Ginger attenuates trimethylamine-N-oxide (TMAO)-exacerbated disturbance in cholesterol metabolism and vascular inflammation

Journal of Functional Foods 52 (2019) 25–33

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Ginger attenuates trimethylamine-N-oxide (TMAO)-exacerbated disturbance in cholesterol metabolism and vascular inflammation

T

Zouyan Hea, Lin Leia,b, Erika Kweka, Yimin Zhaoa, Jianhui Liua, Wangjun Haoa, Hanyue Zhua, ⁎ Ning Lianga, Ka Ying Maa, Hing Man Hoc, Wen-Sen Hea,d, Zhen-Yu Chena, a

Food & Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China College of Food Science, Southwest University, 2 Tiansheng Road, Beibei District, Chongqing 400715, China c School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China d School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: TMAO Ginger extract Cholesterol Inflammation Heart disease

Trimethylamine-N-oxide (TMAO) has recently been identified as an independent risk factor for atherosclerosis. The present study was to investigate the effects of ginger extract (GE) on plasma cholesterol and inflammation in TMAO-exacerbated hypercholesterolemic mice. Forty-five mice were assigned into five groups and fed a noncholesterol diet, a high-cholesterol diet, or one of the three experimental high-cholesterol diets containing 1% GE, 0.2% TMAO, or 0.2% TMAO plus 1% GE for 12 weeks. Results showed that dietary TMAO increased plasma total cholesterol (TC). GE decreased plasma TC in both non-TMAO-fed and TMAO-fed mice, by up-regulating the expression of hepatic cholesterol 7α-hydroxylase (CYP7A1) and promoting fecal excretion of total acidic sterols. GE also lowered plasma levels of proinflammatory cytokines including interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and monocyte chemotactic protein 1 (MCP-1). It was concluded that GE could alleviate the TMAO-aggravated elevation in plasma TC and vascular inflammation in high cholesterol diet-fed mice.

1. Introduction Cardiovascular disease (CVD) has become a leading cause of deaths worldwide. Atherosclerosis, a primary cause of myocardial infarction and stroke, is associated with vascular inflammation and elevated plasma total cholesterol (Lusis, 2000). Blood trimethylamine-N-oxide (TMAO) has recently been identified as an independent risk factor for atherosclerosis in humans (He & Chen, 2017; Tang et al., 2013; Ussher, Lopaschuk, & Arduini, 2013; Wang et al., 2011). TMAO is an odorless and colorless compound, which is naturally found in the muscle of marine invertebrates and sharks to regulate osmotic pressure, adjust buoyancy and protect the folding of proteins in the deep sea (Kelly & Yancey, 1999; Yancey, 2005). Although sea foods derived from marine invertebrates and sharks contain TMAO, the association between sea food intake and changes in plasma or urinary TMAO concentrations in humans has not been thoroughly investigated. In fact, most plasma TMAO is derived from dietary trimethylamine (TMA)-containing nutrients such as choline, phosphatidylcholine and L-carnitine (Koeth et al., 2013; Wang et al., 2011). The production of TMAO from dietary sources depends on two factors with the first being the liberation of TMA moiety from dietary precursors by gut microbiota in the intestine,

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and the second being the oxidation of TMA to TMAO by flavin monooxygenases (FMOs) in the liver (Zeisel & Warrier, 2017). Dietary TMAO, choline or L-carnitine supplements have been shown to elevate the plasma TMAO levels in mice, accompanied by development of larger atherosclerotic lesions (Chen et al., 2016, 2017; Geng et al., 2018; Koeth et al., 2013; Wang et al., 2011; Zhao et al., 2018). TMAO is atherogenic partly attributable to its proinflammatory effect on blood vessels. It has been reported that dietary choline or TMAO upregulates the expressions of inflammatory cytokines in the aorta of mice (Chen et al., 2017; Geng et al., 2018; Seldin et al., 2016). Similar inductive effects of TMAO on inflammasome formation and subsequent expressions of inflammatory cytokines were observed in human umbilical vascular endothelial cells (HUVECs) and mouse carotid artery endothelial cells (CACEs) (Boini, Hussain, Li, & Koka, 2017; Chen et al., 2017). Disturbance of cholesterol homeostasis by TMAO may also account for its promoting effect on atherogenesis. Reverse cholesterol transport (RCT), a pivotal pathway by which excess cholesterol is transported from peripheral tissues to the liver for excretion, is inhibited in TMAO-fed ApoE−/− mice (Koeth et al., 2013). In addition, TMAO adversely affects the hepatic bile acid synthesis from cholesterol at multiple levels, resulting in a smaller bile acid pool size and less fecal

Corresponding author. E-mail address: [email protected] (Z.-Y. Chen).

https://doi.org/10.1016/j.jff.2018.10.022 Received 10 September 2018; Received in revised form 20 October 2018; Accepted 24 October 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.

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bitartrate, 2.5; DL-methionine, 2.3. A high-cholesterol diet (HCD) consisted of 1.25% cholesterol and 40% kcal of fat and the three experimental diets were similarly prepared by adding 1.0% GE into HCD diet (HCD + GE), adding 0.2% TMAO into HCD diet (HCD + TMAO), and adding 0.2% TMAO plus 1.0% GE into HCD diet (HCD + TMAO + GE), respectively. Phytosterol is a most popular cholesterol-lowering supplement. The rationale for adding 1.0% GE into HCD diet was based on the recommended intake of dietary supplement of phytosterol. According to the National Cholesterol Education Program, 2 g/d of phytosterol supplement could reduce blood LDL-cholesterol level (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001), translating to a dose of 0.8 g phytosterol/ 1000 kcal provided that human consume about 2000–2500 kcal energy daily. In view that the energy density of HCD diet was 4500 kcal/kg and the purity of GE was ≥35%, 10 g of GE should be added into 1 kg of HCD diet (1.0% GE) was equivalent to the recommended amount of phytosterol.

bile acid excretion (Chen et al., 2016; Koeth et al., 2013). Ginger, the rhizome of Zingiber officinale, is widely used as a spice as well as a folk medicine to alleviate nausea, arthritis and pain (Semwal, Semwal, Combrinck, & Viljoen, 2015). Ginger extract (GE) has been demonstrated to possess various biological benefits including antioxidant, anti-inflammatory and anti-carcinogenetic activities (Hsiang et al., 2015; Sang et al., 2009; Si, Chen, Zhang, Chen, & Chung, 2017). It is believed that gingerols and shogaols are the major pungent active compounds accountable for such activities (Semwal et al., 2015). Studies showed that GE was cardio-protective mediated by lowering plasma cholesterol level (Fuhrman, Rosenblat, Hayek, Coleman, & Aviram, 2000; Lei et al., 2014; Nammi, Sreemantula, & Roufogalis, 2009; Verma, Singh, Jain, & Bordia, 2004), suppressing the oxidation and aggregation of low-density lipoprotein (LDL) in macrophage (Fuhrman et al., 2000), decreasing platelet activity (NurtjahjaTjendraputra, Ammit, Roufogalis, Tran, & Duke, 2003), and inhibiting vascular smooth muscle cell (VSMC) proliferation (Liu et al., 2015). However, whether GE is able to suppress the TMAO-related alterations in cholesterol homeostasis and inflammation remains unknown. The present study was therefore conducted to investigate the effects of GE on TMAO-exacerbated disturbance in cholesterol metabolism and vascular inflammation in mice fed a high-cholesterol diet.

2.4. Mice Forty-five male C57BL/6J mice (8 weeks of age, body weight = 21–24 g) were obtained from the Laboratory Animal Service Center, The Chinese University of Hong Kong. After one-week acclimation, mice were randomly divided into five groups (n = 9 each) and fed their respective diets for 12 weeks. They were housed at 23 °C with a 12/12 h light-dark cycle and had free access to food and water. Food intake and body weight were recorded twice a week. Fecal outputs were collected at week 12. At the end of week 12, all the mice were fasted overnight and sacrificed after an inhalational anesthesia of isoflurane (100%). Blood sample was obtained by cardiac puncture into tubes containing heparin and plasma was then collected. Liver, kidney, testis, epididymal and peripheral adipose tissues were removed, weighed, frozen in liquid nitrogen and stored at −80 °C. The first 4 cm of duodenum was discarded, and the epithelial cells of the next 24 cm of the small intestine were kept and stored at −80 °C. All the experimental protocols were approved by the Animal Experimental Ethical Committee, The Chinese University of Hong Kong (Ref No. 16-012MIS).

2. Materials and methods 2.1. Chemicals and diet ingredients Diet ingredients including casein, powdered cellulose, mineral mix, and vitamin mix were purchased from Harlan Teklad (Madison, WI, USA). Choline bitartrate, DL-methionine, and cholesterol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lard was purchased from American Food Provision Co. Ltd. (Hong Kong, China). Corn starch, sucrose and gelatin were obtained from Knorr Hong Kong Ltd. (Hong Kong, China). Trimethylamine-N-oxide (TMAO) (purity ≥ 99%) was purchased from Jinan Shangda Chemical Reagent Co. Ltd. (Jinan, Shandong, China). Ginger extract (GE, purity ≥ 35%) was obtained as a kind gift from Sabinsa Corp (Piscataway, NJ, USA). 2.2. HPLC analysis of ginger extract

2.5. Measurement of plasma TMAO Relative concentrations of gingerols and shogaols in GE were analyzed by high performance liquid chromatography (HPLC). GE was dissolved in methanol (1 mg/mL) and then injected onto a BEH C18 column (2.1 × 100 mm, 1.7 μm; Waters, Milford, MA, USA) in an Agilent 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA) at a flow rate of 0.35 mL/min. The mobile phases consisted of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The injection volume of the sample was 1 µL. The ratio of A to B was held at 15:85 (vol/vol) for the first 20 min, then changed to 100% solvent B in the next 5 min, and changed to 85:15 (vol/vol) in the next 3 min. The eluted peaks were monitored at 230 nm and were detected from m/z 200 to 900 using an Agilent 6540 qTOF high definition mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Individual gingerols and shogaols were identified by comparing the retention times and mass spectral fingerprints with published data (Lei et al., 2014; Park & Jung, 2012). Relatively, GE consisted of 31.26% [6]-gingerol, 5.46% [8]-gingerol, 30.26% [8]-shogaol, 9.52% [10]-gingerol, 7.63% [8]-shogaol, and 7.68% [10]-shogaol (Fig. 1).

Plasma TMAO was quantified by liquid chromatography with tandem mass spectrometry (LC/MS/MS) according to the method described by Wang et al. (2011) with modifications. Briefly, 80 μL of methanol was added into 20 μL of plasma sample for protein precipitation. After centrifugation at 20,000g for 10 min, the supernatant was collected and injected onto a HILIC column (2.1 × 100 mm, 1.7 μm; Waters, Milford, MA, USA) in an Agilent 1290 UHPLC system equipped with an Agilent 6460 Triple Quadrupole MS (Agilent Technologies, Santa Clara, CA, USA). Water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) were used as the mobile phase at a flow rate of 0.25 mL/min. The ratio of solvent B was changed from 85% to 60% during the first 6 min, then changed back to 85% in the next 1.6 min, and maintained for 4.4 min. TMAO was monitored in positive multiple reaction monitoring (MRM) mode using characteristic precursor-product ion transitions m/z 76 → 58. Various concentrations of TMAO standards were added into mouse control plasma to construct a calibration curve for quantification of plasma TMAO.

2.3. Preparation of diets 2.6. Measurement of plasma lipids and inflammatory cytokines Five semi-purified diets were prepared (Table 1). A non-cholesterol diet (NCD) was prepared according to AIN-93 M diet with minor modifications by mixing the following ingredients (g/kg): corn starch, 600.2; casein, 140.0; sucrose 100.0; powdered cellulose 50.0; lard, 40.0; mineral mix, 35.0; gelatin, 20.0; vitamin mix, 10.0; choline

Plasma total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triacylglycerol (TG) at week 12 were measured with commercial enzymatic kits (Stanbio Laboratories, Boerne, TX, USA). Non-HDL-C was determined by deducting HDL-C from TC. Plasma 26

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Fig. 1. HPLC chromatogram at 230 nm of ginger extract (GE). Peaks: 1, [6]-gingerol; 2, [8]-gingerol; 3, [6]-shogaol; 4, [10]-gingerol; 5, [8]-shogaol; 6, [10]-shogaol; 7 and 8, unknown compounds.

lyophilized and ground into powder. 5α-Cholestane (1.0 mg) and hyodeoxycholic acid (1.2 mg) were added into 150 mg of fecal sample as internal standards for quantification of neutral and acidic sterols, respectively. The fecal sample was firstly saponified with 8 mL of 1 mol/L NaOH in 90% ethanol at 90 °C for 1 h. Total neutral sterols were extracted with cyclohexane and converted to their TMS derivatives at 60 °C. The remaining aqueous phase was used for acidic sterol analysis. Similarly, acidic sterols were saponified, extracted with diethyl ether, and converted to their corresponding TMS derivatives. Both TMS-derivatives of neutral and acidic sterols were analyzed on a Shimadzu GC2010 mentioned above.

levels of inflammatory cytokines including interleukin (IL)-1β, IL-6, IL10, tumor necrosis factor α (TNF-α), and monocyte chemotactic protein 1 (MCP-1) were determined by ELISA kits (ExCell Biotech Co. Ltd, Taicang, Jiangsu, China) according to the manufacturer’s instructions. 2.7. Analysis of hepatic cholesterol Cholesterol in the liver was determined by gas chromatography (GC) as we previously described (Lei et al., 2014; Zhao et al., 2017). Basically, 1.0 mg of 5α-cholestane as an internal standard was added into 100 mg of liver sample, followed by extraction of liver lipids with chloroform/methanol (2:1, v/v). Cholesterol was then saponified and converted to its trimethylsilyl (TMS) ether derivative. A Shimadzu GC2010 equipped with a fused silica capillary column (SACTM-5, 30 m × 0.25 mm, i.d.; Supelco, Inc., Bellefonte, PA, USA) with a flame ionization detector was applied to quantify the cholesterol-TMS derivatives.

2.9. Quantitative real-time PCR Total RNA was extracted from liver tissue and intestinal epithelial cells with Trizol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) in a thermocycler (Veriti 96 Well Thermal Cycler, Life Technologies Ltd.). Quantitative real-time PCR was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using SYBR green as a fluorophore and the primer

2.8. Analysis of fecal neutral and acidic sterols Fecal neutral and acidic sterols were determined as described before (Lei et al., 2014; Zhao et al., 2017). In brief, fecal samples were

Table 1 Composition of five diets namely a non-cholesterol diet (NCD), a high-cholesterol diet containing 1.25% cholesterol and 40% kcal from fat (HCD), a HCD diet containing 1.0% GE (HCD + GE), a HCD diet containing 0.2% TMAO (HCD + TMAO), and a HCD diet containing 0.2% TMAO plus 1.0% GE (HCD + TMAO + GE). Ingredient (g)

NCD

HCD

HCD + GE

HCD + TMAO

HCD + TMAO + GE

Corn starch Casein Sucrose Powdered cellulose Lard AIN 76 mineral Mix Gelatin AIN 93 vitamin Mix Choline bitartrate DL-Methionine Cholesterol Ginger extract TMAO Total kcal/g % kcal Carbohydrate Protein Fat

600.2 140.0 100.0 50.0 40.0 35.0 20.0 10.0 2.5 2.3 0.0 0.0 0.0 1000.0 3.9

410.2 165.3 68.4 59.1 202.1 41.3 23.6 11.8 3.0 2.7 12.5 0.0 0.0 1000.0 4.5

410.2 165.3 68.4 59.1 202.1 41.3 23.6 11.8 3.0 2.7 12.5 10.0 0.0 1010.0 4.5

410.2 165.3 68.4 59.1 202.1 41.3 23.6 11.8 3.0 2.7 12.5 0.0 2.0 1002.0 4.5

410.2 165.3 68.4 59.1 202.1 41.3 23.6 11.8 3.0 2.7 12.5 10.0 2.0 1012.0 4.5

73.7 16.9 9.4

43.1 16.9 40.0

43.1 16.9 40.0

43.1 16.9 40.0

43.1 16.9 40.0

27

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sequences shown in Table S1. The mRNA levels of hepatic cholesterol 7α-hydroxylase (CYP7A1), farnesoid X receptor (FXR), small heterodimer partner (SHP), liver X-receptor α (LXRα), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA-R), low-density-lipoprotein receptor (LDL-R), and sterol-regulatory element-binding protein 2 (SREBP2) were normalized with β-actin, while those of intestinal Niemann-Pick C1 like 1 (NPC1L1), acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2), microsomal-triacylglycerol-transport protein (MTP), ATPbinding cassette sub-family G member 5 (ABCG5) and member 8 (ABCG8) were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

3.2. Plasma TMAO levels

2.10. Western blot

Mice in the HCD group had higher plasma TC, HDL-C and non-HDLC but similar TG levels as those in the NCD group (Table 3). Addition of TMAO in diet increased plasma TC concentration in mice maintained on HCD + TMAO diet compared with that in mice maintained on HCD control diet, indicating that TMAO had a potency in raising plasma cholesterol. Compared with HCD group, mice given HCD + GE diet had plasma TC concentration decreased by 25.1%. Compared with those maintained on HCD + TMAO diet, mice on diet of HCD + TMAO + GE had plasma TC reduced by 21.2%, indicating GE was effective in reducing plasma cholesterol (Table 3). Addition of TMAO in HCD diet significantly increased cholesterol in the liver by 27.9%, while dietary GE could partially prevent this TMAO-induced increase in hepatic cholesterol (Table 3).

No differences in plasma TMAO concentrations were seen among three groups of mice given diets of NCD, HCD and HCD + GE (Fig. 2A). Addition of TMAO in diet led to a 3-fold increase in plasma TMAO concentrations. No differences in plasma TMAO concentrations were seen between two groups of mice fed HCD + TMAO diet and HCD + TMAO + GE diet, suggesting that GE did not affect the absorption of dietary TMAO. 3.3. Plasma TC, TG, HDL-C and non-HDL-C, and liver cholesterol

Total proteins from the liver and intestinal epithelial cells were extracted with radio-immunoprecipitation assay (RIPA) buffer. Protein concentration was determined using a Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Total proteins were separated on a 7–10% sodium dodecyl sulfate polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) membranes, blocked in 3% non-fat milk, and then incubated with protein’s specific primary antibody and corresponding secondary antibody. The membrane was developed with enhanced chemiluminescence (ECL) agents and visualized on a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). Densitometry of proteins was quantified with Image Lab software (Bio-Rad, Hercules, CA, USA). Data on abundance of hepatic and intestinal proteins were normalized with β-actin.

3.4. Plasma inflammatory cytokines

2.11. Statistical analysis

3. Results

Plasma levels of proinflammatory cytokines IL-6, IL-1β and TNF-α increased while anti-inflammatory cytokine IL-10 decreased in HCD mice compared with those in NCD group, indicating a high-cholesterol diet could induce the inflammatory reactions (Fig. 2B). Addition of TMAO in diet raised plasma IL-6 and TNF-α concentrations, but it did not affect plasma MCP-1, IL-1β or IL-10 levels in mice. Addition of GE in diet reduced plasma proinflammatory cytokine levels, suggesting that TMAO exacerbated the vascular inflammation, whereas GE was able to alleviate the TMAO-aggravated inflammation.

3.1. Food intake, body weight and organ weight

3.5. Fecal neutral and acidic sterols

Mice in the NCD control group had a higher food intake than mice in the other four groups (Table 2). Mice on diets of HCD + GE or HCD + TMAO + GE had a lower food intake and a lesser body weight gain compared to the corresponding mice on diets of HCD and HCD + TMAO. Mice given GE diet had higher relative weights of liver, and lower relative weights of epididymal and perirenal adipose tissues which indicates a less fat accumulation (Table 2).

The fecal sterol analyses showed that the HCD diet notably elevated total fecal neutral sterol excretion compared to the NCD diet (Table 4). Total fecal neutral sterol content was similar between HCD and HCD + GE groups, while that in HCD + TMAO + GE mice was greater than in HCD + TMAO group. Regarding the individual neutral sterols, two groups fed diets containing GE excreted a less amount of comprostanol but higher amount of cholesterol and dihydrocholesterol

Data were expressed as the mean ± standard deviation (SD). All data were analyzed using one-way analysis of variance (ANOVA) followed by post hoc LSD test in a statistical program (SPSS Inc., version 21.0, Chicago, IL, USA). Significance was defined as p value less than 0.05.

Table 2 Growth parameters in C57BL/6J mice fed a non-cholesterol diet (NCD), a high-cholesterol diet (HCD) or one of the three experimental high-cholesterol diets containing 1% ginger extract (HCD + GE), 0.2% TMAO (HCD + TMAO), or 0.2% TMAO plus 1% ginger extract (HCD + TMAO + GE) for 12 weeks. NCD

HCD

HCD + GE

HCD + TMAO

HCD + TMAO + GE

Food intake Daily food intake (g/d) Daily food intake/body weight (g/d/g body weight)

3.72 ± 0.13a 0.12 ± 0.01a

2.96 ± 0.08b 0.10 ± 0.01bc

2.72 ± 0.08c 0.10 ± 0.00b

2.99 ± 0.08b 0.09 ± 0.00c

2.70 ± 0.09c 0.10 ± 0.01b

Body weight (g) Initial Final

22.8 ± 0.8 29.3 ± 1.5c

22.7 ± 0.7 31.0 ± 1.9b

22.8 ± 0.8 26.8 ± 1.1d

22.8 ± 0.7 33.2 ± 1.6a

22.6 ± 0.7 26.3 ± 1.1d

Organ weight (% body weight) Liver Testis Kidney Epididymal fat Perirenal fat

3.73 0.64 1.00 2.34 0.78

± ± ± ± ±

0.30c 0.09b 0.06 0.23c 0.14c

3.99 0.62 1.04 3.60 1.24

± ± ± ± ±

0.30bc 0.05bc 0.04 0.73b 0.19b

4.59 0.74 1.02 1.90 0.61

± ± ± ± ±

0.21a 0.04a 0.05 0.23c 0.06d

4.21 0.57 0.98 4.27 1.45

± ± ± ± ±

0.28b 0.05c 0.06 0.84a 0.23a

Data are expressed as mean ± SD, n = 9. Means in the same row with different superscript letters differ significantly at p < 0.05. 28

4.66 0.70 1.02 1.97 0.68

± ± ± ± ±

0.26a 0.06ab 0.06 0.39c 0.15 cd

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Fig. 2. Plasma levels of trimethylamine-N-oxide (TMAO) and inflammatory cytokines in C57BL/6J mice fed a non-cholesterol diet (NCD), a high-cholesterol diet (HCD) or one of the three experimental high-cholesterol diets containing 1% ginger extract (HCD + GE), 0.2% TMAO (HCD + TMAO), or 0.2% TMAO plus 1% ginger extract (HCD + TMAO + GE) for 12 weeks. (A) Plasma TMAO levels in mice, n = 9. (B) Plasma levels of inflammatory cytokines interleukin (IL)-6, monocyte chemotactic protein 1 (MCP-1), IL-1β, tumor necrosis factor α (TNF-α), and IL-10 in mice, n = 9. Data are expressed as mean ± SD. Means with different superscript letters differ significantly at p < 0.05.

Table 3 Plasma lipids and liver cholesterol in C57BL/6J mice fed a non-cholesterol diet (NCD), a high-cholesterol diet (HCD) or one of the three experimental high-cholesterol diets containing 1% ginger extract (HCD + GE), 0.2% TMAO (HCD + TMAO), or 0.2% TMAO plus 1% ginger extract (HCD + TMAO + GE) for 12 weeks.

Plasma lipids TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) Non-HDL-C (mg/dL) Non-HDL-C/HDL-C HDL-C/TC Liver cholesterol (mg/g)

NCD

HCD

HCD + GE

HCD + TMAO

HCD + TMAO + GE

130.13 ± 13.54e 46.94 ± 6.04ab 115.34 ± 14.10c 14.79 ± 10.19c 0.14 ± 0.10c 0.89 ± 0.08a 2.80 ± 0.93d

201.67 ± 18.6b 47.23 ± 12.56ab 143.41 ± 15.00ab 58.26 ± 9.98a 0.41 ± 0.08ab 0.71 ± 0.04b 15.09 ± 3.88bc

150.96 ± 16.85d 41.22 ± 8.48bc 117.99 ± 9.87c 32.98 ± 18.43b 0.29 ± 0.16b 0.79 ± 0.11b 13.24 ± 2.70c

219.87 ± 18.35a 55.79 ± 7.98a 152.63 ± 10.62a 67.24 ± 17.66a 0.44 ± 0.13a 0.71 ± 0.07b 19.30 ± 2.25a

173.16 ± 13.02c 34.49 ± 5.52c 131.19 ± 17.44bc 41.96 ± 6.84b 0.33 ± 0.10ab 0.76 ± 0.05b 17.12 ± 2.63ab

TC, total cholesterol; HDL-C, high density lipoprotein cholesterol; TG, triacylglycerol. Non-HDL-cholesterol (non-HDL-C) was determined by deducting HDL-C from TC. Data are expressed as mean ± SD, n = 9. Means in the same row with different superscript letters differ significantly at p < 0.05.

3.6. Gene expression of CYP7A1, FXR, SHP, LXRα, HMG-CoA-R, LDL-R and SREBP2

compared with their corresponding controls HCD and HCD + TMAO groups (Table 4). HCD induced a 10-fold increase in fecal acidic sterol excretion compared with NCD diet (Table 4). Addition of GE in HCD diet conspicuously increased the excretion of total fecal bile acids (44.41 versus 29.81 mg/day). In contrast, addition of TMAO in HCD diet significantly decreased the excretion of total fecal bile acids (9.26 versus 29.81 mg/ day). Whereas addition of GE could partially reverse the TMAO-induced reduction in the excretion of total fecal bile acids (17.67 versus 9.26 mg/day). Results clearly demonstrated that GE was effective in promoting the fecal acidic sterol excretion in the absence or presence of dietary TMAO.

CYP7A1 is the rate-limiting enzyme for converting cholesterol to bile acid for its biliary excretion in the liver (Chen, Jiao, & Ma, 2008). Quantitative real-time PCR analysis and western blot analysis clearly demonstrated that GE could up-regulate the gene expression of hepatic CYP7A1 in both the absence and presence of dietary TMAO (Fig. 3). One of FXR’s functions is to suppress the gene expression of hepatic CYP7A1 (Kong et al., 2012). Results showed that GE could down-regulate FXR mRNA in both absence or presence of dietary TMAO, however, it did not affect the protein level of FXR (Fig. 3). Similarly, SHP functions to inhibit the bile acid synthesis from cholesterol (Kong et al., 2012). Quantitative real-time PCR analysis and western blot analysis 29

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Table 4 Fecal contents of neutral and acidic sterols at week 12 in five groups of C57BL/6J mice fed a non-cholesterol diet (NCD), a high-cholesterol diet (HCD) or one of the three experimental high-cholesterol diets containing 1% ginger extract (HCD + GE), 0.2% TMAO (HCD + TMAO), or 0.2% TMAO plus 1% ginger extract (HCD + TMAO + GE) for 12 weeks. NCD

HCD

HCD + GE

HCD + TMAO

HCD + TMAO + GE

0.76 ± 0.52b 1.63 ± 0.54ab 18.25 ± 1.10a 20.64 ± 0.73a

Week 12 fecal neutral sterols (mg/day) Coprostanol Coprostanone Cholesterol + dihydrocholesterol Total neutral sterols

0.02 0.04 0.03 0.09

± ± ± ±

0.01c 0.01c 0.02d 0.02c

3.35 ± 0.46a 1.28 ± 0.30b 13.67 ± 1.08c 18.30 ± 0.92b

0.82 ± 0.54b 1.99 ± 0.75a 16.40 ± 1.51b 19.20 ± 0.64b

3.29 ± 1.49a 1.59 ± 0.75ab 13.23 ± 2.86c 18.12 ± 2.17b

Week 12 fecal acidic sterols (mg/day) Lithocholic acid Deoxycholic acid Chenodeoxycholic acid + cholic acid Total acidic sterols

0.74 0.57 1.58 2.89

± ± ± ±

0.36e 0.22c 0.60bc 0.75e

26.71 ± 1.54b 1.33 ± 0.04b 1.78 ± 0.45b 29.81 ± 1.44b

39.16 ± 1.48a 2.12 ± 0.41a 3.14 ± 0.73a 44.41 ± 2.57a

7.84 0.63 0.79 9.26

± ± ± ±

0.72d 0.11c 0.10c 0.84d

15.60 ± 3.12c 0.74 ± 0.19c 1.33 ± 0.24bc 17.67 ± 3.23c

Data are expressed as mean ± SD, n = 9. Means in the same row with different superscript letters differ significantly at p < 0.05.

Fig. 3. Relative mRNA and protein expression of hepatic cholesterol 7α-hydroxylase (CYP7A1), farnesoid X receptor (FXR), small heterodimer partner (SHP), liver Xreceptor α (LXRα), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA-R), low-density-lipoprotein receptor (LDL-R), and sterol-regulatory element-binding protein 2 (SREBP2) in C57BL/6J mice fed a non-cholesterol diet (NCD), a high-cholesterol diet (HCD), or one of the three experimental high-cholesterol diets containing 1% ginger extract (HCD + GE), 0.2% TMAO (HCD + TMAO), or 0.2% TMAO plus 1% ginger extract (HCD + TMAO + GE) for 12 weeks. Data are expressed as mean ± SD, n = 9. Means with different superscript letters differ significantly at p < 0.05. 30

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Fig. 4. Relative mRNA and protein expression of intestinal Niemann-Pick C1 like 1 (NPC1L1), acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2), microsomaltriacylglycerol-transport protein (MTP), ATP-binding cassette sub-family G member 5 (ABCG5) and member 8 (ABCG8) in C57BL/6J mice fed a non-cholesterol diet (NCD), a high-cholesterol diet (HCD), or one of the three experimental high-cholesterol diets containing 1% ginger extract (HCD + GE), 0.2% TMAO (HCD + TMAO), or 0.2% TMAO plus 1% ginger extract (HCD + TMAO + GE) for 12 weeks. Data are expressed as mean ± SD, n = 9. Means with different superscript letters differ significantly at p < 0.05.

and is an independent risk factor for cardiovascular disease in humans (Wang et al., 2011; Zeisel & Warrier, 2017). It has been shown that TMAO is atherogenic by causing cardiovascular dysfunction and impairing cholesterol metabolism (Chen et al., 2016; Koeth et al., 2013; Seldin et al., 2016). Previous studies have demonstrated that GE is cardio-protective (Fuhrman et al., 2000; Lei et al., 2014; Liu et al., 2015). To the best of our knowledge, the present study was the first to investigate the effect of GE on TMAO-exacerbated disturbance of cholesterol metabolism in mice fed a high-cholesterol diet (HCD). Results clearly demonstrated that TMAO elevated plasma TC and altered cholesterol metabolism. The present results are in agreement with two previous reports of Chen et al. (2016) and Ren, Liu, Zhao, and Yang (2016) who observed that 1% choline in diet and 3% choline in drinking water as a precursor of TMAO could raise serum TC in ApoE−/ − mice, respectively. The present study evidently showed that dietary GE suppressed the TMAO-aggravated elevation of plasma cholesterol in mice. This is consistent with the study of Lei et al. (2014), who reported that GE was able to decrease plasma TC in hypercholesterolemic hamsters. The mechanism by which TMAO elevated plasma TC was possibly attributable to its reducing effect on fecal excretion of bile acids. In general, the excessive cholesterol is eliminated mainly via two pathways. The first pathway is to convert excessive cholesterol in the liver to its metabolites bile acids and then eliminated in feces (Chen et al., 2008). The fecal sterol analysis showed that addition of TMAO in HCD diet remarkably reduced the excretion of fecal bile acids by 68.9% (Table 4). CYP7A1, as a key enzyme in bile acid synthesis, is responsible

found that GE suppressed both mRNA and protein level of SHP only in the presence of dietary TMAO (Fig. 3). SREBP2 and LXRα coordinately regulate the cholesterol metabolism with the former up-regulating genes involved in cholesterol biosynthesis and uptake, whereas the latter up-regulating cholesterol efflux genes (Chen et al., 2008; Miserez et al., 2002). LDL-R is to remove LDL cholesterol from circulation while HMG-CoA-R is the key enzymes regulating cholesterol synthesis in the liver (Chen et al., 2008). Results demonstrated that addition of GE did not affect the gene expression of LXRα, HMG-CoA-R, LDL-R or SREBP2 (Fig. 3). 3.7. Gene expression of intestinal NPC1L1, ACAT2, MTP, ABCG5/8 Cholesterol absorption requires several proteins including NPC1L1, ACAT2, MTP and ABCG5/8 (Wang, 2007). NPC1L1 is a sterol transporter responsible for transporting sterol from the intestinal lumen into enterocytes, while ACAT2 is to esterify cholesterol before being packed in chylomicrons by MTP (Wang, 2007). ABCG5 and 8 are responsible for effluxing the unabsorbed cholesterol form enterocyte to the lumen of intestine (Wang, 2007). Results from real-time PCR showed that GE down-regulated only mRNA of NPC1L1, MTP, ABCG5/8 but their protein mass in both absence and presence of dietary TMAO (Fig. 4). GE neither affected the mRNA nor protein of ACAT2 (Fig. 4). 4. Discussion TMAO is derived from various TMA-containing precursors in food 31

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5. Conclusion

for converting cholesterol to bile acids in the liver (Chen et al., 2008). To further investigate the underlying mechanism at a molecular level, we investigated effect of TMAO on gene expression of CYP7A1, finding TMAO significantly down-regulated hepatic CYP7A1 expression (Fig. 3). Together with the study of Chen et al. (2016), the present results suggested that TMAO-associated elevation in plasma TC was mediated by suppression on gene expression of hepatic CYP7A1. The second pathway to remove excessive cholesterol from the liver is via bile duct where cholesterol is directly eliminated to the lumen of small intestine followed by fecal excretion (Chen et al., 2008). When cholesterol reached the large intestine, it is converted to its microbial metabolites mainly including coprostanol, coprostanone and dihydrocholesterol (Gérard, 2013). Therefore, total fecal neutral sterols are usually regarded as a biomarker of this pathway. However, it was unlikely that TMAO-induced elevation of plasma TC was associated with this pathway because addition of TMAO in HCD diet did not affect the total fecal excretion of neutral sterols (Table 4). GE possessed a plasma TC lowering activity at both the absence and presence of TMAO (Table 3). The fecal sterol analysis clearly demonstrated that dietary GE remarkably increased the excretion of fecal bile acids by 49.0% in the absence of TMAO (Table 4). In the presence of TMAO, dietary GE could raise the excretion of fecal bile acids by 90.8%. At a molecular level, dietary GE could up-regulate the gene of hepatic CYP7A1 by one fold (Fig. 3). The present result was in agreement with the report of Lei et al. (2014), who found that dietary GE could increase the fecal excretion of bile acids and up-regulate the CYP7A1 in hypercholesterolemic hamsters. To take this further, FXR and SHP are two negative regulators in gene expression of CYP7A1 (Kong et al., 2012). The present study was the first time to observe that dietary GE downregulated SHP at both transcriptional and translational levels, while it could down-regulate only mRNA but its protein of FXR in the liver. It was likely that plasma TC-lowering activity associated with dietary GE was mediated by down-regulation of hepatic SHP and up-regulation of hepatic CYP7A1, thus leading to an increase in the synthesis and excretion of fecal bile acids. Dietary GE slightly increased the excretion of total fecal neutral sterols in the presence of TMAO but it had no effect in the absence of TMAO (Table 4). However, it was interesting to notice that dietary GE proportionally decreased coprostanol while it proportionally increased cholesterol and dihydrocholesterol contents (Table 4). Although this study was not to investigate the effect of dietary GE on microbiota in the large intestine, the present results suggested that dietary GE was able to modulate the microbial community in the large intestine, reduce the number of coprostanol-producing bacterial species and thus change the spectrum of fecal neutral sterols. Atherosclerosis is a chronic inflammatory disease caused by the accumulation of fat deposits and fibrous elements in the large arteries (Ross, 1999). Vascular inflammation, characterized by elevations in plasma levels of proinflammatory cytokines and declines in plasma concentrations of anti-inflammatory cytokines, may cause vascular injury, promote the recruitment of monocytes, and facilitate the atherosclerotic plaque development (Lind, 2003; Lusis, 2000; Sprague & Khalil, 2009). The present study clearly demonstrated that HCD diet raised the plasma concentrations of proinflammatory cytokines (IL-6, IL-1β and TNF-α), while it reduced plasma anti-inflammatory IL-10 (Fig. 2B). Addition of TMAO in HCD diet increased plasma IL-6 and TNF-α concentrations (Fig. 2B), suggesting that TMAO could aggravate the HCD-induced vascular inflammation. This was in agreement with previous studies which showed that TMAO exacerbated inflammation in high-fat-fed mice (Gao et al., 2014, 2015). The present study demonstrated that dietary GE possessed an anti-inflammatory activity as reflected by reductions in plasma levels of proinflammatory cytokines IL-6, MCP-1, IL-1β and TNF-α (Fig. 2B).

In summary, we found that TMAO raised plasma TC, hepatic cholesterol and proinflammatory cytokines in mice fed a high-cholesterol diet. Plasma TC raising activity of TMAO was mediated by decreasing the fecal excretion of bile acids via down-regulation of CYP7A1. Dietary GE could partially reverse the TMAO-aggravated adverse effects on cholesterol metabolism and reduce plasma TC by up-regulation of hepatic CYP7A1 expression via down-regulation of FXR and SHP. It was concluded that GE was cardio-protective against TMAO because it was not only hypocholesterolemic but also anti-inflammatory. 6. Animal experiment ethical file All experimental procedures were approved by the Animal Experimental Ethical Committee, The Chinese University of Hong Kong. This has been stated in the text. Conflict of interest We have no conflict of interest in this research. Acknowledgements This project was partially supported by a grant from the Health and Medical Research Fund, The Food and Health Bureau, The Government of the Hong Kong Special Administrative Region, China (Project No. 13140111). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2018.10.022. References Boini, K. M., Hussain, T., Li, P. L., & Koka, S. (2017). Trimethylamine-N-oxide instigates NLRP3 inflammasome activation and endothelial dysfunction. Cellular Physiology and Biochemistry, 44, 152–162. Chen, Z. Y., Jiao, R., & Ma, K. Y. (2008). Cholesterol-lowering nutraceuticals and functional foods. Journal of Agricultural and Food Chemistry, 56, 8761–8773. Chen, M. L., Yi, L., Zhang, Y., Zhou, X., Ran, L., Yang, J., et al. (2016). Resveratrol attenuates trimethylamine-N-oxide (TMAO)-induced atherosclerosis by regulating TMAO synthesis and bile acid metabolism via remodeling of the gut microbiota. mBio, 7, e02210–e2215. Chen, M. L., Zhu, X. H., Ran, L., Lang, H. D., Yi, L., & Mi, M. T. (2017). Trimethylamine-Noxide induces vascular inflammation by activating the NLRP3 inflammasome through the SIRT3-SOD2-mtROS signaling pathway. Journal of the American Heart Association, 6, e006347. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (2001). Executive summary of the third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). The Journal of the American Medical Association, 285, 2486–2497. Fuhrman, B., Rosenblat, M., Hayek, T., Coleman, R., & Aviram, M. (2000). Ginger extract consumption reduces plasma cholesterol, inhibits LDL oxidation and attenuates development of atherosclerosis in atherosclerotic, apolipoprotein E-deficient mice. The Journal of Nutrition, 130, 1124–1131. Gao, X., Liu, X., Xu, J., Xue, C., Xue, Y., & Wang, Y. (2014). Dietary trimethylamine Noxide exacerbates impaired glucose tolerance in mice fed a high fat diet. Journal of Bioscience and Bioengineering, 118, 476–481. Gao, X., Xu, J., Jiang, C., Zhang, Y., Xue, Y., Li, Z., et al. (2015). Fish oil ameliorates trimethylamine N-oxide-exacerbated glucose intolerance in high-fat diet-fed mice. Food & Function, 6, 1117–1125. Geng, J., Yang, C., Wang, B., Zhang, X., Hu, T., Gu, Y., et al. (2018). Trimethylamine Noxide promotes atherosclerosis via CD36-dependent MAPK/JNK pathway. Biomedicine & Pharmacotherapy, 97, 941–947. Gérard, P. (2013). Metabolism of cholesterol and bile acids by the gut microbiota. Pathogens, 3, 14–24. He, Z., & Chen, Z. Y. (2017). What are missing parts in the research story of trimethylamine-N-oxide (TMAO)? Journal of Agricultural and Food Chemistry, 65, 5227–5228. Hsiang, C. Y., Cheng, H. M., Lo, H. Y., Li, C. C., Chou, P. C., Lee, Y. C., et al. (2015). Ginger and zingerone ameliorate lipopolysaccharide-induced acute systemic inflammation in mice, assessed by nuclear factor-κB bioluminescent imaging. Journal of Agricultural and Food Chemistry, 63, 6051–6058.

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