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Bioactive Constituents, Metabolites, and Functions
Cereal Fiber Ameliorates High Fat/Cholesterol Diet-Induced Atherosclerosis by modulating NLRP3-Inflammasome Pathway in ApoE-/- Mice Ru Zhang, Shufen Han, Zheng Zhang, Weiguo Zhang, Jing Yang, Zhongxiao Wan, and Liqiang Qin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00380 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018
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Journal of Agricultural and Food Chemistry
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Cereal Fiber Ameliorates High Fat/Cholesterol Diet-Induced Atherosclerosis by modulating
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NLRP3-Inflammasome Pathway in ApoE-/- Mice
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Ru Zhang,† Shufen Han,*,† Zheng Zhang,† Weiguo Zhang,‡ Jing Yang,† Zhongxiao Wan† and
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Liqiang Qin*,†
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†
Department of Nutrition and Food Hygiene, Jiangsu Key Laboratory of Preventive and
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Translational Medicine for Geriatric Disease, School of Public Health, Soochow University, 199
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Renai Road, Suzhou, 215123, Jiangsu, P.R. China
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‡
DSM Nutritional Products Human Nutrition and Health, 1-3 Xinyuan South Road, 100027,
Beijing, P.R. China
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*Corresponding author:
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E-mails:
[email protected] (S.Han) ;
[email protected] (L. Qin)
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Phone: +86 512 65880075. Fax: +86 512 65880023
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ABSTRACT
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Cereal fiber is associated with decreasing the risk of cardiovascular diseases. However, whether
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cereal fiber modulates inflammatory response and improves atherosclerosis remains unclear. This
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study evaluated the anti-atherosclerotic effect of cereal fibers from oat or wheat bran and explored
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the potential anti-inflammatory mechanisms. Male ApoE-/- mice were given a high fat/cholesterol
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(HFC) diet, HFC diet supplemented with 0.8% oat fiber or wheat bran fiber. After 18 weeks of
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feeding period, serum lipids and inflammatory cytokines were measured. The relative protein levels
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of the nod-like receptor family pyrin domain containing 3 (NLRP3)-inflammasome pathways and
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nuclear factor kappa B (NF-kB) were determined
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Pathologically, oat fiber and wheat fiber significantly reduced atherosclerotic plaques by 43.3% and
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27.1%, respectively. Biochemically, cereal fiber markedly decreased the protein levels of myeloid
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differentiation factor 88 (MyD88) and toll-like receptor 4 (TLR4) in aortic tissues. The expression
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of NF-kB was similarly inhibited by both cereal fibers. Compared with wheat bran fiber, oat fiber
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had greater effects in reducing the plague size and inhibiting TLR4/MyD88/NF-ĸB pathways. Such
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differences might come from modulation of NLRP3-inflammasome pathway because the expression
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of the cleavage of caspase-1 and interleukins (IL)-1β were inhibited only by oat fiber. The present
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study demonstrates that cereal fibers can attenuate inflammatory response and atherosclerosis in
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ApoE-/- mice. Such effects are pronounced with oat fiber and likely mediated by specific inhibition
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of oat fiber on NLRP3-inflammasome pathway.
by western blot method in aortas tissues.
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KEYWORDS: cereal fiber, oat, wheat bran, NLRP3-inflammasome, atherosclerosis
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■ INTRODUCTION
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Cardiovascular disease is a universal public problem nowadays. Atherosclerosis is the leading cause
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of cardiovascular disease resulting in high mortality and disability worldwide.1 Atherosclerosis
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comes from an imbalance in lipid metabolism and a maladaptive immune response driven by the
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accumulation of cholesterol-laden macrophages in the arterial wall. However, inflammation has also
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emerged as a causal factor in forming atherosclerotic plaques, according to the newest hypotheses.
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Chronic inflammation of the arterial wall is a pivotal driving force in the initiation of atherosclerotic
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plaques and the eventual destabilization of atherosclerotic lesions.2-4 Substantial evidence suggests
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that a network of inflammatory cytokines and signaling pathways are related with atherosclerosis
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progression.5
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Interleukins (IL)-1β is a crucial driver in the pathogenesis of atherosclerosis, which contains two
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steps: priming signals facilitate the transcription of immature IL-1β, and then endogenous “danger”
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signals activate innate immune signaling complexes named inflammasomes to promote IL-1β for
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secretion.6 Amounts studies demonstrated that activation of the nod-like receptor family pyrin
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domain containing 3 (NLRP3) inflammasome-mediating IL-1β secretions has emerged as a
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significant component in the processes of inflammatory of atherosclerosis.7,8 A recent study showed
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that lentivirus-mediated NLRP3 silencing inhibited inflammation, retarded atherosclerosis and
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stabilized plaques in ApoE-/- mice.9 These studies indicate that NLRP3-inflammasome is involved
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in the development of atherosclerosis.
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Cereal fiber is associated with decreasing the risk of atherosclerosis and cardiovascular
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disease.10-12 Epidemiological studies have confirmed that an increased consumption of dietary fiber
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can reduce the inflammatory response,13 particularly in some chronic diseases,14 such as
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hypertension,15 diabetes.16 By inhibiting chronic inflammation, high fiber diet holds promise as a
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lifestyle/non-pharmacologic approach for control and prevention of cardiovascular disease.17
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Despite these progresses in the field, the underlying mechanisms of cereal fiber on atherosclerosis
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progression, especially its effects on inflammatory pathway remain largely unknown. Our previous
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studies showed that cereal fiber can ameliorate high-fat diet-induced obesity and reduce serum lipid
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levels in C57BL/6J mice.18,19 According to solubility, the main component of oat fiber is β-glucan,
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which can affect the reabsorption of bile acid in the gut, and then decrease plasma cholesterol levels
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and improve hypercholesterolemia. Insoluble fiber, mainly found in brown rice and wheat bran, can
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increase fecal bulking and food transit time. And the two parameters maximally control colon
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cancer by diluting carcinogens and by decreasing the time at which these carcinogens are in contact
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with epithelial cells. Accordingly, the aim of the present research explores how the
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NLRP3-inflammasome pathway in atherosclerosis progression was modulated by cereal fiber from
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oat or wheat bran in ApoE-/- mice.
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■ MATERIALS AND METHODS
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Animals. Thirty 7-week-old male ApoE-/- mice (18–21 g) were purchased from Vital River
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Laboratory Animal Technology Company (Beijing, China), and five male C57BL/6 mice were
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purchased from SLAC Laboratory Animal Company (Shanghai, China). The ApoE-/- mouse is
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an acknowledged animal model for studying the relationship between atherosclerosis and various
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dietary interventions.20 All animal studies were disposed in accordance with the Guidelines in the
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Care and Use of Animals and with the approval of the Soochow University Animal Welfare
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Committee. The mice were housed in standard cages (four mice per cage) with a constant
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temperature of 22±2 oC, an artificial 12 h light-dark cycle, and 60% relative humidity. The mice had
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free access to food and water during the whole experiment. All possible efforts were made to
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minimize the sample size of mice and the animals’ suffering in the present study.
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Diets and Experimental Design.
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arranged to one of three dietary groups with 12 mice each, namely, ApoE-/- mice fed with a
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high-fat/cholesterol (HFC) diet as model group (AS group), HFC diet supplemented with 0.8% oat
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fiber (O-fiber group), and HFC diet supplemented with 0.8% wheat bran fiber (W-fiber group).
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HFC diet which was purchased from Research diets Inc (New Brunswick, NJ, USA), contained 4.77
After a week of acclimatization, ApoE-/- mice were randomly
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kcal/g with 46%, 34.4%, 19.6% calories from fat, carbohydrates and protein, respectively (Table 1).
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Oat fiber was granted by DSM Nutritional Products Ltd (Basel, Swizerland), which contained 44%
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fiber with 22% β-glucan and 22% insoluble fiber, 20% protein, 20% starch, 5% lipids, 5% water
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and 4% ash. Wheat bran fiber was purchased from Romer Science and Technology Ltd (Qingzhou,
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China), which contained 41% fiber with 11% soluble and 30% insoluble fiber, 18% protein, 24%
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starch, 4.8% lipids, 4.7% water and 5.5% ash. Oat fiber or wheat bran fiber was directly mixed with
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HFC diet as described previously.18,19 C57BL/6 mice as a normal control in atherosclerotic lesion
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area analysis were fed a regular diet (Chow group). The feeding experiment lasted for 18 weeks.
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Body weights and food intake were routinely weighted and recorded weekly for the duration of the
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research. At the end of experiment, the animals were deprived of food for 12 hours. Under mild
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anesthesia by an intraperitoneal (i.p.) injection of pentobarbital (80 mg/kg body weight), mice were
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killed and blood were obtained from the angular vein. Serum samples was separated and stored at a
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ultra-low temperature refrigerator until assayed.
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Serum Lipids and Inflammatory Cytokines. The blood samples were used to determine serum
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lipids including total cholesterol and triglycerides (Applygen Technologies Inc., Beijing, China),
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low density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol (Nanjing
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Jiancheng Bioengineering Institute, Nanjing, China). Serum high-sensitivity C-reactive protein level
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（hs-CRP）was measured by ELISA method following manufacturer’s instructions with absorbance
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read on a microplate reader (R&D Systems, Minneapolis, MN, USA). And the detection limits for
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hs-CRP ranged from 0.002-0.015 ng/mL. The coefficient of variation (CV) for intra-assay precision
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was < 7% in our laboratory. Serum levels of interferon γ (IFN-γ), tumor necrosis factor (TNF),
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interleukin-6 (IL-6), IL-10, IL-12p70 and monocyte chemotactic protein-1 (MCP-1) were obtained
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by BDTM CBA Mouse Inflammation kit (BD Biosciences, San Jose, CA, USA). The detection limits
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for IFN-γ, TNF, IL-6, IL-10, IL-12p70 and MCP-1 were 2.5, 7.3, 5, 17.5, 10.7 and 52.7 pg/mL,
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respectively. Serum IFN-γ, IL-10 and MCP concentrations were below the detection limit of the
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assay in the present study. The average CV for duplicates in the assay was < 8%.
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Hearts and aorta of four mice were harvested together and cleaned
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Atherosclerotic Lesions.
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under a dissecting microscope. The adventitial fat and connective tissue were gently removed, and
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the basal portion of hearts and proximal aortic root were excised. The embedded samples with
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optimum cutting temperature compound, was frozen in liquid nitrogen. Cryosections from the aortic
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root were cut into 7-µm slices and mounted on polylysine-handled glass slides. Hematoxylineosin
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(HE) or Oil Red O staining for lesion evaluation was performed. The pathological changes of the
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sampled organs were assessed by a light microscopy (Olympus BX-50, Olympus Optical, Tokyo,
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Japan). The percentage of Oil Red O staining plaques area was calculated, using ImagePro Plus 6.3
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software. Three sections per animal were collected, and the average value was obtained by a single
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observer blinded to the operating instructions and used for further analysis.
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Immunohistochemistry Analysis for CD36.
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separated and fixed with 4% paraformaldehyde overnight. The treated samples were dehydrated and
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embedded in paraffin. Paraffin sections from the aortic root were cut into 5-µm slices, and
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incubated in blocking solution with 10% goat serum. The sections were incubated for 12 h with
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polyclonal antibodies against CD36 (1:100, Proteintech, Wuhan, China), followed by incubation
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with biotinylated goat anti-rabbit IgG (1:200，ZSGB-BIO, Beijing, China) antibody for 30min.
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Antibody reactivity was detected by DAB solution. Sections were counterstained with hematoxylin
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and observed by a light microscope (Olympus BX-50, Olympus Optical, Tokyo, Japan). Five areas
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of each section were randomly selected and quantified using ImagePro Plus 6.3 software. And four
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mice in each group were calculated and averaged for further analysis.
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Western Blot Analysis.
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ultra-low temperature refrigerator for subsequent measurements. Aortas were homogenized in tissue
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lysis buffer (Beyotime Institute of Biotechnology, Nantong, China). Protein concentrations were
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measured using a BCA assay regent kit (Beyotime Institute of Biotechnology, Nantong, China). The
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content of NLRP3 (1:1,000, Cell Signaling Technology, Danvers, MA, USA), Caspase-1 (1:1,000,
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ImmunoWay Biotechnology Company, Plano, TX, USA), IL-1β (1:1,000, ImmunoWay
The aortas of four mice in each group were
After the mice were killed, aortas were quickly separated and stored at a
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Biotechnology Company, Plano, TX, USA), IL-18 (1:1000, Abcam, Cambridge, MA, USA),
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apoptosis-associated speck-like protein containing a CARD (ASC, 1:1,000, Cell Signaling
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Technology, Danvers, MA, USA), TNF-α (1:1,000, Cell Signaling Technology, Danvers, MA, USA),
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Toll like receptors 4 (TLR4, 1:500, Abcam, Cambridge, MA, USA), myeloiddifferentiationfactor88
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(MyD88, 1:1,000, ImmunoWay Biotechnology Company, Plano, TX, USA), nuclear factor kappa B
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(NF-ĸB, 1:1,000, Abcam, Cambridge, MA, USA) and vascular cell adhesion molecule 1 (VCAM-1,
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1:1,000, Cell Signaling Technology, Danvers, MA, USA) were determined by western blot
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according to Han et al,19 with a slight modification. Subsequently, antigen-antibody complexes were
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incubated for 60min at room temperature with Peroxidase AffiniPure goat anti-mouse or anti-rabbit
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IgG antibody (1:2000, Jackson ImmunoResarch Laboratories, West Grove, PA, USA). Signals were
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visualized using chemiluminescene ECL Detection Systems (EMD Millipore, Billerica, MA, USA).
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Bands were quantified by densitometry via Gene Tool according to the manufacturer’s protocol
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(SynGene, ChemiGenius2, PerkinElmer). Corresponding β-actin was used as an internal control.
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Statistical Analysis.
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analysis package (SPSS Inc, Chicago, IL, USA). Data are expressed as the mean and standard
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deviation (SD). For serum lipids, inflammatory cytokines, signaling pathways, comparisons among
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groups were analyzed using a one-way analysis of variance (ANOVA) followed with the LSD post
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hoc test. Statistical significance was established at a P-value
