Effects of LBP on the apoptosis in hippocampus.

<p>(A) The hippocampus homogenates were separated in SDS page and bloted with Bcl2 and Bax antibodies. Bcl2 level decreases after SCO administration and is reversed by LBP treatment, while Bax shows opposite pattern after SCO or LBP treatment. GAPDH is the internal standard. (B) Quantification of the ratio Bax/Bcl2 in A shows that SCO alone increases the ratio of Bax/Bcl2 which is reversed by LBP treatment. Values are means ± SEM (n = 4 in each group). **,<i>P</i><0.01 and ***,<i>P</i><0.001.</p

Schedule of the projects.

<p>LBPs were administered each day by the intragastric (i.g.) route for 2 weeks, when SCO administration was commenced by a subcutaneous (s.c.) osmotic minipump. Administration of LBP and SCO continued for a further 4 weeks. Behavior tests were performed immediately 1 day after SCO/LBP treatment. Some of animals were sacrificed immediately for biochemical analysis after drug treatment. Twenty-four hours after behavioral testing the rest of animals were sacrificed for immunohistochemical analysis.</p

Expression profile of MSK1 and p-MSK1 (Thr-581 and Ser-360) following LPS intracerebral injection.

<p><b>A</b>. Protein levels of t-MSK1, p-MSK1 Thr-581, p-MSK1 Ser-360 were detected before (control) and after injury. GAPDH was also detected by Western blotting. <b>B</b>. Quantification graphs (relative optical density) of the intensity of staining of p-MSK1 (Thr-581) and total MSK1 to GAPDH at each time point. GAPDH was used to confirm that equal amounts of protein were run on the gel. <b>C–H</b>. Immunofluorescence staining of MSK1 and p-MSk1 (Thr581) was performed to assess the staining changes for MSK1 and p-MSK1 immunoreactivity in the cortex at day 1 after LPS-injection. <b>I</b>. Negative control. * and ^# indicate significant differences at P<0.05, compared with normal brain cortex. Scale bars: 40 µm (C–F), 20 µm (G–J).</p

Effects of MSK1 gene silencing and Thr-581 mutation on cytokine production in LPS-treated astrocytes.

<p><b>A</b>. Effects of siRNA for MSK1 and non-specific siRNA on LPS-induced expression of MSK1, p-MSK1 (Thr581) and iNOS were detected by Western blotting. <b>B</b>. The bar chart shows the ratio of total MSK1 and p-MSK1 to β-actin. <b>C–E</b>. ELISA showed that MSK1 gene silencing by siRNA further promoted the LPS-mediated upregulation of inflammatory cytokines. <b>F</b>. Western blot analysis showed the effect of mutation of Thr-581 to an alanine residue on LPS-induced expression of p-MSK1 Thr-581, p-MSK1 Ser-360, and total MSK1. <b>G</b>. The bar chart shows the ratio of p-MSK1 Thr-581 and p-MSK1 Ser-360 to total MSK1. <b>H–J</b>. ELISA showed the effect of mutation of Thr-581 on LPS-induced TNFα, IL-6, and IL1-β production in activated astrocytes.</p

Immunolocalization of MSK1 and p-MSK1 (Thr-581) with different cellular markers in cerebral cortex by double immunofluorescence staining.

<p>In the adult rat brain cortex, within 5(red, <b>A</b> and <b>E</b>) and p-MSK1 (Thr581) (red, <b>I</b> and <b>M</b>) and different cell markers (green, <b>B, F, J, N</b>), such as a neuronal marker (NeuN) and an astrocyte marker (GFAP). The yellow color in the merged images represents colocalization of MSK1 or p-MSK1 (Thr581) with different phenotype-specific markers (<b>C, G, K, O</b>). Colocalization of MSK1 and p-MSK1 (Thr581) with different phenotype-specific markers in the normal group are shown in the brain cortex (<b>D, H, L, P</b>). Quantitative analysis of different phenotype-specific marker-positive cells expressing MSK1 (<b>Q</b>) and p-MSK1 (<b>R</b>) (%) in the unit area (mm²) in the normal group and 1 day after injury. *indicates significant difference at P<0.05, compared with the normal group. Error bars indicate SEM. Scale bars: 20 µm (<b>A–P</b>).</p

LBP protects the processes of newborn neurons in DG of hippocampus.

<p>(<b>A</b>) Representative images of doublecortin (DCX)-positive neuroblasts in the subgranular zone of DG. The lower panels are the enlargement of the frames in the upper panels. The arrows indicates the tertiary neurites of DCX positive neurons in DG. In control vehicle/saline and LBP/SCO groups DCX-immunoreactive neuroblasts have well-developed processes extending to the molecular layer of the DG. SCO treatment (vehicle/SCO group) led to significant reduction of tertiary dendrites. (<b>B</b>) Quantification of number of the DCX-immunoreactive cells with tertiary dendrites in the three groups. Values are means ± SEM (n = 6 animals per groups); ***,<i>P</i><0.001; scale bars, 50 µm.</p

Effect of SCO/LBP treatments on cell proliferation in the hippocampal dentate gyrus (DG).

<p>(<b>A</b>) Representative immunofluorescence staining for Ki67, a marker of cell proliferation. Sections were subjected to Ki67 antibody after SCO or/and LBP treatment. Arrows indicate Ki67-positive cells in hippocampus. The dashed line is the location of subgranular layer in DG. (<b>B</b>) Quantification of Ki67-positive cells in the DG in A. SCO treatment significantly decreased cell proliferation compared to the vehicle/saline group. The LBP/SCO group showed a significantly higher proportion of Ki67-positive cells than the SCO group. Values are means ± SEM (n = 6 animals per groups); *,<i>P</i><0.05; **,<i>P</i><0.01; scale bars, 50 µm.</p

LBP treatment reverses the SCO-induced increase in latency time in the Morris water maze.

<p>(<b>A</b>) The apparatus for Morris water maze. The black circle in the northeast quadrant represents the location of the hidden platform. Animals were introduced into the southwest quadrant. (<b>B</b>) The latency time, swim distance and swim speed to find the submerged platform were recorded over six consecutive days. The animals of three groups have the similar swim speed, but SCO treatment (vehicle/SCO group) increased the latency and swim distance to find the hidden platform whereas LBP administration (LBP/SCO group) restored latency and distance to the levels of the vehicle/saline control group. (<b>E</b>) The percentage of time spent in 4 quadrants during the probe trial. The animals in control and LBP/SCO groups spent more time in the target quadrant while SCO treatment decreased time in target quadrant. Values are means ± SEM (Vehicle/saline, <i>n</i> = 12; vehicle/SCO, <i>n</i> = 10; LBP/SCO, <i>n</i> = 11); *,<i>P</i><0.05 versus the vehicle/SCO group.</p

LBP did not alter the expressions of BDNF and IGF-1.

<p>(A) The hippocampus homogenates were separated in SDS page and bloted with BDNF, IGF-1 and GAPDH antibodies. GAPDH is the internal standard. (B) Quantification of BDNF in A indicates SCO-decreased BDNF was not reversed by LBP treatment. (C) Quantification of IGF-1 indicates both of SCO and LBP did not influence the level of IGF-1 in hippocampus. Values are means ± SEM (n = 4 in each group). **,<i>P</i><0.01.</p

LBP treatment increases neuroblast differentiation in the hippocampal DG.

<p>(<b>A</b>) DCX immunostaining showed the newborn neurons in the subgranular zone of the DG. DCX-immunoreactive neuroblasts (green) were abundant in the DG in the vehicle/saline group. SCO treatment markedly decreased the number of DCX-positive cells. In the LBP/SCO group, the number of DCX-positive cells was restored. Scale bars, 200 µm. (B) Quantification of DCX-positive cells in DG of the three groups in A. (C) The representative images of Calretinin immunostaining. The length of dendrites is markedly injured by SCO compared with control and LBP treatment groups. Scale bars, 100 µm. (D) Quantification of number of Calretinin positive cells in DG in A. Values are means ± SEM (n = 6 animals per groups); **,<i>P</i><0.01.</p