PSPC (purity, >90%) was purchased from Qingdao Pengyuan Natural Pigment Research Institute (Qingdao, China). Unless otherwise noted, all reagents and drugs were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Bioreagents for western blotting were purchased from Promega Corporation (Madison, WI, USA) or Cell Signaling Technology, Inc. (Danvers, MA, USA).

A total of 60 male C57BL/6 mice (age, 8 weeks; weight, 20–24 g; Jackson Laboratory, Ben Harbor, ME, USA) were used in the present study. Mice were housed in a ventilated and temperature-controlled room (humidity, 60%; temperature, 23±1°C) with a 12 h light-dark cycle and free access to rodent chow and tap water; 3 mice were housed per cage. Following acclimation to the laboratory conditions, mice were randomly divided into the following 4 groups (15 mice/group): i) Control group, which were fed a normal diet containing 10 kcal% fat, 20 kcal% protein and 70 kcal% carbohydrate (cat. no. D12450B; Research Diets, Inc., New Brunswick, NJ, USA); ii) HFD group, which were fed a HFD containing 60 kcal% fat, 20 kcal% protein and 20 kcal% carbohydrate (cat. no. D12492; Research Diets, Inc.); iii) HFD + PSPC group, which were fed a HFD and were given PSPC (700 mg/kg body weight/day; in 0.9% normal saline) by oral gavage for 20 weeks, based as previously described (21,23); and iv) PSPC group, which were fed a normal diet and were given the same daily dose of PSPC as the HFD + PSPC group. Following 20 weeks, mice were sacrificed and whole brain tissues were collected for experimental use or stored at −80°C. All animal care and experimental procedures were in accordance with the Chinese legislation on the use and care of laboratory animals, and experiments were approved by the Animal Supervision Committee of Chengdu Medical College (Sichuan, China). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (33).

Open field testing was performed as previously described (27), with minor modifications, to evaluate the effects of HFD and PSPC treatment on mouse behavior. Locomotor activity and exploratory behavior were examined in a circular arena (diameter, 50 cm; height, 30 cm) with a 40 W bulb (3,000 lux). The floor of the arena was divided into 21 equal units by white lines. Mice were allowed to acclimate for 1 min at the center of the arena prior to the experiment, the spontaneous behavioral variables were recorded within 5 min and were classified into 4 mutually exclusive categories, including: i) The distance mice travelled; ii) the speed mice travelled; iii) the number of rearing and leaning events; and iv) the number of grooming sessions.

Passive avoidance performance testing was performed as previously described (34), using an apparatus composed of two compartments, one illuminated and one dark, with a small hole in the wall separating the two. The illuminated compartment was lit with a 25 W bulb and the dark one contained a metal floor to conduct foot shocks (36 V; 50 Hz). Each mouse was placed in the dimly lit room 3 min prior to the experiment to acclimate to the novel environment within the apparatus. Latency was defined as the interval between the initial entry into the hole and fully entering the dark compartment. Mice were placed individually into the illuminated compartment facing away from the hole in the wall. Once the mouse escaped to the dark compartment, a mild foot shock was administered for 5 sec and the mouse was immediately removed from the apparatus. Following 24 h, the mouse was placed in the illuminated compartment once more, following the aforementioned method, to carry out the retention test. The step-through latency within 5 min was recorded. The test was conducted on 2 consecutive days after the open-field testing.

Body fat content (FC) was assessed using nuclear magnetic resonance-magnetic resonance imaging-based technology (EchoMRI LLC, Houston, TX, USA). At 20 weeks after the administration, blood samples were taken by tail venipuncture using heparin-coated capillary tube, and all rats were sacrificed by cervical dislocation without the use of an anesthetic. Blood samples were centrifuged at 1,000 × g for 10 min at 4°C to remove cell debris, and the serum was stored at −20°C until measurements were taken. Serum levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol were determined using the following reagent kits according to the manufacturer's protocol. TC assay kit (cat. no. A111-2), TG assay kit (cat. no. A110-2), HDL assay kit (cat. no. A112-2) and LDL assay kit (cat. no. A113-2), were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All experiments were performed in triplicate.

Following sacrifice, mice were perfused with 50 mM chilled phosphate-buffered saline solution (PBS; pH 7.40). Mouse brains were harvested and the tissues were homogenized in PBS containing 0.1% phenylmethylsulfonyl fluoride (Sigma-Aldrich; Merck KGaA). Following centrifugation at 16,000 × g for 15 min at 4°C, the supernatant was collected. Quantitative measurements of the expression levels of inflammatory cytokines interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α and IL-10 in mouse brains were performed with the following commercially ELISA kits according to the manufacturer's protocol: IL-6 (cat. no. R6000B), IL-1β (cat. no. RLB00), TNF-α (cat. no. RTA00), IL-10 (cat. no. R1000), obtained from R&D Systems, Inc. (Minneapolis, MN, USA). ELISAs were conducted using an Infinite M200 PRO automated microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at a wavelength of 540 nm. All samples were tested in triplicate.

For western blot analysis, whole brain tissues were homogenized with CelLytic M lysis reagent and a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Nuclear and cytoplasmic extracts for western blotting were obtained using the NE-PER Nuclear and Cytoplasmic Extraction reagents according to the manufacturer's protocol (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein concentrations were determined by the Bradford assay method. Samples were prepared with 5X loading buffer, then equal amounts (40 µg) of total protein were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes, which were rinsed with Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and then blocked in TBS-T containing 5% nonfat dry milk for 1 h at room temperature. Membranes were incubated with the anti-iNOS rabbit mAb (cat. no. 13120; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-COX-2 rabbit mAb (cat. no. 12282; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-IL-6 rabbit mAb (cat. no. 12153; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-IL-1β rabbit mAb (cat. no. 12242; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-IL-10 rabbit mAb (cat. no. 12163; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-TNF-α rabbit mAb (cat. no. 11948; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-β-actin rabbit mAb (cat. no. 4970; dilution, 1:5,000; Cell Signaling Technology, Inc.) overnight at 4°C, followed by washes in TBS-T and incubation with the horseradish peroxidase-conjugated secondary antibodies (cat. no. sc-2357; dilution, 1:10,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature. Membranes were washed with TBS-T and bands were visualized using an enhanced chemiluminescence reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The intensity of the bands was analyzed using Quantity One software (version 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The expression levels of all proteins in each sample were normalized to β-actin levels or K-70 protein, and the experiment was repeated three times.

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, Inc., Chicago, IL, USA) software, version 16.0 for Windows. Data are presented as the mean ± standard error of the mean. One-way analysis of variance followed by a Student-Newman-Keuls post hoc test, or an unpaired Student's t-test was used where appropriate for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

A number of studies have demonstrated that a HFD is a risk factor for obesity or obesity-associated disease (6–8). In the present study, mice fed a HFD for 20 weeks exhibited a significant increase in body weight gain (BWG) and FC compared with mice in the Control group that were fed a normal diet (Table I). In addition, serum levels of TG, TC and LDL were significantly elevated, whereas serum levels of HDL were significantly decreased in HFD-treated mice compared with the Control group.

The HFD-induced increases in BWG, FC and serum TG, TC, LDL and LPS levels were all reduced, and serum HDL levels were increased in HFD + PSPC-treated mice, compared with mice in the HFD group. No significant differences were identified between the Control group and mice treated with PSPC alone and fed a normal diet.

To confirm whether locomotor activity and exploratory behavior were affected by HFD, an open field experiment was performed with mice following different treatments. As presented in Fig. 1, mice challenged with HDF for 20 weeks had a significant decrease in the distance and speed travelled (Fig. 1A and B), the number of grooming sessions (Fig. 1C) and the number of rearing and leaning events (Fig. 1D) compared with mice in the Control group, indicating that mice challenged with HFD had impaired locomotor activity and exploratory behavior. When HFD-treated mice were co-treated with PSPC the impaired activities and behaviors significantly improved (Fig. 1A-D). There were no significant differences identified between the Control and PSPC-only groups for all parameters measured.

In the initial part of the step-through test, no significant differences were identified between the latencies among the four groups (Fig. 1E). However, following a 5 sec mild foot shock in the dark compartment, the latency in the 24-h retention test was significantly decreased in HFD-treated mice compared with the Control group. By contrast, PSPC treatment markedly increased the latency in HFD + PSPC mice compared with mice only fed a HFD (Fig. 1F). These results suggest that PSPC may be able to improve the deficits in memory function of mice to a certain degree. However, no significant differences were identified between the mice challenged with PSPC alone and the Control group.

COX-2 and iNOS are important regulators in the progression of inflammation, whose expression levels are relatively low in normal tissues (35). However, when stimulated with endotoxin or inflammatory cytokines, such as IL-6, IL-1β or TNF-α, their expression levels markedly increase. In the present study western blot analysis was performed to determine the effect of a HFD and PSPC treatment on the expression of COX-2 and iNOS proteins. HFD treatment significantly upregulated the expression levels of COX-2 and iNOS compared with Control mice; this elevated expression was significantly reduced in HFD-fed mice co-treated with PSPC (Fig. 2A and B). No significant differences were identified between the Control and PSPC groups.

Neuroinflammatory cytokines are important contributors to the pathogenesis and development of cognitive impairment, and increased levels of expression have been reported to disrupt hippocampal synaptic plasticity (36). Therefore, the effects of a HFD and PSPC treatment on the levels of neuroinflammatory cytokines, including TNF-α, IL-6, IL-1β and IL-10 in the mouse brain were determined by western blotting and ELISA. As shown in Fig. 3A and B, mice fed a HFD exhibited a significant increase in the protein expression levels of the proinflammatory cytokines IL-6, IL-1β and TNF-α, and a decrease in the expression of anti-inflammatory cytokine IL-10, compared with Control mice. However, these effects were significantly reversed by PSPC treatment in the HFD + PSPC group compared with the HFD group. Results from ELISA analysis confirmed the effects of HFD and PSPC treatment on the production of neuroinflammatory cytokines in the mouse brain (Fig. 3C). A significant increase in the expression levels of IL-6, IL-1β and TNF-α was observed, in addition to a marked decrease in the level of IL-10 in the brain of HFD-fed mice, compared with the Control group. PSPC markedly decreased the expression levels of IL-6 by 43.93%, IL-1β by 32.25% and TNF-α by 42.42%, and increased the level of IL-10 by 40.82% in the HFD + PSPC group compared with the HFD-only group (Fig. 3C). No significant differences were identified between the PSPC group and the Control group in either the western blotting or ELISA analyses.

A number of previous studies have demonstrated that MAPK and NF-κB activation serves a crucial role in the progression of inflammation (37,38). To further determine the molecular mechanisms underlying the suppressive effects of PSPC on neuroinflammation in the mouse brain, the effects of PSPC on the expression and phosphorylation levels of the MAPK family members extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were determined by western blot analysis. The brains of mice fed a HFD had significantly increased expression levels of phosphorylated (p)-ERK, p-JNK and p-p38 proteins compared with Control mice (Fig. 4A and B). However, this increase in p-ERK, p-JNK and p-p38 protein expression levels in mice challenged with a HFD was significantly reduced by PSPC treatment (Fig. 4A and B).

NF-κB activation induces its translocation from the cytoplasm to the nucleus. The present study investigated the nuclear and cytoplasmic expression levels of NF-κB p65 (Fig. 4C and D). NF-κB p65 levels in the nuclear fractions were significantly increased in HFD-treated mice compared with Control mice, whereas the cytoplasmic expression levels were significantly decreased in HFD-treated mice. PSPC treatment in mice fed a HFD inhibited the activation of NF-κB, as demonstrated by the increased cytoplasmic expression of NF-κB p65 and the decreased levels in the nuclear fractions comparing the HFD-only group. No statistically significant differences were identified between the PSPC and Control groups for MAPK phosphorylation or NF-κB activation.