8-Hydroxydaidzein Downregulates JAK/STAT, MMP, Oxidative Phosphorylation, and PI3K/AKT Pathways in K562 Cells.

A metabolite isolated from fermented soybean, 8-hydroxydaidzein (8-OHD, 7,8,4'-trihydroxyisoflavone, NSC-678112), is widely used in ethnopharmacological research due to its anti-proliferative and anti-inflammatory effects. We reported previously that 8-OHD provoked reactive oxygen species (ROS) overproduction, and induced autophagy, apoptosis, breakpoint cluster region-Abelson murine leukemia viral oncogene (BCR-ABL) degradation, and differentiation in K562 human chronic myeloid leukemia (CML) cells. However, how 8-OHD regulates metabolism, the extracellular matrix during invasion and metastasis, and survival signaling pathways in CML remains largely unexplored. High-throughput technologies have been widely used to discover the therapeutic targets and pathways of drugs. Bioinformatics analysis of 8-OHD-downregulated differentially expressed genes (DEGs) revealed that Janus kinase/signal transducer and activator of transcription (JAK/STAT), matrix metalloproteinases (MMPs), c-Myc, phosphoinositide 3-kinase (PI3K)/AKT, and oxidative phosphorylation (OXPHOS) metabolic pathways were significantly altered by 8-OHD treatment. Western blot analyses validated that 8-OHD significantly downregulated cytosolic JAK2 and the expression and phosphorylation of STAT3 dose- and time-dependently in K562 cells. Zymography and transwell assays also confirmed that K562-secreted MMP9 and invasion activities were dose-dependently inhibited by 8-OHD after 24 h of treatment. RT-qPCR analyses verified that 8-OHD repressed metastasis and OXPHOS-related genes. In combination with DisGeNET, it was found that 8-OHD's downregulation of PI3K/AKT is crucial for controlling CML development. A STRING protein-protein interaction analysis further revealed that AKT and MYC are hub proteins for cancer progression. Western blotting revealed that AKT phosphorylation and nuclear MYC expression were significantly inhibited by 8-OHD. Collectively, this systematic investigation revealed that 8-OHD exerts anti-CML effects by downregulating JAK/STAT, PI3K/AKT, MMP, and OXPHOS pathways, and MYC expression. These results could shed new light on the development of 8-OHD for CML therapy.

The Lipid-Modulating Effect of Tangeretin on the Inhibition of Angiopoietin-like 3 (ANGPTL3) Gene Expression through Regulation of LXRα Activation in Hepatic Cells.

The excessive accumulation of TG-rich lipoproteins (TGRLs) in plasma is associated with dyslipidemia and atherosclerotic cardiovascular diseases (ASCVDs). Tangeretin is a bioactive pentamethoxyflavone mainly found in citrus peels, and it has been reported to protect against hyperlipidemia, diabetes, and obesity. The aim of this study was to investigate the lipid-modulating effects and the underlying mechanisms of tangeretin action in hepatic cells. Transcriptome and bioinformatics analyses with the Gene Ontology (GO) database showed that tangeretin significantly regulated a set of 13 differentially expressed genes (DEGs) associated with the regulation of lipoprotein lipase (LPL) activity. Among these DEGs, angiopoietin-like 3 (ANGPTL3), an essential inhibitor of LPL catalytic activity that regulates TGRL metabolism in plasma, was markedly downregulated by tangeretin. We demonstrated that tangeretin significantly inhibited the mRNA expression of ANGPTL3 in HepG2 and Huh-7 cells. Tangeretin treatment of hepatic cells also reduced the levels of both intracellular and secreted ANGPTL3 proteins. Moreover, we found that inhibition of ANGPTL3 production by tangeretin augmented LPL activity. We further demonstrated that the transcriptional activity of the ANGPTL3 promoter was significantly attenuated by tangeretin, and we identified a DNA element located between the -250 and -121 positions that responded to tangeretin. Furthermore, we found that tangeretin did not alter the levels of the nuclear liver X receptor α (LXRα) protein, an essential transcription factor that binds to the tangeretin-responsive element, but it can counteract LXRα-mediated ANGPTL3 transcription. On the basis of molecular docking analysis, tangeretin was predicted to bind to the ligand-binding domain of LXRα, which would result in suppression of LXRα activation. Our findings support the hypothesis that tangeretin exerts a lipid-lowering effect by modulating the LXRα-ANGPTL3-LPL pathway, and thus, it can be used as a potential phytochemical for the prevention or treatment of dyslipidemia.

Vernonia patula (Dryand.) Merr. and Leucas chinensis (Retz.) R. Brown exert anti-inflammatory activities and relieve oxidative stress via Nrf2 activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Vernonia patula (Dryand.) Merr. and Leucas chinensis (Retz.) R. Brown have anti-inflammatory properties and are popularly used as complementary and alternative medicine in Asia. AIM OF THE STUDY: To investigate the underlying molecular mechanism and active chemicals in the ethanol extracts of V. patula (VP) and L. chinensis (LC). MATERIALS AND METHODS: The inhibitory activities of VP and LC on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and interleukin-6 (IL-6) production were investigated in RAW264.7 macrophages and BV2 microglia. Downregulation of pro-inflammatory genes and upregulation of Nrf2 (NF-E2 p45-related factor 2)-ARE (antioxidant response element) pathway were investigated using RT-Q-PCR and Western blotting. Direct antioxidant capacities were measured using free radical scavenging and Folin-Ciocalteu assays. The flavonoids and triterpenes in VP and LC were identified by HPLC-ESI-MS. RESULTS: VP and LC inhibited NO and IL-6 production and suppressed iNOS, IL-6, IL-1ß and CCL2 gene expression. VP and LC were potent direct antioxidants and effective indirect antioxidants assayed by Nrf2 activation and induction of heme oxygenase (HO)-1, glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase 1 (NQO1). Three flavonoids including apigenin (1), luteolin (2) and chryseriol (3), and one triterpene betulinic acid (4) were found in VP; while compounds 1-4 and oleanolic acid (5) were in LC. CONCLUSION: Anti-inflammatory and antioxidant activities of VP and LC may be in great part attributed to the identified Nrf2 activating compounds, which induce expression of Phase II enzymes and attenuate the upregulation of pro-inflammatory genes.

The Cholesterol-Modulating Effect of Methanol Extract of Pigeon Pea (Cajanus cajan (L.) Millsp.) Leaves on Regulating LDLR and PCSK9 Expression in HepG2 Cells.

Pigeon pea (Cajanus cajan (L.) Millsp.) is a legume crop consumed as an indigenous vegetable in the human diet and a traditional medicinal plant with therapeutic properties. The current study highlights the cholesterol-modulating effect and underlying mechanisms of the methanol extract of Cajanus cajan L. leaves (MECC) in HepG2 cells. We found that MECC increased the LDLR expression, the cell-surface LDLR levels and the LDL uptake activity in HepG2 cells. We further demonstrated that MECC suppressed the proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein expression, but not affected the expression of other cholesterol or lipid metabolism-related genes including inducible degrader of LDLR (IDOL), HMG-CoA reductase (HMGCR), fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC1), and liver X receptor-α (LXR-α) in HepG2 cells. Furthermore, we demonstrated that MECC down-regulated the PCSK9 gene expression through reducing the amount of nuclear hepatocyte nuclear factor-1α (HNF-1α), a major transcriptional regulator for activation of PCSK9 promoter, but not that of nuclear sterol-responsive element binding protein-2 (SREBP-2) in HepG2 cells. Finally, we identified the cajaninstilbene acid, a main bioactive stilbene component in MECC, which significantly modulated the LDLR and PCSK9 expression in HepG2 cells. Our current data suggest that the cajaninstilbene acid may contribute to the hypocholesterolemic activity of Cajanus cajan L. leaves. Our findings support that the extract of Cajanus cajan L. leaves may serve as a cholesterol-lowering agent.

Xanthohumol Suppresses Mylip/Idol Gene Expression and Modulates LDLR Abundance and Activity in HepG2 Cells.

Xanthohumol, a prenylated flavonoid found in hops (Humulus lupulus L.), exhibits multiple biological activities such as antiatherosclerosis and hypolipidemic activities. In this study, we aim to investigate the hypocholesterolemic effects and molecular mechanisms of xanthohumol in hepatic cells. We found that xanthohumol (10 and 20 µM) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) from 100.0 ± 2.1% to 115.0 ± 1.3% and 135.2 ± 2.7%, and enhanced the LDL uptake activity from 100.0 ± 0.9% to 139.1 ± 13.2% in HepG2 cells (p < 0.01). The mRNA levels of LDLR, HMGCR, and PCSK9 were not altered. Xanthohumol (20 µM) reduced the expression of inducible degrader of the LDL receptor (Mylip/Idol) mRNA and protein by approximately 45% (p < 0.01), which was reported to be associated with increases of LDLR level. We demonstrated that xanthohumol suppressed hepatic Mylip/Idol expression via counteracting liver X receptor (LXR) activation. The molecular docking results predicted that xanthohumol has a high binding affinity to interact with the LXRα ligand-binding domain, which may result in attenuation of LXRα-induced Mylip/Idol expression. Finally, we demonstrated that the Mylip/Idol expression and LDLR activity were synergistically changed by a combination of xanthohumol and simvastatin treatment. Our findings indicated that xanthohumol may regulate the LXR-Mylip/Idol axis to modulate hepatic LDLR abundance and activity.

Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF-κB and JNK Activation in LPS-Stimulated BV2 Microglia.

Cleome rutidosperma DC. and Euphorbia thymifolia L. are herbal medicines used in traditional Indian and Chinese medicine to treat various illnesses. Reports document that they have antioxidant and anti-inflammatory activities; nonetheless, the molecular mechanisms involved in their anti-inflammatory actions have not yet been elucidated. The anti-neuroinflammatory activities and underlying mechanisms of ethanol extracts of Cleome rutidosperma (CR) and Euphorbia thymifolia (ET) were studied using lipopolysaccharide (LPS)-stimulated microglial cell line BV2. The morphology changes and production of pro-inflammatory mediators were assayed. Gene expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1ß, and CC chemokine ligand (CCL)-2, as well as phase II enzymes such as heme oxygenase (HO)-1, the modifier subunit of glutamate cysteine ligase (GCLM) and NAD(P)H quinone dehydrogenase 1 (NQO1), were further investigated using reverse transcription quantitative-PCR (RT-Q-PCR) and Western blotting. The effects of CR and ET on mitogen activated protein kinases (MAPKs) and nuclear factor (NF)-κB signaling pathways were examined using Western blotting and specific inhibitors. CR and ET suppressed BV2 activation, down-regulated iNOS and COX-2 expression and inhibited nitric oxide (NO) overproduction without affecting cell viability. They reduced LPS-mediated tumor necrosis factor (TNF) and IL-6 production, attenuated IL-1ß and CCL2 expression, but upregulated HO-1, GCLM and NQO1 expression. They also inhibited p65 NF-κB phosphorylation and modulated Jun-N terminal kinase (JNK) activation in BV2 cells. SP600125, the JNK inhibitor, significantly augmented the anti-IL-6 activity of ET. NF-κB inhibitor, Bay 11-7082, enhanced the anti-IL-6 effects of both CR and ET. Znpp, a competitive inhibitor of HO-1, attenuated the anti-NO effects of CR and ET. Our results show that CR and ET exhibit anti-neuroinflammatory activities by inhibiting pro-inflammatory mediator expression and production, upregulating HO-1, GCLM and NQO1, blocking NF-κB and modulating JNK signaling pathways. They may offer therapeutic potential for suppressing overactivated microglia and alleviating neurodegeneration.

Antioxidant, Anti-Tyrosinase and Anti-Inflammatory Activities of Oil Production Residues from Camellia tenuifloria.

Camellia tenuifloria is an indigenous Camellia species used for the production of camellia oil in Taiwan. This study investigated for the first time the potential antioxidant, anti-tyrosinase and anti-inflammatory activities of oil production byproducts, specifically those of the fruit shell, seed shell, and seed pomace from C. tenuifloria. It was found that the crude ethanol extract of the seed shell had the strongest DPPH scavenging and mushroom tyrosinase inhibitory activities, followed by the fruit shell, while seed pomace was the weakest. The IC50 values of crude extracts and fractions on monophenolase were smaller than diphenolase. The phenolic-rich methanol fraction of seed shell (SM) reduced nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. It also repressed the expression of IL-1ß, and secretion of prostaglandin E2 (PGE2) and IL-6 in response to LPS. SM strongly stimulated heme oxygenase 1 (HO-1) expression and addition of zinc protoporphyrin (ZnPP), a HO-1 competitive inhibitor, reversed the inhibition of NO production, indicating the involvement of HO-1 in its anti-inflammatory activity. The effects observed in this study provide evidence for the reuse of residues from C. tenuifloria in the food additive, medicine and cosmetic industries.

Anti-inflammatory effect and mechanism of the green fruit extract of Solanum integrifolium Poir.

The green fruit of Solanum integrifolium Poir. has been used traditionally as an anti-inflammatory and analgesic remedy in Taiwanese aboriginal medicine. The goal of this study is to evaluate the anti-inflammatory activity and mechanism of the green fruit extract of S. integrifolium. A bioactivity-guided fractionation procedure was developed to identify the active partition fraction. The methanol fraction (ME), with the highest phenolic content, exhibited the strongest inhibitory effect against LPS-mediated nitric oxide (NO) release and cytotoxicity in RAW264.7 macrophages. ME also significantly downregulated the expression of LPS-induced proinflammatory genes, such as iNOS, COX-2, IL-1ß, IL-6, CCL2/MCP-1, and CCL3/MIP1α. Moreover, ME significantly upregulated HO-1 expression and stimulated the activation of extracellular-signal-regulated kinase 1/2 (ERK1/2). Pretreatment of cells with the HO-1 inhibitor zinc protoporphyrin and MEK/ERK inhibitor U0126 attenuated ME's inhibitory activity against LPS-induced NO production. Taken together, this is the first study to demonstrate the anti-inflammatory activity of green fruit extract of S. integrifolium and its activity may be mediated by the upregulation of HO-1 expression and activation of ERK1/2 pathway.

Effects of citrus flavonoids, 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone and 3,5,6,7,8,3',4'-heptamethoxyflavone, on the activities of macrophage scavenger receptors and the hepatic LDL receptor.

Epidemiological and animal studies point to a possible protective effect of citrus flavonoids against cardiovascular diseases. The aim of this study is to investigate the effects of citrus flavonoids, 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) and 3,5,6,7,8,3',4'-heptamethoxyflavone (HpMF), on the activities and expressions of macrophage scavenger receptors and the hepatic LDL receptor. Treatment of HpMF (20 µM) during THP-1 differentiation successfully attenuated 12-myristate 13-acetate (PMA)-mediated DiI-labeled oxidized low-density lipoprotein (oxLDL) uptake as evidenced by flow cytometry, indicating that the functions of scavenger receptors were blocked. RT-Q-PCR analysis suggests that the decrease in oxLDL uptake was due to the down-regulation of PMA-induced SR-A mRNA expression. In terminally differentiated THP-1 macrophages, 5-OH-HxMF and HpMF could significantly reduce DiI-oxLDL uptake, with the former having a greater effect. 5-OH-HxMF attenuated oxLDL-mediated CD36 and SR-A expression; while HpMF only decreased CD36 expression. The effects of these two flavonoids on the activity and expression of the hepatic LDL receptor (LDLR) were further investigated in HepG2 cells. 5-OH-HxMF (10-20 µM) enhanced DiI-LDL uptake by 1.33-fold due to the enhanced LDLR expression. These results imply that HpMF is better at inhibiting PMA-induced oxLDL uptake during THP-1 differentiation, while 5-OH-HxMF is more powerful in attenuating oxLDL-induced scavenger receptor expression and activity in terminally differentiated THP-1 macrophages. Furthermore, 5-OH-HxMF may have hypolipidemic activity due to its up-regulating hepatic LDLR expression.

Curcuminoids promote neurite outgrowth in PC12 cells through MAPK/ERK- and PKC-dependent pathways.

Curcuminoids, the predominant polyphenolic compounds in the rhizome of Curcuma longa Linn., consist of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). They exhibit multiple desirable characteristics for a neuroprotective agent including antioxidant, anti-inflammatory, and antiamyloid activities. In this work, we report the first investigation of the neurotrophic action and mechanism of curcuminoids in PC12 cells, which respond to nerve growth factor (NGF) and therefore serve as a model system for primary neuronal cells. The percentages of neurite-bearing cells for those treated with 20 µM curcumin, DMC, and BDMC for 72 h reached 21.6 ± 2.0%, 16.3 ± 2.4%, and 19.9 ± 2.5%, respectively, and were significantly higher than that of the negative control (2.0 ± 0.3%, p < 0.05). In parallel, increased expression of the neuronal differentiation markers, growth-associated protein-43 (GAP-43), and neurofilament-L (NF-L) was found in curcuminoid-treated cells. All three curcuminoids (20 µM) activated extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase C (PKC) signalings, and inhibition of these kinases with the respective pharmacological inhibitors effectively attenuated curcuminoid-induced neurite outgrowth. Furthermore, our results show that both curcumin and DMC, but not BDMC, induced phosphorylation of cAMP response element-binding protein (CREB) and CRE-reporter gene activity significantly (p < 0.05). These inductions were markedly attenuated by the addition of MEK/ERK or PKC inhibitor; as a consequence, ERK- and PKC-dependent pathways may be involved in curcuminoid-mediated neuritogenesis in PC12 cells. Moreover, activation of CREB coupling with CRE-dependent gene transcription may play a vital role for curcumin- or DMC-induced PC12 differentiation.

Immunomodulatory effect of Antrodia camphorata mycelia and culture filtrate.

AIM OF THE STUDY: Antrodia camphorata, a precious folkloric medicinal mushroom, has been used to treat tumorigenic diseases in Taiwan. This study was to investigate the innate immunity augmentation effects of different fractions prepared from hot water extracts of submerged cultured Antrodia camphorata (AC). MATERIALS AND METHODS: The cytokine induction potency of AC fraction in diluted peripheral blood culture was measured by ELISA. The effects of AC fraction on phagocytic activity and CD11b expression were measured by the ingestion of FITC-labeled Escherichia coli and by labeling with PE-labeled CD11b monoclonal antibody, respectively, using flow cytometry. The molecular mass of hot water-soluble polysaccharides and content of adenosine in AC fraction were determined by gel permeation chromatography (GPC) and HPLC, respectively. RESULTS: The mycelia fraction, Fr. M II, and culture filtrate fractions, Fr. E II and Fr. E III, showed the strongest TNF-alpha and IL-6 induction effect as a function of their concentration. These fractions (20mug/ml) also showed marked activity in enhancing phagocytosis in human polymorphonuclear neutrophils (PMN) and monocytes. In parallel, the expression of CD11b, an early marker of PMN activation, was also up-regulated dose-dependently. Composition analysis suggested that immunomodulatory effect of mycelia is mainly attributed to the 10-20kDa polysaccharides and adenosine. CONCLUSIONS: These results provide evidences that Antrodia camphorata can modulate innate immunity and may serve as an adjuvant for tumor treatment.

Oral administration of submerged cultivated Grifola frondosa enhances phagocytic activity in normal mice.

Grifola frondosa fruiting body (Maitake) has been used as a dietary supplement due to its antitumour and immunomodulatory properties. The aim of this study was to evaluate the immunomodulatory effects of orally administered submerged cultivated G. frondosa mixture, including both mycelium and culture broth, in a healthy murine model. Composition analyses showed that submerged cultivated G. frondosa mixture contained only 32.48% carbohydrate, which was less than half of fruiting bodies. The content of adenosine, a potential immunomodulatory agent in medicinal mushrooms, was 2.8 mg g(-1). After feeding 8-week-old female BALB/cByJ mice with AIN-93G diet containing 0% (C), 1% (G1), 3% (G3) or 5% (G5) (wt/wt) G. frondosa mixture for 31 days, neither body weight nor the outward appearance of organs showed any significant difference among different diet groups. Splenocyte subpopulation, mitogen-activated cytokine release and splenic NK activity were not affected by G. frondosa administration, either. On the other hand, the phagocytic activity was enhanced in leucocytes of groups G3 and G5, without exerting detectable levels of serum proinflammatory cytokines. These results suggested that oral administration of submerged cultivated G. frondosa mixture may enhance host innate immunity against foreign pathogens without eliciting adverse inflammatory response.

Immunomodulatory effect of exo-polysaccharides from submerged cultured Cordyceps sinensis: enhancement of cytokine synthesis, CD11b expression, and phagocytosis.

Cordyceps sinensis is widely used as a traditional medicine for treatment of a wide variety of diseases or to maintain health. The immunomodulatory activity of polysaccharides prepared from submerged cultured C. sinensis BCRC36421 was investigated in human peripheral blood. Results demonstrated that Fr. A (exo-polysaccharides, 0.025 approximately 0.1 mg/ml) induced the production of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, and IL-10 dose-dependently. Fr. A, as low as 0.025 mg/ml, could significantly augment surface expression of CD11b in monocytes and polymorphonuclear neutrophils. Functional assay revealed that Fr. A (0.05 mg/ml) also elevated phagocytosis in monocytes and PMN. On the other hand, Fr. B (intracellular polysaccharides) only moderately induced TNF-alpha release, CD11b expression, and phagocytosis at the same concentrations. Our results indicate that the immunomodulatory components of submerged cultured C. sinensis mainly reside in the culture filtrate.

Immunomodulatory effect of Glossogyne tenuifolia in murine peritoneal macrophages and splenocytes.

Glossogyne tenuifolia Cass., a medicinal plant native to Taiwan, is traditionally used as an anti-inflammatory remedy. Oleanolic acid and luteolin-7-glucoside have been previously identified as active components of Glossogyne tenuifolia in the murine macrophage-like cell line, RAW264.7. Current study investigates the effect and mechanism of the ethanol extract of Glossogyne tenuifolia (GT) and its major constituents on the release of inflammatory mediators in activated elicited murine peritoneal macrophages and splenocytes. Our results showed that GT (up to 0.15 mg/ml) inhibited the production of proinflammatory mediators, TNF-alpha, IL-1beta, IL-6, nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in LPS-activated macrophages, and IFN-gamma in PHA-activated splenocytes. GT also inhibited LPS-activated murine iNOS and COX-2 promoter activities in transiently transfected RAW264.7 cells. The major constituents, oleanolic acid and luteolin-7-glucoside, as well as its aglycone, luteolin, inhibited the release of NO, PGE(2), TNF-alpha and IL-1beta in activated peritoneal macrophages. However, only luteolin-7-glucoside and luteolin were able to reduce IFN-gamma release in PHA-stimulated splenocytes. To further investigate the possible mechanisms that interfere with LPS- and PHA-signaling, this study focused on nuclear factor-kappaB activation signaling pathways. Our results demonstrate that GT (0.075-0.15 mg/ml) treatment reduces nuclear factor-kappaB (NF-kappaB) DNA binding activity, as demonstrated by electrophoretic mobility shift assay (EMSA). Collectively, the results suggest that GT inhibits proinflammatory mediator synthesis in activated murine peritoneal macrophages and splenocytes, in part through NF-kappaB-dependent pathways.

Ganoderma lucidum mycelia enhance innate immunity by activating NF-kappaB.

Ganoderma lucidum is a popular medicinal mushroom in China and Japan for its immunomodulatory and antitumor effects. The goal of this research is to investigate the effect of dried mycelia of Ganoderma lucidum produced by submerged cultivation on the enhancement of innate immune response. We found that Ganoderma lucidum mycelia (0.2-1.6 mg/ml) stimulated TNF-alpha and IL-6 production after 8h treatment in human whole blood. IFN-gamma release from human whole blood was also enhanced after 3 day-culture with Ganoderma lucidum mycelia (0.2-1.0mg/ml). However, Ganoderma lucidum mycelia did not potentiate nitric oxide production in RAW264.7 cells. To better understand the possible immuno-enhancement mechanisms involved, we focused on nuclear factor (NF)-kappaB activation. Electrophoretic mobility shift assay revealed that the Ganoderma lucidum mycelia (1.6 mg/ml) activated kappaB DNA binding activity in RAW264.7 cells. These results provide supporting evidences for the immunomodulatory effect of Ganoderma lucidum mycelia.

Antioxidant activity of Glossogyne tenuifolia.

Glossogyne tenuifolia is a native traditional anti-inflammatory herb in Taiwan. It has previously been shown that the ethanol extract of G. tenuifolia (GT) inhibited the LPS-induced inflammatory mediator release from murine macrophage cell line and human whole blood. In the present work, the ethanol extracts of G. tenuifolia and its major constituent, luteolin-7-glucoside, were shown to be scavengers of 1,1-diphenyl-2-picrylhydrazyl, superoxide, and hydroxyl radicals. Moreover, copper-induced low-density lipoprotein oxidation was suppressed by GT and luteolin-7-glucoside as measured by decreased formation of malondialdehyde and conjugated diene as well as reduced electrophoretic mobility. GT and luteolin-7-glucoside were also against N-formyl-methionyl-leucyl-phenylalanine-induced reactive oxygen species (ROS) production in human polymorphonuclear neutrophils and peripheral blood mononuclear cells. In summary, these data indicated that GT is a potential ROS scavenger and may prevent atherosclerosis via inhibiting LDL oxidation or ROS production in human leukocytes. Moreover, luteolin-7-glucoside may serve as the active principal of GT.

Immunomodulatory mechanism of the aqueous extract of sword brake fern (Pteris ensiformis Burm.).

Sword brake fern (Pteris ensiformis Burm.) is an ingredient in most of the traditional herbal beverage formulas in Taiwan; however, no information is available to explain its bioactivity. The aim of this study is to elucidate the molecular pharmacological activity in the aqueous extract of sword brake fern (SBF). We found that SBF (0.05-0.25 mg/ml) slightly induced TNF-alpha, IL-6, NO (nitric oxide) and PGE2 (prostaglandin E2) production in unstimualted murine macrophages, RAW264.7 cells. Furthermore, SBF (0.05-0.25 mg/ml) dose-dependently suppressed LPS-induced TNF-alpha, IL-1beta, IL-6, NO and PGE2 in activated RAW264.7 cells without exerting cytotoxicity. Further analysis of molecular mechanisms revealed that SBF prominently repressed LPS-induced iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) promoter activities. Activation of the transcription factor NF-kappaB, which is one of the important pathways for transduction of LPS-stimulated inflammatory mediator producing signals, was suppressed by SBF in a dose-dependent manner, as demonstrated by both electrophoretic mobility shift assay (EMSA) and transfection with pNF-kappaB-Luc plasmid. These results suggest that SBF attenuates inflammatory mediator synthesis of activated macrophages partially through a NF-kappaB-dependent pathway. The immunomodulatory activity of SBF supports its traditional health promotion effect.

Anti-inflammatory and antiviral effects of Glossogyne tenuifolia.

Glossogyne tenuifolia (Hsiang-Ju) is a traditional antipyretic and hepatoprotective herb used in Chinese medicine. The aim of this research is to investigate the pharmacological activities and potent components of the ethanol extract of Glossogyne tenuifolia (GT) in human primary cells and cell line. We found that GT (0.1 approximately 0.25 mg/ml) exerted dose-dependent inhibitions on the release of TNF-alpha and IL-6 in LPS-activated human whole blood and peripheral blood mononuclear cells (PBMC), and IFN-gamma in PHA-stimulated human whole blood. The lack of cytotoxicity indicated that the inhibitory effects of GT on cytokine production were not due to cell death. Luteolin, the deglycosylated derivative of one of the major compositions, luteolin-7-glucoside, exerted inhibitory effects on TNF-alpha, IL-6 and IFN-gamma production in activated human whole blood with estimated IC(50)s of 42.73 microM, 44.86 microM and 3.34 microM, respectively. Furthermore, GT had potent anti-hepatitis B virus (HBV) effects on the human hepatocellular carcinoma cell line, PLC/PRF/5. GT exhibited a dose-dependent inhibition on the release of hepatitis B surface antigen (HBsAg) by repressing the expression of HBsAg with IC(50) of 0.093 mg/ml. We concluded that GT exerted combinatorial anti-inflammatory and antiviral effects, and the multiple actions may underlie its traditional hepatoprotective function.

Glossogyne tenuifolia acts to inhibit inflammatory mediator production in a macrophage cell line by downregulating LPS-induced NF-kappa B.

Glossogyne tenuifolia (hsiang-ju) (GT) is a traditional antipyretic herb used in Chinese medicine; however, no information is available to explain its action. The objective of this research was to elucidate the molecular pharmacological activity and the effective components in the ethanol extract of GT. We found that GT had potent anti-inflammatory effects on the lipopolysaccharide (LPS)-activated murine macrophages, RAW264.7. GT downregulated LPS-induced expression of inducible nitric oxide synthase (iNOS) by blocking its transcription. GT also caused a dose-dependent inhibition of the release of prostaglandin E(2) by repressing the promoter activity of the inducible cyclooxygenase (COX-2) gene. Moreover, GT exerted a dose-dependent inhibition of the LPS-stimulated release of the proinflammatory cytokines, TNF-alpha, IL-1 beta, IL-6, and IL-12. To determine the mechanism by which GT inhibits LPS signaling, we focused on nuclear factor-kappa B (NF-kappa B) activation. Western blot analysis revealed that GT abolished LPS-induced inhibitor-kappa B phosphorylation. The electrophoretic mobility shift assay demonstrated that GT abolished LPS-mediated kappa B DNA binding activity. Moreover, macrophages were transfected with a vector coding for the luciferase reporter gene under the control of NF-kappa B cis-acting elements, and the transfected macrophages showed that the LPS-stimulated luciferase activity was GT-sensitive. These results suggest that GT attenuates inflammatory mediator synthesis of activated macrophages through an NF-kappa B-dependent pathway. The active components of GT were identified as oleanolic acid and luteolin-7-glucoside. Both of these compounds inhibited LPS-stimulated inflammatory mediator production and NF-kappa B activation. We conclude that GT inhibits NF-kappa B-mediated gene expression and downregulates inflammatory mediator production in murine macrophages.

Antioxidant activity of Porcelainberry (Ampelopsis brevipedunculata (Maxim.) Trautv.).

The stem and root of Porcelainberry (Ampelopsis brevipedunculata (Maxim.) Trautv.) (AB) was traditionally used as an anti-inflammatory, diuretic and anti-hepatotoxic agent in folk medicine. In this study, cell-free and cell culture systems were employed to investigate the antioxidant activity of the methanol extract of AB (MEAB). The cell-free system showed that the MEAB exhibited dose-dependent antioxidant activities against linoleic acid peroxidation and plasmid DNA oxidation. We also demonstrated that the MEAB possessed strong reducing power and scavenging effects of hydroxyl radicals and DPPH free radicals. The harmful effects of reactive oxygen metabolites on HepG2 cells and the possible antioxidant effects of the MEAB were also investigated. Pretreatment or cotreatment of HepG2 cells with the MEAB could significantly protect cells from H2O2-induced oxidative stress. This implies that the antioxidant effects in cell culture may result from the direct interaction between the MEAB and exogenous oxidant sources, as these occur in cell free systems, as well as from the induction of cellular stress gene expression. The antioxidant activity of the MEAB may partially explain its anti-inflammatory and anti-hepatotoxic effects.