Olive Biophenols Reduces Alzheimer's Pathology in SH-SY5Y Cells and APPswe Mice

Abstract

Alzheimer's disease (AD) is a major neurodegenerative disease, associated with the hallmark proteinacious constituent called amyloid beta (Aβ) of senile plaques. Moreover, it is already established that metals (particularly copper, zinc and iron) have a key role in the pathogenesis of AD. In order to reduce the Aβ plaque burden and overcome the side effects from the synthetic inhibitors, the current study was designed to focus on direct inhibition of with or without metal-induced Aβ fibril formation and aggregation by using olive biophenols. Exposure of neuroblastoma (SH-SY5Y) cells with Aβ₄₂ resulted in decrease of cell viability and morphological changes might be due to severe increase in the reactive oxygen species (ROS). The pre-treated SH-SY5Y cells with olive biophenols were able to attenuate cell death caused by Aβ₄₂, copper- Aβ₄₂, and [laevodihydroxyphenylalanine (l-DOPA)] l-DOPA-Aβ₄₂-induced toxicity after 24 h of treatment. Oleuropein, verbascoside and rutin were the major anti-amyloidogenic compounds. Transgenic mice (APPswe/PS1dE9) received 50 mg/kg of oleuropein containing olive leaf extracts (OLE) or control diet from 7 to 23 weeks of age. Treatment mice (OLE) were showed significantly reduced amyloid plaque deposition (p < 0.001) in cortex and hippocampus as compared to control mice. Our findings provide a basis for considering natural and low cost biophenols from olive as a promising candidate drug against AD. Further studies warrant to validate and determine the anti-amyloid mechanism, bioavailability as well as permeability of olive biophenols against blood brain barrier in AD.

Keywords: Alzheimer’s disease; SH-SY5Y cells; amyloid beta; oleuropein; olive biophenols; rutin; verbascoside.

Figure 1

The inhibition of Aβ₄₂ (20 µM) fibrils was monitored by transmission electron microscope (TEM) using ThT fluorescence in the (A) absence of biophenols, and presence of (B) oleuropein (OL) (C) quercetin (QU) and (D) olive leaf extract (OLE).

Figure 2

Thioflavin-T assay: Inhibition of Aβ₄₂ fibrils by olive biophenols: (A) Non-flavonoids olive biophenols, (B) flavonoids olive biophenols and (C) extracts olive biophenols. Control: Aβ₄₂ without biophenols. Nordihydroguaiaretic acid (NDGA) used as reference inhibitor. CA: caffeic acid, OL: oleuropein, HT: hydroxytyrosol, VB: verbascoside, QU: quercetin, RU: rutin, LU: luteolin, OLE: olive leaf extract, OFE: olive fruit extract, HTE: hydroxytyrosol extreme, OLP: olivenol plus. The results were mean ± S.D. analysed by one-way ANOVA (Tukey’s test), * p < 0.001 vs. negative control (NDGA).

Figure 3

Congo red assay: Inhibition of Aβ₄₂ fibrils by olive biophenols: (A) Non-flavonoids olive biophenols, (B) flavonoids olive biophenols and (C) extracts olive biophenols. Control: Aβ₄₂ without biophenols. Nordihydroguaiaretic acid (NDGA) used as reference inhibitor. CA: caffeic acid, OL: oleuropein, HT: hydroxytyrosol, VB: verbascoside, QU: quercetin, RU: rutin, LU: luteolin, OLE: olive leaf extract, OFE: olive fruit extract, HTE: hydroxytyrosol extreme, OLP: olivenol plus. The results were mean ± S.D. analysed by one-way ANOVA (Tukey’s test), * p < 0.001 vs. negative control (NDGA).

Figure 4

Aβ₄₂ induced SH-SY5Y cells toxicity and protection by pre-incubation of olive biophenols for 24 h: (A) SH-SY5Y cells were treated with different concentrations of Aβ₄₂ without olive biophenols for 24 h. SH-SY5Y cells were pre-incubated with different concentrations of (B) non-flavonoid olive biophenols, (C) flavonoid olive biophenols and (D) extract olive biophenols for 24 h followed by 25 μM of Aβ₄₂ for 24 h. The results are mean ± SE of each parallel measurements analyzed by one-way ANOVA (Tukey’s test), * p < 0.001 vs negative control. NC: negative control (cells with Aβ₄₂ without biophenols), CA: caffeic acid, OL: oleuropein, HT: hydroxytyrosol, VB: verbascoside, QU: quercetin, RU: rutin, LU: luteolin, OLE: olive leaf extract, OFE: olive fruit extract, HTE: Hydroxytyrosol extreme, OLP: Olivenol plus.

Figure 5

Copper-amyloid (Cu-Aβ₄₂) induced SH-SY5Y cells toxicity and protection by pre-incubation of olive biophenols for 24 h: (A) SH-SY5Y cells were treated with 20 µM of Aβ₄₂ along with different concentrations of copper for 24 h. The SH-SY5Y cells were pre-incubated with various concentration of olive biophenols and treated with 20 µM of Aβ₄₂ and 200 µM of Cu (B) non-flavonoids olive biophenols, (C) flavonoid olive biophenols and (D) extract olive biophenols for 24 h followed by 25 µM of Aβ₄₂ and 200 µM of copper for 24 h. The results are mean ± S.E. of each parallel measurements analysed by one-way ANOVA (Tukey’s test), * p < 0.001 vs. negative control. NS: non-significant. C: positive control (cells with media), NC: negative control (cells with Cu-Aβ₄₂ without biophenols), CA: caffeic acid, OL: oleuropein, HT: hydroxytyrosol, VB: verbascoside, QU: quercetin, RU: rutin, LU: luteolin, OLE: olive leaf extract, OFE: olive fruit extract, HTE: Hydroxytyrosol extreme, OLP: Olivenol plus.

Figure 6

l-DOPA-amyloid (l-DOPA-Aβ₄₂) induced SH-SY5Y cells toxicity and protection by pre-incubation of olive biophenols for 24 h: (A) SH-SY5Y cells were treated with 20 µM of Aβ₄₂ along with different concentrations of l-DOPA for 24 h. The SH-SY5Y cells were pre-incubated with various concentration of olive biophenols and treated with 20 µM of Aβ₄₂ and 200 µM of l-DOPA (B) non-flavonoids olive biophenols, (C) flavonoid olive biophenols and (D) extract olive biophenols for 24 h followed by addition of 25 µM of Aβ₄₂ and 200 µM of l-DOPA for 24 h. The results are mean ± S.E. of each parallel measurements analysed by one-way ANOVA (Tukey’s test), * p < 0.001 vs. control. Control: cells with media, NC: negative control (cells with l-DOPA-Aβ₄₂ without biophenols), CA: caffeic acid, OL: oleuropein, HT: hydroxytyrosol, VB: verbascoside, QU: quercetin, RU: rutin, LU: luteolin, OLE: olive leaf extract, OFE: olive fruit extract, HTE: Hydroxytyrosol extreme, OLP: Olivenol plus.

Figure 7

Amyloid plaque burden and olive biophenols protection. The result was analysed by one-way ANOVA, * p < 0.001.

Figure 8

Schematic representation of the APPswe/PS1dE9 mice study schedule.