PC12 cells (CRL-1721; American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 5% horse serum (HS; Invitrogen; Thermo Fisher Scientific, Inc.), 10% fetal bovine serum (FBS; Invitrogen Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml) (Invitrogen; Thermo Fisher Scientific, Inc.), under a humidified atmosphere containing 5%/95% CO₂/air at 37°C. PC12 cells underwent differentiation for 48 h at 37°C with 50 ng/ml nerve growth factor (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) dissolved in DMEM containing 1% FBS, 1% HS and 100 U/ml of penicillin/streptomycin.

PC12 cells were seeded into 96-well plates (8,000 cells/well/100 µl), pre-treated with 10 or 30 µM of IAB (purity, ≥95%; Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) for 3 h at 37°C, and then co-incubated with 25 mM of L-Glu for another 24 h at 37°C. Cell viability was detected by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-di-phenyl-2-H-tetrazolium bromide (MTT; cat. no. R20228, Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) assay (19), it was used according to the manufacturer's protocols. A total of 100 µl dimethyl sulfoxide was used to solubilize purple formazan crystals, then analyzing the absorbance using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 490 nm. The activity of caspase-3 was analyzed using a commercial kit, it was used according to the manufacturer's protocols (cat. no. G007; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The experiments were repeated eight times.

PC12 cells were seeded into 6-well plates at 5×10⁵ cells/well/ml, and pre-treated with 10 or 30 µM of IAB for 3 h at 37°C, and then co-incubated with 25 mM of L-Glu for another 24 h at 37°C. The rate of cell apoptosis was determined via the early apoptosis and late apoptosis of cells, which was analyzed by propidium iodide/Annexin V staining (EMD Millipore, Billerica, MA, USA) using a Muse™ Cell Analyzer flow cytometer (EMD Millipore, Billerica, MA, USA); the data was analyzed by Muse 1.4 Analysis. The experiments were repeated eight times.

PC12 cells were seeded into 6-well plates at 5×10⁵ cells/well/ml, and pre-treated with 10 and 30 µM of IAB for 3 h, and then co-incubated with 25 mM of L-Glu for another 12 h at 37°C. The ROS levels were analyzed by staining with 2,7-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich; Merck KGaA) according to a previous study (24). The alterations in MMP were analyzed with 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (Sigma-Aldrich; Merck KGaA) staining as described (25). Alterations in fluorescent intensity were analyzed using a fluorescent microscope (magnification, ×200; CCD camera, Axio Observer Z1; Zeiss AG, Oberkochen, Germany). The experiments were repeated eight times. Quantification of data was conducted with Image J software version 1.46 (National Institutes of Health, Bethesda, MD, USA) and expressed as the green fluorescence intensity for intracellular ROS levels, and the ratio of red to green fluorescence intensity for MMP detection.

The present study was approved by the Animal Ethics Committee of Jilin University (no. 20170206). A total of 36 Balb/c male mice (10-weeks old, 23-35 g) were obtained from Bethune Medical College, Jilin University (Changchun, China) were housed in cages under a temperature of 23±1°C and humidity of 40-60% with sufficient water and food, and under a 12 h light/dark cycle.

A total of 24 mice were subcutaneously injected with 120 mg/kg of D-gal and intragastrically administered 20 mg/kg of AlCl₃ once per day for 56 days. At the 29th day, the mice were randomly divided into two groups and intra-gastrically administered normal saline solution (n=12) or 40 mg/kg of IAB (n=12) once daily for 28 days. The other 12 mice were treated with normal saline solution for 56 days and served as control group. On days 54, 55 and 56, behavioral tests, including a Morris water maze test (17), an open field test (17) and the Y maze test (26), were respectively performed as previously described without modifications. At the end of the experiments, blood samples were collected from the caudal vein under anesthesia with 400 mg/kg (10%) chloral hydrate, and the whole brains, kidney and spleen were collected following euthanasia by injection with 150 mg/kg (1.5%) pentobarbital, after standing at 25°C for 30 min, blood samples were centrifuged at 300 × g for 10 min at 25°C twice to obtain serum.

In each group, six mice were randomly selected for the detection of biochemical indexes. The levels of ROS (cat. no. E-20634), superoxide dismutase (SOD; cat. no. E-20347), and glutathione peroxidase (GSH-Px; cat. no. E-20584) and Aβ1-42 (cat. no. E-20118) in the serum and brains, and the levels of acetylcholine (Ach) (cat. no. E-20535), Ach esterase (AchE; cat. no. E-2143) and choline acetyltransferase (ChAT; cat. no. E-21422) in brains were detected by commercialized ELISA kits (Shanghai Yuanye Biological Technology Co., Ltd.).

Similar to a previous study, immuno-histochemical analysis was conducted to analyze the levels of Aβ1-42 and p-Tau (Ser404) in the brain (27). In each group, the brain tissues of six other mice were fixed with 4% formalin solution at 25°C for 1 week, then sliced into 5 µm-thick sections, which prepared for analysis. Briefly, after antigen retrieval via heating in 0.01 mol/l citrate buffer at 98°C, the slides were blocked in 3% hydrogen peroxide for 10 min, and 2% goat serum for 2 h at room temperature, respectively. The slides were then exposed to antibodies of Aβ1-42 (1:200, cat. no. bs-0877R; BIOSS, Beijing, China) and p-Tau (Ser404, 1:200, cat. no. bs-2392R; BIOSS) overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (cat. no. SH-0032; Bejing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) at a dilution of 1:2,000 for 4 h at 4°C. 3,3′-diaminobenzidine and Mayer's hematoxylin were applied for 5 min at 25°C, the slides were solidified with a neutral resin and photographed by the Olympus IX73 optical microscope (Olympus Corporation, Tokyo, Japan).

TUNEL was applied to analyze the occurrence of apoptosis in the brain by observing the number of positive cells in the field of view (19). Following deparaffinization, the brain slides from six mice of each group were washed with PBS and covered with permeabilization reagent (20 µg/ml Proteinase K). The brain slides were incubated with TdT reaction mixture in the dark for 1 h at 37°C, and then analyzed three fields per view under a Nikon Eclipse TE 2000-S fluorescence microscope (magnification, ×200) Images were collected with a CCD camera.

The whole brains, kidney and spleen of six mice from each group were preserved in 10% formaldehyde solution at 25°C for 1 week. Following dehydration using 100, 95, 80, 70 and 50% ethanol and distilled water in sequence, the samples were embedded in paraffin wax and sliced into joint cavity sections of 5 µm thickness. Following H&E staining for 10 min at 25°C, light microscopy was performed at ×100 (for brain slides) or ×200 (for kidney and splenic slides) to analyze histopathological alterations within the tissues, three fields per view were analyzed.

PC12 cells were seeded into 6-well plates at 5×10⁵ cells/well/1 ml, and pre-treated with 10 or 30 µM of IAB for 3 h, and then co-incubated with 25 mM of L-Glu for another 24 h. Radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA) containing 1% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) was used to lyse the treated cells and collected brain tissues. The nuclear fractions of cells and tissue samples to determine Nrf2 expression were obtained using a nuclear pulp separation kit (cat. no. BB-36021-2; BestBio, Shanghai, China), which was used according to the manufacturer's protocols, The protein concentration of the samples was detected by a Bicinchoninic Acid kit; 40 µg of samples were separated via 12% SDS-PAGE, electrotrans-ferred onto 0.45 µm nitrocellulose membranes (Bio Basic, Inc., Toronto, Canada), and then incubated with primary antibodies including B-cell lymphoma 2 (Bcl-2; sc-70411), B-cell lymphoma-extra large (Bcl-xL; sc-8392), Bcl-2-associated X (Bax; sc-7480), cleaved caspase-3 (sc-136219), cleaved caspase-9 (sc-56076), Nrf2 (sc-722), heme oxygenase-1 (HO-1; sc-390991), HO-2 (sc-17786), superoxide dismutase 1 (SOD1; sc-271014), oxidative enzyme catalase (CAT; sc-271803) and GAPDH (sc-365062 Santa Cruz Biotechnology, Inc., Dallas, TX, SA) at dilution of 1:1,000 at 4°C overnight. The membranes were then exposed to horseradish peroxidase-conjugated secondary antibodies (sc-516102; Santa Cruz Biotechnology, Inc.) at dilution of 1:2,000 for 4 h at 4°C. The protein blots were detected using enhanced chemiluminescence detection kits (GE Healthcare, Chicago, IL, USA), and analyzed with ImageJ software version 1.46 (National Institutes of Health, Bethesda, MD, USA).

Data were expressed as the mean ± standard deviation for in vitro experiments and the mean ± standard error of mean for in vivo experiments. Software SPSS 16.0 (SPSS, Inc., Chicago. IL, USA) was used to analyze the data via one-way analysis of variance, followed by a Duncan's multiple range test. P<0.05 was considered to indicate a statistically significant difference.

IAB significantly improved the viability of >30% L-Glu-damaged PC12 cells compared with cells treated with L-Glu alone (P<0.01; Fig. 1B); however, IAB alone exhibited no signifi-cant effects on cell viability in normal PC12 cells (Fig. 1B). Compared with L-Glu-treated PC12 cells, IAB, particularly at 30 µM, significantly reduced caspase-3 activity by 39.1% (P<0.001; Fig. 1C). PC12 cells exposed to 25 mM L-Glu demonstrated an apoptosis rate of 18%, which decreased to 7.98% in 30 µM IAB pre-treated PC12 cells (P<0.05; Fig. 1D).

A significant accumulation of intracellular ROS (P<0.001; Fig. 2A) and dissipation of MMP (P<0.001; Fig. 2B) were observed in PC12 cells exposed to 25 mM L-Glu for 12 h compared with the control. Pre-incubation with IAB significantly suppressed the over-accumulation of ROS, as indicated by the reduced green fluorescence (P<0.05; Fig. 2A), and restored the dissipation of MMP, as indicated by the enhanced red/green fluorescence (P<0.01; Fig. 2B).

Significantly low expression levels of Bcl-2 (P<0.01) and Bcl-xL (P<0.01), and high expression levels of Bax (P<0.001) and cleaved casapase-3 (P<0.001) and -9 (P<0.001) in PC12 cells were noted after 24-h exposure to L-Glu compared with the control (Fig. 2C). Conversely, 3-h pre-incubation with IAB significantly reversed these alterations induced by L-Glu (P<0.01, Fig. 2C).

L-Glu exposure resulted in significant reductions in the expression of Nrf2 (P<0.01), HO-1 (P<0.01), HO-2 (P<0.05), SOD1 (P<0.001) and CAT (P<0.001) in PC12 cells compared with the control, but were strongly enhanced by IAB treatment (10 and 30 µM) after 24-h co-incubation (P<0.01; Fig. 2D).

The experimental protocol conducted with mice was presented in Fig. 3A. In addition, IAB did not exhibit adverse effects on organs, including the brain (Fig. 3B), kidneys (Fig. 3C), and spleen (Fig. 3D) of AD mice as analyzed by H&E staining.

The learning and memory abilities of mice with mice were analyzed by behavioral tests. In an open field test, a significant time spent in the central area with chaotic movement without purpose was observed in AD mice; however, this duration was significantly reduced in IAB-treated mice (P<0.01; Fig. 4A). In the Morris water maze test, compared with heathy mice, AD mice spent more time finding the platform hidden in the water and exhibited more chaotic movement. Conversely, IAB administration led to a 32.8% reduction in escape latency time (P<0.01; Fig. 4B). In the Y maze test, it took AD mice nearly twice as long to find hidden food compared with the heathy mice (P<0.001; Fig. 4C). IAB administration led to a 30.9% reduction in the seeking time for hidden food (P<0.01; Fig. 4C).

The apoptotic status of neurons was detected by TUNEL staining, and a number of TUNEL-positive cells were observed in the brains of the vehicle-treated AD mice, as indicated by the enhanced green fluorescence intensity (Fig. 4D). The number of TUNEL-positive cells was notably reduced following 4 weeks of treatment with IAB (Fig. 4D).

Central to pathogenesis of AD, the levels of Aβ, the principal constituent of neuritic plaques (28), were detected in the present study. Compared with healthy mice, significantly low serum levels of Aβ1-42 and high cerebral levels of Aβ1-42 were noted in the vehicle-treated AD mice compared with in healthy mice (P<0.05; Fig. 4E); however, IAB administration for 4 weeks resulted in a 29.8% increase in the serum levels of Aβ1-42 (P<0.05) and a 22.9% reduction in the cerebral levels of Aβ1-42 (P<0.05; Fig. 4E). Immunohistochemical analysis further confirmed that the suppressive activities of IAB on Aβ1-42 in the mouse brains resulted in fewer neuritic plaques in IAB-treated mice than in AD mice (Fig. 4F). Furthermore, the expression levels of p-Tau were notably enhanced in the brains of vehicle-treated AD mice, but were significantly reduced upon treatment with IAB (Fig. 4G).

The dysregulated levels of cholinergic neurotransmitters is responsible for the characteristic memory impairment associated with AD (29). Significant reductions in the levels of Ach (P<0.05) and ChAT (P<0.01), and increased levels of AchE (P<0.01) were observed in the brains of the vehicle-treated AD mice compared with the control (Fig. 5). After 4 weeks of treatment with IAB, a 24.9 and 25.3% increase in the cerebral levels of Ach (P<0.05; Fig. 5A) and ChAT (P<0.05; Fig. 5C) were observed, respectively, and a 30.6% reduction in the cerebral levels of AchE were detected (P<0.05; Fig. 5B). These findings suggests that may IAB improve cholinergic dysfunction.

The brain is sensitive to oxidative stress; consequently, the over-accumulation of ROS may affect the expression of β-amyloid precursor protein (APP) and mitochondrial DNA, which further leads to neuronal apoptosis (30). Compared with in healthy mice, significantly higher levels of ROS, and notably lower levels of SOD and GSH-Px were detected in the serum and brains of vehicle-treated AD mice (P<0.05; Fig. 6A-F). In the serum of AD mice, IAB significantly reduced ROS levels by 17.9% (P<0.05; Fig. 6A), and enhanced the levels of SOD by 21.4% (P<0.05; Fig. 6B) and GSH-Px by 71.6% (P<0.01; Fig. 6C). In the brains of mice with AD, IAB significantly reduced ROS levels by 29.7% (P<0.01; Fig. 6D), and enhanced the levels of SOD by 94.2% (P<0.001; Fig. 6E) and GSH-Px by 80.7% (P<0.001; Fig. 6F).

In the brains of AD mice, notably increased expression levels of pro-apoptotic proteins, including Bax (P<0.001; Fig. 6G) and cleaved caspases-3 (P<0.01; Fig. 6G) and -9 (P<0.001; Fig. 6G), and the low levels of anti-apoptotic proteins, such as Bcl-2 (P<0.01) and Bcl-xL (P<0.01), were observed (Fig. 6G). The expression levels were similar to the control after 4 weeks of treatment with IAB (P<0.01; Fig. 6G), all were strongly improved to a healthy standard after four-week IAB administration (Fig. 6G). Additionally, the notably reduced levels of proteins associated with antioxidation, including Nrf2 (P<0.001), HO-1 (P<0.01), HO-2 (P<0.01), SOD1 (P<0.001) and CAT (P<0.001), were detected in the brains of mice with AD (Fig. 6G). On the contrary, the suppressed expression of these proteins induced by the occurrence of oxidative stress within the AD process markedly recovered following IAB treatment, which resulted in >50% upregulation (P<0.01; Fig. 6G).