The combination use of 1-O-acetylbritannilactone (ABL) and gemcitabine inhibits cell growth and induces cell apoptosis in lung adenocarcinoma cells.

1-O-acetylbritannilactone (ABL), a natural chemical component obtained from Chinese traditional medicine, Inula britannica, has been demonstrated to have anticancer activities. In the present study, we evaluated the anti-proliferative and the pro-apoptotic abilities of ABL alone or in combination with gemcitabine in human NSCLC cell line. A549 cells were treated, in vitro, with ABL, gemcitabine, and the combination of ABL and gemcitabine for 72 h. Our results showed ABL and gemcitabine inhibited cell growth and induced apoptosis of A549 cells. These effects after the combination of ABL and gemcitabine were superior to those of each alone. Furthermore, signal transduction analysis revealed NF-κB expression was significantly decreased by ABL and the combination treatment. IκBα and Bax levels were up regulated whereas Bcl-2 was substantially downregulated after all treatments. Our findings suggest that ABL combined with gemcitabine elicits a potent apoptosis of lung cancer cell and hence ABL has the potential to be developed as a chemotherapeutic agent.

Z-ligustilide ameliorated ultraviolet B-induced oxidative stress and inflammatory cytokine production in human keratinocytes through upregulation of Nrf2/HO-1 and suppression of NF-κB pathway.

Ultraviolet B (UVB), a harmful environmental factor, is responsible for a variety of skin disorders including skin inflammation through reactive oxygen species (ROS) and inflammatory mediator production. Here, we investigated the effect of Z-ligustilide (Z-lig), an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on UVB-induced ROS generation and inflammatory mediator production in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z-lig significantly rescued UVB-induced NHEKs damage in a dosage-dependent manner. Pretreatment of NHEKs with Z-lig inhibited UVB-induced ROS production in NHEKs. Both silencing the nuclear factor E2-related factor 2 (Nrf2) and the supplement of tin protoporphyrin IX (SnPP), a haeme oxygenase-1 (HO-1) inhibitor, cancelled the inhibitory effect of Z-lig on UVB-induced ROS upregulation in NHEKs. Moreover, pretreatment of NHEKs with Z-lig reduced UVB-induced nuclear factor kappa B (NF-κB)-dependent inflammatory mediators (IL-6, IL-8 and MCP-1) production at both mRNA and protein level. In the presence of Z-lig, UVB-induced NF-κB subunit p65 nuclear translocation was abolished, and the IκBα degradation was suppressed. Taken together, these findings suggest that Z-lig can suppress UVB-induced ROS generation through Nrf2/HO-1 upregulation and inflammation by suppressing the NF-κB pathway, suggesting that Z-lig may be beneficial in protecting skin from UVB exposure.

The quassinoid isobrucein B reduces inflammatory hyperalgesia and cytokine production by post-transcriptional modulation.

Isobrucein B (1) is a quassinoid isolated from the Amazonian medicinal plant Picrolemma sprucei. Herein we investigate the anti-inflammatory and antihyperalgesic effects of this quassinoid. Isobrucein B (1) (0.5-5 mg/kg) inhibited carrageenan-induced inflammatory hyperalgesia in mice in a dose-dependent manner. Reduced hyperalgesia was associated with reduction in both neutrophil migration and pronociceptive cytokine production. Pretreatment with 1 inhibited in vitro production/release of cytokines TNF, IL-1ß, and KC/CXCL1 by lipopolysaccharide-stimulated macrophages. To investigate its molecular mechanism, RAW 264.7 macrophages with a luciferase reporter gene controlled by the NF-κB promoter were used (RAW 264.7-Luc). Quassinoid 1 reduced the luminescence emission by RAW 264.7-Luc stimulated by different compounds. Unexpectedly, NF-κB translocation to macrophage nuclei was not inhibited by 1 when evaluated by Western blotting and immunofluorescence. Furthermore, quassinoid 1 did not change the levels of TNF mRNA transcription in stimulated macrophages, suggesting post-transcriptional modulation. In addition, constitutive expression of luciferase in RAW 264.7 cells transiently transfected with a plasmid containing a universal promoter was inhibited by 1. Thus, isobrucein B (1) displays anti-inflammatory and antihyperalgesic activities by nonselective post-transcriptional modulation, resulting in decreased production/release of pro-inflammatory cytokines and neutrophil migration.

Suppression of inflammatory responses by handelin, a guaianolide dimer from Chrysanthemum boreale, via downregulation of NF-κB signaling and pro-inflammatory cytokine production.

The anti-inflammatory activity of handelin (1), a guaianolide dimer from Chrysanthemum boreale flowers, was evaluated in vivo, and the effects on mediators nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) and the nuclear factor-κB (NF-κB) and ERK/JNK signaling pathways were investigated in vitro. Compound 1 inhibited lipopolysaccharide (LPS)-induced production of NO and PGE2 in cultured mouse macrophage RAW 264.7 cells. The suppression of NO and PGE2 production by 1 was correlated with the downregulation of mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Compound 1 also suppressed the induction of pro-inflammatory cytokines TNF-α and IL-1ß in LPS-stimulated RAW 264.7 cells. To further clarify the transcriptional regulatory pathway in the expression of iNOS and COX-2 by 1, the role of NF-κB was determined in RAW 264.7 cells. Compound 1 inhibits the binding activity of NF-κB into the nuclear proteins. The transcriptional activity of NF-κB stimulated with LPS was also suppressed by 1, which coincided with the inhibition of IκB degradation. Compound 1 also suppressed the activation of mitogen-activated protein kinases, including ERK and JNK signaling. In addition, the LPS-stimulated upregulation of miRNA-155 expression was suppressed by 1. The oral administration of 1 inhibited acute inflammation in carrageenan-induced paw and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear edema models. The serum level of IL-1ß was also inhibited by 1 in a carrageenan-induced paw edema model. These findings suggest that the suppression of NF-κB activation and pro-inflammatory cytokine production may be a plausible mechanism of action for the anti-inflammatory activity of handelin.

Inhibition of interleukin 1ß-stimulated interleukin-6 production by cranberry components in human gingival epithelial cells: effects on nuclear factor κB and activator protein 1 activation pathways.

BACKGROUND AND OBJECTIVE: In periodontitis, gingival epithelial cells can produce interleukin (IL)-6, a regulator of osteoclastic bone resorption, in response to IL-1ß. IL-1ß regulates cytokine expression via signaling pathways, including nuclear factor (NF)-κB and mitogen activated protein kinase (MAPK)/activator protein (AP)-1. Cranberry proanthocyanidins (PACs) inhibit IL-1ß-stimulated IL-6 production, but specific mechanisms are unclear. The objectives of this study were to determine effects of cranberry PACs on NF-κB and MAPK/AP-1 activation of IL-1ß-stimulated IL-6 production in gingival epithelial cells. MATERIAL AND METHODS: Cranberry high molecular weight non-dialyzable material (NDM), rich in PACs, was derived from cranberry juice. Human gingival epithelial cells [Smulow-Glickman (S-G)] were incubated with IL-1ß in the presence or absence of NDM or inhibitors of NF-κB, [nemo-binding domain (NBD) peptide] or AP-1 (SP600125), and IL-6 levels were measured by ELISA. Effects of NDM on IL-1ß-activated NF-κB and AP-1 and phosphorylated intermediates in both pathways were measured in cell extracts via binding to specific oligonucleotides and specific sandwich ELISAs, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß (≥ 0.1 nm) caused a time- and dose-dependent stimulation of S-G epithelial cell IL-6 production (p < 0.005). This was significantly decreased in a dose-dependent manner by NBD peptide or SP600125 [maximum inhibition ~30-40% (p < 0.02)], and together, the two inhibitors decreased IL-6 by ~80%, similar to the inhibition caused by NDM (p < 0.001). IL-1ß stimulated NF-κB and AP-1 activation (p < 0.003), which was inhibited by NDM (p < 0.0001). NDM did not significantly affect IL-1ß-stimulated levels of phosphorylated intermediates in the NF-κB pathway (IκBα) or the AP-1 pathway (c-Jun, ERK1/2). CONCLUSION: In S-G epithelial cells, IL-1ß appeared to upregulate IL-6 production via activation of both NF-κB and MAPK/AP-1 signaling pathways because cranberry NDM decreased nuclear levels of IL-1ß-activated NF-κB (p65) and AP-1 (phospho-c-Jun) and strongly inhibited IL-6 production. Lack of inhibition of phosphorylation of IκBα, c-Jun or ERK1/2 suggested that NDM might affect both pathways downstream from those points in S-G cells, such as ubiquitination and proteosomal degradation of IκBα, or inhibition of nuclear activity of c-Jun and/or ERK1/2. Defining these points of inhibition precisely may help identify molecular targets of cranberry polyphenols.

Carpesium macrocephalum attenuates lipopolysaccharide-induced inflammation in macrophages by regulating the NF-κB/IκB-α, Akt, and STAT signaling pathways.

Carpesium macrocephalum (CM) Fr. et Sav. (Compositae) has been used in Chinese folk medicine as an analgesic, hemostatic, antipyretic, and to suppress inflammatory conditions. In the present study we aimed to provide scientific evidence for the anti-inflammatory properties of CM extract and evaluate the intrinsic mechanisms involved in both in vitro and in vivo experimental models. In in vitro findings, CM significantly inhibited the LPS-stimulated release of proinflammatory mediators such as nitric oxide, tumor necrosis factor-alpha, prostaglandin E2, and interleukin-6 in RAW264.7 macrophages in a concentration-dependent fashion. The attenuation of inflammatory responses in LPS-activated RAW264.7 cells by CM was closely associated with the suppression of nuclear factor-kappa B (NF-κB) phosphorylation, IκB-α degradation, and phosphorylation of Akt. CM treatment also attenuated the phosphorylation of STAT through TRIF dependent pathways in LPS-activated RAW264.7 cells. In vivo studies revealed that CM extract concentration dependently suppressed the acetic acid-induced vascular permeability in mice. Considering the data obtained regulation of multiple signaling mechanisms involving TRIF and Akt/NF-κB pathways might be responsible for the potent anti-inflammatory action of CM, substantiating its traditional use in inflammatory diseases.

Phlorofucofuroeckol A suppresses expression of inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokines via inhibition of nuclear factor-κB, c-Jun NH2-terminal kinases, and Akt in microglial cells.

Microglial activation has been implicated in many neurological disorders for its inflammatory and neurotrophic effects. In this study, we investigated the effects of phlorofucofuroeckol A isolated from Ecklonia stolonifera Okamura on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated microglia. Pre-treatment of phlorofucofuroeckol A attenuated the productions of nitric oxide, prostaglandin E2, and pro-inflammatory cytokines in LPS-stimulated microglia. Profoundly, phlorofucofuroeckol A treatment showed inactivation of nuclear factor-κB (NF-κB) by preventing the degradation of inhibitor κB-α and the nuclear translocation of p65 NF-κB subunit. Moreover, phlorofucofuroeckol A inhibited the activation of c-Jun NH2-terminal kinases (JNKs), p38 mitogen-activated protein kinase (MAPK), and Akt, but not that of extracellular signal-regulated kinase. These results indicate that phlorofucofuroeckol A inhibits the LPS-induced expression of inflammatory mediators through inactivation of NF-κB, JNKs, p38 MAPK, and Akt pathways. These findings suggest that phlorofucofuroeckol A can be considered as a nutraceutical candidate for the treatment of neuroinflammation in neurodegenerative diseases.

Antiinflammatory effects of traditional Korean medicine, JinPi-tang and its active ingredient, hesperidin in HaCaT cells.

A traditional Korean medicine, JinPi-tang (JPT), has been used in dermatological therapeutics and cosmeceuticals including antiaging creams and moisturizers. However, it has not been clarified how JPT and its active ingredient, hesperidin (HES), regulates inflammatory reactions in HaCaT cells. Thus, the mechanisms of action of JPT and HES on inflammatory reactions were investigated for the first time in an experimental model. The antiinflammatory effects of JPT and HES in HaCaT cells were investigated by using an enzyme-linked immunosorbent assay and a reverse transcription-polymerase chain reaction as well as western blot analysis. JPT and HES inhibited the H(2)O(2)-induced interleukin-8 and tumor necrosis factor-α production as well as its mRNA expression. In addition, JPT and HES inhibited the activation of the nuclear factor-κB, the phosphorylation of IκBα and the p38 mitogen-activated protein kinase, as well as the activation of cyclooxygenase-2. The findings suggest that JPT and HES would be helpful in the treatment of UV radiation-induced inflammatory skin diseases.

The Shaggy Inc Cap medicinal mushroom, Coprinus comatus (O.F.Mull.: Fr.) Pers. (Agaricomycetideae) substances interfere with H2O2 induction of the NF-kappaB pathway through inhibition of Ikappaalpha phosphorylation in MCF7 breast cancer cells.

Breast cancer is the most commonly diagnosed cancer among women. Currently, there is no effective therapy for malignant estrogen-independent breast cancer. In our study, we used hydrogen peroxide, a well-known strong oxidative reagent capable of activating the nuclear factor kappa B (NF-kappaB) transcription factor. The IC50 value of the culinary-medicinal Shaggy Inc Cap mushroom Coprinus comatus culture liquid crude extract on MCF7 cell viability was found to be as low as 76 microg/mL, and the IC50 value of C. comatus ethyl acetate extract was only 32 microg/ mL. Our results also showed that both extracts significantly affected IkappaBalpha phosphorylation in a dose-dependent manner. The effect of ethyl acetate extract was comparable to the effect of curcumin, a known NF-kappaB pathway inhibitor, and seemed to be the most active inhibitor of H2O2-dependent IkappaBalpha phosphorylation. In addition, the data obtained showed that only ethyl acetate extract inhibited the activity of IKK complex, at close to 90% as compared to the control of the untreated sample. These results suggest that C. comatus contains potent compounds capable of inhibiting NF-kappaB function and also possibly acts as an antitumor agent.

Solidago chilensis Meyen hydroalcoholic extract reduces JNK/IκB pathway activation and ameliorates insulin resistance in diet-induced obesity mice.

Hydroalcoholic extract of Solidago chilensis (Sc) is employed in popular medicine to treat inflammatory disease. The low-grade proinflammatory state and the activation of serine/threonine kinases in adipose tissue, like c-jun kinase (JNK) and IKK, and transcription factors, have an important role in obesity-associated insulin resistance. The aim of this study was to further investigate the effects of the Sc extract on glucose homeostasis in diet-induced obesity mice. Male Swiss mice were randomized to three groups: a control group (C) fed with standard laboratory chow; a group with an experimental high-fat diet (HFD); and a group fed with a high-fat (45% kcal from fat) diet + extract of Sc (via intraperitoneal, 3 mg/kg) (ScHFD). The dietary treatment lasted for eight weeks. Subsequently, the expression and phosphorylation of proteins of interest in the liver, hypothalamus and skeletal muscle were evaluated by Western blot analysis. Body weight, epididymal fat pad mass and liver triglycerides were higher in HFD than in control mice, but these parameters were reduced by intraperitoneal administration of the extracts (3 mg/kg) to the HFD group. AKT phosphorylation stimulated by insulin in the liver, hypothalamus and skeletal muscle was higher in ScHFD as compared with HFD mice. Additionally, liver expression of phosphoenolpyruvate carboxykinase (PEPCK) and fatty acid synthase were lower in ScHFD as compared with HFD mice. Nuclear factor κB, p-IκB and p-JNK levels were higher in HFD when compared with control mice, but they were lowered by treatment with extract (ScHFD). In addition, in db/db mice, Sc extract also improved liver AKT phosphorylation stimulated by insulin and reduced PEPCK expression. The data presented herein show that Sc improves AKT activation. This effect may be promoted by reduction of the proinflammatory pathway in the liver and hypothalamus. Therefore, systemic action of the Sc components may contribute to improve obesity-associated pathophysiology.

Shikonin inhibits tumor invasion via down-regulation of NF-κB-mediated MMP-9 expression in human ACC-M cells.

OBJECTIVE: The aim of this study was to examine the anti-invasion effect of Shikonin on human high-metastatic adenoid cystic carcinoma (ACC-M) cells and to explain the possible molecular mechanism involved. METHODS: The ACC-M cells were treated with Shikonin (0, 2.5, 5, 10 µM) for 24 h. The protein levels and gelatinolytic activities of MMP-2 and MMP-9 were analyzed using Western blot and Gelatin zymography test, respectively. Matrigel invasion assays were used to investigate tumor invasive potential and electromobility shift assays were used to determine the activity of NF-κB. RESULTS: The invasiveness of ACC-M cells was reduced in a dose dependent manner following 24-h treatment of up to 10 µM of the Shikonin at which concentration no cytotoxicity occurred. The protein levels and gelatinolytic activities of MMP-9 were significantly suppressed by increasing Shikonin concentrations. The down-regulation of MMP-9 appeared to be via the inactivation of NF-κB as the treatment with Shikonin suppressed the protein level of phosphate-IkBa, which was accompanied by a decrease in DNA-binding level of the factor. CONCLUSIONS: Shikonin inhibits tumor invasion via downregulation of MMP-9 expression in ACC-M cells. Pharmacologic inhibition of the NF-κB-mediated MMP-9 expression by Shikonin might be a powerful treatment option for ACC patients in future.

Effect of Serenoa repens, lycopene, and selenium on proinflammatory phenotype activation: an in vitro and in vivo comparison study.

OBJECTIVES: To investigate the antiinflammatory activity of Serenoa repens (SeR), LY, and) on proinflammatory phenotype in rat peritoneal macrophages (Ms) stimulated with Salmonella enteritidis lipopolysaccharide (LPS) and in the prostate of rats with partial bladder outlet obstruction. SeR, combined with other compounds, such as LY and Se is used to relieve symptoms associated with benign prostatic hyperplasia (BPH). Inflammation plays a pivotal role in the pathogenesis of BPH and represents a target for anti-BPH drugs. METHODS: After stimulation with 1 µg/mL of LPS, peritoneal rat MΦs were coincubated with LY (2 µg/mL), Se (0.03 µg/mL), and SeR (10 µg/mL), alone or in association (LY-Se-SeR) and with RPMI. Inducible cyclooxygenase (COX-2), 5-lypoxygenase (5-LOX), inducible nitric oxide synthase (iNOS), and inhibitor κBα (IκB-α) protein were evaluated by Western blot. Nuclear factor-kappa B (NF-κB) binding activity was measured by electrophoretic mobility shift assay. Tumor necrosis factor-α (TNF-α) gene expression was investigated by real-time polymerase chain reaction. We also evaluated malondialdehyde (MDA) and nitrite levels. RESULTS: LPS stimulation produced a proinflammatory phenotype in rat peritoneal MΦs. LY, Se, and SeR inhibited the inflammatory cascade, but the Ly-Se-SeR association caused a greater inhibitory effect on the expression of COX-2, 5-LOX, and iNOS. The Ly-Se-SeR association showed a higher efficacy in reducing the loss of IκB-α, the increased NF-κB binding activity, the enhanced mRNA levels of TNF-α, the elevated MDA, and nitrite content. The LY-Se-SeR association in vivo caused a greater inhibitory effect on prostate inflammation induced in rats by partial bladder outlet obstruction. CONCLUSIONS: The LY-Se-SeR association might be useful in the treatment of BPH.

Tetrahydropalmatine inhibits pro-inflammatory mediators in lipopolysaccharide-stimulated THP-1 cells.

For centuries, Corydalis Rhizoma has been used in Korean traditional medicine. Tetrahydropalmatine is an alkaloid compound and a prominent anti-inflammatory agent found in plants, including Corydalis yanhusuo. However, the therapeutic mechanisms of tetrahydropalmatine are still not well understood. To provide insight into the biological effects of tetrahydropalmatine, we examined its influence on lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in the human monocytic cell line THP-1. In the present study, IL-8 production was measured using enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and mitogen-activated protein kinase (MAPK) activation, as determined by western blot analysis. Tetrahydropalmatine inhibited LPS-induced IL-8 production in a dose-dependent manner. Furthermore, tetrahydropalmatine inhibited extracellular signal-regulated kinase and p38 MAPK phosphorylation, which suggests that tetrahydropalmatine inhibits IL-8 secretion by blocking MAPK phosphorylation. Taken together, these findings may help elucidate the mechanism by which tetrahydropalmatine modulates THP-1 cell activation under inflammatory conditions.

The antiproliferation effect of berbamine on k562 resistant cells by inhibiting NF-kappaB pathway.

Imatinib mesylate is effective against Ph chromosome-positive leukemia; however, resistance has been reported. High expression of bcr-abl in mRNA and protein levels, and other alterations were found in patients who experienced imatinib treatment failures and thus it is important to design alternative treatment strategies. The aim of this study was to evaluate the in vitro effect of berbamine, on imatinib-resistant chronic myelogenous leukemia (CML) K562 (K562-r) cells, and explore the mechanisms. The growth of K562-r cells was examined using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Morphological analysis and DNA agarose electrophoresis were used to detect apoptosis in K562-r cells, and the extent of the cells in the sub-G1 cell cycle phase was measured using flow cytometry. The expression levels of BCR-ABL, phospho-BCR-ABL, and nuclear factor kappaB (NF-kappaB), IkappaBalpha, phospho-IkappaBalpha, IkappaB kinases alpha(IKKalpha), and Survivin were determined by Western blot. bcr-abl mRNA expression was determined by RT-PCR. MTT assays indicated that berbamine significantly inhibited the proliferation of K562-r cells. Cells with characteristics of apoptosis were confirmed by morphology examination and DNA agarose electrophoresis and percentage of apoptosis were increased after treatment with berbamine. The results also showed that berbamine was able to down-regulate BCR-ABL and phospho-BCR-ABL proteins by affecting bcr-abl mRNA expression and decrease expression of nuclear NF-kappaB, phospho-IkappaBalpha, IKKalpha, and Survivin. Collectively, berbamine could inhibit the proliferation of K562-r cells and induce apoptosis. The mechanisms may be related at least in part, to inhibit BCR-ABL and its downstream NF-kappaB signaling. Berbamine may provide an alternative candidate for the treatment of patients with CML resistant to imatinib therapy.

Nuclear factor kappaB-mediated down-regulation of adhesion molecules: possible mechanism for inhibitory activity of bigelovin against inflammatory monocytes adhesion to endothelial cells.

The flowers of Inula britannica L. var. chinensis (Rupr.) Reg. (Compositae) are used in traditional medicine to treat asthma, chronic bronchitis, and acute pleurisy in China and Korea. However, the pharmacological actions of Inula britannica L. var. chinensis on endothelial cells and inflammatory monocytes are not clear. In this study, we investigated whether bigelovin, a sesquiterpene lactone isolated from the flowers of Inula britannica L. var. chinensis, inhibits monocyte adhesion and adhesion molecule expression in brain endothelial cells. We measured tumor necrosis factor-alpha (TNF-alpha)-enhanced Raw264.7 monocyte binding to brain endothelial cells and the levels of cell adhesion molecules, including vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and endothelial-selectin (E-selectin) on the surface of brain endothelial cells. Bigelovin significantly inhibited these in a dose-dependent manner without affecting cell viability. Furthermore, bigelovin suppressed the nuclear factor kappaB (NF-kappaB) promoter-driven luciferase activity, NF-kappaB activation, and degradation of NF-kappaB inhibitor protein alpha (IkappaBalpha). These results indicate that bigelovin inhibits inflammatory monocyte adhesion to endothelial cells and the expression of VCAM-1, ICAM-1, and E-selectin by blocking IkappaBalpha degradation and NF-kappaB activation.

Ent-pimara-8(14), 15-dien-19-oic acid isolated from the roots of Aralia cordata inhibits induction of inflammatory mediators by blocking NF-kappaB activation and mitogen-activated protein kinase pathways.

Macrophages play central roles in the innate immune system. The roots of Aralia cordata are widely used in Oriental medicine as a remedy for arthritis. During our program to screen medicinal plants for potential anti-inflammatory compounds, ent-pimara-8(14), 15-dien-19-oic acid (pimaradienoic acid; PA) was isolated from the roots of A. cordata. We examined the effect of PA on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. PA was found to significantly inhibit the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and interleukin-6 (IL-6), as well as the expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and IL-6. Furthermore, we examined whether mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are involved in LPS-induced RAW 264.7 cells. We found that a p38 inhibitor (SB203580) and an ERK 1/2 inhibitor (PD98059) significantly affected LPS-induced IL-6 production. In contrast, a JNK 1/2 inhibitor (SP600125) and PI3K inhibitor (wortmannin or LY294002) did not block the induction of IL-6 production by LPS. The LPS-induced phosphorylation of p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2) was inhibited by PA, but not the phosphorylation of JNK 1/2 and AKT (Ser473). Moreover, PA suppressed I kappaB alpha degradation, NF-kappaB activation and luciferase activity. These results suggest that PA isolated from A. cordata has a potential regulatory effect on inflammatory iNOS, COX-2 and IL-6 expression through blockade of the phosphorylation of MAPKs following I kappaB alpha degradation and NF-kappaB activation.

[Studies on the effect of Ginkgo biloba extracts on NF-kappaB pathway].

OBJECTIVE: To investigate NF-kappaB and IkappaBalpha activities in HL-60 induced by TNF-alpha in order to understand the molecular mechanism of GbE in asthma treatment. METHODS: The amount of IkappaBalpha in HL-60 cells stimulated by TNF-alpha and GbE was measured by western blotting. Plasmid pNF-kappaB-LuC was transfected and NF-kappaB activity was analyzed by measuring the expression level of luciferase. RESULTS: It showed in the luciferase assay that the activity of NF-kappaB could significantly be suppressed in HL-60 cells after the pretreatment with CGbE. However, the phosphorylation and subsequent degradation of IKBalpha induced by TNF-alpha can not be inhibited in HL-60 cells even we prolonged the treatment time or increased the concentration of GhE. CONCLUSION: GhE can suppress the NF-kappaB gene expression actively on independent of NIK/ IKK/ IkappaBalpha pathway in HL-60 cells.

Fructus Ligustrum lucidi inhibits inflammatory mediator release through inhibition of nuclear factor-kappaB in mouse peritoneal macrophages.

Fructus Ligustrum lucidi (FLL) is a widely used herbal medicine for the treatment of a variety of pathologies. We have investigated the anti-inflammatory mechanism of FLL in mouse peritoneal macrophages. FLL exerted an anti-inflammatory action through inhibition of lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-alpha production in mouse peritoneal macrophages. The maximal inhibition rate of TNF-alpha production by FLL (0.5 mg mL(-1)) was 60.88 +/- 0.30%. In the inflammatory process, nitric oxide (NO) and prostaglandin E(2) (PGE(2)) increased in peritoneal macrophages. FLL decreased the protein level of NO and PGE(2) in LPS-stimulated mouse peritoneal macrophages. In addition, FLL inhibited nuclear factor-kappaB activation and IkappaB-alpha degradation by the decrease in IkappaB-alpha phosphorylation. Our study suggested that FLL reduced inflammation via an important molecular mechanism, which might explain its beneficial effect in the regulation of inflammatory reactions.

Low and high levels of alpha-tocopherol exert opposite effects on IL-2 possibly through the modulation of PPAR-gamma, IkappaBalpha, and apoptotic pathway in activated splenocytes.

OBJECTIVE: We previously demonstrated that a high dose of alpha-tocopheryl succinate inhibits interleukin-2 (IL-2) mRNA and production in autoimmune-prone MRL/lpr mice. In the present study, we investigated the regulation of alpha-tocopherol (alphaTOC) on IL-2 gene expression by examining the mRNA of IL-2, inhibitor kappaBalpha (IkappaBalpha), and peroxisome proliferator-activated receptor-gamma (PPARgamma). METHODS: Messenger RNA expression in active splenocytes of BALB/c mice was investigated with reverse transcriptase polymerase chain reaction. RESULTS: Levels of IL-2 mRNA in phorbol 12-myristate 13-acetate/ionomycin activated splenocytes and cytokine in T-helper-1 cells were increased by 50 microM of alphaTOC but decreased by 1 mM of alphaTOC. In addition, the IkappaBalpha gene expression significantly increased by the high dose (>or=500 microM) of alphaTOC, suggesting an inhibition on nuclear factor-kappaB pathway for activation of IL-2 expression. PPARgamma mRNA level in activated splenocytes was upregulated by 1 mM of alphaTOC. PPARgamma mRNA level in unstimulated splenocytes was upregulated by alphaTOC in a dose-dependent manner, suggesting that alphaTOC might enhance the PPARgamma signaling pathway. High-dose alphaTOC induced apoptosis of splenocytes and inhibited phytohemagglutinin-stimulated T-cell proliferation. Conversely, the proliferative response of splenocytes was enhanced by 5 microM of alphaTOC. Low-dose (50 microM) alphaTOC increased IL-2 expression, which may have been due to the absence of downregulation of PPARgamma and IkappaBalpha on the IL-2 gene. CONCLUSION: The results indicated that low and high doses of alphaTOC exert opposite effects on IL-2, possibly through modulation of PPARgamma, IkappaBalpha, and apoptosis pathways. The present findings support our previous observation of opposite effects of low- and high-dose vitamin E on survival of MRL/lpr mice.