Purple sweet potato color (PSPC) is a natural anthocyanin pigment that is derived from purple sweet potato storage roots. PSPC possesses a variety of biological activities, including antioxidant, anti-inflammatory and neuroprotective effects; however, the detailed effects of PSPC on high-fat diet (HFD)-induced neuroinflammation remain to be determined. The aim of the present study was to investigate whether PSPC has a protective role in HFD-associated neuroinflammation in the mouse brain and to provide novel insight into the mechanisms of the action. C57BL 6J mice were maintained on a normal diet (10 kcal% fat), a HFD (60 kcal% fat), a HFD with PSPC (700 mg/kg/day) or PSPC alone, which was administrated over 20 weeks. Open field and step-through tests were used to evaluate the effects of HFD and PSPC on mouse behavior and memory function. Western blotting and ELISA analyses were used to assess the expression of inflammatory cytokines and the activation of mitogen-activated protein kinase and nuclear factor-κB (NF-κB). The results demonstrated that PSPC treatment was able to significantly improve the HFD-induced impairment of mouse behavior and memory function, and suppressed the increase in body weight, fat content, hyperlipemia and the level of endotoxin. PSPC treatment also markedly decreased the expression of cyclooxygenase-2, inducible nitric oxide synthase, tumor necrosis factor-α, interleukin (IL)-1β and IL-6, and increased the level of IL-10 in the HFD-treated mouse brain. In addition, PSPC inhibited the HFD-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and the activation of NF-κB. These findings indicated that PSPC treatment may alleviate HFD-induced neuroinflammation in the mouse brain by inhibiting ERK, JNK, p38 and NF-κB activation.
Obesity is a common trophic disorder that may be attributed to an imbalance of energy metabolism. Long-term, high-calorie food intake overwhelms energy expenditure, leading to excessive adipose accumulation in the body (
Over the past few decades, our diets have altered substantially, and in modern society the consumption of fatty foods has increased greatly. An epidemiological report revealed that a HFD causes hyperlipemia and is associated with detrimental effects on the brain (
A number of recent studies have focused on the pharmacological properties of natural products such as anthocyanins, which are a group of naturally occurring polyphenol compounds that are widely distributed in flowers, fruits and vegetables and are responsible for their intense color (
Previous studies have demonstrated that PSPC possesses various biological activities, including antioxidant, anti-inflammatory and neuroprotective effects (
Multiple pharmacological effects of PSPC have been studied; however, the detailed effects of PSPC treatment on HFD-induced neuroinflammation have not yet been identified. The aim of the present study was to investigate whether PSPC treatment was able to improve spontaneous behavior, learning and memory functions in HFD-challenged mice. In addition, the present study aimed to determine whether PSPC has a protective role in HFD-associated neuroinflammation in the brain and to provide novel insight into the mechanisms of action.
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 (
Open field testing was performed as previously described (
Passive avoidance performance testing was performed as previously described (
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 (
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
In the initial part of the step-through test, no significant differences were identified between the latencies among the four groups (
COX-2 and iNOS are important regulators in the progression of inflammation, whose expression levels are relatively low in normal tissues (
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 (
A number of previous studies have demonstrated that MAPK and NF-κB activation serves a crucial role in the progression of inflammation (
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 (
An increasing number of studies have demonstrated that a HFD contributed to the development of obesity and fat deposition in a variety of tissues and is associated with hyperlipemia and detrimental effects on the brain (
The results of biochemical analysis revealed that HFD treatment induced hyperlipemia and significantly increased BWG and FC in mice, which was consistent with a previous report (
To characterize the effects of PSPC on neuroinflammation in the mouse brain, the expression profile of two mediators of inflammation, iNOS and COX-2, were detected in mouse brains. Previous studies have implicated iNOS expression in neuronal damage and death in a number of central nervous system-associated diseases (
To further determine the mechanisms underlying the PSPC-induced suppression of neuroinflammation in mouse brains, the present study investigated the effects of PSPC on the expression levels of MAPK family members ERK, JNK and p38 proteins, as MAPK activation is known to be involved in the expression of proinflammatory cytokines (
NF-κB is a transcription regulator that serves important roles in cell proliferation and survival, inflammation and immunity, and is considered the main signaling pathway that is involved in the development of inflammatory diseases, including hepatitis, nerve injury and lung injury (
To clarify whether PSPC was able to affect mouse metabolism and physiological processes, the present study compared the aforementioned characteristics between the experimental group receiving PSPC alone and the Control group; both were fed a normal diet. The results demonstrated that there were no significant differences in the biochemical parameters or in the expression levels of iNOS, COX-2 and the inflammatory cytokines, or in the phosphorylation/activation of MAPKs and NF-κB, indicating that PSPC itself may not alter metabolism and physiological processes in mice. These results further confirmed that the observed behavior and biochemical alterations may be due to the ameliorating effect of PSPC on HFD-induced neuroinflammation in mouse brains. The present study confirmed the anti-inflammatory and neuroprotective effects of PSPC.
In conclusion, the present study demonstrated that PSPC attenuates HFD-induced neuroinflammation, and thereby improves behavior and memory function impairments in mouse brains by downregulating the expression of iNOS, COX-2, IL-1β, IL-6 and TNF-α, and upregulating the expression of IL-10, through the suppression of ERK, JNK and p38 MAPK phosphorylation and NF-κB signaling pathway activation. These findings about the pharmacological efficacy of PSPC may offer a novel therapeutic approach to ameliorate obesity-associated neuroinflammation.
The present study was funded by a Special Grant from the Key Laboratory of Sichuan Province for Developmental Regeneration (grant no. SYS10-006).
body weight gain
enzyme-linked immunosorbent assay
extracellular signal-regulated kinase
fat content
high-fat diet
c-Jun N-terminal kinase
mitogen-activated protein kinase
nuclear factor-κB
purple sweet potato color
Effects of PSPC treatment on spontaneous behavior and memory function in HFD-treated mouse (n=15). Mice in each experimental group were evaluated for (A) distance and (B) speed travelled, (C) the number of rearing and leaning events, and (D) the number of grooming sessions. Latency in the (E) initial trial and (F) 24-h retention trial of each group in the step-through test. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. Control group; #P<0.05 and ##P<0.01 vs. HFD group. HFD, high fat diet; PSPC, purple sweet potato color.
Effects of PSPC treatment on COX-2 and iNOS protein expression levels in HFD-treated mouse brains (n=5). (A) Western blots demonstrating the expression levels of iNOS and COX-2 in brain tissues from mice in each of the indicated experimental conditions. (B) COX-2 and iNOS expression levels were normalized to β-actin; expression levels in the Control group were set to 1.0. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. Control group; #P<0.05 and ##P<0.01 vs. HFD group. COX-2, cyclooxygenase-2; HFD, high fat diet; iNOS, inducible nitric oxide synthase; PSPC, purple sweet potato color.
Effects of PSPC treatment on the expression of neuroinflammatory cytokines in HFD-treated mouse brains (n=5). (A) Western blots revealing the expression levels of the proinflammatory cytokines IL-6, IL-1β and TNF-α and the anti-inflammatory cytokine IL-10 in the brain tissues of mice from each group under the indicated experimental conditions. (B) Expression levels of the neuroinflammatory cytokines were normalized to β-actin; expression levels in the Control group were set to 1.0. (C) ELISA analysis of the levels of neuroinflammatory cytokines produced in mouse brains. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. Control group; #P<0.05 and ##P<0.01 vs. HFD group. HFD, high fat diet; IL, interleukin; PSPC, purple sweet potato color; TNF-α, tumor necrosis factor-α.
Effects of PSPC treatment on HFD-induced MAPK phosphorylation and NF-κB activation in mouse brains (n=5). (A) Western blots revealing the expression levels of p-ERK, ERK, p-p38, p38, p-JNK and JNK. (B) Densitometric analysis of the proteins in (A); expression levels were normalized to β-actin and expressions in the Control group were set to 1.0. (C) Western blots revealing the expression levels of NF-κB p65 protein in nuclear and cytoplasmic fractions in the brain tissues of mice from each group under the indicated experimental conditions. (D) Densitometric analysis of the proteins in (C); nuclear and cytoplasmic expression levels were normalized to β-actin or K-70, respectively; expressions in the Control group were set to 1.0. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. Control group; #P<0.05 and ##P<0.01 vs. HFD group. C, cytoplasm; ERK, extracellular signal-regulated kinase; HFD, high fat diet; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; N, nuclear; NF-κB, nuclear factor-κB; p-, phosphorylated; PSPC, purple sweet potato color.
Effects of purple sweet potato color on the different parameters in each group of mice.
Parameter | Control | HFD | HFD + PSPC | PSPC |
---|---|---|---|---|
Body weight gain (g) | 15.62±2.54 | 28.23±3.81 |
19.78±3.66 |
14.83±3.04 |
FC |
9.78±1.33 | 20.45±1.98 |
13.57±1.19 |
10.15±1.28 |
Serum TC (mmol/l) | 2.98±0.48 | 3.84±0.37 |
3.21±0.24 |
3.15±0.33 |
Serum TG (mmol/l) | 1.04±0.15 | 1.75±0.25 |
1.29±0.18 |
1.20±0.17 |
Serum HDL (mmol/l) | 1.58±0.36 | 0.96±0.28 |
1.34±0.42 |
1.46±0.45 |
Serum LDL (mmol/l) | 1.37±0.26 | 2.67±0.42 |
1.75±0.28 |
1.58±0.37 |
Data are presented as the mean ± standard error of the mean (n=15/group).
Nuclear magnetic resonance-magnetic resonance imaging-based technology was used to assess body fat content and biochemical analysis of mouse blood samples was performed to investigate the other parameters.
P<0.01 vs. Control group
P<0.05
P<0.01 vs. HFD group. FC, fat content; HDL, high-density lipoprotein; HFD, high-fat diet; LDL, low-density lipoprotein; PSPC, purple sweet potato color; TC, total cholesterol; TG, triglyceride.