Koshu GSE augments cell survival of cultured hippocampal neurons after excitotoxic treatment.

<p>Hippocampal neurons plated at 2.0×10⁵ cells/cm² were treated with 50 µM glutamate in the presence or absence of 1.0 ng/ml KOS GSE for 10 min and allowed to recover in conditioned medium for 24 hr. (A) Representative images of hippocampal neurons after treatment. Bar, 100 µm. (B) The mean density of MAP2-positive neurons surviving 24 hr after glutamate treatment (<i>n</i> = 8). ***, <i>P</i><0.001; *, <i>P</i><0.05. (C) Quantification of the mean dendrite length by morphometric analysis (<i>n</i> = 8). ***, <i>P</i><0.001; *, <i>P</i><0.05.</p

Typical liquid chromatography profile of Koshu and MBA grape seed extracts.

<p>Polyphenols in the eluate were detected by absorbance at 280 nm. The peaks were assigned by TOF-MS as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014575#pone-0014575-t001" target="_blank">Table 1</a>.</p

Koshu GSE protects dendrite processing of cultured hippocampal neurons exposed to a toxic concentration of glutamate.

<p>(A) Representative image of untreated hippocampal neurons at 8 DIV immunostained for MAP2. Bar, 100 µm. (B) Representative binary images of cultured hippocampal neurons immunostained for MAP2 with no treatment or after a 30 min treatment with 50 µM glutamate alone, 50 µM glutamate plus 1.0 ng/ml KOS, or 50 µM glutamate plus 1.0 ng/ml MBA. Bar, 100 µm. (C) Quantification of the mean dendrite length by morphometric analysis (<i>n</i> = 10). ***, <i>P</i><0.001 versus untreated controls.</p

Effect of Koshu GSE on active caspase-3.

<p>(A) Hippocampal neurons treated with 50 µM glutamate alone or in the presence of the indicated concentrations (in ng/ml) of KOS GSE for 30 min were analyzed for active-caspase-3 by Western blot. Numbers below the active caspase-3 bands indicate their relative intensities quantified by densitometric analysis. (B) Hippocampal neurons treated with 50 µM glutamate in the presence or absence of 1.0 ng/ml KOS GSE for 30 min and incubated in normal culture medium for 0, 3, or 6 hr were analyzed for active-caspase-3 by Western blot. Numbers below the active caspase-3 bands indicate their relative intensities quantified by densitometric analysis.</p

Koshu GSE alleviates glutamate-induced inactivation of Erk1/2 in cultured hippocampal neurons.

<p>(A) <i>Top</i>, detection of phosphorylated Erk1/2 in cultured hippocampal neurons by western blot. Neurons were treated with 50 µM glutamate alone or in the presence of indicated concentrations (in ng/ml) of KOS or MBA for 30 min. The immunoblot for total Erk1/2 shows equal loading. <i>Bottom</i>, quantification of phospho-Erk1/2 levels by image analysis (<i>n</i> = 5). The relative values to the untreated controls (set to 100%) are shown. *, <i>P</i><0.05 versus untreated controls. (B) Hippocampal neurons were treated with 50 µM glutamate with or without indicated concentrations (in ng/ml) of KOS GSE for 30 min, and 200 µg of total protein were loaded in each lane for western blot analyses for phospho- and total Akt. The basal Akt phosphorylation appeared slightly diminished upon glutamate insult. KOS GSE did not show any protective effect on basal Akt phosphorylation levels. (C) The effect of KOS GSE alone on Erk1/2 phosphorylation in cultured hippocampal neurons. Cells were treated with indicated concentrations (in ng/ml) of KOS GSE for 30 min. <i>Top</i>, representative western blot images of phospho- and total-Erk1/2. <i>Bottom</i>, a quantification of the phospho-Erk1/2 levels by image analysis (<i>n</i> = 3).</p

LC/TOF-MS data of phenolic compounds.

<p>Asterisk indicates those only detected in KOS GSE.</p

Resveratrol inhibits early T cell activation and co-stimulatory molecule expression in APCs <i>in vitro</i>.

<p>DO11.10 mice-derived splenocytes were un-stimulated (no treat) or stimulated with 300 µg/ml OVA plus 12 pM CT and were cultured in the presence or absence of 10 or 30 µM resveratrol for 1 day. Then the analysis described below was performed. A. After 1 day-culture, whole splenocytes were stained with PE-conjugated anti-KJ1–26 and APC-conjugated anti-CD25 antibody. Representative FACS plots are shown. Numbers indicate the proportion of the KJ1–26⁺ CD25⁺ cells. B. Quantitative analysis of A (n = 3 per group). C. After 1 day-culture, the supernatants were collected and IL-2 concentrations were measured by ELISA (n = 9 per group). D. After 1 day-culture, RNA samples were extracted from the splenocytes and the cDNA samples were synthesized using reverse transcriptase system. Quantitative real-time PCR analysis was then performed to assess CD80 and CD86 mRNA levels. Relative expression levels are shown (n = 3 per group). Values represent the mean ± SD. *P<0.05 in comparison to the corresponding controls. (ND: non-detected).</p

Resveratrol inhibits OVA plus CT-induced Th1/2 cell differentiation <i>in vitro</i>.

<p>DO11.10 mice-derived splenocytes were un-stimulated or stimulated with 300 µg/ml OVA plus 12 pM CT and were cultured in the presence or absence of 10 or 30 µM resveratrol for the indicated times. Then the analysis described below was performed. A. After 3 day-culture, RNA samples were extracted from the cells and the cDNA samples were synthesized using reverse transcriptase system. Quantitative real-time PCR analysis was then done for T-bet, GATA3, and Foxp3 mRNAs. Relative expression levels are shown (n = 3 per group). B. After 3 day-culture, the supernatants were collected and IFN-ã, IL-4, and IL-13 concentrations were measured by ELISA (n = 9 per group). C. After 3 day-culture, the cells were subjected to a WST assay for evaluation of the cell viability (n = 4 per group). Values represent the mean ± SD. *P<0.05 in comparison to the corresponding controls. (ND: non-detected).</p

Dietary resveratrol protects mice against a lethal dose of CT.

<p>Survival of 8-week-old C57BL/6 mice orally challenged with CT at a dose of 150 µg monitored until day 10 after challenge (n = 5 per group). Representative results from 2 independent experiments with similar results are shown. (S: standard diet-fed mice, S/R: standard diet plus resveratrol-fed mice).</p

Resveratrol inhibits CT-driven mucosal sensitization to OVA in mice.

<p>Mice were fed the standard diet or standard diet plus resveratrol (22.4 mg/kg diet, 0.01% resveratrol) for 5 weeks (day 0–35). The mice were orally administered 50 mg of OVA with 10 µg of CT 1 week after the start of resveratrol feeding and 4 times a week for 4 weeks and sacrificed at the 5 weeks (on day 35) for the analysis below. A. Experimental protocol. B. Serum samples were collected on day 35 from the standard diet-fed and standard diet plus resveratrol-fed mice with or without OVA plus CT sensitization. OVA-specific serum IgE concentrations were measured by ELISA (n = 7 per group). C. The standard diet-fed and standard diet plus resveratrol-fed mice sensitized with OVA plus CT were challenged intraperitoneally with OVA on day 35 and rectal temperatures were measured with a digital thermometer at 0, 5, 30, and 60 minutes after the challenge (n = 10 per group). D. Splenocytes (SPL) and mesenteric lymph node (MLN) cells were obtained on day 35 from the standard diet-fed and standard diet plus resveratrol-fed mice with or without OVA plus CT sensitization and the cells were re-stimulated <i>in vitro</i> with OVA for 3 days (n = 5 per group). The culture supernatants were then collected and IL-13 and IFN-ã concentrations were measured by ELISA. (S: standard diet-fed mice, S/R: standard diet plus resveratrol-fed mice) Values represent the mean ± SD. *P<0.05 in comparison to the corresponding controls. Representative results from 2 independent experiments with same results are shown.</p