Abstract
Taxifolin is a potent flavonoid that exerts anti-oxidative effect, and cilostazol increases intracellular cAMP levels by inhibiting phosphodiesterase 3 that shows antiinflammatory actions. BACE1 (β-site APP cleaving enzyme 1) is the rate-limiting enzyme responsible for the β-cleavage of amyloid precursor proteins to Aβ peptides. In this study, endogenous Aβ and C99 accumulation was explored in N2a Swe cells exposed to 1% FBS medium. Increased Aβ and C99 levels were significantly attenuated by taxifolin alone and in combination with cilostazol. Increased phosphorylated JAK2 at Tyr1007/1008 (P-JAK), phosphorylated STAT3 at Tyr 705 (P-STAT3) expressions and increased expressions of BACE1 mRNA and protein in the activated N2a Swe cells were significantly attenuated by taxifolin (10~50 μM), cilostazol (10~50 μM) alone and in combination at minimum concentrations. In these cells, decreased cytosol IκBα expression was elevated, and increased nuclear NF-κB p65 level and nuclear NF-κB p65 DNA binding activity were significantly reduced by taxifolin and cilostazol in a similar manner. Following STAT3 gene (~70% reduction) knockdown in N2a cells, Aβ-induced nuclear NF-κB and BACE1 expressions were not observed. Taxifolin, cilostazol, or resveratrol significantly stimulated SIRT1 protein expression. In SIRT1 gene-silenced (~50%) N2a cells, taxifolin, cilostazol, and resveratrol all failed to suppress Aβ1-42-stimulated P-STAT3 and BACE1 expression. Consequently, taxifolin and cilostazol were found to significantly decrease lipopolysaccharide (1–10 μg/ml)-induced iNOS and COX-2 expressions, and nitrite production in cultured BV-2 microglia cells and to increase N2a cell viability. In conclusion, taxifolin and cilostazol strongly inhibited amyloidogenesis in a synergistic manner by suppressing P-JAK2/P-STAT3-coupled NF-κB-linked BACE1 expression via the up-regulation of SIRT1.
Highlights
Alzheimer’s disease (AD) is characterized by increased amyloid β (Aβ)-containing extracellular plaque and intracellular neurofibrillary tangles, which are associated with synaptic failure and cognitive deficits [1]
Increased P-Janus kinase 2 (JAK2) expression determined at 3 hr in medium containing 1% fetal bovine serum (FBS) was concentration-dependently decreased by taxifolin (10 ~ 50 μM; F3,12 = 39.40, P < 0.0001), by cilostazol (10 ~ 50 μM; F3,12 = 47.02, P < 0.0001), and by 20 μM AG490 (Fig 1C & 1D)
In line of phosphorylated JAK2 at Tyr1007/1008 (P-JAK2) expression, when N2a Swe cells were exposed to depleted FBS in culture medium, the expression of P-signal transducer and activator of transcription 3 (STAT3) at Tyr 705 (P-STAT3) in cytosol was significantly elevated at 3 hr (2.14 ± 0.42 fold, P < 0.001) and declined (F4,15 = 16.63, P < 0.0001) (Fig 2A), which posed the question: Where did phosphorylated STAT3 at Tyr 705 (P-STAT3) move to? we investigated the time-dependent nuclear translocation of P-STAT3
Summary
Alzheimer’s disease (AD) is characterized by increased amyloid β (Aβ)-containing extracellular plaque and intracellular neurofibrillary tangles, which are associated with synaptic failure and cognitive deficits [1]. Enhanced amyloidogenic processing of amyloid precursor protein (APP) by β- and γ-secretase increases intracellular level of soluble oligomeric Aβ, which results in pronounced synaptic failure and eventually in memory decline [2,3]. A membrane-associated C-terminal fragment of APP, C99, is liberated by the action of β-secretase, and this is subsequently cleaved by γ-secretase to produce Aβ peptide [4]. BACE1 (β-secretase, a membrane-bound aspartyl protease β-site APP cleaving enzyme 1) is a rate-limiting enzyme for β-amyloid production [5]. The expression of BACE1 protein and its activity have been demonstrated to be elevated in the brains of AD patients [6,7]. Aβ has been shown to activate nuclear transcription factor NF-κB [9,10], which is activated during the early stages of AD, where RelA/p65 plays a critical role in neurons and astrocytes surrounding amyloid plaques in the brain, and elevates oxidative stress [11]
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