Figure 1.
Miyabenol C treatment inhibits Aβ secretion.
(A) Chemical structure of miyabenol C. (B) N2a695 cells were treated with indicated doses of miyabenol C for 10h. Cell viability was measured using CCK-8 assay, n = 3. Error bars indicate standard deviation. (C, D) N2a695 cells were treated with DMSO (negative control), β-secretase inhibitor II (positive control, 2μM) or indicated doses of miyabenol C for 10h. Extracellular Aβ40 (C) and Aβ42 (D) levels were quantified by ELISA (One-way ANOVA followed by Tukey’s post hoc test, n = 3, *: p< 0.05, **: p< 0.01, ***: p< 0.001).
Figure 2.
Miyabenol C does not inhibit γ-secretase activity.
N2aWT cells were first transfected with NotchΔE. After splitting equally, cells were treated with DMSO (negative control), the γ-secretase inhibitor compound E (positive control, 0.5μM) or indicated doses of miyabenol C for 10h. Equal protein amounts of cell lysates were subjected to SDS-PAGE and Western blot. NICD levels were quantified by densitometry using image J for comparison (One-way ANOVA followed by Tukey’s post hoc test, n = 3, ns: p> 0.05, ***: p< 0.001).
Figure 3.
Miyabenol C treatment reduces APP β-CTF and sAPPβ levels and increases sAPPα levels.
N2a695 cells were treated with DMSO (negative control), β-secretase inhibitor II (2μM) or indicated doses of miyabenol C for 10h. (A) Conditioned media and (B) cellular lysates were subjected to SDS-PAGE and Western blot. sAPPα, sAPPβ and β-CTF levels were quantified by densitometry using image J for comparison (One-way ANOVA followed by Tukey’s post hoc test, n = 3, ns: p>0.05, *: p<0.05, **: p<0.01).
Figure 4.
Miyabenol C inhibits β-secretase activity.
(A) N2aWT and (B) SH-SY5Y cells were treated with DMSO (negative control), miyabenol C (10μM) or a β-secretase inhibitor (positive control, 2μM, provided in the kit) for 10h. Cell lysates were assayed for β-secretase activity by using a commercial kit from Millipore and subjected to comparison. (C) Indicated amounts of miyabenol C and β-secretase inhibitor II were incubated with BACE1 and its substrate provided by a commercial kit from Sigma, for 2h at 37°C. BACE1 activity was measured for comparison. (D) SY5Y cells were treated with DMSO (negative control), miyabenol C (10μM) or the α-secretase inhibitor TAPI-1 (positive control, 10μM) for 10h. Cell lysates were assayed for the α-secretase (TACE) activity for comparison (One-way ANOVA followed by Tukey’s post hoc test, n = 3, ns: p> 0.05, *: p< 0.05, **: p< 0.01, ***: p< 0.001).
Figure 5.
Short term miyabenol C treatment reduces sAPPβ and soluble Aβ levels in APP/PS1 AD mouse brain.
APP/PS1 AD mice (12 month old) were treated with miyabenol C (0.6μg/g) or vehicle (45% DMSO in artificial cerebrospinal fluid) through intracerebroventricular injection (4μl) for 72h. (A) Cortex and hippocampus of treated mice were dissected and lysed. Equal protein amounts of lysates were analyzed by Western blot. Levels of TBSX-soluble Aβ42 (B) and Aβ40 (D), as well as TBSX-insoluble (re-dissolved in GuHCl) Aβ42 (C) and Aβ40 (E) in the cortex and hippocampus of treated mice were measured by ELISA assays (Student’s t test, n = 6, ns: p>0.05, **: p<0.01).