A Scutellaria baicalensis root was provided from Jilin Northeast Asia Pharmaceutical Co., Ltd. (Yanji, China). Extract standards were obtained from the Jilin Institutes for Food and Drug Control (China). The Scutellaria baicalensis root was dried and crushed, soaked for 1.5 h in a 10-fold water volume (w/v), and subsequently refluxed three times for 30 min. Polyamide column chromatography was used to separate Scue and monitored using high-performance liquid chromatography (HPLC). Measurements were performed using HPLC U3000 with a Corona^® charged aerosol detector (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A XB-C18 (250×4.6 mm; 5 µm) column (Welch Materials, Inc., Hurst, TX, USA) was used for sample separation at 30°C. A 10 µl injection volume with 20:80 gradient was used with acetonitrile as mobile phase A and 0.1% (v/v) formic acid in water as mobile phase B. The flow rate was set to 0.8 ml/min.

Rat pheochromocytoma (PC12) cells were purchased from The Type Culture Collection of The Chinese Academy of Medical Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, Inc.) supplemented with 8% fetal bovine serum and 8% horse serum (Gibco; Thermo Fisher Scientific, Inc.), and maintained at 37°C in a humidified atmosphere of 5% CO². For the experimental treatments, cells were pre-incubated with different concentrations (0, 5, 10, 15 and 30 µg/ml) of the test compound (Scue) and (0, 5, 10, 15 and 30 µg/ml) of the test compound (Scu) dissolved in 0.2% dimethyl sulfoxide (DMSO) in DMEM for 4 h and subsequently treated with 2 µg/ml Aβ for 24 h (7).

Cell viability was determined using a MTT assay. Cells were seeded in 96-well microplates at a density of 2×10³ cells/well and incubated for 24 h to allow the cells to adhere. Subsequently, MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a final concentration of 0.2 mg/ml was added to each well and incubated at 37°C for 72 h. Following incubation, the cell supernatants were removed and the remaining formazan crystals were dissolved in 200 µl DMSO for 15 min. The optical density of each well was determined using an ELX-800 spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) at 490 nm.

A flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) and an apoptosis detection kit (Biogot Technology Co., Ltd., Nanjing, China) were used according to the manufacturer's protocol to detect apoptotic cells. Cells were harvested, centrifuged at 2–8°C for 10 min at 1,500 × g), washed and subsequently resuspended in 400 µl binding buffer. A total of 5 µl Annexin V-fluorescein isothiocyanate was added to each cell suspension and the suspension was incubated at 2–8°C for 15 min in the dark. Subsequently, 10 µl propidium iodide was added and the suspension was incubated at 2–8°C for 5 min in the dark. Flow cytometry was performed within 1 h of the final step. FlowJo software (version 7.6; FlowJo LLC, Ashland, OR, USA) was used for analysis.

Cells were fixed in 4% paraformaldehyde for 15 min at room temperature and subsequently gently rinsed in PBS. To permeabilize the cells, they were incubated for 30 min in PBS containing 0.1% Triton X-100. Cells were blocked with 8% goat serum (Gibco; Thermo Fisher Scientific, Inc.) for 15 min at room temperature and each slide was incubated with NF-κB p65 primary antibody (1:100; cat. no. bs-3543R; BIOSS, Beijing, China) overnight at 4°C, and Cy3-labeled goat anti-rabbit secondary antibody (1:200; cat. no. A0516; Beyotime Institute of Biotechnology, Haimen, China) in the dark at room temperature for 60 min. The cells were counterstained with DAPI for 10 min at room temperature. Cells were viewed under a fluorescent microscope (Olympus Corporation, Tokyo, Japan; magnification, ×400) subsequent to adding anti-fading reagent.

Healthy male Wistar rats (n=24; age, 6–8 weeks; weight, 200 g) were purchased from the Laboratory Animal Center of Jilin University (Changchun, China). Animals were housed in laboratory facilities under the following conditions: Temperature, 21.0–25.0°C; relative humidity, 40.0–70.0%; complete air replacement 15 times/h; and 12-h light/dark cycle. Prior to the experiments, the rats were group-housed in a pathogen-free grade animal room with ad libitum access to food and water and allowed to acclimate for 1 week. The Animal Ethics Committee of Jilin University approved the experimental protocol.

Each rat was randomly assigned to one of four groups: Control, Aβ, Scue (50 mg/kg; intragastrically) or Scu (50 mg/kg; intraperitoneally). Non-control rats were anesthetized with sodium pentobarbital (60 mg/kg; intraperitoneally) and injected in the deep frontal cortex (3.2 mm anteroposterior, 2 mm dorsoventral relative to bregma; depth 3 mm) with 3 µl Aβ (10 ng/µl) (28), whereas, control rats received an injection of 3 µl saline. Following these injections, rats were subjected to treatment and testing over a period of 4 weeks.

All the mice were sacrificed with CO² (the displacement rate of CO² was 10% chamber volume/min) for 0.5 h subsequent to behavioral testing (7 days), and hippocampal tissues were dissected. Subsequently, one-half of the hippocampal tissues were frozen in liquid nitrogen and stored at −70°C for future protein analyses. The other tissues were fixed for 12 h in 4% paraformaldehyde at room temperature, and embedded in paraffin for sectioning and staining.

The MWM test includes an acquisition period and a special probe trial, and is a well-established method for the evaluation of learning and memory in animals (29). In the present study, the acquisition period lasted for 6 days, with two training sessions per day and a 15 min interval between sessions. Each animal was placed at a designated starting point in a different quadrant of the maze, facing the tank wall. In one quadrant, a platform was hidden just below the surface of the water. If a rat identified and climbed onto the platform, and remained there for >3 sec, the swimming time was documented as the latency to locate the platform. If a rat failed to locate the platform within 120 sec, it was manually placed onto the platform for 30 sec and the latency was recorded as 120 sec.

The spatial probe trial was only performed once. The platform was removed from the pool and the rat was placed at any starting point that was not adjacent to the platform. The number of times that each rat swam across the original location of the platform within 120 sec was documented.

Cells [cytoplasmic and nuclear NF-κB were separated by the membrane protein separation method (30)] were lysed in nonyl-phenoxypolyethoxylethanol-40 lysate (Beyotime Institute of Biotechnology), and total protein and nucleoproteins from the hippocampal tissues were extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using the bicinchoninic acid method (Beyotime Institute of Biotechnology). For each sample, 40 µg total protein was run on an SDS-PAGE gel (15%), followed by transfer to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked for 1.5 h in 5% BSA (cat. no. A8806; Sigma-Aldrich; Merck KGaA) at 2–8°C. The membranes were incubated with primary antibodies against nuclear factor of κ-light polypeptide gene enhancer in B cells inhibitor α [IκBα; cat. no. YS-(kt)-0907, phospho (p)-IκBα (cat. no. YS-KT1551) (Shanghai Yansheng Biochemical Reagent Co., Ltd., Shanghai, China), NF-κB, cleaved caspase-3 (cat. no. bs-0081R), B cell lymphoma-2 (Bcl-2; cat. no. bs-20351R), apoptosis regulator BAX (Bax; cat. no. bs-4564R), β-actin (cat. no. bs-0061R) and lamin-A (cat. no. bs-1839R) (1:1,000; BIOSS) overnight at 4°C. Subsequently, the membranes were incubated with goat anti-rabbit immunoglobulin G-horseradish peroxidase antibodies (1:5,000; cat. no. A0208; Beyotime Institute of Biotechnology) at room temperature for 1 h, followed by chromogenic detection using an enhanced chemiluminescence reagent (cat. no. WP20005; Thermo Fisher Scientific, Inc.) Films were scanned and analyzed using Gel-Pro-Analyzer 4.0 software (Media Cybernetics, Inc., Rockville, MD, USA), and the optical densities of the target bands were quantified relative to β-actin.

Paraffin-embedded hippocampal tissues were cut into 5 µm sections, processed with conventional Nissl staining (31), and examined under a light microscope (magnification, ×400).

Commercially available ELISA kits were used to detect interleukin-6 (IL-6; cat. no. ml002293), tumor necrosis factor-α (TNF-α; cat. no. ml002095), interferon-γ (IFN-γ; cat. no. ml027464), superoxide dismutase (SOD; cat. no. ml643059), malondialdehyde (MDA; cat. no. ml077384), acetylcholine (Ach; cat. no. ml401805) (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) and Aβ (cat. no. JL14446-48T; Shanghai Jianglai Industrial Ltd., Shanghai, China) expression levels in the hippocampal tissues, according to the manufacturer's protocols.

Data are presented as the mean ± standard deviation. In vitro experiments were repeated three times, whereas in vivo experiments were conducted in six rats. Comparisons between groups were performed using one-way analysis of variance and Bonferroni post hoc tests. GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used to analyze all the data and images. P<0.05 was considered to indicate a statistically significant difference.

Scue was obtained as described above. The results demonstrated that a single symmetrical peak was identified by HPLC; the shape of the peak was sharp and steep, and the retention time was the same as the reference substance (Fig. 1).

Cell viability was assessed by an MTT assay in order to determine the effects of Scue on Aβ-induced cell death. To determine the proper concentration ranges for the study of neuroprotective effects, the cytotoxicity of each test compound was evaluated using PC12 cells. Scue did not demonstrate any detectable toxicity at any of the concentrations tested (Fig. 2A). The protective effects of Scue on Aβ-induced cell death were measured. Treatment with 2 µg/ml Aβ for 24 h decreased cell viability by 31%, whereas, pretreatment with Scue (30 µg/ml) significantly attenuated Aβ-induced cell death (Fig. 2B; P<0.01). Aβ-induced cell death was further investigated by flow cytometry. Treatment with Aβ (2 µg/ml) significantly increased apoptosis compared with the control group; however, pretreatment with Scue (30 µg/ml) significantly decreased the number of apoptotic cells compared with treatment with Aβ alone (P<0.001). Apoptotic ratios with respect to the control group were as follows: 24.0% in the Aβ (2 µg/ml) treatment group; 13.4% in the Scue (30 µg/ml) treatment group; 10.8% in the Scu (30 µg/ml) treatment group. Therefore, Scue and Scu decreased the apoptotic ratios (Fig. 2C and D).

Western blotting was used to evaluate the expression of NF-κB p65 in cells. Compared with the control group, cytoplasmic NF-κB was significantly decreased by 54% (P<0.01; Fig. 3A) and nuclear NF-κB was significantly increased by 1.549-fold (P<0.001; Fig. 3B) in the Aβ group. Scue and Scu increased the expression level of cytoplasmic NF-κB compared with the Aβ group (P<0.01 and P<0.05, respectively; Fig. 3A), and decreased the expression level of nuclear NF-κB compared with the Aβ group (P<0.01 and P<0.05, respectively; Fig. 3B). This suggested that Scu and Scue inhibited the NF-κB signaling pathway.

Immunofluorescence staining demonstrated that NF-κB p65 was primarily expressed in the cytoplasm of the cells, and the NF-κB p65 expression in the nucleus was increased in the Aβ group compared with the control group (Fig. 3C). Treatment with Scu and Scue reversed the Aβ-induced alterations in NF-κB p65 localization. These results suggested that Scue may inhibit activation of the NF-κB signaling pathway in Aβ-induced injury of PC12 cells.

The MWM task was used to investigate the effects of Scue on Aβ-induced spatial memory impairments. Rats in the Aβ group at day 6 demonstrated significantly longer latencies to locate the platform (68.25±8.67 sec) compared with the control group (31.90±5.68 sec; P<0.001; Fig. 4A). Treatment with Scue significantly attenuated the effects of Aβ-induced latency to locate the platform during the acquisition period and increased the number of platform crossings in the spatial probe trial (Fig. 4B; P<0.001).

ELISA was used for the analysis of hippocampal tissues from each of the groups. The Aβ content was increased 1.45-fold in the Aβ group relative to the control group (Fig. 4C; P<0.01), and decreased 0.05-fold in the Scue group compared with in the Scu group (P<0.01). SOD expression was decreased 0.35-fold in the Aβ group (Fig. 4D; P<0.01), and increased 0.04-fold in the Scue group compared with in the Scu group (P<0.05).

The MDA content in the hippocampal tissues was significantly increased in the Aβ group by 118% relative to the control (P<0.01; Fig. 4E), and decreased 0.05-fold in the Scue group compared with in the Scu group (P<0.05). Hippocampal Ach content was significantly decreased by 79% relative to the control (P<0.01; Fig. 4F), and increased 0.06-fold in the Scue group compared with in the Scu group (P<0.05). These observations were significantly reversed by treatment with Scu/Scue, with Scue presenting more prominent effects (P<0.05).

Nissl staining was used to assess neurons in the hippocampus. Fewer neurons were labeled in the Aβ group compared with the control group. Furthermore, numerous cells demonstrated an irregular arrangement and loss of cytomorphology in samples from the Aβ group. Treatment with Scu/Scue improved the cytomorphology of the hippocampal neurons (Fig. 4G).

Western blotting was used to evaluate the hippocampal expression of proteins associated with apoptosis, including Bcl-2, Bax and cleaved caspase-3. The expression level of Bcl-2 in the Aβ group was reduced to 57% of that in the control group (P<0.01; Fig. 4H), whereas, expression levels of Bax (1.34-fold; P<0.01; Fig. 4H) and cleaved caspase-3 (2.60-fold; P<0.001; Fig. 4H) were increased. In the Scue 50 mg/kg group, expression levels of Bcl-2, Bax and cleaved caspase-3 were similar to those observed in the control group (Fig. 4H). In general, treatment with Scu/Scue attenuated the Aβ-induced alterations in the hippocampus, with Scue presenting more prominent effects compared with Scu.

To assess the mechanism of the effects of Scue on Aβ-induced alterations in the hippocampus, the expression levels of TNF-α, IFN-γ and IL-6 were determined by ELISA. Compared with the control group, the expression levels of TNF-α (P<0.01; Fig. 5A), IFN-γ (P<0.05; Fig. 5B) and IL-6 (P<0.01; Fig. 5C) in the hippocampi of Aβ-treated rats were significantly increased; however, these increases were significantly decreased by treatment with Scue (all P<0.05; Fig. 5A-C) compared with treatment with Aβ alone. Subsequently, the NF-κB signaling pathway was investigated: Hippocampal expressions levels of IκBα and p-IκBα in addition to expression levels of NF-κB in the cytoplasm and the nucleus were determined by western blotting. Compared with the control group, in the Aβ group, the phosphorylation of IκBα level was significantly increased and the expression of hippocampal IκBα was significantly decreased (P<0.001; Fig. 5D and E). The ratio of p-IκBα/IκBα was 83.3% in the control group and 300% in the Aβ group (P<0.001; Fig. 5F), and cytoplasmic NF-κB was reduced to 64% of control (P<0.01; Fig. 5G). Nuclear NF-κB was significantly increased by 1.54-fold (P<0.01; Fig. 5H). Scue significantly increased the expression of IκBα (P<0.05) and decreased the phosphorylation of IκBα (P<0.01; Fig. 5D and E). Scue additionally increased the cytoplasmic NF-κB (P<0.05; Fig. 5G) and decreased the nuclear NF-κB (P<0.05; Fig. 5H) compared with the Aβ group. Aβ-associated alterations in NF-κB were significantly attenuated by treatment Scue, suggesting that Scue decreased the phosphorylation of IκBα and subsequently inhibited activation of NF-κB signaling in the hippocampus.