealed that several pro‐inflammatory cytokines, including tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6), were reduced by anomalin, and this reduction correlated with the down‐regulation of the NF‐κB signaling pathway. In addition, anomalin suppressed the LPS‐induced phosphorylation and degradation of IκBα. To further study the mechanisms underlying its anti‐inflammatory activity, an electrophoretic mobility shift assay (EMSA) using a 32P‐labeled NF‐κB probe was conducted. LPS‐induced NF‐κB DNA binding was drastically abolished by anomalin. The present data suggest that anomalin is a major anti‐inflammatory agent and may be a potential therapeutic candidate for the treatment of inflammatory disorders. J. Cell. Biochem. 112: 2179–2188, 2011.

Inhibitory effects of kaurenoic acid from Aralia continentalis on LPS-induced inflammatory response in RAW264.7 macrophages

This study investigates the anti-inflammatory effects of a diterpenoid, kaurenoic acid, isolated from the root of Aralia continentalis (Araliaceae). To determine its anti-inflammatory effects, LPS-induced RAW264.7 macrophages were treated with different concentrations of kaurenoic acid and carrageenan-induced paw edema mice model was used in vivo. Kaurenoic acid (ent-kaur-16-en-19-oic acid) dose-dependently inhibited nitric oxide (NO) production, prostaglandin E(2) (PGE(2)) release, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression at micromolar concentrations in LPS-induced RAW264.7 macrophages with IC(50) (the half maximal inhibitory concentration) values of 51.73 (±2.42) μM and 106.09 (±0.27) μM in NO production and PGE(2) release, respectively. Kaurenoic acid also dose-dependently inhibited LPS-induced activation of NF-κB as assayed by electrophorectic mobility shift assay (EMSA) and it almost abolished NF-κB DNA binding affinity at 100μM. Furthermore, the in vivo anti-inflammatory effect of kaurenoic acid was examined in a carrageenan-induced paw edema model. Eight ICR mice in each group were injected with carrageenan and observed hourly, compared with the control group. Kaurenoic acid dose-dependently reduced paw swelling up to 34.4% at 5h after induction, demonstrating inhibition in an acute inflammation model. Taken together, our data suggest that kaurenoic acid, a major diterpenoid from the root of A. continentalis shows anti-inflammatory activity and the inhibition of iNOS and COX-2 expression might be one of the mechanisms responsible for its anti-inflammatory properties.

Anti-inflammatory effects of (Z)-ligustilide through suppression of mitogen-activated protein kinases and nuclear factor-κB activation pathways

AbstactThe roots of Angelica tenuissima have been commonly used for the treatment of cardiovascular diseases and menstrual discomfort in Asian countries, such as China and Korea. The primary volatile flavor components are essential oil ingredients, phthalide lactones. In this study, (Z)-ligustilide was tested for its anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We found that (Z)-ligustilide strongly inhibitis the induction of LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both the mRNA and protein levels in a dose-dependent manner. The transcriptional activity of nuclear factor kappa B (NF-B) was also down-regulated in a concentration-dependent manner. Further study revealed that (Z)-ligustilide inhibited the phosphorylation and subsequent degradation of IBα, an inhibitor protein of NF-B. In addition, (Z)-ligustilide inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in a dose-dependent manner. Taken together, these data suggest that (Z)-ligustilide can exert its antiinflammatory effects by regulating the NF-B and MAPK signal pathways.

Molecular mechanism of capillarisin-mediated inhibition of MyD88/TIRAP inflammatory signaling in in vitro and in vivo experimental models

ETHNOPHARMACOLOGICAL RELEVANCE Artemisia capillaris Thunberg (Compositae) have been used as traditional medicine as a diuretic, liver protective agent, and for amelioration of inflammatory and analgesic disorders. The present study was carried out to establish the scientific rationale for treating inflammation and to find active principles from A. capillaris. The aim of the present study is to investigate the possible anti-inflammatory mechanism of the major component (capillarisin) isolated from A. capillaris via inhibition of MyD88/TIRAP inflammatory signaling both in vitro and in vivo models. MATERIALS AND METHODS The nitrite, PGE(2), and TNF-α productions were evaluated by Griess reagent and ELISA kits. The protein and mRNA expression levels were investigated by Western blot and RT-PCR. The NF-κB and AP-1 DNA-binding was performed by electrophoretic mobility shift assay. The CFA- and carrageenan-induced paw edema was performed in ICR mice in which 20 and 80 mg/kg body weight of capillarisin was administered intraperitoneally (i.p.). RESULTS The results demonstrated that pretreatment with capillarisin effectively inhibited the LPS-induced activation of NF-κB, Akt, and MAP kinase-activated inflammatory genes, which is mediated by MyD88 and TIRAP. Treatment with capillarisin reduced the mRNA and protein levels of iNOS and COX-2 in RAW 264.7 cells as assessed by RT-PCR and Western blot. Capillarisin suppressed LPS-induced inhibitory kappa kinase (IKK) phosphorylation and the degradation of inhibitory kappa B (IκBα) and prevented the nuclear translocation of p65 and p50. Capillarisin also exhibited a promising inhibitory effect on the LPS-induced NF-κB and AP-1 DNA binding activity based on an electrophoretic mobility shift assay. The LPS-induced activation of p-JNK, p-p38, p-ERK, and p-Akt was significantly inhibited. In addition, the TNF-α level in the media was effectively reduced by capillarisin. In vivo experimental analysis revealed that capillarisin (20 and 80 mg/kg, i.p.) inhibited complete Freunds adjuvant (CFA)-and carrageenan-induced paw edema, nitrite production in plasma, and TNF-α, a pro-inflammatory cytokine production. CONCLUSION The results presented here demonstrate that capillarisin has consistent anti-inflammatory properties and acts by inhibiting inflammatory mediators in in vitro and in vivo experimental models, and suggest its potential utility in the control of inflammatory disorders.

Anti-inflammatory properties of anthraquinones and their relationship with the regulation of P-glycoprotein function and expression

There is a growing interest in natural products that potentially have anti-inflammatory properties and inhibit P-glycoprotein (P-gp) function. In this report, we assessed the effects of anthraquinone derivatives from rhubarb on LPS-induced RAW 264.7 macrophages to determine their anti-inflammatory potential. The derivatives were also tested in Caco-2 cell lines to evaluate the inhibition of the drug efflux function of P-gp. The transport abilities were examined and the cellular accumulation of rhodamine-123 (R-123) was also measured. Electorphoretic mobility shift assay (EMSA) was performed to check the activator protein-1 (AP-1) DNA binding affinity. Five anthraquinones were tested to determine their inhibitory activities on NO production and the protein and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, the level of prostaglandin E(2) (PGE(2)) was determined in LPS-induced RAW264.7 macrophages. Emodin was found to be the most potent inhibitor, and it also reduced paw swelling in the mouse model of carrageenan-induced paw edema. In Caco-2 cells, emodin elevated the accumulation of R-123 and decreased the efflux ratio of R-123, which indicates the inhibition of P-gp function. The inhibition of COX-2 protein by emodin paralleled the decrease in P-gp expression. In addition, mitogen-activated protein kinase (MAPK) expression was decreased through the prevention of AP-1 DNA binding, which leads to downregulation in the expression of P-gp. Our data indicate that the decrease of P-gp expression is caused by the decreased expression of COX-2 through the MAPK/AP-1 pathway. Based on our results, we suggest that anti-inflammatory drugs with COX-2 inhibitory activity might be used to modulate P-gp function and expression.

Mechanism of anti-inflammatory activity of umbelliferone 6-carboxylic acid isolated from Angelica decursiva

AIMS OF THE STUDY We recently reported the potential antioxidant and anti-inflammatory activities of umbelliferone 6-carboxylic acid (UMC) isolated from the whole plants of Angelica decursiva. In this study, we elucidated the anti-inflammatory mechanisms of UMC in vitro and in vivo. METHODS The inhibitory effects of UMC on the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and tumor necrosis factor-α (TNF-α), the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the activation of nuclear factor kappa B (NF-κB) were evaluated using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The reactive oxygen species (ROS) generation inhibitory activity of UMC was evaluated using t-butyl hydroperoxide (t-BHP)-induced RAW 264.7 cells. Furthermore, the in vivo anti-inflammatory activity of UMC was evaluated using carrageenan induced mouse paw edema model. RESULTS UMC dose-dependently inhibited NO and PGE(2) production by down-regulating iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 macrophages. UMC also suppressed the production of the proinflammatory cytokine TNF-α in LPS stimulated RAW 264.7 cells in a concentration dependent manner. In addition, UMC dose-dependently prevented LPS-induced nuclear translocation of NF-κB in RAW 264.7 macrophages. Furthermore, UMC exhibited the inhibitory activity against t-BHP-induced ROS generation in RAW 264.7 cells with an IC(50) value of 705.1 μg/ml. Moreover, UMC inhibited λ-carrageenan induced mouse paw edema by 70.40 and 60.20% at doses of 50 and 25 mg/kg body weight, respectively. CONCLUSION The combined results of this study indicate that UMC is an important anti-inflammatory constituent of A. decursiva and its anti-inflammatory effect was due to its ability to inhibit the production of inflammatory mediators via inhibition of NF-κB activation pathway.

Desoxyrhapontigenin, a potent anti-inflammatory phytochemical, inhibits LPS-induced inflammatory responses via suppressing NF-κB and MAPK pathways in RAW 264.7 cells.

This study investigates the anti-inflammatory effects of a stilbene compound, desoxyrhapontigenin, which was isolated from Rheum undulatum. To determine the anti-inflammatory effects of this compound, lipopolysaccharide (LPS)-induced RAW 264.7 macrophages were treated with different concentrations of six stilbene derivatives. The results indicated that compared with other stilbene compounds, desoxyrhapontigenin (at 10, 30 and 50μM concentrations) significantly inhibited nitric oxide (NO) production, nuclear factor kappa B (NF-κB) activation, the protein expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression. Therefore, the anti-inflammatory mechanism of desoxyrhapontigenin was investigated in detail. The results of this investigation demonstrated that desoxyrhapontigenin suppressed not only LPS-stimulated pro-inflammatory cytokine secretions, including the secretions of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), but also PGE2 release. As assayed by electrophoretic mobility shift assays (EMSAs), desoxyrhapontigenin also produced the dose-dependent inhibition of the LPS-induced activation of NF-κB and AP-1. Moreover, desoxyrhapontigenin inhibited the protein expression of myeloid differentiation primary response gene 88 (MyD88), IκB kinase (IKK) phosphorylation and the degradation of IκBα. Activations of p-JNK1 and p-Akt were also significantly inhibited, and phosphorylation of p38 and ERK was down-regulated. A further study revealed that desoxyrhapontigenin (5 and 25mg/kg, i.p.) reduced paw swelling in carrageenan-induced acute inflammation model in vivo. On the whole, these results indicate that desoxyrhapontigenin showed anti-inflammatory properties by the inhibition of iNOS and COX-2 expression via the down-regulation of the MAPK signaling pathways and the inhibition of NF-κB and Akt activation.

Anti-hyperalgesic and anti-allodynic activities of capillarisin via suppression of inflammatory signaling in animal model

ETHNOPHARMACOLOGICAL RELEVANCE Artemisia capillaris has widespread traditional and pharmacological applications such as analgesic, anti-inflammatory, anti-pyretic, enhance immunity and anti-tumor activity properties. To evaluate the pharmacological activities of this plant, capillarisin, one of the potent constituent of Artemisia capillaris was studied based on anti-hyperalgesic and anti-allodynic effects with detailed mechanism. It can be assumed that measurement of anti-nociceptive effects of capillarisin is one of the parameter for the evaluation of this herb. Capillarisin has extensive pharmacological properties and has been considered to have promising ant-inflammatory and anti-nociceptive activities. The aim of the current study is to investigate the effect of capillarisin and underlying molecular mechanisms of action in preventing acute and subchronic inflammatory pain. MATERIALS AND METHODS The inflammatory pain was induced after 40 min or 1h of administration of vehicle, 70% EtOH extract of Artemisia capillaris (100mg/kg) or capillarisin (20 and 80 mg/kg) by intraplantar (i.p.l.) injections of CFA and carrageenan in ICR mice, respectively. Mechanical hyperalgesia and allodynia were evaluated in both acute and subchronic models. Further analysis was performed in CFA-induced mice exploring various molecular and signaling pathways such as NF-κB, AP-1, and ERK-CREB involved in the persistent pain sensations. RESULTS In acute model, mechanical hyperalgesia and allodynia were evaluated after every 2h until 6h of CFA and after 4h of carrageenan injections. Whereas, in subchronic inflammatory pain model, mechanical hyperalgesia and paw edema were measured after 4h of CFA injection and every day after 4h of daily treatment until 5 days with interval of day four in order to assess the tolerance effect of capillarisin. Further analysis was performed in CFA-induced mice exploring various molecular and signaling pathways such as NF-κB, AP-1 and ERK-CREB involved in the persistent of pain sensations. Pre-treatment of capillarisin strongly inhibited NF-κB mediated genes (iNOS, COX-2), involved in pain. The plasma leading nitrite production was significantly reduced by capillarisin. Moreover, i.p. administration of capillarisin markedly suppressed the adenosine 5׳-triphosphate (ATP) in plasma and substance P in CFA-induced paw tissue. CONCLUSIONS The present study indicates that capillarisin possessed promising anti-hyperalgesic and anti-allodynic effects through the inhibition of various inflammatory pain signaling, suggesting that capillarisin constitutes a significant component for the treatment of inflammatory pain.