3,5,4′-trihydroxy-6,7,3′-trimethoxyflavone protects against beta amyloid-induced neurotoxicity through antioxidative activity and interference with cell signaling

Aβ_25–35, crystal violet, and 2′,7′-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma Chemical Co. (St Louis, MO, USA); Dulbecco’s modified Eagle’s medium (DMEM) and Opti-MEM were purchased from Gibco (Paisley, UK); glutamine, antibiotics (10,000 IU/mL penicillin and 10,000 μg/mL streptomycin), fetal bovine serum (FBS) and trypin-EDTA were purchased from Biological Industries (Beit Haemek, Israel); dimethyl sulfoxide (DMSO) was obtained from Applichem (Darmstadt, Germany).

Isolation of TTF was carried out as previously described [14]. Briefly: the sun-dried A. fragrantissima plant was homogenized and extracted with ethyl acetate twice and with ethyl acetate:methanol (9:1) once. The organic extracts were combined and evaporated. The latter residue was chromatographed on a Sephadex LH-20 column that was eluted with methanol: CH₂Cl₂ (1:1). A fraction yielded by the Sephadex LH-20 column, which was monitored by thin layer chromatography (TLC) and ¹H–nuclear magnetic resonance (NMR) and that protected astrocytes from H₂O₂-induced cell death, was further purified by repeated chromatography over silica gel, with hexane that contained increasing proportions of ethyl acetate used as eluent. The active compound was afforded by elution with 50% ethyl acetate in hexane.

The original medium was aspirated from the cells and replaced with fresh medium. Fresh dilutions of TTF, first in DMSO and then in the growth medium, were prepared from stock solution just prior to each experiment, and were used immediately. The final concentration of DMSO in the medium was 0.2%. The Aβ_25–35 peptide was dissolved in DDW and incubated at 37 °C for 48 h. Fresh dilutions of Aβ in the growth medium were prepared just prior to each experiment and were used immediately. Each treatment was performed in replicates.

Cytotoxicity – N2a cells were grown in a medium containing 43% DMEM (high glucose), 50% Opti-MEM, 5% FBS, 2 mM glutamine, penicillin at 100 U/mL, and streptomycin at 100 μg/mL. The cells were re-plated in 96-well plates at a density of 5 × 10³ cells/well, in a similar medium; Aβ and/or TTF were added concomitantly 24 h later, and cytotoxicity was determined 20 h later with a commercial colorimetric assay (Roche Applied Science, Germany) based on the measurement of lactate dehydrogenase (LDH) activity released into the incubation medium from the cytosol of damaged cells. The absorbance was measured at 492 nm in a Synergy2 multi-detection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The percentage of cytotoxicity was calculated according to:

in which the term “A_{Triton-x treated cells}” is the maximum releasable LDH from the cells.

Cell viability – N2a cells were grown and treated as in the cytotoxicity assay, except that replating was at a density of 5 × 10³ cells/well. Cell viability was determined by a modification of the crystal violet assay [15], as follows. At the end of their treatments the cells were fixed with 150 μL of 5% (v/v) formaldehyde in PBS for 15 min at room temperature. The plates were washed by immersion in deionized water, dried and stained for 15 min with 150 μL of a 1% crystal violet solution. Following aspiration of the crystal violet solution the plates were washed with deionized water and dried, and the bound dye was solubilized with 150 μL of 33% aqueous solution of glacial acetic acid. The optical density was measured at 540 nm, by comparison with a 690-nm reference filter, in a Synergy2 multi-detection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

The percentage of ROS levels was calculated according to:

in which F is the fluorescence.

N2a cells were treated with TTF concomitantly with addition of Aβ_25–35. The cells were lysed after 40 or 30 min, for SAPK/JNK or ERK 1/2, respectively, in lysis buffer that was part of the PathScan Sandwich ELISA kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s protocol. Protein concentrations in cell lysates were determined with Bradford reagent (Bio-Rad, Hercules, CA, USA), and equal amounts of proteins were subjected to ELISA. To measure the amounts of total and of phosphoSAPK/JNK in cell lysates of the N2a cells, ELISA was performed according to the manufacturer’s protocols with the PathScan total SAPK/JNK sandwich ELISA kit and the PathScan phosphoSAPK/JNK (Thr183/Tyr185) sandwich ELISA kit, respectively (both kits from Cell Signaling Technology, Danvers, MA, USA). To measure the amounts of total and phosphoERK 1/2, i.e., phospho-p44/42 MAPK, in cell lysates, ELISA was performed according to the manufacturer’s protocol with Cell Signaling Technology’s PathScan total p44/42 MAPK (ERK 1/2) sandwich ELISA kit and the PathScan phospho-p44/42 MAPK (Thr202/Tyr204) sandwich ELISA kit, respectively. The optical density was measured at 450 nm with a Synergy2 multi-detection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

The results were subjected to one-way ANOVA followed by Tukey-Kramer multiple comparison tests, by means of Graph Pad InStat 3 for Windows (GraphPad Software, San Diego, CA, USA).

Phytochemicals that can protect neuronal cells against Aβ toxicity and oxidative stress may assist in coping with Alzheimer’s disease. In order to assess the ability of TTF (The structure of TTF is presented in Fig. 1) to counteract Aβ toxicity in N2a neuroblastoma cells, we have used the Aβ_25–35 peptide, which represents the neurotoxic fragments of Aβ_1–40 and Aβ_1–42, and is used in cell models to mimic their toxicity. Exposure of neuronal cells to Aβ_25–35 resulted in their death after 20 h, as reflected in a fivefold increase in the LDH assay (Fig. 2a), and a 55% reduction in the crystal violet assay (Fig. 2b). To assess the ability of TTF to protect neuronal cells against Aβ_25–35, and to determine the optimal concentrations of TTF required to induce a protective effect, cells were treated with Aβ_25–35 at 25 μM and with various concentrations of TTF. Cytotoxicity and viability were determined after 20 h by means of the LDH assay (Fig. 2c) and the crystal violet assay (Fig. 2d), respectively. Our results show that TTF exhibited a protective effect against Aβ_25–35-induced cell death, with maximal efficacy (96% protection) at a concentration of 140 nM (Fig. 2c, d). It should be noted that at all concentrations tested, up to 700 nM, the crystal violet assay did not show TTF alone to be cytotoxic to neuronal cells (Fig. 2e).

The role of free radicals in AD has been reported in many studies [16]. Moreover, it has been shown that Aβ induced generation of reactive oxygen species (ROS), leading to neuronal death [3]. To investigate the effect of TTF on ROS levels, which are elevated in response to Aβ treatment, the levels of intracellular ROS were determined. Treatment of cells with Aβ_25–35 for 20 h resulted in a twofold increase in intracellular ROS levels (Fig. 3), but no significant elevation in ROS levels was observed one or five hours after stimulation (Fig. 3). We therefore tested the possibility that TTF could protect neuronal cells from Aβ_25–35-induced cell death by inhibiting the Aβ_25–35-induced production of ROS. Cells were treated with various concentrations of TTF, simultaneously with application of Aβ_25–35. Changes in intracellular levels of ROS were detected with the ROS indicator DCF-DA, and ROS formation was determined by examining fluorescence after 20 h. Our results show that treatment with TTF at a concentration of 25 nM – similar to that used to protect cells from Aβ_25–35-induced cell death – inhibited the intracellular levels of Aβ_25–35-induced ROS by 60% (Table 1).

Mitogen-activated protein kinases (MAPKs) – a family of serine/threonine protein kinases – which initiate many cellular responses to external stress signals, were shown to be modulated by flavonoids [12, 17]. Enhanced activation of SAPK/JNK and ERK 1/2, which belong to the MAPK cascade, was observed in AD-affected brains [18‐20]. Moreover, Aβ has been reported to stimulate SAPK/JNK and ERK 1/2 [21‐24]. Therefore, we determined the effect of TTF on Aβ_25–35-induced phosphorylation of SAPK/JNK and ERK 1/2.

As can be seen in Fig. 4, treatment with Aβ_25–35 increased cellular levels of phosphorylated SAPK/JNK and ERK 1/2 by 2.5-fold. Treatment of cells with TTF at 3 and 32 nM inhibited the Aβ_25–35-induced phosphorylation of ERK 1/2 and SAPK/JNK, respectively, by 84 and 60%, respectively (Figs 4a and b, respectively). The total amounts of these proteins in the cells were not affected by TTF. It should be noted that TTF was more effective in inhibiting phosphorylation of ERK 1/2 than that of SAPK/JNK: at 3 nM it inhibited induced phosphorylation of ERK 1/2 by 84%, whereas at this concentration it inhibited that of SAPK/JNK by only 26%. These results suggest that inhibition of Aβ-induced phosphorylation of SAPK/JNK and ERK 1/2 is part of the mechanism by which TTF protects cells against Aβ_25–35 toxicity.