Inhibitory effects of ChondroT and its constituent herbs on RANKL-induced osteoclastogenesis.

BACKGROUND: ChondroT is a complex herbal medicine consisting of water extracts of Ostericum koreanum (Maxim.) Kitag., Lonicera japonica Thunb., Angelica gigas Nakai, Clematis manshurica Rupr., and Phellodendron amurense Rupr. (6:4:4:4:3). Previous studies have reported that ChondroT possesses chondroprotective and anti-inflammatory, anti-osteoarthritic, and anti-hyperuricemic activities. The study is aim to demonstrate the effects of ChondroT and its five constituent herbs on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and the underlying mechanisms. METHODS: Osteoclastogenesis was identified in bone marrow-derived macrophages (BMDMs) by tartrate-resistant acid phosphatase (TRAP) staining assay, actin ring formation assay and the bone resorption assay. For the molecular mechanisms, activation of RANKL-induced NF-κB and MAPK signaling pathways and the expression levels of osteoclast-specific proteins were investigated by Western blotting. Cell viability was assessed by MTT assay. Actin ring formation and NF-κB translocation were evaluated by immunostaining. RESULTS: ChondroT and each of its constituent herbs significantly suppressed osteoclast differentiation dose dependently, and decreased actin ring formation as well as bone-resorbing capacity. Mechanistically, ChondroT and its constituent herbs downregulated the expressional levels of osteoclast-specific proteins such as NFATc1, c-Fos, Cathepsin K, and matrix metalloproteinase 9 (MMP9) by suppressing NF-κB translocation to nucleus and MAPKs phosphorylation at different levels. Compared to its five constituent herbs, ChondroT exhibited the best inhibitory efficiency against osteoclastogenesis. CONCLUSIONS: Taken together, ChondroT has anti-osteoclastogenesis properties by inhibiting NF-κB and MAPKs pathways. It could be considered as a potential therapeutic candidate for the treatment of osteoclast-related bone diseases.

Effects of ChondroT on potassium Oxonate-induced Hyperuricemic mice: downregulation of xanthine oxidase and urate transporter 1.

BACKGROUND: ChondroT, a new herbal medication, consists of the water extracts of Osterici Radix, Lonicerae Folium, Angelicae Gigantis Radix, Clematidis Radix, and Phellodendri Cortex (6:4:4:4:3). We previously reported that ChondroT showed significant anti-arthritis and anti-inflammatory effects. METHODS: This study was designed to evaluate the effect of ChondroT on hyperuricemia. First, the effect of ChondroT was evaluated on xanthine oxidase (XOD) activity in vitro. The anti-hyperuricemic effect of ChondroT was also studied in potassium oxonate (PO)-induced hyperuricemic model mice. Uric acid (UA) and XOD were evaluated in the serum, urine, and liver of the mice. In addition, we measured serum creatinine (Cr) and blood urea nitrogen (BUN) levels as well as mRNA expression of the mouse urate transporter 1 (mURAT1) to evaluate kidney function and urate excretion in hyperuricemic mice. RESULTS: ChondroT showed in vitro XOD inhibitory activity in a dose-dependent manner (P < 0.05). We demonstrated that ChondroT (37.5, 75 and 150 mg/kg) significantly reduced serum UA (P < 0.01 and P < 0.001, respectively), and upregulated urinary UA (P < 0.001, respectively) in PO-induced hyperuricemic mice. In addition, ChondroT (75 and 150 mg/kg) significantly reduced Cr (P < 0.05 and P < 0.01, respectively), BUN (P < 0.05 and P < 0.001, respectively), GOT (P < 0.05 and P < 0.01, respectively), and GPT (P > 0.05 and P < 0.05, respectively) levels in PO-induced hyperuricemic mice. ChondroT (75 and 150 mg/kg) also significantly downregulated serum (P < 0.05) and liver (P < 0.05) XOD activity. Compared to the hyperuricemic mice, the ChondroT (37.5, 75, and 150 mg/kg)-treated mice showed decreased mURAT1 protein expression level. CONCLUSION: ChondroT displayed anti-hyperuricemic effects by regulating XOD activity and kidney mURAT1.

Biotransformed Metabolites of the Hop Prenylflavanone Isoxanthohumol.

A metabolic conversion study on microbes is known as one of the most useful tools to predict the xenobiotic metabolism of organic compounds in mammalian systems. The microbial biotransformation of isoxanthohumol (1), a major hop prenylflavanone in beer, has resulted in the production of three diastereomeric pairs of oxygenated metabolites (2⁻7). The microbial metabolites of 1 were formed by epoxidation or hydroxylation of the prenyl group, and HPLC, NMR, and CD analyses revealed that all of the products were diastereomeric pairs composed of (2S)- and (2R)- isomers. The structures of these metabolic compounds were elucidated to be (2S,2"S)- and (2R,2"S)-4'-hydroxy-5-methoxy-7,8-(2,2-dimethyl-3-hydroxy-2,3-dihydro-4H-pyrano)-flavanones (2 and 3), (2S)- and (2R)-7,4'-dihydroxy-5-methoxy-8-(2,3-dihydroxy-3-methylbutyl)-flavanones (4 and 5) which were new oxygenated derivatives, along with (2R)- and (2S)-4'-hydroxy-5-methoxy-2"-(1-hydroxy-1-methylethyl)dihydrofuro[2,3-h]flavanones (6 and 7) on the basis of spectroscopic data. These results could contribute to understanding the metabolic fates of the major beer prenylflavanone isoxanthohumol that occur in mammalian system.

Chondroprotective and anti-inflammatory effects of ChondroT, a new complex herbal medication.

BACKGROUND: Ganghwaljetongyeum (GHJTY) is a complex herbal decoction comprising 18 plants; it is used to treat arthritis. In order to develop a new anti-arthritic herbal medication, we selected 5 out of 18 GHJTY plants by using bioinformatics analysis. The new medication, called ChondroT, comprised water extracts of Osterici Radix, Lonicerae Folium, Angelicae Gigantis Radix, Clematidis Radix, and Phellodendri Cortex. This study was designed to investigate its chondroprotective and anti-inflammatory effects to develop an anti-arthritic herb medicine. METHODS: ChondroT was validated using a convenient and accurate high-performance liquid chromatography-photodiode array (HPLC-PDA) detection method for simultaneous determination of its seven reference components. The concentrations of the seven marker constituents were in the range of 0.81-5.46 mg/g. The chondroprotective effects were evaluated based on SW1353 chondrocytes and matrix metalloproteinase 1 (MMP1) expression. In addition, the anti-inflammatory effects of ChondroT were studied by Western blotting of pro-inflammatory enzymes and by enzyme-linked immunosorbent assay (ELISA) of inflammatory mediators in lipopolysaccharides (LPS)-induced RAW264.7 cells. RESULTS: ChondroT enhanced the growth of SW1353 chondrocytes and also significantly inhibited IL-1ß-induced MMP-1 expression. However, ChondroT did not show any effects on the growth of HeLa and RAW264.7 cells. The expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was induced by LPS in RAW264.7 cells, which was significantly decreased by pre-treatment with ChondroT. In addition, ChondroT reduced the activation of NF-kB and production of inflammatory mediators, such as IL-1ß, IL-6, PGE2, and nitric oxide (NO) in LPS-induced RAW264.7 cells. CONCLUSIONS: These results show that ChondroT exerted a chondroprotective effect and demonstrated multi-target mechanisms related to inflammation and arthritis. In addition, the suppressive effect was greater than that exhibited by GHJTY, suggesting that ChondroT, a new complex herbal medication, has therapeutic potential for the treatment of arthritis.

Cholera toxin breakdowns oral tolerance via activation of canonical NF-κB.

The mechanisms of mucosal immunogenicity and adjuvanticity of bacterial exotoxins remains unknown. In this study, we investigated the role of the transcription factor nuclear factor-κB (NF-κB) in cholera toxin (CT)-induced alteration of oral tolerance. Feeding CT abrogated ovalbumin (OVA)-induced oral tolerance, as evaluated by OVA-specific serum antibody responses, and CD4(+) T cell proliferation. CT feeding activated canonical NF-κB (one heterodimer type, p50-p65) and mRNA expression of NF-κB-dependent proinflammatory cytokines in mesenteric lymph node (MLN) and Peyer's patch (PP) cells. CT no longer showed abrogation of oral tolerance in mice pretreated with p50 small interfering RNAs (siRNAs). ADP-ribosylation inhibitors inhibited CT-induced NF-κB activation. These data suggest that CT induces canonical NF-κB activation in intestinal lymphoid cells, which plays a key role in mucosal immunogenicity and adjuvanticity.

Picropodophyllotoxin Inhibits Cell Growth and Induces Apoptosis in Gefitinib-Resistant Non-Small Lung Cancer Cells by Dual-Targeting EGFR and MET.

Patients with non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) amplification or sensitive mutations initially respond to the tyrosine kinase inhibitor gefitinib, however, the treatment becomes less effective over time by resistance mechanism including mesenchymal-epithelial transition (MET) overexpression. A therapeutic strategy targeting MET and EGFR may be a means to overcoming resistance to gefitinib. In the present study, we found that picropodophyllotoxin (PPT), derived from the roots of Podophyllum hexandrum, inhibited both EGFR and MET in NSCLC cells. The antitumor efficacy of PPT in gefitinib-resistant NSCLC cells (HCC827GR), was confirmed by suppression of cell proliferation and anchorage-independent colony growth. In the targeting of EGFR and MET, PPT bound with EGFR and MET, ex vivo, and blocked both kinases activity. The binding sites between PPT and EGFR or MET in the computational docking model were predicted at Gly772/Met769 and Arg1086/Tyr1230 of each ATP-binding pocket, respectively. PPT treatment of HCC827GR cells increased the number of annexin V-positive and subG1 cells. PPT also caused G2/M cell-cycle arrest together with related protein regulation. The inhibition of EGFR and MET by PPT treatment led to decreases in the phosphorylation of the downstream-proteins, AKT and ERK. In addition, PPT induced reactive oxygen species (ROS) production and GRP78, CHOP, DR5, and DR4 expression, mitochondrial dysfunction, and regulated involving signal-proteins. Taken together, PPT alleviated gefitinib-resistant NSCLC cell growth and induced apoptosis by reducing EGFR and MET activity. Therefore, our results suggest that PPT can be a promising therapeutic agent for gefitinib-resistant NSCLC.

Ets-1, a functional cofactor of T-bet, is essential for Th1 inflammatory responses.

To mount an effective type 1 immune response, type 1 T helper (Th1) cells must produce inflammatory cytokines and simultaneously suppress the expression of antiinflammatory cytokines. How these two processes are coordinately regulated at the molecular level is still unclear. In this paper, we show that the proto-oncogene E26 transformation-specific-1 (Ets-1) is necessary for T-bet to promote interferon-gamma production and that Ets-1 is essential for mounting effective Th1 inflammatory responses in vivo. In addition, Ets-1-deficient Th1 cells also produce a very high level of interleukin 10. Thus, Ets-1 plays a crucial and unique role in the reciprocal regulation of inflammatory and antiinflammatory Th responses.

Runx3 inhibits IL-4 production in T cells via physical interaction with NFAT.

Interleukin (IL)-4 plays a key role in T helper 2 (Th2) cell differentiation favoring humoral immune response. Regulation of IL-4 gene expression, therefore, is critically important for Th2 dependent responses and Th2 dominant disorders. In T cells, IL-4 gene expression is regulated positively or negatively by a combination of several transcription factors. Recently, enhanced IL-4 production was reported in Runx3 knockout mice; this implies negative regulation of IL-4 by Runx3. Runx proteins are transcription factors that have a Runt domain and have essential functions in development. In this study, the molecular mechanism that downregulates IL-4 expression was investigated. Runx3 inhibited IL-4 production in EL-4 T cells stimulated with PMA/ionomycin. Runx3-mediated IL-4 inhibition was NFAT-dependent, and Runx3 was physically associated with NFAT. Therefore, our results suggest that the interaction between NFAT and Runx3 is a mechanism that causes the negative regulation of IL-4, along with previously reported repression by T-bet.

Protective effect of bromocriptine against BH4-induced Cath.a cell death involving up-regulation of antioxidant enzymes.

Previously, we suggested that tetrahydrobiopterin (BH4), an obligatory cofactor for dopamine synthesis, as an intrinsic contributor to dopaminergic neuron vulnerability. The BH4 toxicity is observed in dopamine-producing cells, including Cath.a cells, but not in non-dopaminergic cells. Furthermore, the dopaminergic cell death induced by BH4 is apoptotic in nature and involves oxidative stress, similar to that observed in Parkinson's disease. Accordingly, various antioxidants have been found to protect dopaminergic cells from BH4. This study was undertaken to evaluate protective effects of the dopamine receptor agonist bromocriptine on BH4-induced Cath.a cell death, because bromocriptine has been reported to be an antioxidant with a neuroprotective activity. In the presence of bromocriptine, the increase in LDH activity and mitochondrial cytochrome c release induced by BH4 were significantly abolished. This cytoprotective effect was phosphatidylinositol 3-kinase (PI3K)/Akt pathway-dependent. In addition, bromocriptine was found to up-regulate the expressions of nuclear factor-E2-related factor-2 and antioxidant enzymes including NAD(P)H quinone oxidoreductase 1. Our findings show that bromocriptine stimulates antioxidant defense mechanisms in Cath.a cells and suggest a potential use of bromocriptine as a neuroprotectant.

Increased IL-2 production in T cells by xanthohumol through enhanced NF-AT and AP-1 activity.

Xanthohumol (XN) is a major chalcone found in hop, which is used to add bitterness and flavor to beer. In this study, we investigated the effects of XN on the production of interlukin-2 (IL-2), a potent T cell growth factor. Treatment with XN significantly increased IL-2 production in mouse EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io) in a dose-dependent manner. To further characterize its regulatory mechanism of XN on increased IL-2 production, the effects of XN on IL-2 promoter activity and the activity of several transcription factors modulating IL-2 expression were analyzed. XN enhanced activity of the IL-2 promoter, which contains distal and proximal regulatory elements in PMA/Io-activated EL-4 T cells. Furthermore, the activity of NF-AT and AP-1 was enhanced but NF-kappaB activity was not influenced by XN in PMA/Io-activated EL-4 T cells. These results suggest that XN increased IL-2 production at the transcriptional levels via the up-regulation of NF-AT and AP-1 in PMA/Io-activated EL-4 T cells.

Anti-Inflammatory Effects of Canavalia gladiata in Macrophage Cells and DSS-Induced Colitis Mouse Model.

Canavalia gladiata, known as sword bean, has been used as a Chinese traditional medicine for anti-inflammatory effects. However, the action mechanisms of sword bean have not yet been clearly defined. In the present study, the whole parts of a ripened sword bean (RSB) and the green sword bean (GSB) containing bean pod were extracted with ethanol by reflux extraction. The two crude extracts (RSBE and GSBE) from RSB and GSB were validated by a liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis of gallic acid as a reference chemical. The anti-inflammatory effects of two sword bean extracts were extensively investigated using LPS-stimulated macrophage cells. First, RSBE and GSBE significantly inhibited the production of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandinE2 (PGE2), and nitric oxide (NO) in LPS-induced RAW264.7 cells. RSBE and GSBE showed no cytotoxicity to RAW264.7 cells and mouse peritoneal macrophage cells. In addition, the overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) induced by LPS in RAW264.7 cells was significantly decreased by RSBE and GSBE. Western blotting and immunostaining analysis showed that RSBE and GSBE inhibited the nuclear translocation of NF-κB subunits, which correlated with the inhibitory effects on inhibitor kappa B (IκB) degradation. In dextran sulfated sodium (DSS)-induced colitis mice model, RSBE restored body weight, colon length, and the levels of pro-inflammatory cytokines, such as TNF-α, IL-6, interleukin-1ß (IL-1ß), and interferon-γ (IFN-γ). In addition, RSBE significantly suppressed the expression of COX-2, iNOS, and NF-κB.

Sirt2 interacts with 14-3-3 beta/gamma and down-regulates the activity of p53.

Sirt2 is a mammalian member of the Sirtuin family of NAD(+) (nicotinamide adenine dinucleotide)-dependent protein deacetylases. Although Sir-2.1 (a Caenorhabditis elegans Sirt2 ortholog) has been reported to interact with PAR-5/FTT-2 (a C. elegans 14-3-3 homolog), the molecular significance of the interaction between Sirt2 and 14-3-3 proteins in mammalian cell is not understood. Here, we report that Sirt2 interacts with 14-3-3 beta and gamma among various 14-3-3 isoforms, and that this interaction is strengthened by AKT. Furthermore, Sirt2 deacetylates and down-regulates the transcriptional activity of p53, and 14-3-3 beta/gamma augment deacetylation and down-regulation of the p53 transcriptional activity by Sirt2 in an AKT-dependent manner. Treatment of cells with nicotinamide, an inhibitor of Sirtuins, relieves the inhibition of p53 by Sirt2 and 14-3-3 beta/gamma. Therefore, our results suggest that the interaction between Sirt2 and 14-3-3 beta/gamma is a novel mechanism for the negative regulation of p53 beside the well-characterized Mdm2-mediated repression.

Acetylation of Sirt2 by p300 attenuates its deacetylase activity.

Histone deacetylases (HDACs) are subdivided into three classes--HDAC I, HDAC II, and Sir2. Sirt proteins are mammalian members of the Sir2 family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. The balance between acetylation and deacetylation of histone and non-histone proteins, regulated by protein acetyltransferases and deacetylases, affects the expression of genes involved in a variety of cellular processes. In addition, HDAC1 is acetylated and regulated by p300, a transcriptional co-activator with protein acetyltransferase activity, suggesting that protein acetyltransferases and deacetylases they control the activities of each other. Although the regulation of HDAC1 by p300 is well characterized, the relationship between Sir2 homologs and p300 is not understood. Here, we report that p300 interacts with Sirt2, a member of the Sir2 family, and triggers the acetylation and subsequent down-regulation of the deacetylation activity of Sirt2, and that the acetylation of Sirt2 by p300 relieves the inhibitory effect of Sirt2 on the transcriptional activity of p53. These observations demonstrate that p300 can inactivate Sirt2 by acetylation and that p300 may regulate the activity of p53 indirectly through Sirt2 in addition to its direct modification of p53.

Cyclooxygenase-2 is involved in oxidative damage and alpha-synuclein accumulation in dopaminergic cells.

Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the formation of prostaglandins from arachidonic acid. The inducible isoform COX-2 is upregulated in the dopaminergic neurons of the substantia nigra of postmortem Parkinson's disease (PD) patients and in neurotoxin-induced Parkinsonism models. COX-2 has attracted significant attention as an important source of oxidative stress in dopaminergic neurons due to its potential to oxidize catechols including dopamine. However, the role of COX-2 in the pathogenesis of PD has not been fully evaluated. Here, we show that COX-2 induces dopamine oxidation, as evidenced by the findings that COX-2 can facilitate dopamine oxidation in a cell-free system and in COX-2-overexpressing SH-SY5Y cells, and that this can be completely abolished by the selective COX-2 inhibitor meloxicam. Increased COX-2 expression causes oxidative protein modification and alpha-synuclein accumulation in dopaminergic cells. These data suggest that an abnormal increase in COX-2 expression causes dopamine oxidation and contributes to the preferential vulnerability of dopaminergic cells as in PD.

ROG negatively regulates T-cell activation but is dispensable for Th-cell differentiation.

ROG, a transcriptional repressor, is a direct target gene of NF-AT and a putative negative regulator of T-cell activation. In addition, overexpression of ROG suppresses the activity of GATA-3, implying a role of ROG in the differentiation and function of Th cells. Despite these observations, the function of ROG has yet to be confirmed by loss-of-function approaches. Here we report that ROG-deficient T cells are hypersensitive to anti-CD3 stimulation and produce more interleukin-2 (IL-2) due to enhanced NF-kappaB activity. ROG-deficient dendritic cells also produce more IL-12p40, another NF-kappaB target gene. However, ROG-deficient Th cells are capable of differentiating into Th1 and Th2 cells, and ROG-deficient mice have no defect in mounting appropriate Th immune responses in vivo. Thus, ROG is dispensable for the differentiation and function of Th cells but serves as a mediator of NF-AT-initiated suppression of NF-kappaB. Its mechanism of action and its expression pattern are distinct from those of other transcription factors negatively regulating the activation of T cells.

Differential anti-inflammatory pathway by xanthohumol in IFN-gamma and LPS-activated macrophages.

Macrophages are the main cells responsible for the innate immunity, and their activation by lipopolysaccharide (LPS) from Gram-negative bacteria or interferon (IFN)-gamma from host immune cells is important for controlling infections. However, the overwhelming activation of macrophages can cause a severe inflammatory state. This study investigated the inhibitory mechanism of xanthohumol (XN) against the inflammatory effectors (IL-1beta, TNF-alpha, and iNOS) in activated RAW264.7 macrophages by using different stimuli such as LPS, IFN-gamma, or LPS plus IFN-gamma. XN is a major prenylated chalcone found in hops, which is used to add bitterness and flavor to beer. XN reduced the expression of the LPS receptor components such as TLR4 and MD2 resulting in the suppression of NF-kappaB activation in LPS-activated RAW264.7 cells. In the IFN-gamma stimulated RAW264.7 cells, the binding activity of STAT-1alpha and IRF-1 was inhibited by XN. This suggests that differential signaling pathways are used by XN for the inhibition of excess inflammatory mediators depending on the stimuli in macrophages.

Microbial metabolites of 8-prenylnaringenin, an estrogenic prenylflavanone.

Microbial metabolism studies of the phyto-estrogen (+/-)-8-prenylnaringenin (8-PN) (1) has led to the isolation of three pairs of metabolites (2-4). The structures of these compounds were identified as 5,4'-dihydroxy-7,8-[2-(1-hydroxy-1-methylethyl)-2,3-dihydrofurano]flavanones (2), 8-prenylnaringenin 7-O-beta-D-glucopyranosides (3), and 8-prenylnaringenin 7-O-beta-D-(6'''-O-alpha-hydroxypropionyl)-glucopyranosides (4) on the basis of the spectroscopic analysis.

Hydroquinone, a major component in cigarette smoke, reduces IFN-gamma production in antigen-primed lymphocytes.

Exposure to cigarette smoke is known to suppress immune responses and to increase the incidence and severity of respiratory infections. In this study, we determined the effect of hydroquinone (HQ), which is found at high concentrations in cigarette smoke, on interferon-gamma (IFN-gamma) production by lymphocytes. HQ significantly inhibited IFN-gamma secretion by keyhole limpet hemocyanin-primed lymphocytes in a dose-dependent manner. In addition, HQ inhibited IFN-gamma secretion in effector CD4+ T cells and Th1-differentiated CD4+ T cells. The mRNA expression of IFN-gamma and the IFN-gamma gene promoter activity were inhibited by HQ. These results suggest that the inhibitory effect of HQ on IFN-gamma secretion may occur at the transcriptional level. Furthermore, the effects of HQ on transcription factors were investigated. HQ inhibited the transcriptional activity of activator protein-1 and nuclear factor-kappa B, which are known to be involved in IFN-gamma transcriptional activation. These findings provide evidence that HQ might suppress immune responses by reducing the production of IFN-gamma and may explain the susceptibility to microbial infections caused by cigarette smoking.

Application of coupling reaction between lithiated toluamide and benzonitrile for the synthesis of phenolic benzo[c]phenanthridine alkaloid, oxyterihanine.

The total synthesis of the natural phenolic benzo[c]phenanthridine alkaloid, oxyterihanine, was accomplished via substituted 3-arylisoquinoline intermediate. The key reaction was a coupling between the o-toluamide 4 and the benzonitrile 5.

Ethyl Acetate Fraction from Dendropanax morbifera Leaves Increases T Cell Growth by Upregulating NF-AT-Mediated IL-2 Secretion.

Dendropanax morbifera Leveille (Araliaceae) is an endemic species that grows in Southwestern Korea and has been used as a folk medicine. Several studies reported that D. morbifera leaves have diverse therapeutic potentials. We found that the water extract of D. morbifera leaves increased the growth of EL-4 T cells. The water extract was divided into five fractions: [Formula: see text]-hexane, chloroform, ethyl acetate, [Formula: see text]-butanol, and water layers. The ethyl acetate (W-EA) fraction showed a more significant effect than the other fractions on the growth of EL-4 T cells, splenocytes, and isolated murine CD4[Formula: see text] T cells. We evaluated the W-EA fraction for its immunomodulatory effects focusing on T cell functions. First, we tested the effect of the W-EA fraction on the regulation of interleukin-2 (IL-2), a potent T cell growth factor. The W-EA fraction significantly increased IL-2 secretion in EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). In addition, the W-EA fraction increased interferon-gamma (IFN-[Formula: see text] production in isolated murine splenocytes activated with Concanavalin A (ConA). Next, we examined the effect of the W-EA fraction on the regulation of transcriptional factors related to IL-2 production in T cells. The W-EA fraction significantly increased PMA/Io-induced promoter activity of a nuclear factor of activated T cells (NF-AT) in EL-4 T cells, but did not show any significant effects on the promoters of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-[Formula: see text]B). These results indicate that the W-EA fraction from water extract of D. morbifera leaves enhances IL-2 production at the transcriptional levels via the up-regulation of NF-AT in PMA/Io-activated EL-4 T cells.