Schematic diagram for experimental procedures.

<p>All experiments were performed on the indicated day.</p

Inhibitory effects of taxifolin, cilostazol, and of taxifolin plus cilostazol on LPS-induced iNOS, COX-2 expressions, and nitrite levels in BV-2 microglia cells.

<p><b>A</b>. Concentration-dependent LPS (1 ~ 20 μg/ml)-induced increase in nitrite formation. <b>B</b>. Inhibitory effect of taxifolin (3, 10, 30 μM) on LPS-stimulated nitrite levels. <b>C</b> and <b>D</b>. Concentration-dependent inhibition of taxifolin (3, 10, 30 or 100 μM) and cilostazol (3, 10, or 30 μM) on LPS (10 μg/ml)-stimulated iNOS expression, and synergistic inhibition of iNOS by 3 μM cilostazol (C3) plus 10 μM taxifolin (T10). <b>E</b>. Inhibitory effect of taxifolin (3, 10, 30 or 100 μM) on LPS-stimulated COX-2 expression. <b>F</b>. Inhibitory effect of cilostazol (3, 10, or 30 μM) on LPS-stimulated COX-2 expression, and of 3 μM cilostazol (C3) plus 10 μM taxifolin (T10) on the inhibition of COX2. Results are the means ± SEMs of 4–6 experiments. **<i>P</i> < 0.01, ***<i>P</i> < 0.001 vs. None; ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01, ^###<i>P</i> < 0.001 vs. Vehicle; ^$<i>P</i> < 0.05; ^$ $$<i>P</i> < 0.01 vs. 3 μM of Cilostazol.</p

Effects of EA treatment on neurogenesis at 14 days after MCAO.

<p>Schematic diagram shows the regions of the hippocampus (★, in and around the hippocampus), SVZ (*, including the striatum) and cortex (#, the whole cortex) of the brain for immunohistochemical and further Western blot analysis (A). Photomicrograph (B) and its histogram for BrdU/Dcx double-positive cells in the hippocampus (C), SVZ (D) and cortex (E) of the brain. BrdU/Dcx double-positive cells were significantly increased by EA treatment in the hippocampus and SVZ of the ipsilateral hemisphere. ^#<i>P</i><0.05, ^##<i>P</i><0.01 and ^###<i>P</i><0.001 versus the contralateral hemisphere; *<i>P</i><0.05 and **<i>P</i><0.01 versus MCAO mice. Scale bars = 100 µm.</p

RT-PCR analysis for growth and neurotropic factors at 14 days after MCAO.

<p>Expression of each mRNA is expressed as a percentage of contralateral hemisphere of MCAO. Panel (A) its densitometric analysis (B). Panel represents a typical result from three independent experiments. EA treatment significantly increased the mRNA level of BDNF and VEGF in the ipsilateral hemisphere. *<i>P</i><0.05 and ***<i>P</i><0.001 versus MCAO mice.</p

Effects of EA treatment on neurogenesis in the whole brain at 26 days after MCAO.

<p>Serial sections of the whole brain showing the regions for immunohistochemical analysis (A, cresyl violet stain) and its analysis for double-positive cells BrdU with neuroblast marker Dcx (B) or neuronal marker NeuN (C). Photomicrograph represents a typical result in the cortex. The number of proliferated NSCs indicated by BrdU positive was increased in both hemispheres of MCAO mice and EA treatment significantly increased the number of these cells in the posterior region of the brain. Total number of BrdU and Dcx or NeuN double-positive cells was significantly increased by EA stimulation. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 versus MCAO mice. Scale bars = 100 µm.</p

Concurrent Treatment with Taxifolin and Cilostazol on the Lowering of β-Amyloid Accumulation and Neurotoxicity via the Suppression of P-JAK2/P-STAT3/NF-κB/BACE1 Signaling Pathways - Fig 6

<p>Stimulation of SIRT1 protein expression (<b>A</b>) and SIRT1 deacetylase activity in the absence and presence of sirtinol (<b>B</b>) by taxifolin (TFL), cilostazol (CSZ), and resveratrol (RES) in N2a cells. <b>C</b>. Analysis of the effects of SIRT1gene-knockdown in N2a cells. In N2a cells subjected to SIRT1 siRNA-gene silencing, SIRT1 protein expression reduced to ~50% by 200 nM SIRT1 siRNA. <b>D</b> and <b>E</b>. Failure of SIRT1 gene-silenced cells to inhibit Aβ_1-42-stimulated P-STAT3 and BACE1 expressions as compared with the inhibitions observed in scrambled siRNA duplex (negative control) transfected cells. Results are the means ± SEMs of 4 experiments. **<i>P</i> < 0.01, ***<i>P</i> < 0.001 vs. Vehicle (Veh); ^###<i>P</i> < 0.001 vs. Absence of sirtinol; ^$<i>P</i> < 0.05, ^$ $$<i>P</i> < 0.01 vs. Negative control.</p

Aβ-induced BACE1 mRNA and protein expressions in activated N2a Swe cells and their reductions by taxifolin and cilostazol.

<p>Time (3 ~ 24 hr)-dependent changes in BACE1 mRNA (<b>A</b>) and protein (<b>B</b>) expressions. <b>C</b>. Significant decreases in BACE1 protein expression by taxifolin (50 or 100 μM), cilostazol (30 μM), and by 30 μM quercetin (a flavonoid) after 24 hr of treatment. <b>D</b>. Synergistic suppression of BACE1 expression by 30 μM of taxifolin plus 10 μM cilostazol as compared with the suppressions induced by monotherapies. Results are the means ± SEMs of 4 experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 vs. Zero time; ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01, ^###<i>P</i> < 0.001 vs. Vehicle (Veh); ^@@ <i>P</i> < 0.01 vs. N2a cells; ^††† <i>P</i> < 0.001 vs. Vehicle (Veh) of N2a Swe cells.</p

Western blot and immunohistochemical analysis for VEGF at 14 days after MCAO.

<p>Western blot (A) and its densitometric analysis (B) showed that EA treatment significantly improved the expression of VEGF in the ipsilateral hippocampus and cortex. Photomicrograph (C) and its histogram (D) showed that VEGF positive cells were significantly increased by EA treatment in the hippocampus and ipsilateral SVZ. ^#<i>P</i><0.05 and ^##<i>P</i><0.01 versus contralateral hemisphere; *<i>P</i><0.05 and ***<i>P</i><0.001 versus MCAO mice. Scale bars = 100 µm.</p

Cerebral perfusion responses elicited by EA abolished in eNOS KO.

<p>(A) Cerebral blood flow (CBF) time course after EA (arrow) from a representative experiment from the EA group of C57BL/6J and from the EA group of eNOS KO, and (B) the average of ten experiments. The control groups received the same electrical stimulation at non-acupuncture points. The horizontal bar represents the EA stimulation period. CBF measurement was conducted for 5 min before EA stimulation, 20 min during EA and 20 min after EA, lasting a total of 45 min. The cerebral perfusion response elicited by EA was significantly attenuated in eNOS KO (<b>**</b>, <i>P</i><0.01 vs. control group; ^##, <i>P</i><0.01 vs. EA group of C57BL/6J, two-way ANOVA for repeated measures). Vertical error bars indicate ± SEM.</p

EA improved tissue outcome in moderate ischemic injury, but not severe ischemic injury.

<p>Mice were subjected to 60 min and 90 min MCA occlusion followed by 24-h reperfusion. The mice received 20 min-EA stimulation immediately after the onset of occlusion. (A) Representative photographs of coronal brain sections following infarction stained with 2,3,5-triphenyltetrazolium chloride. The red area is healthy tissue and the white area is infarct tissue. (B) Quantification of indirect infarct volume at 24 h after ischemia. Infarct volume was calculated by integrating the infarct area in 2-mm-thick coronal slices. Results are expressed as means ± SEM. <b>*</b>, <i>P</i><0.05 vs. control group, unpaired t-test, N = 6.</p