IFN-γ production induced by GD-1 is dependent on NF-κB activity.

<p><b>A</b>. GD-1 was treated onto NK-92 cells at 1 to 100 ng/ml concentrations, and several down-stream targets for PKC signaling were analyzed by Western blotting. The phosphorylation of IKK was detected with the concomitant degradation of IκB. ERK and MEK were also phosphorylated by GD-1 treatment. The bands were quantified using Quantity One 1-D Analysis Software (Bio-Rad Laboratories) and p-IKK and IκB expression normalized to control their protein expression. And p-ERK and p-MEK expressions normalized to total their protein expression. The values are given above the blots. <b>B</b>. Inhibitors of NF-κB, Bay11-7082, was treated with a serial dilution from 100 µM with 10 0 ng/ml GD-1, and IFN-γ production was analyzed by ELISA. The addition of Bay11-7082 has showed an inhibitory effect on GD-1 activity to increase IFN-γ production. All ELISA data are representative of at least three independent experiments. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. *<i>P</i><0.05. <b>C</b>. The nuclear translocation of p65 (an NF-κB subunit) was observed in GD-1-stimulated NK-92 cells. The modulation of cytoskeleton was observed through Phase contrast images and DAPI was used to visualize nuclei. GD-1 treatment onto NK-92 cells induced nuclear localization of cytosolic p65 transcription factor, which was inhibited by the co-addition of Rottlerin or Bay11-7082.</p

Activation of PKC isoforms in the GD-1 treated NK-92 cells.

<p><b>A</b>. 200 ng of GD-1 was treated onto NK-92 cells for 20 min. The cell lysates were analyzed by Western blotting with various antibodies against PKC isoforms, including PKD1. <b>B</b>. Phosphorylation of PKD1 was observed at serially diluted GD-1 concentrations with specific antibody to phosphorylated PKD1. <b>C</b>. Phosphorylation of PKD1 was observed to increase in a time-dependent manner within 15 min of GD-1 treatment. <b>D</b>. The addition of Rottlerin, a PKC inhibitor, and CID755673, a PKD inhibitor, has showed inhibitory effect on GD-1-mediated IFN-γ production, which support the increase of IFN-γ production by GD-1 is dependent of PKC signaling pathway. All ELISA data are representative of at least three independent experiments. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. *<i>P</i><0.05.</p

STAT1 signaling pathway is activated by GD-1 treatment.

<p><b>A</b>. The phosphorylation of STAT1 was induced within 1 hr after GD-1 treatment. <b>B</b>. The increase of phosphorylated STAT1 was detected at high concentration of GD-1 (>1 µg/ml). <b>C</b>. The addition of STAT1 inhibitor blocked IFN-γ production induced by GD-1 treatment on a concentration-dependent manner. All ELISA data are representative of at least three independent experiments. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. *<i>P</i><0.05. <b>D</b>. The induction of IFN-γ production in GD-1-treated NK-92 cells was inhibited by the addition of STAT1 and NF-κB inhibitors but by STAT3 inhibitor.</p

The effects of the flower bud extracts on IFN-γ production and morphological changes. A

<p>. The structure of GD-1 and yuanhuacine isolated from the extract of <i>Daphne genkwa</i> plant. <b>B</b>. ELISA-based assay for IFN-γ production. Total extract (TE) and twenty fractions (1 to 20) were treated on NK-92 and IFN-γ secretion was analyzed by ELISA. Fraction number 10 (#10) and 11 (#11) indicate GD-1 and yuanhuacine, respectively. All ELISA data are representative of at least three independent experiments. <b>C</b>. NK-92 cell morphologies were analyzed microscopically after the treatment of flower extracts. The addition of GD-1 (#10), yuanhuacine (#11) and TE have induced the morphological changes of NK-92 cells, which matches with the pattern of IFN-γ production.</p

Genkwadaphnin Induces IFN-γ via PKD1/NF-κB/STAT1 Dependent Pathway in NK-92 Cells

<div><p>The flower buds of <i>Daphne genkwa</i> Sieb. et Zucc. have been used as a traditional Chinese medicine although their functional mechanisms have not been discovered yet. We have studied the potential effects of the plant extracts on natural killer (NK) cell activation, and isolated an active fraction. Genkwadaphnin (GD-1) displayed a potent efficacy to induce IFN-γ transcription in NK cells with concentration- and time-dependent manners. GD-1 treatment triggered the phosphorylation of PKD1, a member of PKC family, MEK and ERK, resulting in IKK activation to induce IκB degradation, and the nuclear localization of p65, an NF-κB subunit, which regulates IFN-γ transcription. GD-1 effect on IFN-γ production was blocked by the addition of Rottlerin, a PKC inhibitor, CID 755673, a PKD inhibitor, or Bay11-7082, an IKKα inhibitor. The nuclear localization of p65 was also inhibited by the kinase inhibitors. Secreted IFN-γ activates STAT1 phosphorylation as autocrine-loops to sustain its secretion. GD-1 induced the phosphorylation of STAT1 probably through the increase of IFN-γ. STAT1 inhibitor also abrogated the sustained IFN-γ secretion. These results suggest that GD-1 is involved in the activation of PKD1 and/or ERK pathway, which activate NK-κB triggering IFN-γ production. As positive feedback loops, secreted IFN-γ activates STAT1 and elongates its production in NK-92 cells.</p></div

GD-1 regulates the transcription of IFN-γ.

<p><b>A</b>. The effect of GD-1 on IFN-γ production was analyzed by Western blotting of the culture medium after GD-1 treatment at various concentrations (0∼200 ng/ml). The production of IFN-γ increased with the increase of GD-1 concentration. <b>B</b>. The time-dependent increase of IFN-γ secretion was analyzed by ELISA, which showed the production of IFN-γ reached plateau around 3-hr treatment of GD-1. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. All ELISA data are representative of at least three independent experiments. *<i>P</i><0.05. <b>C</b>. The production of IFN-γ was increased in a time-dependent manner. 100 ng/ml of GD-1 was treated onto NK-92 cells, and the culture supernatant was taken at various time points for Western blot analysis. IFN-γ production was seemed to begin within 1 hr. Golgistop treatment has showed that GD-1 does not affect the secretory pathway for IFN-γ production. <b>D</b>. Time dependent transcriptional activity for IFN-γ in NK-92 cell after GD-1 treatment was evaluated by RT-PCR. The transcription of IFN-γ was increased within 1 hr after GD-1 treatment.</p

Rapid Identification of Cholinesterase Inhibitors from the Seedcases of Mangosteen Using an Enzyme Affinity Assay

Enzyme binding affinity has been recently introduced as a selective screening method to identify bioactive substances within complex mixtures. We used an assay which identified small molecule binders of acetylcholinesterase (AChE) using the following series of steps: incubation of enzyme with extract; centrifugation and filtration; identification of small molecule content in the flow through. The crude extract contained 10 peaks in the UPLC chromatogram. However, after incubation the enzyme, six peaks were reduced, indicating these compounds bound AChE. All these isolated compounds (<b>2</b>, <b>3</b>, and <b>5</b>–<b>8</b>) significantly inhibited human AChE with IC₅₀s = 5.4–15.0 μM and butyrylcholinsterase (IC₅₀s = 0.7–11.0 μM). All compounds exhibited reversible mixed kinetics. Consistent with the binding screen and fluorescence quenching, γ-mangostin <b>6</b> had a much higher affinity for AChE than 9-hydroxycalabaxanthone <b>9</b>. This validates this screening protocol as a rapid method to identify inhibitors of AChE

Cytotoxic Triterpenoids from the Rhizomes of <i>Astilbe chinensis</i>

Six new triterpenoids (1−6) with a carboxylic acid functionality at C-27 were isolated from the rhizomes of a Korean native perennial herb, Astilbe chinensis, along with nine known triterpenoids. The structures of 1−6 were elucidated on the basis of spectroscopic data interpretation. All compounds isolated were evaluated for cytotoxic effects against a small panel of human cancer lines

Image_3_Topical Skullcapflavone II attenuates atopic dermatitis in a mouse model by directly inhibiting associated cytokines in different cell types.tif

Skullcapflavone II (SFII), a flavonoid derived from Scutellaria baicalensis, is an anticancer agent. We aimed to validate SFII for atopic dermatitis (AD) therapy by demonstrating the anti-inflammatory effects of SFII in an AD mouse model produced by the topical application of the vitamin D3 analog MC903. We showed that topical treatment with SFII significantly suppressed MC903-induced serum IgE levels compared with topical hydrocortisone (HC) treatment. Topical SFII also prevents MC903-induced pruritus, skin hyperplasia, and inflammatory immune cell infiltration into lesional skin comparable to topical HC. In addition, MC903-induced immune cell chemoattractants and AD-associated cytokine production in skin lesions were effectively suppressed by topical SFII. The production of MC903-induced effector cytokines influencing T helper (Th)2 and Th17 polarization in lesioned skin is significantly inhibited by topical SFII. Furthermore, we showed that SFII can directly inhibit the production of AD-associated cytokines by human primary keratinocytes, mouse bone marrow-derived cells (BMDCs), and mouse CD4+ T cells in vitro. Lastly, we demonstrated that topical SFII more effectively suppressed serum IgE levels, the production of IL-4 and thymic stromal lymphopoietin (TSLP), and infiltration of CD4+ T cells and Gr-1+ cells (neutrophils) into lesion skin compared to topical baicalein (a flavonoid derived from Scutellaria baicalensis), which has anti-inflammatory effects. Taken together, our findings suggest that SFII may have promising therapeutic potential for this complex disease via the regulation of multiple AD-associated targets. </p

Image_1_Topical Skullcapflavone II attenuates atopic dermatitis in a mouse model by directly inhibiting associated cytokines in different cell types.tif

Skullcapflavone II (SFII), a flavonoid derived from Scutellaria baicalensis, is an anticancer agent. We aimed to validate SFII for atopic dermatitis (AD) therapy by demonstrating the anti-inflammatory effects of SFII in an AD mouse model produced by the topical application of the vitamin D3 analog MC903. We showed that topical treatment with SFII significantly suppressed MC903-induced serum IgE levels compared with topical hydrocortisone (HC) treatment. Topical SFII also prevents MC903-induced pruritus, skin hyperplasia, and inflammatory immune cell infiltration into lesional skin comparable to topical HC. In addition, MC903-induced immune cell chemoattractants and AD-associated cytokine production in skin lesions were effectively suppressed by topical SFII. The production of MC903-induced effector cytokines influencing T helper (Th)2 and Th17 polarization in lesioned skin is significantly inhibited by topical SFII. Furthermore, we showed that SFII can directly inhibit the production of AD-associated cytokines by human primary keratinocytes, mouse bone marrow-derived cells (BMDCs), and mouse CD4+ T cells in vitro. Lastly, we demonstrated that topical SFII more effectively suppressed serum IgE levels, the production of IL-4 and thymic stromal lymphopoietin (TSLP), and infiltration of CD4+ T cells and Gr-1+ cells (neutrophils) into lesion skin compared to topical baicalein (a flavonoid derived from Scutellaria baicalensis), which has anti-inflammatory effects. Taken together, our findings suggest that SFII may have promising therapeutic potential for this complex disease via the regulation of multiple AD-associated targets. </p