Time-course effects of 1-methoxyoctadecan-1-ol on calpain activation, STEP cleavage, and p38 MAPK phosphorylation after glutamate exposure.

<p>Cortical neurons were treated with glutamate (300 µM, 15 min) (A and C) and pretreatment with 1-methoxyoctadecan-1-ol (0.1 µg/ml, 24 h) followed by exposure to glutamate (B and D) and both were maintained in the original medium for the specified time. Equal amounts of proteins and each sample were subjected to Western blot assays using the indicated antibodies. Equal protein loading was confirmed by actin expression.</p

Effects of 1-methoxyoctadecan-1-ol on infarct volume, neurological evaluation, and wire-grip test in a photothrombotic ischemic model.

<p>Mice received intraperitoneal administration of DMSO or 1, 3/kg of 1-methoxyoctadecan-1-ol at 30 min before the ischemic insult. Representative photographs of coronal brain sections stained with TTC in vehicle (Veh)- and 1-methoxyoctadecan-1-ol-treated mice (A). White indicates the infarct area. Quantification of the infarct volume, neurological score, and wire-grip test (B). *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. vehicle group. Data are expressed as mean±SEM of three separate experiments.</p

Flow cytometry analysis for neuronal death.

<p>Cortical neurons were pretreated with 1-methoxyoctadecan-1-ol (0.01 and 0.1 µg/ml) for 24 h, followed by exposure to 200 µM glutamate for 6 h. Cells were harvested and stained with Annexin V-FITC/PI, as described under methods and analyzed using flow cytometry. Annexin V⁺PI⁻ cells indicate early apoptotic cells, whereas Annexin V⁺PI⁺ cells are late apoptotic cells. The estimates (%) of gated cells in different compartments are given for each dot blot. Representative flow cytometry analysis scatter-grams of Annexin V/PI staining (A) and quantitative analysis of the histograms (B and C).^#<i>P</i><0.05 and ^##<i>P</i><0.01 <i>vs</i>. control; *<i>P</i><0.05 <i>vs</i>. treatment with glutamate alone. Data are represented as the mean±SEM of three independent experiments.</p

Effects of 1-methoxyoctadecan-1-ol on calpain activation, STEP cleavage and p38 MAPK phosphorylation.

<p>The significant calpain1 activation (A), STEP cleavage (B) and p38 MAPK phosphorylation (C) were shown in the ipsilateral (Ipsil) cerebral hemisphere of photothrombotic ischemic mice compared with the contralateral (Con). **<i>P</i><0.01, ***<i>P</i><0.001, <i>vs</i>. vehicle group, Data are expressed as mean±SEM of three separate experiments.</p

¹H (500 MHz in CDCl₃), ¹³C NMR (125 MHz in CDCl₃) data and key HMBC correlations of 1-methoxyoctadecan-1-ol.

<p>¹H (500 MHz in CDCl₃), ¹³C NMR (125 MHz in CDCl₃) data and key HMBC correlations of 1-methoxyoctadecan-1-ol.</p

Effects of calpeptin on neuroprotection of 1-methoxyoctadecan-1-ol.

<p>Cortical neurons were pretreated with calpeptin (10 µM) for 1 h and subsequently treated with 1-methoxyoctadecan-1-ol (0.1 µg/ml) for 24 h, followed by exposure to 200 µM glutamate for 6 h. Representative flow cytometry analysis scatter-grams of Annexin V/PI staining (A) and its quantitative analysis of the histograms (B). Cell lysates were subjected to Western blot assays using calpain1, STEP, and p38 MAPK (C). Equal protein loading was confirmed by actin expression.^###<i>P</i><0.001 <i>vs</i>. control; ***<i>P</i><0.001 <i>vs</i>. treatment with glutamate alone; ⁺⁺⁺<i>P</i><0.001 <i>vs</i>. 1-methoxyoctadecan-1-ol treatment with glutamate. Data are represented as the mean±SEM of three independent experiments.</p

Effects of SB203580 on neuroprotection of 1-methoxyoctadecan-1-ol.

<p>Cortical neurons were pretreated with SB203580 (5 µM) for 30 min and subsequently treated with 1-methoxyoctadecan-1-ol (0.1 µg/ml) for 24 h, followed by exposure to 200 µM glutamate for 6 h. Representative flow cytometry analysis scatter-grams of Annexin V/PI staining (A) and quantitative analysis of the histograms (B–C).^##<i>P</i><0.01, ^###<i>P</i><0.001 <i>vs</i>. control; **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i>. treatment with glutamate alone; ⁺⁺<i>P</i><0.01 <i>vs</i>. 1-methoxyoctadecan-1-ol treatment with glutamate. Data are represented as the mean±SEM of three independent experiments.</p

Time-course effects of 1-methoxyoctadecan-1-ol on NMDAR and AMPAR subunit after glutamate exposure.

<p>Cortical neurons were treated with glutamate (300 µM, 15 min) (A) and pretreatment with 1-methoxyoctadecan-1-ol (0.1 µg/ml, 24 h) followed by exposure to glutamate (B), then both were maintained in the original medium for the specified time. Densitometric analysis (C) showed that 1-methoxyoctadecan-1-ol treatment significantly decreased the phosphorylation of the NMDAR pGluN2B subunit. Equal amounts of proteins in each sample were subjected to Western blot assays using the indicated antibodies. Equal protein loading was confirmed by actin expression. ^#<i>P</i><0.05 <i>vs</i>. control; **<i>P</i><0.01 and ***<i>P</i><0.001 <i>vs</i>. treatment with glutamate alone. Data are represented as the mean±SEM of three independent experiments.</p

Effects of 1-methoxyoctadecan-1-ol on glutamate-induced apoptosis in cultured cortical neurons.

<p>Cortical neurons were pretreated with 1-methoxyoctadecan-1-ol (0.01 and 0.1 µg/ml) for 24 h, followed by exposure to 200 µM glutamate for 6 h. Quantitative analysis of the histograms for Hoechst 33342 (A) and TUNEL staining (B). ^#<i>P</i><0.05 and ^##<i>P</i><0.01 <i>vs</i>. control; *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. treatment with glutamate alone. Data are represented as the mean±SEM of three independent experiments.</p

Identification of 1-methoxyoctadecan-1-ol from <i>U. sinensis</i>.

<p>Structure of 1-methoxyoctadecan-1-ol (A). Key HMBC correlations of 1-methoxyoctadecan-1-ol (B). HPLC profile showed that the active compound had a purity of >96% (C).</p