A total of 40 Wistar rats were selected (age, 10–11 weeks; weight, 240–260 g) from the laboratory animal room of Liaoning University of Traditional Chinese Medicine. The rats fed and drank freely at room temperature (20-22°C) with 40–50% humidity, and were maintained under a 12-h light/dark cycle. The present study was performed according to the principles and procedures of the National Institutes of Health's Guide for the Care and Use of Laboratory Animals (16). This study was approved by the Animal Care and Use Committee of the Second Hospital of Hebei Medical University.

The rat model of AD was established using Aβ1–42 (Sigma-Aldrich; Merck KGaA) as in previous studies (17–19). The Wistar male rats were randomly divided into 4 groups with 10 rats in each group: Sham, model, model + vehicle (saline) and model + CPCGI (1 ml/kg/d). Rats in the sham group were treated with saline by a gradual intracerebroventricular (icv) injection (1 µl/min) into the lateral ventricle. Rats in the model group were treated with Aβ1–42 (400 pmol/3 µl/rat) by gradual intracerebroventricular (icv) injection (1 µl/min) into the lateral ventricle (17). The AD model rats received CPCGI treatment (1 ml/kg/d; intraperitoneal injection) for 15 consecutive days starting 1 h after AD induction. Rats in the model + vehicle group received an equal amount of saline. At the end of the experiment, the rats were anaesthetized with pentobarbital (40 mg/kg, intraperitoneal injection) before being sacrificed through cervical dislocation (rats without a heartbeat that were not breathing were confirmed as dead). Subsequently, the hippocampal tissues of rats from the different groups were collected. No rats died during the experiment. Tests were terminated when the rats lost more than 15% of their body weight and every effort was made to alleviate their suffering.

On day 12 after CPCGI treatment, to assess anhedonic behavior of rats, the sucrose preference test was performed as in a previous study (17). Briefly, rats were acclimated to the two-bottle choice paradigm (two identical bottles were placed on the cages) for three days. In order to avoid withdrawal symptoms in rats, each rat was given two bottles, one containing a 2% sucrose solution and the other containing tap water. The two bottles were changed every 12 h to avoid a ‘side’ bias. The amount of the sucrose solution or tap water consumed was detected by weighing the bottles immediately before and after the test. The sucrose preference ratio was calculated as following: Sucrose preference value (%)=sucrose intake (g) ×100%/[sucrose intake (g) + water intake (g)].

Following the final CPCGI treatment, the tail suspension test was performed as previously described (17). In brief, every rat was individually suspended by the tail using a clamp, 3–4 cm from the end, in a gray wooden enclosure (60×30×20 cm). A square platform was placed under the rat's forepaws and lightly touching them to avoid hemodynamic stress and limb pain. The immobility time was recorded (in seconds) during the 5-min test period.

Rat adrenal pheochromocytoma (PC12) cells were purchased from American Type Culture Collection (ATCC, cat. no. CRL-1721). CPCGI was obtained from Jilin Buchang Pharmaceutical Co., Ltd. DMEM high-sugar medium, horse serum, and fetal bovine serum were purchased from Gibco (Thermo Fisher Scientific, Inc.).

PC12 cells were cultured in DMEM containing 10% fetal bovine serum and 5% horse serum, and cultured at 37°C with 95% humidity and 5% CO₂ (20). PC12 cells were divided into control, model, model + vehicle (PBS) and model + CPCGI. In the model + vehicle and model + CPCGI groups, PC12 cells were pre-treated with PBS and CPCGI separately for 1 h before stimulation with 50 µM Aβ25–35 (Sigma-Aldrich; Merck KGaA) for 24 h. PC12 cells in the model group were stimulate with 50 µM Aβ25–35 for 24 h (21). Cells in the control group were not subjected to any treatment. Subsequently, PC12 cells in each group were subjected to the following experiments.

Total RNA from tissues (100 mg) was collected by using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using PrimeScript RT Reagent Kit (Takara Bio, Inc.) according to the manufacturer's protocols. The temperature protocol for the reverse transcription reaction was 25°C for 5 min, 42°C for 60 min and 80°C for 2 min. qPCR was performed to analyze gene expression using the cDNA the SYBR RT-PCR kit (Takara Bio, Inc.) according to the manufacturer's protocols. The thermocycling conditions were as follows: 95°C for 5 min, followed by 38 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 30 sec. Primer sequences were: GAPDH, forward 5′-CTTTGGTATCGTGGAAGGACTC-3′; reverse 5′-GTAGAGGCAGGGATGATGTTCT-3′; Aβ42, forward 5′-ATGGCGAGCAAAGTCTCGATC-3′; reverse 5′-CGCAATCACCACGCCGCCCAC-3′. GAPDH was used as the internal control, and the gene expression was quantified by the 2^−ΔΔCq method (22).

Protein expression was detected using western blotting. Radioimmunoprecipitation assay buffer (Auragene Bioscience) was used to extract the proteins from hippocampus or PC12 cells. A bicinchoninic acid protein quantitative kit (Thermo Fisher Scientific, Inc.) was used to detect protein concentrations in line with the manufacturer's instructions. 10% SDS-PAGE gel electrophoresis was used to isolate proteins (30 µg/lane), and then the proteins were transferred onto PVDF membranes (EMD Millipore). 5% skimmed milk was used to block the membrane at room temperature for 1 h, and then the membranes were incubated with primary antibodies: β-Amyloid (for Aβ42 and Aβ40 detection; cat no. 8243; 1:1,000; Cell Signaling Technology, Inc.), Bcl-2 (cat no. ab196495; 1:1,000; Abcam), Bax (cat no. ab32503; 1:1,000; Abcam), cleaved caspase3 (cat no. ab49822; 1:1,000; Abcam), pro-caspase3 (cat no. ab183179; 1:1,000; Abcam), phosphorylated (p)-p38 (cat no. ab4822; 1:1,000; Abcam), p38 (cat no. ab170099; 1:1,000; Abcam), p65 (cat no. ab16502; 1:1,000; Abcam), p-p65 (cat no. ab86299; 1:1,000; Abcam), and β-actin (cat no. ab179467; 1:1,000; Abcam) at room temperature for 3 h. Subsequently, the PVDF membranes were hybridized with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) for 1 h. Finally, ECL reagent (Applygen Technologies, Inc.) was used to visualize protein bands. The band density was semi-quantified with Gel-Pro Analyzer densitometry software (version 6.3, Media Cybernetics, Inc.).

The expression of TNF-α (cat. no. PT516) and IL-1β (cat. no. PI303) in the hippocampus of rats from different groups were detected using ELISA kits (Beyotime Institute of Biotechnology) according to the manufacturers instructions.

To determine SOD activity (cat. no. S0101) and MDA content (cat. no. S0131) in the hippocampus of rats, commercial colorimetric detection kits (Beyotime Institute of Biotechnology) were performed following the manufacturer's instructions.

A Reactive Oxygen Species Assay kit (cat. no. S0033, Beyotime Institute of Biotechnology) was used to determine the production of ROS in the hippocampus of rats according to the manufacturer's instructions. The ROS level was expressed as the percentage of the dichlorofluorescein (DCF) fluorescence level in control group whose DCF level was set to 100%.

Cell viability was determined by the conventional MTT assay. Following treatment, PC12 cells were seeded in 96-well plates (10⁴ cells/well) and incubated at 37°C for 24 h. Then MTT solution (10 µl) was added to each well and the cells were incubated for further 4 h at 37°C. Subsequently, 100 µl DMSO (Nanjing KeyGen Biotech Co., Ltd.) was used to dissolve the formazan crystals. Finally, the absorbance was measured at the wavelength of 490 nm by using a micro-plate reader (Synergy2, BioTek Instruments, Inc.).

Flow cytometry (BD Accuri Flow Cytometer, BD Biosciences) was performed to analyze cell apoptosis. PC12 cells were treated with or without CPCGI for 48 h. Then, the cell apoptosis was determined by using the Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit [cat. no. 70-AP101-100; Multisciences (Lianke) Biotech Co., Ltd.] in accordance with the manufacturer's protocols. FlowJo 7.6 software (FlowJo LLC) was used to analyze the cell apoptosis rate.

Statistical analysis was performed using SPSS 18.0 (SPSS, Inc.). All experiments were performed three times. Data are presented as mean ± standard deviation. The differences between groups were analyzed by one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To study the therapeutic effect of CPCGI on AD, the rat model of AD was constructed and then treated with CPCGI. It was found that the reduced the percentage of sucrose preference of rats induced by Aβ1–42 was significantly increased by CPCGI treatment (Fig. 1A). The enhanced immobility time of rats caused by Aβ1–42 administration was significantly reduced by CPCGI treatment (Fig. 1B).

Results suggested that compared with sham group, the protein expression of Aβ40 and Aβ42 in the hippocampus of AD model rats was markedly increased, while CPCGI significantly decreased the expression of Aβ40 and Aβ42 (Fig. 2A). Compared with the sham group, the mRNA expression of Aβ40 and Aβ42 in the hippocampus of AD model rats was significantly increased, while CPCGI significantly decreased the mRNA expression of Aβ40 and Aβ42 (Fig. 2B and C). Compared with the rats from the sham group, the production of TNF-α and IL-1β in the hippocampus of AD model rats was significantly increased, and these increases were inhibited by CPCGI treatment (Fig. 2D and E). In addition, the content of MDA in the hippocampus of Aβ1–42 induced rats was significantly increased (Fig. 2F), the activity of SOD was significantly decreased (Fig. 2G), and the ROS level significantly enhanced (Fig. 2H). CPCGI treatment significantly reduced the content of MDA, increased the activity of SOD, and decreased ROS level in the hippocampus of AD model rats (Fig. 2F-H).

In order to detect the protein expression of Aβ40 and Aβ42, western blot analysis was used. The results demonstrated that the protein expression of Aβ40 and Aβ42 in Aβ25–35 induced PC12 cells increased significantly compared with the control group, and that CPCGI could decrease the protein expression of Aβ40 and Aβ42 in Aβ25–35 induced PC12 cells (Fig. 3A-C).

MTT assay was used to investigate the effect of CPCGI on the viability of PC12 cells. The results demonstrated that compared with the control group, the viability of PC12 cells was significantly decreased in Aβ25–35 treated PC12 cells. CPCGI treatment could significantly increase the viability of Aβ25–35 induced PC12 cells (Fig. 4A).

Flow cytometry was used to analyze the effect of CPCGI on apoptosis in PC12 cells. Compared with the control group, the apoptosis of PC12 cells in AD cell model group increased significantly. The apoptosis of Aβ25–35 induced PC12 cells (Fig. 4B and C) was significantly reduced by CPCGI treatment. In addition, the protein expression of Bcl2 (Fig. 4D and E) and pro-caspase3 decreased (Fig. 4D and H), while the protein level of Bax (Fig. 4D and F) and cleaved-caspase3 (Fig. 4D and G) increased significantly in Aβ25–35 induced PC12 cells, and these changes were inhibited by CPCGI treatment (Fig. 4D-H).

To explore the mechanism by which CPCGI affected AD, the p38 mitogen-activated protein kinase (MAPK)/NF-κB pathway was analyzed. In addition, the protein expression of p38, p65, p-p38 and p-p65 in PC12 cells was detected using western blot analysis. The results demonstrated that the protein expression of p-p38 and p-p65 and the ratio of p-p38/p38 and p-p65/p65 in Aβ25–35 induced PC12 cells were significantly higher than that in the control group. CPCGI treatment significantly reduced the protein expression of p-p38 and p-p65 and the ratio of p-p38/p38 and p-p65/p65 in Aβ25–35 induced PC12 cells (Fig. 5A-C), indicating the inhibitory effect of CPCGI on p38MAPK/NF-κB pathway in Aβ25–35 induced PC12 cells.