Curcumin Protects against Atherosclerosis in Apolipoprotein E

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Curcumin attenuates atherosclerosis in apolipoprotein Eknockout mice by inhibiting Toll-like receptor 4 expression Shanshan Zhang, Jun Zou, Peiyang Li, Xiumei Zheng, and Dan Feng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04260 • Publication Date (Web): 10 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Journal of Agricultural and Food Chemistry

ApoE-/- mice were fed a high-fat diet supplemented with or without curcumin (0.1 % w/w) for 16 weeks. Curcumin supplementation significantly reduced TLR4 exprssion and macrophages infiltration in atherosclerosis plaque, suppressed aortic IL-1β, TNF-α,VCAM-1 and ICAM-1 expression and NF-κB activation, reduced plasma IL-1β, TNF-α, sVCAM-1 and sICAM-1 levels, and then inhibited the formation of early atherosclerotic lesions in the ApoE-/- mice. 202x193mm (300 x 300 DPI)

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Curcumin protects against atherosclerosis in apolipoprotein E-knockout mice by inhibiting Toll-like receptor 4 expression

Shanshan Zhang, ‡ † Jun Zou, # † Peiyang Li,‡ Xiumei Zheng,‡ and Dan Feng ‡ *

Affiliation ‡

Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Preventive Medicine, School of Public Health, Sun Yat-sen University, 510080, China

#

Department of Cardiology, Affiliated NanHai Hospital of Southern Medical University, 528200, China

Corresponding Author Dr. Dan Feng, School of Public Health, Sun Yat-sen University (Northern Campus) 74 Zhongshan Road 2, Guangzhou, Guangdong Province 510080, China. E-mail: [email protected] .

Phone: +86 20 87332527

Fax: +86 20 87330446.

ORCID Dan Feng: 0000-0002-0311-8990 †

Shanshan Zhang and Jun Zou contributed equally to this work.

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ABSTRACT

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Toll-like receptor 4 (TLR4) has been reported to play a critical role in the

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pathogenesis of atherosclerosis, the current study aimed to investigate whether

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curcumin suppresses atherosclerosis development in ApoE-knockout (ApoE-/-) mice

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by inhibiting TLR4 expression. ApoE-/- mice were fed a high-fat diet supplemented

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with or without curcumin (0.1% w/w) for 16 weeks. Curcumin supplementation

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significantly reduced TLR4 expression and macrophage infiltration in atherosclerotic

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plaques. Curcumin also reduced aortic interleukin-1β (IL-1β), tumour necrosis

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factor-α (TNF-α), vascular cell adhesion molecule -1(VCAM-1) and intercellular

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adhesion molecule- 1(ICAM-1) expression, nuclear factor-κB (NF-κB) activity, and

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plasma IL-1β, TNF-α, soluble VCAM-1 and ICAM-1 levels. In addition, aortic sinus

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sections revealed that curcumin treatment reduced the extent of atherosclerotic lesions

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and inhibited atherosclerosis development. In vitro, curcumin inhibited NF-κB

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activation in macrophages and reduced TLR4 expression induced by

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lipopolysaccharide. Our results indicate that curcumin protects against atherosclerosis

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at least partially by inhibiting TLR4 expression and its related inflammatory reaction.

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KEYWORDS

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Atherosclerosis; ApoE-/- mice; Curcumin; TLR4; NF-κB.

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INTRODUCTION

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Atherosclerosis is a progressive immunoinflammatory disease characterized by

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plaques composed of lipids and inflammatory cells including

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monocytes/macrophages 1, 2. Toll-like receptor 4 (TLR4) is a pattern recognition

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receptor of the innate immune system that plays a pivotal role in the innate immunity

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and inflammatory response 3. TLR4 can recognize exogenous or endogenous

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molecular patterns 3. Lipopolysaccharide (LPS) from gram-negative bacteria is a

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natural ligand of TLR4 4. Additionally, several pro-atherogenic ligands, such as

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saturated fatty acid(SFA), oxidized low-density lipoprotein (ox-LDL) and fibronectin,

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have been shown to activate TLR4 5-7. TLR4 has been reported to participate in the

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pathogenesis of atherosclerosis 8. Lack of TLR4 diminishes atherosclerotic plaque

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formation 9. Stimulation of TLR4 results in activation of nuclear factor-κB (NF-κB)

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transcription factor and proinflammatory proteins10, which propel the inflammatory

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reaction and cause the initiation of atherogenesis as well as the destabilization of

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atherosclerotic plaques 8. In addition, macrophages play a crucial role in the

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pathogenesis of atherogenesis, and TLR4 is largely expressed by macrophages 11.

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There is accumulating evidence that the expression of TLR4 is abundant in

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macrophages infiltrating lipid-rich atherosclerotic lesions12.

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Curcumin is a hydrophobic polyphenol present in turmeric13, and possesses various

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biological functions such as anti-inflammatory, antioxidant, and anti-atherosclerosis

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properties 14, 15. Briefly, curcumin administration has been previously associated with

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the regulation of different inflammatory cytokines such as vascular cell adhesion

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molecule -1(VCAM-1) , intercellular adhesion molecule- 1(ICAM-1) ,

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interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) 16, 17. Furthermore, the

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effect of curcumin is related with inhibiting the activation of different signalling

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pathways, including the TLR4, mitogen-activated protein kinase (MAPK) pathways

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and NF-κB 18, 19. Based on these findings, here we hypothesized that curcumin can

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suppress atherosclerosis development in ApoE-knockout (ApoE-/-) mice by inhibiting

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TLR4 expression and its related inflammatory reaction. ApoE−/− mice is a

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genetically modified strain of mice that is commonly used as the animal model for

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spontaneous atherosclerosis. Therefore, we evaluated the effect of curcumin on

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atherogenesis induced by high fat diet in ApoE−/− mice and investigated the

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mechanisms by which curcumin attenuated atherosclerosis.

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MATERIALS AND METHODS

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Chemicals

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Curcumin (purity≥98%) and LPS (Escherichia coli 0111:B4) were obtained from

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Sigma-Aldrich(St. Louis, MO, USA). TLR4 antibody was purchased from Santa

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Cruz Biotechnology (Santa Cruz, CA). p65 and β-actin antibodies were purchased

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from Cell Signalling Technology (Danvers, MA). CD68 antibody was obtained from

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Abcam (Abcam, Cambridge, UK). SYBR Green-based real-time PCR kit was

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purchased from Applied Biosystems (Foster City, CA, USA). TRIzol reagent and the

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cDNA synthesis kit were obtained from Invitrogen Life Technology (Carlsbad, CA,

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USA). ELISA kits for sVCAM-1 and sICAM-1 quantification were purchased from

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Pierce Chemical Co. (Rockford, IL, USA) and R&D Systems (Minneapolis, MN,

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USA). TransAM NF-κB p65 ELISA kit was purchased from Active Motif (Carlsbad,

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CA, USA).

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Animals and diets

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Eight-week-old male ApoE-/- mice were obtained from the Beijing Vital River

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Laboratory Animal Technology Co. Ltd. (Beijing, People’s Republic of China).

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ApoE-/- mice were individually fed in stainless steel metabolic cages and were

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randomly divided into two groups after 1 week of adaptation. The mice in control

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group were fed a high-fat diet (41% of total calories from fat; 0.15% cholesterol) (n =

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10), and the mice in curcumin group were fed a high-fat diet supplemented with

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curcumin (0.1% w/w) (n= 10). The compositions of the different diets were analysed

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chemically (Table 1). Following16 weeks of feeding, the ApoE-/- mice were fasted

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overnight and then anesthetized. Serum was collected, aortas and whole hearts were

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extracted 20. All animal procedures conducted in this study were approved by the

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Animal Care and Use Committee of Sun Yat-sen University.

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Analysis of atherosclerotic lesions in aortic sinus

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Atherosclerotic lesions at the aortic sinus were measured as previously described 20.

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Briefly, the hearts from ApoE-/- mice were embedded in optimal cutting temperature

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(OCT) compound and frozen at -20℃. Every 10 µm frozen section throughout the

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aortic sinus (400 µm) was taken using Leica cryostats (Leica, Rijswijk, The

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Netherlands). Cryostat sections were stained with Oil Red O and then were evaluated

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for fatty streak lesions using computer-assisted imaging and the Optimas Image

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Analysis software package (Bioscan, Inc., Edmonds, WA).

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Immunohistochemistry

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To measure TLR4 expression and macrophage content in aortic sinus plaques,

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immunohistochemical staining was used as previously described 21. Briefly, aortic

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sinus sections were fixed in cold acetone for 10 min, washed with PBS for three

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times, and blocked with 3% BSA for 30 min at room temperature. After that,

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sections were incubated with specific antibodies (TLR4 or CD68, a macrophage

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marker) overnight at 4°C, then incubating with biotin-conjugated secondary

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antibodies, avidin-biotin complex, and DAB as a substrate. Finally, sections were

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counterstained with haematoxylin and then analysed with a Leica microscope.

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Inflammatory cytokine analysis

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The plasma-soluble VCAM-1 (sVCAM-1) and ICAM-1 (sICAM-1) levels of the

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mice were assayed by the corresponding ELISA kits, following the manufacturer’s

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instructions.

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Quantitative real-time PCR

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Total RNA was extracted using Trizol reagents from mouse aortic tissues and cell

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lysates. The levels of TLR4, VCAM-1 and ICAM-1 mRNA were quantified by

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quantitative real-time PCR as previously described 22. The primer sequences were

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shown in Table 2. The internal control used GAPDH mRNA.

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Western blotting

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The methods for western blotting was previously described 22. Proteins (50 µg) from

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mouse aortic tissues or nuclear extracts were subjected to 7.5% SDS-PAGE and

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electrotransferred to a nitrocellulose membrane. The membrane was then

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immunoblotted with specific antibodies and secondary antibodies conjugated with

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horseradish peroxidase. The loading control used β-actin antibody.

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NF-κB activity assay

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NF-κB activity was assayed as previously described 23. Briefly, nuclear

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extracts from mouse aortic tissues and cultured cell lines were prepared, and the

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binding of p65 to DNA was measured with the TransAM NF-κB p65 ELISA kit,

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according to manufacturer’s instructions.

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Cell culture and treatments

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The methods for preparation of mouse peritoneal macrophages (MPMs) from adult

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C57BL/6J mice (Jackson Laboratories) were performed as previously described 24.

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MPMs were cultured in DMEM medium containing 10% (v/v) foetal bovine serum,

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100 U/mL penicillin and 100 µg/mL streptomycin. Upon stimulation with LPS, the

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cells were preincubated with different concentrations (10, 25, 50 µM) of curcumin

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for 1 h, then stimulated by 1 µg/mL LPS for different times. MTT assay showed that

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curcumin at the tested concentrations (10, 25, 50 µM) did not affect cell viability

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(data not shown).

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Statistical analysis

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Data are presented as the mean ± standard error of the mean (SEM), and data were

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analysed using either unpaired Student’s t-test or one-way analysis of variation

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(ANOVA) followed by the Bonferroni post-test for multiple comparisons. A value of

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P < 0.05 was considered significant.

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RESULTS

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Curcumin decreased macrophage infiltration in atherosclerosis plaque

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Macrophages in plaque not only form foam cells, but they secrete lots of

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inflammatory factors 11. To determine the effect of curcumin on macrophage

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infiltration in atherosclerosis, immunohistochemical staining with anti-CD68 was

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performed on acetone-embedded aortic sections to evaluate the macrophage content

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in atherogenic plaque. As shown in Figure 1, macrophage infiltration of the lesions

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was attenuated in the curcumin-treated mice. Compared with the control group,

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curcumin treatment reduced CD68+ macrophage accumulation in atherogenic plaque

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by 45% (46.3% ± 11.5% vs.25.4% ± 8%, P

Curcumin Protects against Atherosclerosis in Apolipoprotein E

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