Available online at www.sciencedirect.

com

ScienceDirect
Journal of Nutritional Biochemistry 32 (2016) 20 – 28

RESEARCH ARTICLES

Pomegranate extract and exercise provide additive benefits on improvement of

immune function by inhibiting inflammation and oxidative stress in
high-fat-diet-induced obesity in rats☆

Fei Zhao a, 1 , Wentao Pang a, 1, Ziyi Zhang a , Jialong Zhao a , Xin Wang c , Ye Liu b , Xun Wang b, Zhihui Feng b ,
Yong Zhang a, b , Wenyan Sun d,⁎, Jiankang Liu a, b,⁎
a
Tianjin Key Laboratory of Exercise Physiology and Sports Medicine, Tianjin University of Sport, Tianjin, 300381, China
b
Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology and Frontier
Institute of Science and technology, Xi'an Jiaotong University, Xi'an, 710049, China
c
Department of Central Laboratory, Shaanxi Provincial People's Hospital, Xi'an, 710068, China
d
Department of Nutrition and Food Security, School of Public Health, Xi'an Jiaotong University, Xi'an, 710061, China

Received 28 May 2015; received in revised form 12 January 2016; accepted 8 February 2016

Abstract

Background: Obesity is reported to be associated with immune dysfunction and a state of low-grade, chronic inflammation. Either pomegranate extract (PomE)
or exercise (Ex) has been shown to have antiobesity, anti-inflammatory and antioxidant effects. Nevertheless, no study has addressed the additive benefits of
PomE and Ex on the restoration of obesity-induced immune defects.
Objective: The present work aims to study the effect of PomE and Ex as a combined intervention on immune function and the underlying mechanism involved in
inflammation and oxidative stress in rats with high-fat-diet (HFD)-induced obesity.
Results: Our results demonstrate that the combination of PomE and Ex showed additive benefits on inhibition of HFD-induced body weight increase and
improvement of HFD-induced immune dysfunction, including (a) attenuating the abnormality of histomorphology of the spleen, (b) increasing the ratio of the
CD4 +:CD8+ T cell subpopulations in splenocytes and peripheral blood mononuclear cells (PBMC), (c) inhibition of apoptosis in splenocytes and PBMC, (d)
normalizing peritoneal macrophage phenotypes and (e) restoring immunomodulating factors in serum. We also find that immune dysfunction in HFD-fed rats
was associated with increased inflammatory cytokine secretion and oxidative stress biomarkers, and that the combination of PomE and Ex effectively inhibited
the inflammatory response and decreased oxidative damage.
Conclusions: The effect of PomE and Ex as a combined intervention is greater than the effect of either PomE or Ex alone, showing that PomE and Ex may be
additively effective in improving immune function in HFD-fed rats by inhibiting inflammation and decreasing oxidative stress.
© 2016 Elsevier Inc. All rights reserved.

Keywords: Pomegranate extract; Exercise; Immune function; Inflammation; Oxidative stress; Obesity

1. Introduction

Abbreviations: Con, control rats fed a standard chow; HFD, rats fed an high-
fat-diet (HFD); HFD + PomE, rats fed an HFD and administered a daily oral
gavage of pomegranate extract (PomE); HFD + Ex, rats fed an HFD and
exercised; HFD + PomE + Ex, rats fed an HFD and administered a daily oral
gavage of PomE and exercised.
☆
Funding sources: This work was supported by the Tianjin Science and
Technology Planning Major Project (12JCZDJC34400), the Tianjin Education
Committee Sci-Tech Development Major Project (20112D05), the Tianjin Key
Labs and Tech-Platform Project (10SYSYJC28400), the Tianjin Science and
Technology Planning (09JCYBJC12000) and the 973 Plan Project of the
Ministry of Science and Technology of China (2015CB553602).
⁎ Corresponding authors at: Department of Nutrition and Food Security, School of
Public Health, Xi'an Jiaotong University, No. 76 Yanta West Road, Xi'an, 710061, China.
E-mail addresses: wenyan2014@mail.xjtu.edu.cn (W. Sun),
j.liu@mail.xjtu.edu.cn (J. Liu).
1
Co-first author.
Obesity is often associated with an increased risk of dyslipidemia,
hypertension, atherosclerosis, diabetes, nonalcoholic fatty liver dis-
ease and certain cancers [1]. Obesity has recently been reported to be
associated with immune dysfunction. Changes in immune function
have been proposed to underlie the pathogenesis of all these diseases.
In obese individuals, reduced lymphocyte numbers and reduced
responsiveness to mitogen have been observed [2]. Obesity induces
immune defects that lead to attenuated host responses to bacterial
infection [3,4]. Defects in specific immunity such as reduced
lymphocyte numbers in the spleen, thymus and peripheral blood
have been reported in Ob/Ob mice, db/db mice and Zucker rats [5],
indicating that obesity may suppress the immune response. Con-
versely, innate immune dysfunction, associated with the absence of
interleukin (IL)-6, granulocyte-macrophage colony-stimulating fac-
tor, IL-1R1 and IL-18, could be induced by obesity [6–8]. And

http://dx.doi.org/10.1016/j.jnutbio.2016.02.003
0955-2863/© 2016 Elsevier Inc. All rights reserved.
 F. Zhao et al. / Journal of Nutritional Biochemistry 32 (2016) 20–28
substantial evidence has indicated that the obesity-induced immune
dysfunction may contribute to the progression of several diseases,
such as type II diabetes and cardiovascular disease [9]. Given the
involvement of adipose tissue in the inflammatory process, one of the
clinical objectives is to identify lifestyle factors that may affect the
obesity–immune system dynamic. For instance, exercise and nutrition
have shown to be significant lifestyle factors influencing the inflam-
matory cytokines associated with the state of obesity [10]. Particularly,
it is well documented that obesity with chronic inflammation is also
highly correlated to nutritional factors such as the type and amount of
carbohydrates, proteins and fats that are consumed in the diet [11–12].
Thus, to evaluate the impact of physical activity and nutrition on
obesity-related immune function is of significance.
Pomegranate extract (PomE) contains an array of compounds that
have been attributed antiobesity effects. We have previously reported
that pomegranate-extract-enriched punicalagin may be a useful
nutrient for the treatment of obesity-associated nonalcoholic fatty
liver disease that promotes mitochondrial function and eliminates
oxidative stress and inflammation [13]. Dietary pomegranate-seed-
oil-enriched punicic acid ameliorates high-fat-diet (HFD)-induced
obesity and insulin resistance in mice [14]. Diet, in particular,
combined with exercise (Ex) has been proven to be more effective
than diet alone on improving obesity-related vascular dysfunction in
obese children [15]. Nevertheless, no study has addressed the additive
benefits of PomE and Ex on the restoration of obesity-induced immune
defects. In our current work, we present the beneficial effects of PomE
and Ex on improving immune function in HFD-induced obesity rats.
2. Materials and methods
2.1. Animals and experimental design
Sprague–Dawley male rats (180–220 g) were purchased from SLAC Laboratory
Animal Co. Ltd. (Shanghai, China). Animal rooms were maintained on a 12-h light–dark
cycle at 20°C±3°C and 60% relative humidity. After 1 week of acclimatization, the rats
were randomly assigned into the following five groups: (a) control rats fed a standard
chow (Con, 12% kcal fat content); (b) rats fed an HFD (HFD, 45% kcal fat content); (c) rats
fed an HFD and administered a daily oral gavage of PomE (HFD + PomE, 150 mg/kg/day);
(d) rats fed an HFD and exercised (HFD + Ex, 20 m/min for 60 min at five times per week);
(e) rats fed an HFD and administered a daily oral gavage of PomE and exercised
(HFD + PomE + Ex, 150 mg/kg/day, 20 m/min for 60 min at five times per week).
Punicalagin-enriched PomE was purchased from Tianjin Jianfeng Natural Products Research
and Development Co., Ltd., China. The growing area of pomegranate is in Shaanxi province of
China. The main composition is polyphenol, and punicalagin accounts for 40%. The extract
preparation method is mainly column chromatography. The standard chow for control group
contained 27.5% protein, 12% fat and 60.5% carbohydrate. The high-fat diet contained 19%
protein, 45% fat and 36% carbohydrate and a premix to confirm the essential nutrients. Both
diets have the same content of antioxidant (tertiary butylhydroquinone).
The exercise training was according to Bedford's protocol [16]. The training protocol we used
is as follows: Rats in Ex groups commit 8 weeks of treadmill running (20 m/min, 60 min/day for
5 days per week). The exercise intensity was about 45% maximum oxygen uptake. Before these
8 weeks, there was 1 week of adaptive training for rats; the running time was increased day by
day from 15 min to 60 min. Each group contained 10 rats. Body weight and food intake were
measured twice weekly. After 8 weeks of PomE feeding and/or training, all rats were fasted
overnight (about 12–16 h) and then sacrificed before afternoon. There was more than 48 h
between the last time exercise or PomE feeding and sacrifice. This study involving animals was
conducted according to the guidelines in the Declaration of Helsinki, and all procedures were
approved by the Institutional Animal Care Committee of Xi'an Jiaotong University.
2.2. Body weight and immune organ weight
Body weight was measured and recorded every week. The weights of thymus and spleen
were measured when the rats were sacrificed, and the relative weights of spleen and thymus
for each rat were calculated by the formula organ weight (mg)/body weight (g).
2.3. Blood sample and peripheral blood mononuclear cells (PBMC) preparation
After the rats were sacrificed, blood samples were obtained by cardiac puncture,
and the serum was separated by centrifugation (3000 rpm, 10 min). Serum samples
were stored at−80°C.
PBMC were isolated from the peripheral blood of rats by density centrifugation
followed by dextran sedimentation and lysis of contaminating red blood cells. Briefly,
blood was collected in EDTA-treated tubes, diluted 1/2 with RPMI 1640 medium and
21
carefully layered onto a density gradient Ficoll-Paque (Tianjin Haoyang, Tianjin, China).
After centrifugation, the bands of PBMC were aspirated; PBMC were washed three times
with RPMI 1640 medium and then preserved with frozen solution containing 90% fetal
calf serum and 10% dimethyl sulfoxide (DMSO). Frozen PBMC were used for flow
cytometry analysis.
Serum levels of C-reactive protein (CRP), leptin, adiponectin, immunoglobulin
(Ig)A, IgG, IgM, tumor necrosis factor (TNF)-α, IL-1β, IL-6 and IL-4 were measured using
commercial enzyme-linked immunosorbent assay (ELISA) kits according to the
manufacturer's standards and protocols (R&D Systems, Minneapolis, MN, USA). The
levels of triglyceride (TG) and total cholesterol (CHO) were analyzed using an
automated biochemistry analyzer (Hitachi Ltd., Tokyo, Japan).
2.4. Splenocyte preparation
Spleens were removed aseptically at the time of sacrifice, and single-cell
suspensions were prepared by forcing spleens through 200-mesh stainless steel.
Splenocytes were washed with phosphate-buffered saline (PBS), and then erythrocytes
in spleen were completely lysed using lyse/fix buffer (BD Bioscience, San Jose, CA, USA).
This red blood cell (RBC) lysis buffer is formulated for optimal lysis of erythrocytes in
single-cell suspensions of spleen and peripheral blood. After washing with PBS, the cells
were resuspended in RPMI 1640 medium and then preserved with frozen solution
containing 90% fetal calf serum and 10% DMSO. Frozen splenocytes were used for flow
cytometry analysis.
2.5. Peritoneal macrophage preparation
Resident peritoneal macrophages were prepared by washing the peritoneal cavity
with ice-cold PBS. After washing twice, the peritoneal cells were suspended in RPMI
1640 medium, and 2 × 106 cells were plated. Peritoneal macrophages were purified by
2-h adherence to dishes. More than 95% of the adherent cells were consistently found to
be peritoneal macrophages, as assessed by their morphology after staining with Giemsa.
Resident preparations were 95% macrophages. The cells were frozen for subsequent
experiments.
2.6. Histopathological analysis of thymuses and spleens
At the time of sacrifice, thymi and spleens were collected, trimmed of fat and
weighed before being stored in buffered 10% formalin. After fixation, one middle cross
section from the spleen and one lobe of the thymus were embedded in paraffin, and five
5–6-μm sections were prepared and stained with hematoxylin and eosin for
histopathological evaluation by light microscopy.
2.7. Analysis of T lymphocyte subpopulations in PBMC and splenocytes
The PBMC and splenocytes were suspended in RPMI 1640 medium at a
concentration of 5 × 106 cells/ml, then recentrifuged and suspended in 100 μl PBS.
The suspensions were incubated with 5 μl PerCP-conjugated anti-CD8 monoclonal
antibody, 2 μl PE-conjugated anti-CD4 monoclonal antibody and 2 μl FITC-conjugated
anti-CD3 monoclonal antibody (BioLegend, San Diego, CA, USA) for 30 min and then
examined by flow cytometry. Data analysis was performed using EXPO32 ADC analysis
software.
2.8. Analysis of PBMC and splenocyte apoptosis
To determine the extent of early apoptosis and necrosis in PBMC and splenocytes, cells
were stained with annexin V and propidium iodide (PI). PBMC and splenocytes were
collected as described above when the rats were sacrificed, adjusted to 2 × 106/ml,
washed with cold PBS three times, centrifuged and incubated with 5 μl annexin V–
fluorescein isothiocyanate (FITC) and 5 μl PI for 15 min in the dark at room temperature,
and 400 μl of binding buffer was added to each sample. Samples were collected using a
flow cytometer (Beckman Coulter), and data were analyzed using EXPO32 ADC analysis
software. At least three independent experiments were performed.
2.9. Macrophage phagocytosis assay
For phagocytosis assays, macrophages were washed with PBS. After washing, FITC-
labeled zymosan particles (100 g/ml) (Molecular Probes, Life Technologies) were
added to the cells, and the cells were incubated at 37°C for the indicated times. The cells
were then put on ice and washed thoroughly with PBS to remove unbound particles. The
macrophages were detached and analyzed by flow cytometry immediately.
2.10. Assessment of antioxidant status
Rat serum was used for the measurement of biomarkers of oxidative stress. The
total antioxidant capacities (T-AOC), lipid peroxidation malonaldehyde (MDA),
superoxide dismutase (SOD), glutathione (GSH) and oxidized glutathione (GSSG) kits
(Nanjing Jiancheng, Nanjing, China) were used according to the instructions of the
manufacturer.
22 F. Zhao et al. / Journal of Nutritional Biochemistry 32 (2016) 20–28
2.11. Statistical analysis
All data were expressed as mean ± S.E.M. Each experiment was performed three to
five times, and the best representative data from among each experimental set are
presented. Differences between groups were evaluated by one-way analysis of variance.
Pb.05 was considered to be significant.
3. Results
3.1. The additive benefits of PomE and Ex on body weight and splenic
index and histopathology in HFD rats
Obesity was induced by the administration of an HFD over an 8-
week period. As shown in Fig. 1A and B, the HFD significantly increased
body weight gain. The PomE or Ex treatment effectively reduced the
body weight and body weight gain of HFD rats. The combined
supplement of PomE and Ex further decreased the body weight and
body weight gain of HFD rats (Fig. 1A and B). The thymi and spleens
were used for histological and pathological examinations. The splenic
index of the combined PomE and Ex group was increased to a greater
extent than the splenic index of the PomE or Ex alone group as
compared to the HFD group (Fig. 1C). The HFD group exhibited blurry
boundaries of the marginal zone, reduction of white pulp and germinal
center disappearance. PomE or Ex or combined PomE and Ex
treatment alleviated these abnormalities, mainly indicated by mitiga-
tion of red pulp congestion, emergence of the germinal center and
increasing of the white pulp nearly to the control group level (Fig. 1D).
Our results showed that there was no significant difference in the
thymus index and the histopathology of the thymus among each
group (data not shown).
PomE treatment had no significant effects on HFD-induced
increase of fat mass (Fig. 2A, B, C). Combination of PomE and exercise
training showed lower fat mass compared to Ex group (Fig. 2A, B, C).
However, there were no significant differences in total caloric intake
(kcal) and total food intake (g) between all the HFD groups, although
there was difference in total caloric intake (kcal) between control
group and HFD groups (Fig. 2D, E, F, G).
Fig. 1. The additive benefits of PomE and Ex on body weight, spleen index and splenic architecture in HFD rats. After treatment with the combined PomE and Ex or PomE or Ex alone in
HFD rats for 8 weeks, the rats were then sacrificed. Body weight was collected, and spleen tissues were used for analysis. (A) Body weight of each group of rats. (B) Body weight gain of
each group of rats. (C) Splenic index in each group. (D) Histopathology of spleen (×100). In spleen, R = red pulp, W = white pulp. n=6–10; data are means ± S.E.M. **Pb.01,
***Pb.001.
3.2. The additive benefits of PomE and Ex on PBMC and splenocytes
based on T cell subpopulation analysis in HFD rats
The percentages of and the ratio between CD4 + and CD8 + cells
in PBMC and splenocytes are shown in Figs. 3 and 4. We found that
the level of CD4 + T cells in PBMC was significantly decreased and
that PBMC CD8 + T cell levels were higher in the HFD group than in
control rats. Consequently, the PBMC CD4 +/CD8 + ratio decreased
significantly in the HFD group. Meanwhile, when the rats were
treated with PomE and Ex simultaneously, the PBMC CD4+/CD8+
T cell ratio was observably increased compared to that in the HFD group
(Fig. 3A and B).
An HFD dramatically decreased splenic CD4+ lymphocytes without
affecting the mean values of splenic CD8+ T cells. Accordingly, the
splenic CD4+/CD8+ ratio decreased in the HFD group, whereas the
mean value of the CD4+/CD8+ ratio increased significantly when the
rats were treated with PomE and Ex simultaneously compared with the
HFD group (Fig. 4A and B).
3.3. The additive benefits of PomE and Ex on apoptosis of PBMC and
splenocytes in HFD rats
The effects of PomE or Ex or combined PomE and Ex treatment on
cell death in the PBMC and splenocytes of HFD rats were examined by
flow cytometry analysis. The PBMC and splenocytes were stained with
annexin V and PI, respectively.
As a major damage marker, the percentages of apoptotic PBMC and
splenocytes were all increased after HFD feeding. This outcome was
shown by the increased numbers of annexin V+ and annexin V +/PI+
cells in the HFD group compared to those in the control group (Fig. 5A
and B). The percentages of apoptotic PBMC and splenocytes were all
decreased with PomE or Ex treatment compared with the HFD group,
respectively (Fig. 5C and D). Additionally, the percentages of apoptotic
cells showed a trend toward normal levels in rat PBMC and
splenocytes with combined PomE and Ex treatment, respectively
(Fig. 5C and D).
 F. Zhao et al. / Journal of Nutritional Biochemistry 32 (2016) 20–28 23

Fig. 2. The additive benefits of PomE and Ex on tissue weight. After treatment with the combined PomE and Ex or PomE or Ex alone in HFD rats for 8 weeks, the rats were then sacrificed.
(A) Perirenal fat mass, (B) epididymal fat mass, (C) VFAT %, (D) total caloric intake, (E) total food intake, (F) average daily food intake per week, (G) average daily caloric intake per week.
n=6–10; data are means ± S.E.M. *Pb.05, **Pb.01.

Fig. 3. The additive benefits of PomE and Ex on the subsets of PBMC T cells in HFD rats. After treatment with the combined PomE and Ex or PomE or Ex alone in HFD rats for 8 weeks, the
rats were then sacrificed, and peripheral blood was collected for PBMC analysis. (A) Flow cytometric analysis of population percentages of CD4+ and CD8+ cells in PBMC. (B) The
percentages of CD3+CD4+ cells, CD3+CD8+ cells and the ratio of CD4+/CD8+ in PBMC. n=5–6; data are means ± S.E.M. *Pb.05, **Pb.01.
24 F. Zhao et al. / Journal of Nutritional Biochemistry 32 (2016) 20–28

Fig. 4. The additive benefits of PomE and Ex on the subsets of splenic T cells in HFD rats. After treatment with the combined PomE and Ex or PomE or Ex alone in HFD rats for 8 weeks, the
rats were then sacrificed, and spleen tissues were collected for splenocyte preparation and analysis. (A) Flow cytometric analysis of population percentages of CD4+ and CD8+ cells in
splenocytes. (B) The percentages of CD3+CD4+ cells, CD3+CD8+ cells and the ratio of CD4+/CD8+ in splenocytes. n=5–6; data are means ± S.E.M. *Pb.05, **Pb.01.

3.4. The additive benefits of PomE and Ex on peritoneal macrophages in
HFD rats
In our current study, a significantly low rate of peritoneal
macrophage phagocytosis was induced by HFD. However, the rate of
phagocytosis was significantly increased by the PomE or Ex or
combined PomE and Ex treatment in HFD group rats (Fig. 6A and C).
Meanwhile, the percentages of apoptotic peritoneal macrophage in
the HFD group were higher than those in the control group. The HFD-
induced increase in the levels of apoptotic peritoneal macrophages
was reduced by PomE or Ex treatment. In addition, when the HFD rats
were treated with PomE and Ex simultaneously, the levels of apoptotic
peritoneal macrophages showed significant reduction (Fig. 6B and D).
3.5. The additive effects of PomE and Ex on serum inflammatory
cytokines in HFD rats
The HFD-induced obesity model is usually accompanied by
abnormal cytokine expression levels. The serum levels of CRP
(Fig. 7A), leptin (Fig. 7B), IgA (Fig. 7D), IgG (Fig. 7E), IgM (Fig. 7F),
TNF-α (Fig. 8A), IL-1β (Fig. 8B), IL-6 (Fig. 8C) and IL-4 (Fig. 8D) were all
significantly increased after HFD feeding. The level of serum adiponectin
(Fig. 7C) in HFD rats was significantly lower than that in control rats.
These results indicate an increase in inflammation in HFD rats. As
expected, supplementation with PomE or Ex significantly inhibited the
increase in cytokine levels. Meanwhile, HFD induced significant
increases of serum TG and total CHO, which were all efficiently restored
to normal levels in the PomE group or Ex group (Fig. 7G, H). The
combined PomE and Ex treatment showed additive benefits by
regulating all these factors.
3.6. The additive benefits of PomE and Ex on oxidative stress markers in
the serum of HFD rats
To test whether decreased immune function is associated with an
increase in oxidative damage, several markers of oxidative stress in rat
serum were examined. The T-AOC level (Fig. 8E) and MDA/SOD ratio
(Fig. 8F) in serum were significantly increased and the GSH/GSSG ratio
(Fig. 8G) was dramatically decreased in the HFD group compared with
the control rats. PomE or Ex treatment attenuated these markers
levels, and combined PomE and Ex treatment generated the additive
effect of restoring oxidative stress markers compared with HFD group.
4. Discussion
Obesity is accompanied by numerous physiological changes that
may directly or indirectly influence the immune system. Studies on the
immune system have mainly focused on analyzing immune organs,
including thymus, spleen and peripheral blood. The genetically obese
Zucker rats, which lack the leptin receptor, showed lymphopenia (low
levels of CD4 + and CD8 + T cells) in the thymus, spleen and
peripheral blood [17]. Results of the present study indicated that the
CD4 +/CD8 + T cell ratio of PBMC and splenocytes was decreased in
the HFD group. Recent reports have showed that Ex effectively
 F. Zhao et al. / Journal of Nutritional Biochemistry 32 (2016) 20–28 25

Fig. 5. The additive benefits of PomE and Ex on apoptosis of PBMC and splenocytes in HFD rats. After treatment with the combined PomE and Ex or PomE or Ex alone in HFD rats for
8 weeks, the rats were then sacrificed. PBMC and splenocytes were used for analysis. (A) The PBMC and (B) splenocytes were isolated, and cell distribution was analyzed with annexin V
and PI. The annexin V and PI fluorescence was measured using a flow cytometer. Results were expressed as dot plots representing one of the five or six independent values. (C) Statistical
result of PBMC apoptosis. (D) Statistical result of splenocyte apoptosis. n=5–6; data are means ± S.E.M. *Pb.05, **Pb.01, ***Pb.001.

prevents the development of obesity and obesity-related diseases.
Dietary pomegranate seed oil may enhance B cell function in vivo in
C57BL/6 N mice [18]. Therefore, we speculated that the combined
PomE and Ex treatment may exert additive benefits on obesity-
induced immune defects. In our study, combined PomE and Ex
treatment effectively reduced the body weight of HFD rats and
significantly increased the CD4+/CD8+ T cell ratio of PBMC and
splenocytes when compared with the HFD group. The percentages of
apoptotic PBMC and splenocytes were all significantly decreased with
combined PomE and Ex treatment compared to the HFD group. This
effect is greater than the effect of either PomE or Ex alone, showing
that PomE and Ex may be additively effective in improving immune
function.
Activated macrophages have been defined as two main macro-
phage phenotypes: M1 proinflammatory and M2 anti-inflammatory
macrophages [19]. It appeared that macrophages were the source of
the elevated inflammatory cytokines reported in obesity [20].
However, our results showed that the percentages of apoptotic
peritoneal macrophage in the HFD group were higher than those in
the control group. Shaul et al. demonstrated that, in HFD-fed animals,
classical M1 macrophage accumulation is observed after 8 weeks of
diet; however, after 12 weeks of diet, HFD-fed animals exhibited
increased levels of M2 macrophage, demonstrating that, in response to
HFD, mixed M1/M2 macrophages are recruited and become more M2-
like with extended HFD feeding [21]. Most adipose tissue macro-
phages arising in situ stained positive for the M2 markers CD206 and
CD301 [22]. M2 macrophages are able to secrete proinflammatory
mediators under specific conditions [23]. Given that macrophages are
able to alter their phenotype and that most studies assess adipose
tissue macrophage content at a single time, we speculated that the
higher percentages of apoptotic peritoneal macrophages may be
primarily attributed to M2 macrophages in the HFD group. Addition-
ally, phagocytosis is a main function of macrophages that allows them
to contribute to tissue homeostasis. In our study, a significantly low
rate of peritoneal macrophage phagocytosis was induced by the HFD.
The PomE or Ex group, especially the combined PomE and Ex
treatment group, restored the balance of M1/M2 and towarded to
M2 macrophage compared with the HFD group rats.
Obesity is characterized by a state of low-grade, chronic
inflammation. Particularly, both adipocytes and adipose-tissue-
resident immune cells (primarily lymphocytes and macrophages)
contribute to increased circulating levels of the proinflammatory
cytokines TNF-α, IL-1β, IL-6, eating-related peptide leptin and CRP
[24]. Similar findings have been reported in obese individuals with
low levels of adiponectin and increased levels of circulating TNF-α
[25–26]. Thus, the inflammatory cytokines were assayed in our
study. We observed that serum levels of the inflammatory cytokines
CRP, leptin, adiponectin, TNF-α, IL-1β, IL-6 and IL-4 and immuno-
globulins IgA, IgG and IgM were all attenuated by combined PomE
and Ex treatment in the HFD group, and the results illustrate
that attenuating inflammation may improve immune function
in HFD rats.
It has been reported that obesity may induce systemic oxidative
stress, and in turn, oxidative stress is associated with irregular
production of several cytokines, which contribute to the development
of metabolic syndrome [27]. Supplementation with antioxidants
26 F. Zhao et al. / Journal of Nutritional Biochemistry 32 (2016) 20–28

Fig. 6. The additive benefits of PomE and Ex on HFD rat peritoneal macrophage function. After treatment with the combined PomE and Ex or PomE or Ex alone in HFD rats for 8 weeks, the
rats were then sacrificed, and peritoneal macrophage was prepared for analysis. (A) Peritoneal macrophage phagocytosis rate was assayed using a flow cytometer. (B) Peritoneal
macrophage apoptosis was analyzed with annexin V and PI using a flow cytometer. (C) Statistical result of cellular phagocytosis rate. (D) Statistical result of cell apoptosis. n=5–6; data
are means ± S.E.M. *Pb.05, **Pb.01.

would reduce the risk of complications related to oxidative stress in
obesity [28]. Therefore, total antioxidant capacity, the MDA/SOD ratio
and the GSH/GSSG ratio in serum were examined to explain the effect
of combined PomE and Ex treatment on oxidative stress. The
combined PomE and Ex treatment generated synergism to further
inhibit oxidative stress compared with the HFD group, indicating
that oxidative stress may be the main reason of immune dysfunction
and be the primary target of combined PomE and Ex in obesity. In our
current study, the PomE administration at a dosage of 150 mg/kg/day
(containing 60 mg/kg/day punicalagin) is the same as the dose in our
previous study [13]. After PomE gavage (150 mg/kg), serum
punicalagin concentration increased time dependently and reached
17.5 μg/ml at 2 h, which suggested that PomE may exert the
improving effects of immune function with its active component
punicalagin in our study.
In summary, our study demonstrates that immune dysfunction
may play a critical role in HFD rats and that supplementation with
PomE and Ex may improve immune function by inhibiting inflamma-
tory cytokine secretion and decreasing oxidative stress. The present
findings expand our current understanding of immune dysregulation
induced by the HFD and provide an interventional strategy with
additive benefits by supplementation with PomE and Ex.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by the Tianjin Science and Technology
Planning Major Project (12JCZDJC34400), the Tianjin Education
Committee Sci-Tech Development Major Project (20112D05), the
Tianjin Key Labs and Tech-Platform Project (10SYSYJC28400), the
Tianjin Science and Technology Planning (09JCYBJC12000) and the
973 Plan Project of the Ministry of Science and Technology of China
(2015CB553602).
 F. Zhao et al. / Journal of Nutritional Biochemistry 32 (2016) 20–28 27

Fig. 7. The additive benefits of PomE and Ex on the serum parameter levels and immunoglobulins in HFD rats. After treatment with the combined PomE and Ex or PomE or Ex alone in HFD
rats for 8 weeks, the rats were then sacrificed and serum were collected. (A) CRP; (B) leptin; (C) adiponectin levels; (D) the immunoglobulins IgA, (E) IgG and (F) IgM; (G) triglyceride
(TG) and (H) total CHO levels were assayed with commercial kits by ELISA. n=6–10; data are means ± S.E.M. *Pb.05, **Pb.01.

Fig. 8. The additive effects of PomE and Ex on inflammatory cytokines and oxidative stress markers. After treatment with the combined PomE and Ex or PomE or Ex alone in HFD rats for
8 weeks, the rats were then sacrificed and serum were collected. The inflammatory cytokines (A) TNF-α, (B) IL-1β, (C) IL-6 and (D) IL-4 levels in rat serum were measured using ELISA
kits, respectively. (E) T-AOC, (F) lipid peroxidation MDA/SOD ratio and (G) GSH/GSSG ratio were assayed using commercial kits, respectively. n=6–10; data are means ± S.E.M. *Pb.05,
**Pb.01, ***Pb.001.

References
[1] Flegal KM, Graubard BI, Williamson DF, Gail MH. Cause-specific excess deaths
associated with underweight, overweight, and obesity. JAMA 2007;298:
2028–37.
[2] Tanaka S, Isoda F, Ishihara Y, Kimura M, Yamakawa T. T lymphopaenia in relation
to body mass index and TNF-alpha in human obesity: adequate weight reduction
can be corrective. Clin Endocrinol (Oxf) 2001;54:347–54.
[3] Amar S, Zhou Q, Shaik-Dasthagirisaheb Y, Leeman S. Diet-induced obesity in mice
causes changes in immune responses and bone loss manifested by bacterial
challenge. Proc Natl Acad Sci U S A 2007;104:20466–71.
[4] Smith AG, Sheridan PA, Harp JB, Beck MA. Diet-induced obese mice have increased
mortality and altered immune responses when infected with influenza virus. J
Nutr 2007;137:1236–43.
[5] Marti A, Marcos A, Martinez JA. Obesity and immune function relationships.
obesity reviews: an official journal of the international association for the study of.
Obesity 2001;2:131–40.
[6] Wallenius V, Wallenius K, Ahren B, Rudling M, Carlsten H, Dickson SL, et al.
Interleukin-6-deficient mice develop mature-onset obesity. Nat Med 2002;8:
75–9.
[7] Chida D, Osaka T, Hashimoto O, Iwakura Y. Combined interleukin-6 and
interleukin-1 deficiency causes obesity in young mice. Diabetes 2006;55:
971–7.
28 F. Zhao et al. / Journal of Nutritional Biochemistry 32 (2016) 20–28
[8] Netea MG, Joosten LA, Lewis E, Jensen DR, Voshol PJ, Kullberg BJ, et al. Deficiency
of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance. Nat
Med 2006;12:650–6.
[9] Li ZY, Wang P, Miao CY. Adipokines in inflammation, insulin resistance and
cardiovascular disease. Cli Exp Pharmacol P 2011;38(12):888–96.
[10] Pedersen BK, Febbraio MA. Muscles, exercise and obesity: skeletal muscle as a
secretory organ. Nat Rev Endocrinol 2012;8(8):457–65.
[11] Kasim-Karakas SE, Tsodikov A, Singh U, Jialal I. Responses of inflammatory
markers to a low-fat, high-carbohydrate diet: effects of energy intake. Am J Clin
Nutr 2006;83(4):774–9.
[12] Baumgarner KM, Setti S, Diaz C, Littlefield A, Jones A, Kohman RA. Diet-induced
obesity attenuates cytokine production following an immune challenge. Behav
Brain Res 2014;1(267):33–41.
[13] Zou X, Yan C, Shi Y, Cao K, Xu J, Wang X, et al. Mitochondrial dysfunction in
obesity-associated nonalcoholic fatty liver disease: the protective effects of
pomegranate with its active component punicalagin. Antioxid Redox Sign 2014;
21:1557–70.
[14] Vroegrijk IO, van Diepen JA, van den Berg S, Westbroek I, Keizer H, Gambelli L,
et al. Pomegranate seed oil, a rich source of punicic acid, prevents diet-
induced obesity and insulin resistance in mice. Food Chem Toxicol 2011;49:
1426–30.
[15] Woo KS, Chook P, Yu CW, Sung RY, Qiao M, Leung SS, et al. Effects of diet and
exercise on obesity-related vascular dysfunction in children. Circulation 2004;
109:1981–6.
[16] Bedford TG, Tipton CM, Wilson NC, Oppliger RA, Gisolfi CV. Maximum oxygen
consumption of rats and its changes with various experimental procedures. J Appl
Physiol Respir Environ Exerc Physiol 1979;47(6):1278–83.
[17] Tanaka S, Isoda F, Yamakawa T, Ishihara M, Sekihara H. T lymphopenia in
genetically obese rats. Clin Immunol Immnopathol 1998;86:219–25.
[18] Yamasaki M, Kitagawa T, Koyanagi N, Chujo H, Maeda H, Kohno-Murase J, et al.
Dietary effect of pomegranate seed oil on immune function and lipid metabolism
in mice. Nutrition 2006;22:54–9.
[19] Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol
2005;5:953–64.
[20] Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante Jr AW.
Obesity is associated with macrophage accumulation in adipose tissue. J Clin
Invest 2003;112:1796–808.
[21] Shaul ME, Bennett G, Strissel KJ, Greenberg AS, Obin MS. Dynamic, M2-like
remodeling phenotypes of CD11c + adipose tissue macrophages during high-fat
diet-induced obesity in mice. Diabetes 2010;59:1171–81.
[22] Haase J, Weyer U, Immig K, Kloting N, Bluher M, Eilers J, et al. Local proliferation of
macrophages in adipose tissue during obesity-induced inflammation. Diab
Tologia 2014;57:562–71.
[23] Gundra UM, Girgis NM, Ruckerl D, Jenkins S, Ward LN, Kurtz ZD, et al. Alternatively
activated macrophages derived from monocytes and tissue macrophages are
phenotypically and functionally distinct. Blood 2014;123:e110–22.
[24] Ouchi N, Parker JL, Lugus JJ, Walsh K. Adipokines in inflammation and metabolic
disease. Nat Rev Immunol 2011;11:85–97.
[25] Misra A, Garg A. Clinical features and metabolic derangements in acquired
generalized lipodystrophy: case reports and review of the literature. Medicine
(Baltimore) 2003;82:129–46.
[26] Tilg H, Hotamisligil GS. Nonalcoholic fatty liver disease: cytokine–adipokine
interplay and regulation of insulin resistance. Gastroenterology 2006;131:934–45.
[27] Esposito K, Ciotola M, Schisano B, Misso L, Giannetti G, Ceriello A, et al. Oxidative
stress in the metabolic syndrome. J Endocrinol Invest 2006;29:791–5.
[28] Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al.
Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin
Invest 2004;114:1752–61.