SH-SY5Y cell line was purchased from the American Type Culture Collection (Manassas, VA, USA; catalog number CRL-2266). Cell culture reagents, including penicillin/streptomycin were from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). DMEM/F12 medium was from Thermo Fisher Scientific, Inc. and fetal bovine serum (FBS) from Gemini Bio-Products (West Sacramento, CA, USA). Non-essential amino acids and all-trans retinoic acid (RA) were from Merck KGaA (Darmstadt, Germany). The ROS detection kit was also obtained from Merck KGaA. The catalase activity kit was from Biovision Inc. (Milpitas, CA, USA), and the superoxide dismutase (SOD) activity kit from Cayman Chemical Company (Ann Arbor, MI, USA). The lactate dehydrogenase (LDH) kit, together with Aβ25–35 and 3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Merck KGaA. The LDH assay kit was from Thermo Fisher Scientific, Inc., and the caspase 3/7 activity kit was from Promega Corporation (Madison, WI, USA). Antibodies against total- and phosphor(p)-Akt, and p-cAMP-responsive element binding protein (CREB) were from Cell Signaling Technology, Inc., (Danvers, MA, USA). Secondary anti-rabbit or anti-mouse antibodies and ECL detection kits were from GE Healthcare (Chicago, IL, USA). RIPA buffer, protease and phosphatase inhibitor cocktail, calcein-AM and quercetin were obtained from Merck KGaA. The BCA protein assay was from Thermo Fisher Scientific, Inc.

Fruits of Oroxylum indicum were collected from Maha Sarakham Province, Thailand, in July 2016. Species identification was performed by members of the Applied Thai Traditional Medicine Department, Faculty of Medicine, Mahasarakham University. A specimen was deposited at the herbarium at the Faculty of Science, Mahasarakham University (specimen no. MSUT_7234). An ethanolic extract of Oroxylum indicum was prepared by drying the fruits, then weighing and chopping them, and macerating them in 95% (v/v) ethanol for 7 days at room temperature (RT). The extract was then filtered, concentrated using a rotary evaporator and lyophilized. The % yield of extract was 12.89% per dry weight of Oroxylum indicum fruits.

The total flavonoid content of Oroxylum indicum crude extract was determined by the aluminum chloride colorimetric method. In brief, 100 µl of 1 mg/ml Oroxylum indicum crude extract was mixed with 0.9 ml flavonoid mixture (10% aluminum hydroxide, 1 M potassium acetate; dilution, 0.1, 0.1 and 4.3 ml). The mixture was incubated for 30 min at RT in the dark, and the absorbance intensity was measured at 450 nm. The total flavonoid content was calculated from a calibration curve and the result was expressed as mg rutin equivalent per g dry weight.

The total phenolic content of the Oroxylum indicum extract was determined using the Folin-Ciocalteu method. In brief, 100 µl of 1 mg/ml Oroxylum indicum crude extract was mixed thoroughly with 0.45 ml Folin-Ciocalteu reagent for 5 min, followed by the addition of 0.45 ml of 60 g/l sodium bicarbonate. The mixture was kept in the dark for a further 1 h at RT, and the absorbance intensity was measured at 650 nm. The total phenolic content was calculated from the calibration curve, and the results were expressed as mg of gallic acid equivalent per g dry weight.

Human neuroblastoma SH-SY5Y cells were maintained in DMEM/F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin and 1% non-essential amino acids at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium was changed every 3 days. Cells were plated at an appropriate density according to each experiment. Cells were differentiated with 10 µM all-trans retinoic acid over 6 days prior to treatments. At the beginning of each experiment, the culture medium in each well was completely removed and replaced with fresh medium containing Aβ with or without Oroxylum extract.

Aβ25–35 peptide was dissolved in deionized distilled water as a 1 mM stock solution and incubated at 37°C for 3 days. The solution was aliquoted into 1-ml tubes, kept at −20°C and thawed for subsequent use.

The in vitro cytotoxicity of Aβ25–35 and Oroxylum indicum were determined using the MTT assay. SH-SY5Y cells were plated in 96-well plates at a density of 1×10⁴ cells/well and cultured as described above. Cells were treated with various concentrations of Aβ25–35 (10–30 µM) and Oroxylum indicum (0–100 µg/ml) in 1% FBS for 24 h. To determine whether Oroxylum indicum protects against Aβ-induced neurotoxicity, cells were treated with Aβ25–35 with or without of Oroxylum indicum extract in 1% FBS for 24 h. Following 24 h of treatment, the medium was removed and replaced with MTT reagent at a final concentration of 0.5 mg/ml. Cells were then incubated for 4 h at 37°C in 5% CO₂ in an incubator. Following incubation, MTT reagent was aspirated and 100 µl dimethyl sulfoxide was added to dissolve the insoluble purple formazan product. Absorbance was determined at 570 nm using a Synergy-4 plate reader (BioTek Instruments, Inc Winooski, VT, USA). Results were expressed as a percentage of the control.

Cells were seeded at a density of 1×10⁴ cells/well in 96-well plates and cultured as described above. After 24 h, intracellular ROS levels were measured using the fluorescent probe 2′,7′-dichlorofluorescein, as previously described (13). Data were expressed as the percentage of ROS relative to untreated controls.

Cells were plated at a density 1×10⁴ cells/well in 96-well plates. Cells were then cultured and treated as described above. Following 24 h of treatment, Aβ-induced cell injury was measured by determining how much of the intracellular enzyme LDH had been released into the culture medium. Culture medium (100 µl) was collected from each well and transferred to a new 96 well plate, and 100 µl reaction mixture was added to each well and incubated for 30 min at 37°C. Absorbance was measured at 492 nm using a microplate reader. The quantity of LDH released was then expressed as a percentage of the untreated control.

Cells were plated at a density 1×10⁵ cells/well in 6-well plates. Overnight cultured cells were treated as described above. Following 24 h of treatment, cells were harvested with a rubber policeman and collected by centrifugation (2,000 × g for 10 min at 4°C). The cell pellets were homogenized in cold assay buffer and centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was also collected for the assay. Catalase activity was determined using a commercially available assay kit (Biovision Inc.), according to the manufacturer's instructions.

Cells (1×10⁵ cells/well) were plated in 6-well plates and were then cultured and treated as described above. Following 24 h of treatment, cells were harvested as described for the CAT activity assay. Cell pellets were then homogenized in 20 mM cold HEPES buffer, and centrifuged at 1,500 × g for 5 min at 4°C. The supernatant was also collected for the assay. Superoxide dismutase activity was measured using an assay kit from Cayman Chemical, according to the manufacturer's instructions. Results were expressed as a percentage of the untreated control.

Cells (1×10⁵ cells/well) were plated in 6-well plates, and then cultured and treated as described above. Following treatment, cells were collected and total protein concentration was determined using a BCA kit. Equal amounts of proteins were separated by 4–20% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were then incubated with primary antibodies against Bcl-2 (1:1,000), p-Akt, total Akt (1:1,000), p-CREB (1:1,000) and actin (1:5,000) overnight at 4°C. The membranes were then washed with TBST (Tris-buffered saline, 0.1% Tween 20), and probed with secondary antibody conjugated with HRP for 1 h at RT. Protein bands were detected using an enhanced chemiluminescence detection kit, and results were expressed as a fold change of the untreated control.

Caspase-3/7 activity was measured using Caspase-Glo^® 3/7 kits from Promega Corporation, according to the manufacturer's instructions. Briefly, the caspase-GloR 3/7 buffer and lyophilized caspase-GloR 3/7 substrate were equilibrated at RT. The contents of the caspase-GloR 3/7 buffer were transferred into the bottle containing caspase-GloR3/7 substrate. Equal volumes of reaction mixture were added to samples and incubated for 30 min-2 h prior to the luminescence measurement. Results were expressed as a percentage of the untreated control.

All data are expressed as the mean ± SEM from at least three independent experiments performed in triplicate. Multiple comparisons of data were evaluated by one-way ANOVA followed by Bonferroni post-hoc test. A P<0.05 was considered to indicate a statistically significant difference.

Since the SH-SY5Y cell line shares only a few properties with mature neurons (23), it is important to differentiate these cells with retinoic acid, so that they are comparable to in vivo models. Undifferentiated SH-SY5Y cells have been used as a model of cytotoxicity in several studies (24–26), but the comparative cytotoxic effects of Aβ on cell survival of RA-differentiated and undifferentiated SH-SY5Y cells has not yet been reported. Therefore, the purpose of this study was to compare the in vitro cytotoxicity of Aβ25–35 between differentiated and undifferentiated SH-SY5Y cells. Both differentiated and undifferentiated cells were treated with various concentrations of Aβ25–35 (0–30 µM). As shown in Fig. 1A, Aβ25–35 treatments were toxic to both cell groups, beginning at 10 µM for undifferentiated cells and 20 µM for differentiated cells. Although undifferentiated SH-SY5Y cells were more susceptible to Aβ25–35 than differentiated cells, the differentiated cells possess more neuron-like properties, including neurite outgrowth and morphological changes of neurons in the brain. Therefore, RA-differentiated SH-SY5Y cells were selected for subsequent assays, and Aβ25–35 was used at a concentration of 20 µM.

To determine whether Oroxylum indicum has any effect on the viability of SH-SY5Y cells, the cells were treated with various concentrations of the extract (0–100 µg/ml). The results indicated that Oroxylum indicum extract at concentrations of 25 and 50 µg/ml increased cell viability (139.45±7.89 and 130.61±5.83% of control value, respectively) and that concentrations of up to 100 µg/ml were non-toxic to SH-SY5Y cells (Fig. 1B). Therefore, the highest non-toxic concentrations of Oroxylum indicum (50 and 100 µg/ml) were used in subsequent assays.

To determine the effect of Oroxylum indicum on Aβ25-35-induced cytotoxicity, SH-SY5Y cells were challenged with 20 µM Aβ25–35 in the presence or absence of 50 and 100 µg/ml Oroxylum indicum extract for 24 h. As shown in Fig. 2A, treatment of SH-SY5Y cells with 20 µM Aβ25–35 for 24 h induced cytotoxicity, as demonstrated by a cell viability reduction to 76.83±0.67%, when compared with the control group. When the cells were treated with Oroxylum indicum extract at concentrations of 50 and 100 µg/ml, cell viability was restored to 94.14±2.79 and 98.35±3.74%, respectively, indicating a concentration-dependent cytoprotective effect.

To further confirm the cytoprotective effect of Oroxylum indicum, LDH, another indicator of cell toxicity, was also examined. The results were similar to those determined by the MTT assay. The exposure of SH-SY5Y cells to 20 µM Aβ25–35 resulted in a 1.45-fold increase in LDH release in the medium, when compared to that in the control group. Treatment of cells with 50 and 100 µg/ml Oroxylum indicum extract reduced Aβ25-35-induced LDH release in a concentration-dependent manner (Fig. 2B).

ROS play a critical role in Aβ-dependent cell death. Therefore, in the present study, the effect of Oroxylum indicum on Aβ25-35-induced intracellular ROS production was examined. SH-SY5Y cells exposed to Aβ25–35 for 24 h exhibited elevated ROS levels (152.4±3.53%, as compared to the control group) (Fig. 3A). When the cells were treated with Oroxylum indicum extract at concentrations of 50 and 100 µg/ml, there was a significant and concentration-dependent inhibition of Aβ25-35-induced intracellular ROS levels (89.17±2.46 and 64.25±6.21%, respectively).

To determine whether the cytoprotective effect of Oroxylum indicum is associated with the activity of anti-oxidative enzymes, SOD and CAT activity was determined. Following exposure to Aβ25–35 for 24 h, SOD activity was significantly decreased to 76.48±0.6% of the control value. Treatment of cells with 50 and 100 µg/ml Oroxylum indicum extract for 24 h restored SOD activity to 87.30±0.81 and 100.76±1.19%, respectively (Fig. 3B). Following exposure to Aβ25-35, CAT activity was significantly increased to 122.45±0.029% of the control value. CAT activity following treatment with Oroxylum indicum extract was significantly increased beyond that of the Aβ treatment and control groups (144.15 and 148.4%, respectively) (Fig. 3C).

It has been reported that Aβ-induced neuronal cell death through the activation of the caspase pathway (27–31). To study the protective mechanism of Oroxylum indicum, caspase-3/7 activity was measured. Following Aβ25–35 treatment, caspase-3/7 activity significantly increased to levels that were 1.4-fold higher than that of the control group. However, the induction of caspase-3/7 activity was blocked in the presence of Oroxylum indicum (Fig. 3D)

The activation of Akt has been associated with the inhibition of the apoptotic cleavage of caspases, as well as the promotion of neuronal survival (32–35). We therefore hypothesized that Oroxylum indicum can modulate the signaling of Akt. The results showed that treatment with Aβ25–35 for 15 min significantly decreased p-Akt, as compared to the untreated control (Fig. 4A). Oroxylum indicum treatments significantly increased the phosphorylation of Akt, as compared to Aβ25–35 treatment.

It has been reported that one element of the downstream signaling of Akt is CREB the transcription factor that regulates neuronal survival (35). We therefore examined whether CREB phosphorylation increased in SH-SY5Y cells following treatment with Aβ25–35 in the presence of Oroxylum indicum. When cells were treated with Aβ25–35 for 1 h, CREB phosphorylation was significantly decreased, when compared with the untreated control. The phosphorylation of CREB was significantly increased following treatment with Oroxylum indicum, when compared to both the Aβ25–35 treatment and control groups (Fig. 4B).

The transcription factor CREB has been identified as a positive regulator of Bcl-2 (an apoptosis suppressor gene) expression (36,37). Therefore, these results demonstrated that Aβ25–35 ameliorated Bcl-2 expression, as compared with the control group. Treatment with Oroxylum indicum extract for 24 h caused a significant increase in Bcl-2 expression, as compared to the Aβ25–35 group (Fig. 4C).

The Oroxylum indicum fruit extract was standardized using colorimetric methods to quantify the total phenolic content using gallic acid as a standard, and the total flavonoid content using rutin as a standard. The values obtained were 10.50±0.68 and 17.08±0.85 mg/g of dried extract, respectively (Table I).