FLZ inhibited Aβ production in APP/PS1 mice and SH-SY5Y (APPwt/swe) cells.

<p>APP/PS1 double transgenic mice were orally treated with FLZ 150 mg/kg for 20 weeks. SH-SY5Y (APPwt/swe) cells were grown in “stimulating medium” containing 50% DMEM, 50% Opti-MEM, 0.5% FBS, 200 µg/ml G418 and 10 mM butyric acid sodium salt for 12 h to induce the transgene expression. FLZ (0.1, 1 and 10 µM) were incubated with cells for 24 h. (A) Immunohistochemistry of Aβ deposits in hippocampus. Representative sections of hippocampus from 5 mice were shown. Scale bar: 500 µm. (B) Western blot assay of Aβ expression in hippocampus. A representative immunoblot from 4 mice was shown. Results were expressed as mean±SD. **<i>P</i><0.01 <i>vs</i>. WT mice; ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. APP/PS1 mice. (C) Western blot assay and (D) ELISA assay of Aβ_1–40 in SH-SY5Y (APPwt/swe) cells. The results were obtained from 4–6 independent experiments. Results were expressed as mean±SD. **<i>P</i><0.01 <i>vs</i>. Neo SH-SY5Y cells, ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. solvent-treated SH-SY5Y (APPwt/swe) cells.</p

FLZ attenuated tau phosphorylation through regulating Akt/GSK3β pathway.

<p>APP/PS1 double transgenic mice were orally treated with FLZ 150 mg/kg for 20 weeks. SH-SY5Y (APPwt/swe) cells were grown in “stimulating medium” containing 50% DMEM, 50% Opti-MEM, 0.5% FBS, 200 µg/ml G418 and 10 mM butyric acid sodium salt for 12 h to induce the transgene expression. FLZ (0.1, 1 and 10 µM), Ly294002 10 µM combined with FLZ 10 µM or alone were incubated with cells for 24 h. (A) Western blot assay of p-Akt (Ser473), Akt, GSK3β and p-GSK3β (Ser9) in hippocampus of APP/PS1 mice. A representative immunoblot from 4 mice was shown. Results were expressed as mean ± SD. *<i>P</i><0.05 <i>vs</i>. WT mice; ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. APP/PS1 mice. (B) Western blot assay of p-Akt (Ser473), Akt, GSK3β and p-GSK3β (Ser9) in SH-SY5Y (APPwt/swe) cells. A representative immunoblot from four independent experiments was shown. Results were expressed as mean±SD. **<i>P</i><0.01 <i>vs</i>. Neo SH-SY5Y cells, ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. solvent-treated SH-SY5Y (APPwt/swe) cells. (C,D) Ly294002 was added to test the effect of Akt on GSK3β activity and tau phosphorylation in SH-SY5Y (APPwt/swe) cells treated with FLZ. A representative result of three independent experiments was shown. Results were expressed as mean±SD. **<i>P</i><0.01 <i>vs</i>. Neo SH-SY5Y cells, ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. solvent-treated SH-SY5Y (APPwt/swe) cells; ^Δ<i>P</i><0.05, ^ΔΔ<i>P</i><0.01 <i>vs</i>. FLZ-treated SH-SY5Y (APPwt/swe) cells.</p

FLZ ameliorated learning and memory deficits of APP/PS1 mice.

<p>APP/PS1 double transgenic mice were orally treated with FLZ 150 mg/kg for 20 weeks, and the learning and memory ability was accessed by passage water maze test. (A) The latencies of mice to find the destination. (B) Number of platform crossing. (C) Time in the target quadrant. (D) Travel distance. Results were expressed as mean±SD. *<i>P</i><0.05,**<i>P</i><0.01 <i>vs,</i> WT mice, ^#<i>P</i><0.05 <i>vs</i>. APP/PS1 transgenic mice. n = 10 in each group.</p

FLZ attenuated tau phosphorylation.

<p>APP/PS1 double transgenic mice were orally treated with FLZ 150 mg/kg for 20 weeks. SH-SY5Y (APPwt/swe) cells were grown in “stimulating medium” containing 50% DMEM, 50% Opti-MEM, 0.5% FBS, 200 µg/ml G418 and 10 mM butyric acid sodium salt for 12 h to induce the transgene expression. FLZ (0.1, 1 and 10 µM) were incubated with cells for 24 h. Antibodies against p-tau (Ser396), p-tau (Ser202/Thr205), p-tau (Thr231) and p-tau (Ser404) were subjected to Western blot assay. A representative immunoblot from 4 mice was shown. Results were expressed as mean±SD. (A) Western blot result in hippocampus of APP/PS1 mice. *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. WT mice; ^#<i>P</i><0.05,^##<i>P</i><0.01 <i>vs</i>. APP/PS1 mice. (B) Western blot result of SH-SY5Y (APPwt/swe) cells. **<i>P</i><0.01 <i>vs</i>. Neo SH-SY5Y cells, ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. solvent-treated SH-SY5Y (APPwt/swe) cells.</p

FLZ decreased the expression of BACE1 and did not affect the expression of IDE.

<p>APP/PS1 double transgenic mice were orally treated with FLZ 150 mg/kg for 20 weeks. SH-SY5Y (APPwt/swe) cells were grown in “stimulating medium” containing 50% DMEM, 50% Opti-MEM, 0.5% FBS, 200 µg/ml G418 and 10 mM butyric acid sodium salt for 12 h to induce the transgene expression. FLZ (0.1, 1 and 10 µM) were incubated with cells for 24 h. (A) Western blot assay of BACE1 and IDE in the hippocampus of APP/PS1 mice treated with FLZ. A representative immunoblot from 4 mice was shown. Results are expressed as mean±SD. *<i>P</i><0.05 <i>vs</i>. WT mice; ^#<i>P</i><0.05 <i>vs</i>. APP/PS1 mice. (B) Western blot assay of BACE1 and IDE in SH-SY5Y (APPwt/swe) cells treated with FLZ. A representative immunoblot from four independent experiments was shown. Results were expressed as mean ± SD. **<i>P</i><0.01 <i>vs</i>. Neo SH-SY5Y cells, ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. solvent-treated SH-SY5Y (APPwt/swe) cells.</p

FLZ protected SH-SY5Y (APPwt/swe) cells from apoptosis.

<p>SH-SY5Y (APPwt/swe) cells were grown in “stimulating medium” containing 50% DMEM, 50% Opti-MEM, 0.5% FBS, 200 µg/ml G418 and 10 mM butyric acid sodium salt for 12 h to induce the transgene expression. FLZ (0.1, 1 and 10 µM) were incubated with cells for 24 h. (A) Cell viability of SH-SY5Y (APPwt/swe) cells. (B) Apoptosis of SH-SY5Y (APPwt/swe) cells measured by flow cytometry with Annexin-V/PI staining. Bar chart is the statistical of the sum of early and late cell apoptosis. (C) Caspase-3 activity of SH-SY5Y (APPwt/swe) cells. Results were expressed as mean ± SD from 6 independent experiments. **<i>P</i><0.01 <i>vs</i>. Neo SH-SY5Y cells, ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. solvent-treated SH-SY5Y (APPwt/swe) cells.</p

FLZ decreased the expression of phospho-APP and APP-CTF.

<p>APP/PS1 double transgenic mice were orally treated with FLZ 150 mg/kg for 20 weeks. SH-SY5Y (APPwt/swe) cells were grown in “stimulating medium” containing 50% DMEM, 50% Opti-MEM, 0.5% FBS, 200 µg/ml G418 and 10 mM butyric acid sodium salt for 12 h to induce the transgene expression. FLZ (0.1, 1 and 10 µM) were incubated with cells for 24 h. Antibodies against p-APP, APP-CTF and APP were subjected to Western blot assay. A representative immunoblot from 4 mice was shown. Results were expressed as mean ±SD. (A) Western blot assay of APP, phospho-APP and APP-CTF in hippocampus of APP/PS1 mice. *<i>P</i><0.05,**<i>P</i><0.01 <i>vs</i>. WT mice; ^#<i>P</i><0.05 <i>vs</i>. APP/PS1 mice. (B) Western blot assay of APP, phospho-APP and APP-CTF in SH-SY5Y (APPwt/swe) cells. **<i>P</i><0.01 <i>vs</i>. Neo SH-SY5Y cells, ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. solvent-treated SH-SY5Y (APPwt/swe) cells.</p

Lycojaponicumins D and E: Two New Alkaloids from <i>Lycopodium japonicum</i>

Two new alkaloids, lycojaponicumins D (<b>1</b>) and E (<b>2</b>), were isolated from the club moss <i>Lycopodium japonicum</i>. Their structures were elucidated by spectroscopic methods, calculated ECD, CD experiments and X-ray diffraction analysis. Lycojaponicumin D (<b>1</b>) possesses an unprecedented 5/7/6/6 tetracyclic skeleton formed by an unusual C3–C13 linkage, which is first reported in <i>Lycopodium</i> alkaloids. The plausible biogenetic pathway of <b>1</b> is proposed

Lycojaponicumins D and E: Two New Alkaloids from <i>Lycopodium japonicum</i>

Two new alkaloids, lycojaponicumins D (<b>1</b>) and E (<b>2</b>), were isolated from the club moss <i>Lycopodium japonicum</i>. Their structures were elucidated by spectroscopic methods, calculated ECD, CD experiments and X-ray diffraction analysis. Lycojaponicumin D (<b>1</b>) possesses an unprecedented 5/7/6/6 tetracyclic skeleton formed by an unusual C3–C13 linkage, which is first reported in <i>Lycopodium</i> alkaloids. The plausible biogenetic pathway of <b>1</b> is proposed

Lupane Triterpenoids from the Stems of <i>Euonymus carnosus</i>

Fifteen new lupane-type triterpenoids (<b>1</b>–<b>15</b>) and 10 known triterpenoids (<b>16</b>–<b>25</b>) were isolated from the stems of <i>Euonymus carnosus</i>. The structures of the new compounds were elucidated on the basis of spectroscopic analyses, and the absolute configuration of compound <b>1</b> was confirmed by X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation. In addition, the compounds were tested for their cytotoxic activity against five human cancer cell lines and their ability to inhibit LPS-induced nitric oxide production in the murine microglia BV2 cell line. Compound <b>11</b> exhibited moderate cytotoxicity against several human cancer cell lines, and compounds <b>1</b>, <b>2</b>, <b>4</b>, <b>5</b>, <b>20</b>, and <b>25</b> showed neuritis inhibitory activity against microglial inflammation factor, with IC₅₀ values of 7.39, 7.48, 7.80, 3.48, 2.54, and 6.09 μM, respectively