All the experiments were performed with the approval of the Ethics Committee of the Tianjin Medical University (Tianjin, China). APPswe/PS1ΔE9 mice (n=20; 3 months, male, 26–30 g) and C57BL/6J mice (n=20; 3 months, male, 23–26 g) in clean grade were obtained from Beijing Huafukang Biological Technology company (Beijing, China) and raised in the temperature of 25±2°C, relative humidity 70% and in a 12-h light/dark room at with free access to food and water. Following 1 week of adaption, all APPswe/PS1ΔE9 mice were randomly divided into either the AD model group or the salidroside + AD model group (n=10 in each group). C57BL/6J mice were also randomly divided into either the normal control (NC) group or the salidroside + NC group (n=10 in each group). The mice in the salidroside + NC and salidroside + AD model groups were administered salidroside (30 mg/kg) orally once daily for 3 consecutive months. Following treatment, behavioral tests and biochemical experiments were performed.

Following the 3 months of salidroside treatment, the cognitive behavior of all of the mice was investigated using MWM (17), which measures spatial learning and memory ability. The animals were subjected to a daily session of four training trials for 4 consecutive days. In each training session, mice were placed into the pool at four different starting points (different quadrants). The mice were then permitted to find the platform within a maximum time period of 120 sec and following that the mice were allowed to remain on the platform for a further 30 sec. If a mouse was unable to find the platform within the 120 sec time period, the mouse would then be guided to the platform by the experimenter, and the escape latency was recorded as 120 sec. At day 5 of spatial testing, the platform was removed and mice were placed into water at any starting point. The time period that the mouse remained in the quadrant where the platform had previously been placed was then recorded.

Detection of hippocampal neuronal apoptosis was carried out using the TUNEL assay. The hippocampal tissue was fixed in 4% formalin at 4°C for 48 h and then embedded in paraffin wax. Apoptosis rates were detected using the In Situ Cell Death Detection kit (Roche Applied Science, Rotkreuz, Switzerland) according to the manufacturer's instructions. Apoptotic rate changes were measured via a light microscope (Olympus Corporation, Tokyo, Japan). Hippocampal tissue was fixed in 4% paraformaldehyde for 48 h at 4°C and embedded into paraffin wax. In Situ Cell Death Detection kit was used to detect the apoptosis according to the manufacturer's instructions. The images were taken using a light microscope (Olympus Corporation). Apoptotic rates were then determined according to the method previously detailed by Soslow et al (18). A positive expression rate of 0–1% was defined as score 0 (negative), 1–10% as score 1 (weakly positive), 10–50% as score 2 (positive), 50–80% as score 3 (medium positive) and 80–100% as score 4 (strongly positive). Three sections were used for each group, and five fields were randomly selected from each section (magnification, ×400).

Following the MWM test, all of the mice were anaesthetized, immediately sacrificed, and brain tissues were quickly isolated. Following this, the brains were separated into two cerebral hemispheres via incision along the midline. Hippocampal tissue of CA1 region was then isolated from one of the cerebral hemispheres of each animal and then immediately frozen in liquid nitrogen for subsequent SOD and MDA testing. Hippocampal SOD (cat. no. ab65354) and MDA (cat. no. ab65354) (both from Abcam, Cambridge, UK) activity were then detected usinga microplate reader (wavelength, 210 nm).

Colorimetric reaction using Griess reagent was used to investigate the nitrate concentration in the hippocampal tissue of the mouse. The Nitric Oxide Assay kit was purchased from Abcam, and all procedures were performed out in accordance with the manufacturer's instructions. The results of the hippocampal nitrate concentrations of the different groups were expressed as µg/mg protein.

Firstly, the hippocampal rat samples were precipitated by the addition of 5% sulfosalicylic acid and protein-free supernatant was removed by centrifugation at 2,000 × g for 10 min. A sample of 100 µl protein-free supernatant of the cell lysate was then added to 800 µl 0.3 mM Na₂HPO₄ and 100 µl 0.04% 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) in 0.1% sodium citrate at 4°C overnight. Following this, the absorbance of DTNB was monitored using a spectrophotometer at 412 nm for 5 min. A standard curve of GSH was established, and the sensitivity of measurement was determined to be between 1–100 µM. The results of the GSH concentrations in the samples were expressed as µg/mg protein.

The concentrations of TNF-α and IL-6 in the hippocampal tissue of the mice were determined using ELISA. ELISA kits for TNF-α (cat. no. ab46070) and IL-6 (cat. no. ab100772) were purchased from Abcam. All procedures were performed in accordance with the manufacturer's instructions. The results of the determined hippocampal concentrations of TNF-α and IL-6 were expressed as µg/mg protein.

Statistical analysis was performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). All data were expressed as the mean ± standard deviation of the mean. One-way analysis of variance was used to compare differences among three or more groups, and this was followed by Student Newman-Keuls-test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

MWM was used to investigate the effects of salidroside administration on the learning and memory abilities of mice. As revealed in Fig. 1A, the escape latency time of the AD group was longer than that of the normal group, and there was no significant difference between the normal and the salidroside + NC groups. Furthermore, the escape latency of the mice in the salidroside + AD model group was significantly reduced compared with the escape latency of the AD model group. As demonstrated in Fig. 1B, the time period that the AD model group spent in the target quadrant was significantly less than that spent by the normal group. Additionally, there was no significant difference in the time period spent in the target quadrant between the NC and the salidroside + NC groups. Furthermore, the time period that the mice of the salidroside + AD model group spent in the target quadrant was significantly higher than the time spent by the AD group, but significantly less than the time spent by the NC group. Results in the salidroside + AD group, the escape latency was significantly shortened, the time of swim in the platform quadrant significantly increased, suggesting that mice significantly improved learning and memory to escape the incubation period (P<0.05; Fig. 1).

As revealed by Fig. 2, the number of neurons undergoing apoptosis in the CA1 hippocampal region was significantly increased in the AD group compared with the other groups. However, the administration of salidroside reduced this effect, as there was a significant reduction in neuronal apoptotic rates between the salidroside + AD group and the AD group.

The activity of SOD and the concentration of GSH in hippocampal tissue were investigated. As demonstrated in Fig. 3A, the relative SOD activity was significantly decreased in the AD group compared with the NC group; whereas SOD activity was upregulated in the salidroside + AD group compared with the AD group. Furthermore, there was no significant difference between the SOD activity of the NC group and of the salidroside + NC group. Compared with the NC group, the GSH concentration was decreased in the AD group; whereas GSH concentration was upregulated in the salidroside + AD group compared with the AD group. In addition, there was no significant difference between the GSH concentration of the NC group and the salidroside + NC group (Fig. 3B).

The hippocampal concentrations of MDA and GSH in mice were also investigated. As revealed in Fig. 4, in comparison with the NC group, MDA and nitrate concentrations were both increased in the AD group; whereas both MDA and nitrate concentrations were downregulated in the salidroside + AD group compared with the AD group. Furthermore, there was no significant difference between the MDA and nitrate concentrations of the NC group and the salidroside + NC group.

The effects of salidroside administration with regards to induced inflammation were investigated. The expressions of TNF-α and IL-6 inflammatory factors decreased significantly in the hippocampus of the salidroside + AD group compared with mice belonging to the AD group (Fig. 5). There was no significant difference between the expression levels of TNF-α and IL-6 of the NC group and the salidroside + NC group.