- Email: [email protected]
Effect of embryo-remaining oat rice on the lipid profile and intestinal microbiota in high-fat diet fed rats
Journal Pre-proofs Effect of embryo-remaining oat rice on the lipid profile and intestinal microbiota in high-fat diet fed rats Kai Huang, Wenwen Yu, Sen Li, Xiao Guan, Jing Liu, Hongdong Song, Dandan Liu, Ruiqian Duan PII: DOI: Reference:
S0963-9969(19)30702-1 https://doi.org/10.1016/j.foodres.2019.108816 FRIN 108816
To appear in:
Food Research International
Received Date: Revised Date: Accepted Date:
3 August 2019 8 November 2019 10 November 2019
Please cite this article as: Huang, K., Yu, W., Li, S., Guan, X., Liu, J., Song, H., Liu, D., Duan, R., Effect of embryo-remaining oat rice on the lipid profile and intestinal microbiota in high-fat diet fed rats, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108816
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.
Effect of embryo-remaining oat rice on the lipid profile and intestinal microbiota in high-fat diet fed rats Kai Huanga, Wenwen Yua, Sen Lia, Xiao Guana*, Jing Liub, Hongdong Songa, Dandan Liua, Ruiqian Duana aSchool
of Medical Instruments and Food Engineering, University of Shanghai for
Science and Technology, Shanghai, P.R. China bCollege
of Information Engineering, Shanghai Maritime University, Shanghai
200135, P.R. China *Corresponding author: Xiao Guan Telephone number: 86-021-55396993 E-mail: [email protected]
Abstract Embryo-remaining oat rice (EROR), as a newly developed oat product, is popular in China for its good taste, but little is known about its healthy functions. In this study, the effects of EROR on lipid metabolism regulation were investigated in in vitro and in vivo models. The results showed that the oat ethanol extracts significantly alleviated lipid accumulation, total cholesterol and triglyceride levels in HepG2 cells. EROR supplementation dramatically improved the lipid profile in the serum and liver and downregulated the expression levels of HMGCR, SREBP-1C and FAS, which are related to lipid metabolic disorder in high-fat diet (HFD) fed rats. A HFD decreases the production of short-chain fatty acids (SCFAs) in the cecum, which are related to intestinal microbiota dysbiosis. The intake of EROR significantly increased the total SCFAs, acetate and propionate and promoted the abundance of SCFA-producing bacteria. Furthermore, the intake of EROR led to abundant increases in Bifidobacterium and Akkermansia and decreases of Rombutsia, Fusicatenibacter, Holdemanella and Turicibacter, which were negatively and positively correlated with the lipid metabolism-related indices. These results provide evidence that EROR is a good functional food candidate to ameliorate lipid metabolic disorder and hyperlipidemia.
Keywords: EROR, Lipid metabolism regulation, Hyperlipidemia, SCFAs, Intestinal microbiota
1. Introduction Oat (Avena sativa L.), as an important grain grown around the word, has been recognized as a valuable foodstuff and numerous oat products have been developed (Miller & Fulcher, 2011). Because of eating habits, Chinese people prefer oat rice (OR), which is produced just from the oat kernel with the hull removed or just the naked oat. OR is composed of three distinct anatomical parts: bran, the endosperm and the embryo (germ). Although oat bran is rich in vitamins, minerals, polysaccharides, lipids and proteins (Wood, 2010; Leão, Aquino, Dias, & Koifman, 2019), complete bran in OR leads to a tough taste and a long cooking time (Chakraborty et al., 2019). To solve these problems, embryo-remaining oat rice (EROR), as a welcomed oat product in China, has been newly developed based on traditional OR with deeper processing techniques. The bran in EROR is partially removed while the complete embryo, as the important resource of proteins and lipids, remains. Compared with traditional OR, EROR has the advantages of good taste, better appearance and a short cooking time. In addition to taste, people pay attention to nutritional quality and healthy functions. Oat provides abundant, well-balanced protein, soluble dietary fiber, lipids, starch, and B vitamins (Wood, 2010). Consumption of oat products has health benefits associated with coronary heart disease, blood and liver cholesterol levels, blood glucose levels and diabetes (Rasane, Jha, Sabikhi, Kumar, & Unnikrishnan, 2015; Chakraborty et al., 2019). Many studies have suggested that oat products, regarding the ability of oat βglucan, can reduce blood cholesterol and the risk of cardiovascular diseases (Dong, Cai, Shen, & Liu, 2011; Tiwari & Cummins, 2011). The evidence for these beneficial effects of oat β-glucan has been accepted by the European Food Safety Authority (EFSA) and the Food and Drug Administration (FDA) (Grundy et al., 2017). However, the mechanisms of the lowering of blood cholesterol are still unclear. The beneficial effects of oats have been attributed not only to the presence of β-glucan but also to other bioactive compounds. Several studies have shown that oat oil and protein also significantly reduce blood cholesterol (Tong et al., 2014; Tong et al., 2016). Currently, increasing attention has been paid to the effects of investigations on the properties of individual oat constituents, and these effects would have impact on the extent of their
bioactivity (Grundy et al., 2017; Leão, Aquino, Dias, & Koifman, 2019). However, little attention has been paid to the impact of oat kernel processing. Different processes affect the characteristics of the oat matrix on a macroscale and the structural integrity (Albert Lihong, Nancie, Giovanni, & Michael, 2015). Furthermore, these characteristics impact the digestion and adsorption of oat products (Rasane et al., 2015). EROR is regarded as a whole grain product with good taste and special processing. However, little information is available about the health benefits of this whole grain food. Hence, investigations on its nutrition and function are crucial in determining its future development. The aim of this study was to investigate the healthy functions of EROR and its extract to gain a better understanding of how EROR affects serum and liver lipid levels. It has been reported that intestinal microbiota plays an important role in the regulation of lipid metabolism (Danneskiold-Samsøe et al., 2019; Li et al., 2019a), and previous studies have collectively suggested that cecal short chain fatty acids (SCFAs) effectively reduce plasma cholesterol concentrations (Gong et al., 2018; Koh et al., 2016). However, the effects of EROR on intestinal microbiota and cecal SCFAs and the regulatory functions in lipid synthesis remain insufficiently explained. In this study, the effects of EROR on the lipid profile, cecal SCFAs and intestinal microbiota in high-fat diet (HFD) fed rats were investigated. Additionally, the role of the ethanol extract of EROR on cell lipid accumulation in vitro was evaluated. 2. Materials and methods 2.1 Materials and reagents EROR was provided by Yan Gufang Co., Ltd. (Huhehaote, Inner Mongolia, China), and the compositions were measured by China Agricultural University (Supplementary Table 1). Acetic acid, propionic acid, and butyric acid were purchased from Aladdin Co., Ltd. (Shanghai, China). Oil Red O was purchased from Sigma-Aldrich (St. Louis, MO, USA) and other chemical reagents used in this study were of analytical grade. 2.2 Preparation of EROR extracts The dried EROR was ground into a powder using a pulverizer (HK-820, Xulang Machinery Equipment Co., Ltd., Guangzhou, Guangdong, China) at room temperature.
The lipid extract of EROR (OL) was obtained by extraction with n-hexane, and the ratio of grain flour to n-hexane was 1:4. Residue fractions were dried and mixed with deionized water (1:10 ratio) at 50 ℃ and then incubated for 2 h. The solution was centrifuged, and the supernatant was freeze-dried. The dried powder was regarded as the water extract of EROR (OW). The precipitate was dried and then dissolved in a 10fold volume of 80% ethanol for 2 h at 50 °C. The solution was centrifuged, and the supernatant was evaporated at 60 °C to remove the ethanol using a rotatory evaporator. The obtained fractions were freeze-dried, and the powder was regarded as the ethanol extract of EROR (OE). All the EROR extracts were stored at -20 °C and dissolved in DMSO for the following experiments. 2.3 EROR treatments on HepG2 cells HepG2 cells were cultured in 10% fetal bovine serum with Dulbecco’s minimal Eagle medium (DMEM) at 37 °C and 5% CO2. Cells were inoculated into 96-well plates (5 × 103 cells/well) and handled as follows: (1) control group: cultured in the stated medium without OA and extracts for 24 h; (2) model group: pretreated with 1 mM OA for 24 h and then cultured in the stated medium without extracts for 24 h; or (3) pretreated with 1 mM OA for 24 h and then cultured in the stated medium with extracts at low-dose level (100 and 200 µg/ml) or high-dose level (400 µg/ml) for 24 h. Cultured cells were fixed with 4% paraformaldehyde for 30 min and then stained with Oil Red O to measure lipid droplet accumulation with a MicroQ6 plate reader (BioRad, Hercules, CA, USA) at 510 nm. Stained cells were visualized using a microscope (Olympus, Tokyo, Japan) at a final magnification of 200×. Cell viability was determined using the MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime Biotech, Nanjing, Jiangsu, China) with various concentrations of EROR extract (0-400 µg/ml) for 24 h. The absorbance of formazan was measured at 570 nm. 2.4 Animal and sample collection A total of 24 male SD rats (weighing 180-220 g) were purchased from Shanghai Jie Si Jie Laboratory Animal Co., Ltd. (Shanghai, China). The rats were housed in stainless steel screen-bottomed cages. The room was illuminated with a dark/light cycle (8:00 am on - 8:00 pm off) at a constant temperature of 22 ± 2 °C and a humidity of 40-60%.
After adaptive feeding for a week, rats were discretionarily divided into four groups (n = 6 per group, 3 rats in the same group housed per cage): a normal diet group (ND), a high-fat diet group (HFD), a HFD supplemented with 10% EROR group (HFD-LO) and a HFD supplemented with 50% EROR group (HFD-HO). The composition of the experimental diet is presented in Supplementary Table 2. All rats were allowed free access to tap water and the corresponding diet mentioned above. Food intake and body weights were recorded weekly. At the end of 4 weeks, the rats were fasted for 12 h and then anesthetized with pentobarbital sodium (50 mg/kg). The rats were dissected, and blood samples were collected from the heart. Samples were centrifuged at 4000 rpm and 4 °C for 3 min, and the serum was stored at -80 °C until analysis. The livers were weighed and stored at -80 °C, and the liver index was calculated as liver weight (g)/body weight (g). The cecum of each rat was removed, and 2 g of fresh cecal contents were collected and stored at -80 °C. All animal experiments were conducted according to the guidelines of the Laboratory Animal Ethics Association of the University of Shanghai for Science and Technology (approval opinion: SCXK (SH) 20130006). 2.5 Biochemical analysis in the serum and liver The levels of TG, TC, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured with commercial reagent kits (Jiancheng Biotech, Naning, Jiangsu, China). The activities of serum alanine transaminase (ALT) and aspartate transaminase (AST) were assayed according to the manufacturer’s instructions for the colorimetric enzymatic kits (Jiancheng Biotech, Nanjing, Jiangsu, China). 2.6 Histological analysis of liver tissue A portion of the right liver lobe was fixed with 4% paraformaldehyde overnight. The fixed liver tissues were paraffin-embedded, cut into 5 μm sections and then stained with hematoxylin eosin (H&E) according to the standard method. The stained sections were observed and photographed with a microscope (Olympus, Tokyo, Japan) at a final magnification of 400×. 2.7 Gene expression analysis in the liver Liver RNA was extracted using the TRIzol Reagent Kit (Life Technologies, Carlsbad,
CA, USA). Reverse transcription reactions were conducted using the HiScript II Q RT SuperMix for qPCR Reagent Kit (Vazyme Biotech Co., Ltd, Nanjing, Jiangsu, China). The obtained product was regarded as the template for real-time PCR, which was performed with the ChamQ™ Universal SYBR qPCR Master Mix Regent Kit (Vazyme Biotech Co., Ltd, Nanjing, Jiangsu, China) and a CFX96 real-time PCR detection system (BioRad, Hercules, CA, USA). The primer sequences involved in our research are presented in Supplementary Table 3. The relative mRNA levels were normalized to the level of the β-actin gene in each sample and expressed as values of relative expression compared to that of the ND group. Relative levels of target mRNAs were determined using the 2-ΔΔCt method and normalization. 2.8 Cecal SCFA measurement The cecal SCFA concentrations were measured using a 7890 gas chromatograph (GC, Agilent Technologies, Palo Alto, CA, USA) with a DB-WAX capillary column (1227032, 30 m × 0.25 μm × 0.25 mm, Agilent Technologies) as in a previous report (Li et al., 2019a). Briefly, 50 mg of cecal contents were diluted with 500 μl of distilled water and then centrifuged at 1000 rpm for 5 min. The obtained supernatant was mixed with 25% metaphosphoric acid solution (9:1), incubated overnight at 4 °C, and then centrifuged at 10000 rpm for 5 min. The prepared sample (1 μl) was injected into the GC, and the procedures were as follows: 80 °C for 30 s, raised to 130 °C at a rate of 5 °C/min and held for 2 min, raised to 240 °C at a rate of 20 °C/min and held for 1 min. 2.9 Analysis of gut microbiota Bacterial genomic DNA was extracted from 300 mg of cecal contents from each rat with the DNA Isolation Kit (Takara Co., Dalian, Liaoning, China). The 16S rDNA V3V4
region
was
amplified
ACTCCTACGGGAGGCAGCAG-3′)
using
the and
primers 806R
338F
(5′(5′-
GGACTACHVGGGTWTCTAAT-3′). DNA concentration was quantified using a QuantiFluor™-ST fluorescent quantitative system (Promega Co., Madison, WI, USA). High-throughput sequencing was performed on an Illumina MiSeq platform (Illumina Co., San Diego, CA, USA) according to the standard proposed by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw sequence data were deposited into the
NCBI sequence read archive database (https://www.ncbi.nlm.nih.gov/sra) with accession No. SRP171421. 2.10 Bioinformatics and statistical analysis The composition and richness of the intestinal microbiota were analyzed using the online platform provided by the Majorbio I-Sanger Cloud Platform (www.isanger.com). Operational taxonomic units (OTUs) were clustered with 97% identity by UPARSE (http://drive5.com/uparse/). Alpha diversity parameters such as ACE and Shannon index were analyzed using mothur software (http://mothur.org/). A heat map at the genus level was generated using the R package heat map followed by relative abundance calculation at the genus level by the R software package (https://www.rproject.org/). The relationships between the intestinal microbiota and biochemical indicators were all preformed using the heat map package by Spearman’s rank correlation. Data were analyzed with GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) by one-way ANOVA. Significant differences were evaluated by Tukey’s post hoc test. The values are expressed as the mean ± SEM. The significance level was set at P < 0.05. 3. Results 3.1 Effects of EROR extracts on lipid accumulation in HepG2 cells Different EROR extracts were prepared with a sequential extraction strategy. A total of 6.7 g of OL, 20.5 g of OW and 3.5 g of OE were extracted from 100 g of EROR. As shown in Fig. 1a, compared with the ND group, OA treatment significantly induced lipid accumulation. OE treatment inhibited lipid accumulation (up to 20%) in a dosedependent manner. However, the OL and OW treatments showed no significant difference at any concentration level (data not shown). As shown in Fig. 1b-e, MTT analysis showed that the OE did not have cytotoxic effects at concentrations ranging from 0 to 400 μg/ml. Furthermore, OE treatment significantly decreased the intracellular TG and TC levels in OA-induced HepG2 cells. 3.2 Effect of EROR supplementation on body weight, liver index, and hepatic lipid accumulation As shown in Fig. 2a, compared with the HFD group, the intake of EROR did not affect
the body weights of the rats. However, EROR supplementation decreased the liver index (Fig. 2b). As shown in Fig. 2c, compared with the ND group, the hepatocytes were swollen and their volume increased significantly in the HFD group, and the cytoplasm was filled with fat vacuoles. Moreover, the cell boundary was not clear, and the hepatocytes showed severe steatosis. Compared with the HFD group, the HFD-LO and HFD-HO groups had smaller fat vacuoles and clearer cell boundaries. 3.3 Rat lipid profile analysis As shown in Fig. 3a-d, compared with the ND group, the serum TG, TC, and LDL-C levels in the HFD, HFD-LO and HFD-HO groups were significantly higher, while the HDL-C levels were lower. Compared with the HFD group, the intake of EROR significantly decreased the serum TG, TC and LDL-C levels and increased the HDL-C levels. The liver lipid distributions are shown in Fig. 3e-f, which suggests that the rat model of non-alcoholic fatty liver was successfully established. Compared with the HFD group, the HFD-HO group showed significantly lower hepatic TG and TC levels, while the HFD-LO group only showed a significantly lower TC level but no significant difference in the TG level. 3.4 Analysis of hepatic dysfunction markers and gene expression in liver The effects of EROR supplementation on liver enzymes were investigated. As shown in Fig. 4a-b, the activities of ALT and AST in the HFD group were significantly higher than those in the ND group. Compared with the HFD group, supplementation with EROR significantly decreased the activities of ALT and AST. To assess the mechanism of lipid metabolism in the liver, the expression levels of the genes SREBP-1C, FAS, and HMGCR were determined. As shown in Fig. 4c-e, compared with the ND group, the FAS gene in the HFD group was significantly upregulated, and the other two genes were upregulated slightly. Compared with the HFD group, the expression levels of all hepatic lipid metabolism-, fatty acid- and cholesterol synthesis-related genes were significantly downregulated in the HFD-LO and HFD-HO groups. 3.5 SCFA analysis The generated SCFAs (the concentrations of total SCFAs, acetate, propionate and
butyrate) in the cecal contents of each rat were determined. As shown in Fig. 5, compared with the ND group, there were significant decreases in the contents of total SCFAs, acetate and butyrate in the HFD group. The concentrations of total SCFAs, acetate and propionate in the HFD-HO group were significantly higher than those in the HFD group. Interestingly, the concentration of propionate in the HFD-HO group was significantly higher than that in the ND group. Acetate, propionate and butyrate showed no significant difference in the HFD-LO group compared with the HFD group. 3.6 Effect of EROR on the composition of intestinal microbiota To determine the specific changes in intestinal microbiota, the OTUs were obtained by high-throughput sequencing. A total of 16 rat samples from the four groups were evaluated. After the sequence optimization, a total of 1,072,565 reads were generated, corresponding to an average of 268,414 reads per group. After quality filtering, the average length of each read was more than 430 bp. Sequences were clustered into a total of 848 OTUs at a 97% similarity level (data not shown). The results of the ACE and Shannon index showed that the intake of EROR did not affect the intestinal microbiota diversity (Fig. 6a-b). As shown in Fig. 6c-f, compared with the ND group, a HFD mainly led to a higher abundance of Firmicutes (93.0%, phylum level), Bacilli and Clostridia (29.6% and 58.3%, respectively, class level), Clostridiales and Lactobacillales (58.3% and 27.2%, respectively, order level), and Lachnospiraceae and Lactobacillaceae (41.3% and 26.5%, respectively, family level). Compared with the HFD group, supplementation of EROR mainly increased the abundance of Actinobacteria (34.0-34.6%, phylum level), Coriobacteriales, Bacillales and Verrucomicrobiales (26.1-32.3%, 5.7-6.7% and 2.3-4.7%, respectively, order level) and Bifidobacteriaceae, Staphylococcaceae, and Coriobacteriaceae (0.5-2.1%, 5.7-6.7% and 26.1-32.3%, respectively, family level), and decreased the abundance of Firmicutes (60.1-62.7%), Bacilli and Clostridia (11.2-18.5% and 40.9-47.7%, respectively), Clostridiales and Lactobacillales (40.9-47.7% and 5.4-11.6%, respectively) and Lachnospiraceae and Lactobacillaceae (24.9-30.0% and 4.5-10.5% respectively). Furthermore, as shown in Fig. 7, the abundances of 20 key bacteria with significant changes at each level were investigated. At the genus level, intake of EROR decreased
the abundance of Desulfovibrio, Holdemanella, Fusicatenibacter, Faecalitalea, Turicibacter and Bifidobacterium and increased the abundance of Akkermansia, Ruminococcus ssp, Faecalibaculum and Roseburia. 3.7 Correlation between gut microbiota and lipid metabolism related indices The correlations between gut microbiota (the relative abundance of 50 key species at the genus level) and lipid metabolism-related indices were analyzed using Spearman correlation analysis (Fig. 8). The serum TC, TG, LDL-C, hepatic TC, TG and genes SREBP-1C, FAS, and HMGCR were positively correlated with Fusicatenibacter, Faecalitalea,
Blautia,
Eubacterium_hallii_group,
Akkermansia,
Eubacterium_nodatum_group and unclassified Lachnospiraceae but negatively correlated
with
Bifidobacterium,
Lachnospiraceae_UCG006,
norank
Feacalibacterium, Coriobacteriaceae,
Enterohabdus, Romboutsia
and
Staphylococcus. However, the correlation between the serum HDL-C and gut microbiota showed the opposite trend, which was positively correlated with Feacalibacterium, Enterohabdus, norank Coriobacteriaceae, Staphylococcus and Romboutsia
and
negatively
correlated
with
Faecalitalea,
Blautia
and
Eubacterium_hallii_group. In addition, the liver TC also showed a negative correlation with
Tuicibacter
and
a
positive
correlation
with
Roseburia
and
Ruminococcus_torques_group. 4. Discussion Recent studies have reported that the health benefits of whole grains are related to dietary fiber (Xia et al., 2017; Chakraborty et al., 2019). OR is an excellent source of dietary fiber, which usually accounts for 10.2-12.1% of OR (Manthey, Hareland, & Huseby, 1999). Partial oat bran removal leads to decreases in dietary fiber and some other constituents. In this study, the total dietary fiber of EROR was only 6.36%, and nearly 7.04% of the dietary fiber was removed (Supplementary Table 1). However, EROR presents the advantages of cooking properties. Furthermore, we found that EROR alleviated lipid disorder in HFD fed rats in the present study. In addition to dietary fiber, constituent phytochemicals also play important roles in health benefits, such as β-glucan, oil, protein and polyphenol (Grundy et al., 2017; Tong et al., 2014;
Tong et al., 2016). All these constitutes mainly remained in the lipid, water and ethanol extracts. Numerous studies have claimed that natural plant ethanol extracts modulate lipid metabolism in vivo and in vitro, and these functions are mainly attributed to the phenolic compounds, such as ethanol extracts from millet, black adzuki bean, mulberry, Cynara scolymus, and Schisandra chinensis berry (Chung et al., 2017; Kim, Kim, & Cha, 2017; Li et al., 2019a; Safat, Sheibani, Mohammadi, Hasanabadi, & Sakhaee, 2017). Similar to these reports, OE treatment effectively inhibited lipid accumulation and decreased the levels of TC and TG in OA-induced HepG2 cells. Thus, it has been proposed that the effect of OE on lipid modulation is also related to the presence of oat phenolic compounds. Further investigation of oat phenolic compounds on lipid disorder is currently being investigated in our laboratory. A HFD results in an increased risk of hyperlipidemia, which is accompanied by a significant increase in serum TC, TG and LDL-C and a simultaneous decrease in HDLC (Scicchitano et al., 2014). Consumptions of whole grain foods, such as sorghum, purple rice, rhubarb rice, qingke and millet, have been reported to reduce serum lipid concentrations (Arbex et al., 2018; De Sousa et al., 2018; Li et al., 2019a; Shen et al., 2017; Xia et al., 2017). Similar to these reports, EROR supplementation improved the serum lipid profile in HFD fed rats. Disease research has indicated that LDL-C and TG, as the key risk indicators of heart and cerebrovascular diseases, play important roles in the process of intervention and treatment (Li et al., 2019b). The changes in serum biochemical indicators were associated with healthy morphological alterations in rat livers. A HFD promotes the accumulation of fat droplets and then causes fatty liver (Scicchitano et al., 2014). Histopathological analysis showed that EROR could regulate the structure of liver tissue and that the number of lipid droplets were significantly reduced. Furthermore, EROR improved AST and ALT levels, which indicated that EROR ameliorated fatty liver in HFD fed rats. The serine kinase AMPK plays an important role in glucose and lipid metabolism. Lipid metabolism disorder is usually regulated by downstream genes in the AMPK signaling pathway, such as SREBP-1C, HMGCR and FAS (Brahma Naidu et al., 2016). SREBP1C, as a transcription factor that maintains lipid homeostasis, regulates the expression
of genes (FAS and HMGCR) and fat synthesis in the liver (Moreira et al., 2018). The intake of a HFD increased the expression of FAS, which induced the production of C16:0 (Dziadek, Kopeć, & Piątkowska, 2019). Downregulation of FAS showed the regulation of fatty acids in HFD fed rats. HMGCR, the rate-limiting enzyme of cholesterol biosynthesis that transforms HMG-CoA into mevalonic acid, is regulated through a negative feedback mechanism and increases the decomposition rate of plasma LDL (Chen et al., 2019). The results showed that compared with the HFD group, EROR treatment significantly lowered the expression levels of SREBP-1C, FAS and HMGCR. These results suggested that EROR alleviated lipid metabolic disorder by regulating the related genes through the AMPK signaling pathway, which could provide a theoretical basis for the development of EROR. Undigested carbohydrates can be rapidly consumed by the intestinal microbiota to produce SCFAs, which are mainly composed of acetate, propionate and butyrate (Xia et al., 2017). Acetate is the most abundant SCFA, which serves as a substrate for fatty acid synthesis, enhances ileal motility, and downregulates lipogenesis-related gene expression, such as FAS and ACC (Choque Delgado & Tamashiro, 2018). Propionate can lower lipogenesis and serum cholesterol and is regarded as a good precursor for gluconeogenesis and protein synthesis, which decreases food intake via glucagon-like peptide-1- and peptide YY (PYY)-driven mechanisms (Koh et al., 2016). Butyrate is a good energy source for colonocytes and acts as an antitumor or anti-inflammatory agent (Morrison & Preston, 2016; Xia et al., 2017). Treatment of obese rodents with SCFA has been proven to reduce body weight and improve the plasma lipid profile (Lu et al., 2016). The HFD decreased the concentrations of SCFAs; however, the intake of EROR significantly improved the SCFA profile in HFD fed rats. Similarly, many studies have reported that the consumption of whole grains increased the content of the SCFAs (Gong et al., 2018; Li et al., 2019b; Xia et al., 2017). Most of these studies showed that higher SCFA production mainly resulted from dietary fiber (Koh et al., 2016; Maurer et al., 2019). However, some other components, such as tannins and phenolic compounds, also promote SCFA production (Li et al., 2019a; Maurer et al., 2019; Rocchetti et al., 2017). This is a potential reason for EROR having obvious stimulation
of SCFA production in HFD fed rats, even though its dietary fiber was partially removed. The intestinal microbiota is directly associated with lipid metabolic disorder, liver steatosis and obesity (Danneskiold-Samsøe et al., 2019; Li et al., 2019b; Monk et al., 2019). A HFD induces intestinal dysbiosis, which is usually accompanied by an increase in Firmicutes and Verrucomicrobia at the phylum level and an increase in Clostridia and Bacilli at the class level (Gao et al., 2019; Xia et al., 2017). A higher abundance of Firmicutes and a lower abundance of Bacteroidetes indicate an acceleration of energy harvest from food and increased energy storage in adipose tissue (Arbex et al., 2018). This acceleration and storage further suppresses the production of fasting-induced adipose factors and leads to the higher storage of triacylglycerols (Luo et al., 2018). Intake of EROR did not affect the intestinal microbiota diversity, although it decreased the abundance of Firmicutes, Clostridia and Bacilli and increased the abundance of Bacteroidetes (though its proportion was low in each group). At the family level, the intake of EROR increased the abundance of Porphyromonadaceae, Enterobacteriaceae, and Coriobacteriaceae. These bacteria are positively correlated with serum HDL-C and negatively correlated with obesity (Kong, Gao, Yan, Huang, & Qin, 2019). At the genus level, Spearman correlation analysis showed that the levels of TC, TG and LDL-C in the serum and liver and the expression of the related genes were positively correlated with Fusicatenibacter, Faecalitalea, Blautia, Akkermansia, Roseburia and unclassified Lachnospiraceae, while negatively correlated with Bifidobacterium, Feacalibacterium, Enterohabdus, Romboutsia, Tuicibacter and Staphylococcus. Similar to our results, studies have shown that Lachnospiraceae and Blautia contribute to the development of obesity and lipid metabolic disorders in obese mice (Chen et al., 2019; Ju et al., 2019; Lv, Guo, Li, Yu, & Liu, 2019). A HFD leads to a decrease in beneficial bacteria, such as Bifidobacterium and Feacalibacterium (Dziadek et al., 2019; Xia et al., 2017). Turicibacter has a negative impact on the healthy functions of the gut and maintaining the serum metabolic index (Luo et al., 2018; Monk et al., 2019). Furthermore, it has been reported that Rombutsia, Fusicatenibacter and Holdemanella are negatively correlated with serum HDL-C and
positively correlated with TG, TC and LDL-C (Arbex et al., 2018; Chen et al., 2019; Shen et al., 2017). The concentrations of SCFAs are related to the intestinal microbiota community richness, and a growing number of recent studies have shown that more attention should be focused on the lower classification levels of SCFA-producing bacteria (Morrison & Preston, 2016). It has been reported that the increased abundance of Akkermansia, Bifidobacterium, Blautia, Roseburia and Lachnospiraceae promoted the production of SCFAs (De Sousa et al., 2018; Koh et al., 2016; Xia et al., 2017). Interestingly, the intake of EROR increased the abundance of the bacteria mentioned above. The experimental results suggest that EROR may regulate the intestinal microbiota, promote SCFA production and alleviate lipid metabolic disorders against a HFD. In addition to these functions, intestinal microbiota regulation also plays therapeutic roles in other metabolism functions. EROR improved the abundance of Ruminococcus ssp and Faecalibacterium and decreased the abundance of Desulfovibrio. Ruminococcus ssp and Faecalibacterium have been reported as beneficial bacteria related to improving malnutrition and promoting health recovery (Kong, Gao, Yan, Huang, & Qin, 2019; Liang et al., 2018). Desulfovibrio, as an H2S-producing bacterium, is involved in blood pressure and plasmatic insulin levels (Prieto et al., 2018). Conclusions The present study confirmed that the ethanol extract of EROR decreased lipid accumulation in HepG2 cells. Furthermore, the intake of EROR, especially at a high dose, mediated the lipid disorder in HFD fed rats with an improvement in the lipid parameters in the serum and liver, modulated the related gene expression levels in liver, ameliorated cecal SCFAs, and beneficially regulated the intestinal microbiota. The current results contribute to the development of EROR as a potential functional food for dietary strategies to prevent lipid metabolic disorder. Funding This work was supported by the National Key Research and Development Program of China (2017YFD0401202), Yangtze River Delta Cooperation Program (18395810200) and Youth Scientific Talent Program of National Grain Industry (LQ2018204). Conflict of interest
The authors have no conflict of interests Authorship KH, SL, WWY, XG and JL designed the study and had primary responsibility for the content of the paper; WWY, DDL and RQD performed the experiments; HK, WWY analyzed the data and wrote the manuscript. All authors approved the manuscript. Reference Albert Lihong, Z., Nancie, H., Giovanni, R., & Michael, L. (2015). Whole grain oats improve insulin sensitivity and plasma cholesterol profile and modify gut microbiota composition in C57BL/6J mice. Journal of Nutrition, 145(2), 222-230. Arbex, P. M., Moreira, M. E. D. C., Toledo, R. C. L., Cardoso, L. D. M., Pinheiro-Sant'Ana, H. M., Benjamin, L. D. A., . . . Martino, H. S. D. (2018). Extruded sorghum flour (Sorghum bicolor L.) modulate adiposity and inflammation in high fat diet-induced obese rats. Journal of Functional Foods, 42, 346-355. Brahma Naidu, P., Uddandrao, V. V. S., Ravindar Naik, R., Suresh, P., Meriga, B., Begum, M. S., . . . Saravanan, G. (2016). Ameliorative potential of gingerol: Promising modulation of inflammatory factors and lipid marker enzymes expressions in HFD induced obesity in rats. Molecular and Cellular Endocrinology, 419, 139-147. Chakraborty, P., Witt, T., Harris, D., Ashton, J., Stokes, J. R., & Smyth, H. E. (2019). Texture and mouthfeel perceptions of a model beverage system containing soluble and insoluble oat bran fibres. Food Research International, 120, 62-72. Chen, H., Zeng, F., Li, S., Liu, Y., Gong, S., Lv, X., . . . Liu, B. (2019). Spirulina active substance mediated gut microbes improve lipid metabolism in high-fat diet fed rats. Journal of Functional Foods, 59, 215-222. Choque Delgado, G. T., & Tamashiro, W. M. d. S. C. (2018). Role of prebiotics in regulation of microbiota and prevention of obesity. Food Research International, 113, 183-188. Chung, M. Y., Shin, E. J., Choi, H. K., Kim, S. H., Sung, M. J., Park, J. H., & Hwang, J. T. (2017). Schisandra chinensis berry extract protects against steatosis by inhibiting histone acetylation in oleic acid-treated HepG2 cells and in the livers of diet-induced obese mice. Nutrition Research, 46, 1-10. Danneskiold-Samsøe, N. B., Dias de Freitas Queiroz Barros, H., Santos, R., Bicas, J. L., Cazarin, C. B. B., Madsen, L., . . . Maróstica Júnior, M. R. (2019). Interplay between food and gut microbiota in health and disease. Food Research International, 115, 23-31. De Sousa, A. R., de Castro Moreira, M. E., Toledo, R. C. L., dos Anjos Benjamin, L., Queiroz, V. A. V., Veloso, M. P., . . . Martino, H. S. D. (2018). Extruded sorghum (Sorghum bicolor L.) reduces metabolic risk of hepatic steatosis in obese rats consuming a high fat diet. Food Research International, 112, 48-55. Dong, J., Cai, F., Shen, R., & Liu, Y. (2011). Hypoglycaemic effects and inhibitory effect on intestinal disaccharidases of oat beta-glucan in streptozotocin-induced diabetic mice. Food Chemistry, 129(3), 1066-1071. Dziadek, K., Kopeć, A., & Piątkowska, E. (2019). Intake of fruit and leaves of sweet cherry beneficially affects lipid metabolism, oxidative stress and inflammation in Wistar rats fed with high fat-
cholesterol diet. Journal of Functional Foods, 57, 31-39. Gao, J., Ding, G., Li, Q., Gong, L., Huang, J., & Sang, Y. (2019). Tibet kefir milk decreases fat deposition by regulating the gut microbiota and gene expression of Lpl and Angptl4 in high fat diet-fed rats. Food Research International, 121, 278-287. Gong, L., Cao, W., Chi, H., Wang, J., Zhang, H., Liu, J., & Sun, B. (2018). Whole cereal grains and potential health effects: Involvement of the gut microbiota. Food Research International, 103, 84-102. Grundy, M. M. L., Quint, J., Rieder, A., Ballance, S., Dreiss, C. A., Cross, K. L., . . . Ellis, P. R. (2017). The impact of oat structure and β-glucan on in vitro lipid digestion. Journal of Functional Foods, 38(Pt A), 378-388. Ju, M., Liu, Y., Li, M., Cheng, M., Zhang, Y., Deng, G., . . . Liu, H. (2019). Baicalin improves intestinal microecology and abnormal metabolism induced by high-fat diet. European Journal of Pharmacology, 857, 172457. Koh, A., Filipe D. V., Petia K. D., & Fredrik B. (2016). From dietary fiber to host physiology: shortchain fatty acids as key bacterial metabolites. Cell, 165(6), 1332-1345. Kong, C., Gao, R., Yan, X., Huang, L., & Qin, H. (2019). Probiotics improve gut microbiota dysbiosis in obese mice fed a high-fat or high-sucrose diet. Nutrition, 60, 175-184. Leão, L. S. C. d. S., Aquino, L. A. d., Dias, J. F., & Koifman, R. J. (2019). Addition of oat bran reduces HDL-C and does not potentialize effect of a low-calorie diet on remission of metabolic syndrome: A pragmatic, randomized, controlled, open-label nutritional trial. Nutrition, 65, 126130. Li, S., Yu, W., Guan, X., Huang, K., Liu, J., Liu, D., & Duan, R. (2019a). Effects of millet whole grain supplementation on the lipid profile and gut bacteria in rats fed with high-fat diet. Journal of Functional Foods, 59, 49-59. Li, T.-T., Tong, A.-J., Liu, Y.-Y., Huang, Z.-R., Wan, X.-Z., Pan, Y.-Y., . . . Zhao, C. (2019b). Polyunsaturated fatty acids from microalgae Spirulina platensis modulates lipid metabolism disorders and gut microbiota in high-fat diet rats. Food and Chemical Toxicology, 131, 110558. Liang, Y., Liang, S., Zhang, Y., Deng, Y., He, Y., Chen, Y., . . . Yang, Q. (2018). Oral administration of compound probiotics ameliorates HFD-induced gut microbe dysbiosis and chronic metabolic inflammation via the G protein-coupled receptor 43 in non-alcoholic fatty liver disease rats. Probiotics Antimicrob Proteins, 1-11. Lu, Y., Fan, C., Li, P., Lu, Y., Chang, X., & Qi, K. (2016). Short chain fatty acids prevent high-fat-dietinduced obesity in mice by regulating G protein-coupled receptors and gut microbiota. Scientific Reports, 6, 37589. Luo, J., Yuetong, L., Yunshi, M., Lijuan, G., Shiyi, O., Yong, W., . . . Xichun, P. (2018). Flaxseed gum reduces body weight by regulating gut microbiota. Journal of Functional Foods, 47, 136-142. Lv, X. C., Guo, W. L., Li, L., Yu, X. D., & Liu, B. (2019). Polysaccharide peptides from Ganoderma lucidum ameliorate lipid metabolic disorders and gut microbiota dysbiosis in high-fat diet-fed rats. Journal of Functional Foods, 57, 48-58. Manthey, F. A., Hareland, G. A., & Huseby, D. J. (1999). Soluble and insoluble dietary fiber content and composition in oat. Cereal Chemistry, 76(3), 417-420. Maurer, L. H., Cazarin, C. B. B., Quatrin, A., Minuzzi, N. M., Costa, E. L., Morari, J., . . . Emanuelli, T. (2019). Grape peel powder promotes intestinal barrier homeostasis in acute TNBS-colitis: A major role for dietary fiber and fiber-bound polyphenols. Food Research International, 123,
425-439. Miller, S. S., & Fulcher, R. G. (2011). CHAPTER 5 – Microstructure and chemistry of the oat kernel 1. Oats, 77-94. Monk, J. M., Wu, W., Lepp, D., Wellings, H. R., Hutchinson, A. L., Liddle, D. M., . . . Power, K. A. (2019). Navy bean supplemented high-fat diet improves intestinal health, epithelial barrier integrity and critical aspects of the obese inflammatory phenotype. The Journal of Nutritional Biochemistry, 70, 91-104. Moreira, M. E. d. C., de Oliveira Araújo, F., de Sousa, A. R., Toledo, R. C. L., dos Anjos Benjamin, L., Veloso, M. P., . . . Martino, H. S. D. (2018). Bacupari peel extracts (Garcinia brasiliensis) reduces the biometry, lipogenesis and hepatic steatosis in obese rats. Food Research International, 114, 169-177. Morrison, D. J., & Preston, T. (2016). Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes, 7(3), 189-200. Prieto, I., Hidalgo, M., Segarra, A. B., Martã-Nez-Rodrã-Guez, A. M., Cobo, A., Ramã-Rez, M., . . . Martã-Nez-Caã-Amero, M. (2018). Influence of a diet enriched with virgin olive oil or butter on mouse gut microbiota and its correlation to physiological and biochemical parameters related to metabolic syndrome. Plos One, 13(1), e0190368. Rasane, P., Jha, A., Sabikhi, L., Kumar, A., & Unnikrishnan, V. S. (2015). Nutritional advantages of oats and opportunities for its processing as value added foods - a review. Journal of Food Science & Technology, 52(2), 662-675. Rocchetti, G., Lucini, L., Chiodelli, G., Giuberti, G., Gallo, A., Masoero, F., & Trevisan, M. (2017). Phenolic profile and fermentation patterns of different commercial gluten-free pasta during in vitro large intestine fermentation. Food Research International, 97, 78-86. Safat, A. A., Sheibani, H., Mohammadi, P., Hasanabadi, N., & Sakhaee, E. (2017). Evaluation of lipidlowering effect of Cynara scolymus extract-loaded mesoporous silica nanoparticles on ultralipid-fed mice. Comparative Clinical Pathology, 27(6), 1-6. Scicchitano, P., Cameli, M., Maiello, M., Modesti, P. A., Muiesan, M. L., Novo, S., . . . Ciccone, M. M. (2014). Nutraceuticals and dyslipidaemia: Beyond the common therapeutics. Journal of Functional Foods, 6, 11-32. Shen, Y., Song, X., Chen, Y., Li, L., Sun, J., Huang, C., . . . Zhang, H. (2017). Effects of sorghum, purple rice and rhubarb rice on lipids status and antioxidant capacity in mice fed a high-fat diet. Journal of Functional Foods, 39, 103-111. Tong, L. T., Zhong, K., Liu, L., Guo, L., Cao, L., & Zhou, S. (2014). Oat oil lowers the plasma and liver cholesterol concentrations by promoting the excretion of faecal lipids in hypercholesterolemic rats. Food Chemistry, 142, 129-134. Tong, L. T., Guo, L., Zhou, X., Qiu, J., Liu, L., Zhong, K., & Zhou, S. (2016). Effects of dietary oat proteins on cholesterol metabolism of hypercholesterolemic hamsters: Cholesterol-lowering effects of oat proteins. Journal of the Science of Food & Agriculture, 96(4), 1396-1401. Wood, P. J. (2010). Oat Bran. Journal of Food Biochemistry, 16(6), 401-401. Xia, X., Li, G., Ding, Y., Ren, T., Zheng, J., & Kan, J. (2017). Effect of whole grain qingke (Tibetan Hordeum vulgare L. Zangqing 320) on the serum lipid levels and intestinal microbiota of rats under high-fat Diet. Journal of Agricultural & Food Chemistry, 65(13), 2686-2693.
Figure legends Fig. 1 Effects of OE on lipid accumulation and viability of HepG2 cells. HepG2cells were treated
for 24 h with OE in the presence or absence of OA, and then lipid accumulation was measured by Oil-Red O staining (a, b). The effects of OE on intracellular TG and TC levels were determined in OA induced HepG2 cells (c, d). Cytotoxicity was measured by the MTT assay (e). All results are expressed as the mean ± SD. #p < 0.05,
##p<0.01, ###p<0.001
vs. Control; *p < 0.05, **p<0.01,
***p<0.001 vs. OA Fig. 2 Effect of EROR supplementation on body weight (a), liver index (b), liver fat accumulation (c). Data are expressed as the means ± SD. #P<0.05, ##P<0.01, ###P<0.001 compared to the ND group; *P<0.05, **P<0.01, ***P<0.001 compared to the HFD group (n=4 per group). Fig. 3 Effect of EROR supplementation on Lipid profile in serum and liver. Levels of TG, TC, HDL-C, and LDL-C in serum (a-d), and levels of TG and TC in liver (e-f). Data are expressed as the means ± SD. #P<0.05,
##P<0.01, ###P<0.001
compared to the ND group; *P<0.05, **P<0.01,
***P<0.01 compared to the HFD group (n=4 per group). Fig. 4 Effect of EROR supplementation on the activity of ALT (a) and AST (b), and the expression of SREBP-1C (c), FAS (d), HMGCR (e). One-way ANOVA was used for statistical analysis. The expression of hepatic lipid metabolism genes in liver was determined by real-time PCR analysis. Multiple comparison of two-way ANOVA was employed for statistical analysis (n=4 per group). Fig. 5 Effect of EROR supplementation on cecal SCFAs. Total SCFA (a), Acetate (b), Propionate (c) and Butyrate (d). Values are presented as the means ± SD. #P<0.05,
##P<0.01, ###P<0.001
compared to the ND group; *P<0.05, **P<0.01, ***P<0.001 compared to the HFD group (n=4 per group). Fig. 6 Effect of EROR supplementation on microbial community richness and Diversity. The α-diversity index of Ace and Shannon (a-b), and relative the abundance at the phylum, class, order and family level (c-f). Fig. 7 Detailed proportions for the specific bacteria at each classification levels. Firmicutes and Bacteroidetes (at the phylum level), Clostridia and Bacilli (at the class level), Porphyromonadaceae, Enterobacteriaceae, Lachnospiraceae and Coriobacteriaceae (at the family level), Akkermansia, Bifidobacterium,
Blautia,
Roseburia,
Fusicatenibacter,
Desulfovibrio,
Turicibacter,
Faecalibaculum, Holdemanella, Faecalitalea and Ruminococcus ssp (at the genus level). The ND group (), the HFD group (), the HFD-LO group () and the HFD-HO group () were
represented with different colors. Fig. 8 Heatmap of Spearman’s correlation between gut microbiota (the relative abundances of 50 key phylotypes at genus level) and lipid metabolism related indices. The colors range from blue (negative correlation) to red (positive correlation). Significant correlations are noted by *P<0.05, **P<0.01, ***P<0.001.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Conflict of Interest form
The authors have no conflict of interest/financial disclosure.
Highlights: 1. EROR is popular in China for its good taste, welcomed appearance and short cooking time 2. Ethanol extract of EROR could alleviate the lipid accumulation in OA induced HepG2 cells 3. EROR could dramatically modulate the lipid profile in serum and liver in HFD fed rats 4. Supplementation of EROR could significantly increase total SCFAs, acetate and propionate 5. EROR is beneficial for the regulation of intestinal microbiota dysbiosis against HFD
S0963-9969(19)30702-1 https://doi.org/10.1016/j.foodres.2019.108816 FRIN 108816
To appear in:
Food Research International
Received Date: Revised Date: Accepted Date:
3 August 2019 8 November 2019 10 November 2019
Please cite this article as: Huang, K., Yu, W., Li, S., Guan, X., Liu, J., Song, H., Liu, D., Duan, R., Effect of embryo-remaining oat rice on the lipid profile and intestinal microbiota in high-fat diet fed rats, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108816
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.
Effect of embryo-remaining oat rice on the lipid profile and intestinal microbiota in high-fat diet fed rats Kai Huanga, Wenwen Yua, Sen Lia, Xiao Guana*, Jing Liub, Hongdong Songa, Dandan Liua, Ruiqian Duana aSchool
of Medical Instruments and Food Engineering, University of Shanghai for
Science and Technology, Shanghai, P.R. China bCollege
of Information Engineering, Shanghai Maritime University, Shanghai
200135, P.R. China *Corresponding author: Xiao Guan Telephone number: 86-021-55396993 E-mail: [email protected]
Abstract Embryo-remaining oat rice (EROR), as a newly developed oat product, is popular in China for its good taste, but little is known about its healthy functions. In this study, the effects of EROR on lipid metabolism regulation were investigated in in vitro and in vivo models. The results showed that the oat ethanol extracts significantly alleviated lipid accumulation, total cholesterol and triglyceride levels in HepG2 cells. EROR supplementation dramatically improved the lipid profile in the serum and liver and downregulated the expression levels of HMGCR, SREBP-1C and FAS, which are related to lipid metabolic disorder in high-fat diet (HFD) fed rats. A HFD decreases the production of short-chain fatty acids (SCFAs) in the cecum, which are related to intestinal microbiota dysbiosis. The intake of EROR significantly increased the total SCFAs, acetate and propionate and promoted the abundance of SCFA-producing bacteria. Furthermore, the intake of EROR led to abundant increases in Bifidobacterium and Akkermansia and decreases of Rombutsia, Fusicatenibacter, Holdemanella and Turicibacter, which were negatively and positively correlated with the lipid metabolism-related indices. These results provide evidence that EROR is a good functional food candidate to ameliorate lipid metabolic disorder and hyperlipidemia.
Keywords: EROR, Lipid metabolism regulation, Hyperlipidemia, SCFAs, Intestinal microbiota
1. Introduction Oat (Avena sativa L.), as an important grain grown around the word, has been recognized as a valuable foodstuff and numerous oat products have been developed (Miller & Fulcher, 2011). Because of eating habits, Chinese people prefer oat rice (OR), which is produced just from the oat kernel with the hull removed or just the naked oat. OR is composed of three distinct anatomical parts: bran, the endosperm and the embryo (germ). Although oat bran is rich in vitamins, minerals, polysaccharides, lipids and proteins (Wood, 2010; Leão, Aquino, Dias, & Koifman, 2019), complete bran in OR leads to a tough taste and a long cooking time (Chakraborty et al., 2019). To solve these problems, embryo-remaining oat rice (EROR), as a welcomed oat product in China, has been newly developed based on traditional OR with deeper processing techniques. The bran in EROR is partially removed while the complete embryo, as the important resource of proteins and lipids, remains. Compared with traditional OR, EROR has the advantages of good taste, better appearance and a short cooking time. In addition to taste, people pay attention to nutritional quality and healthy functions. Oat provides abundant, well-balanced protein, soluble dietary fiber, lipids, starch, and B vitamins (Wood, 2010). Consumption of oat products has health benefits associated with coronary heart disease, blood and liver cholesterol levels, blood glucose levels and diabetes (Rasane, Jha, Sabikhi, Kumar, & Unnikrishnan, 2015; Chakraborty et al., 2019). Many studies have suggested that oat products, regarding the ability of oat βglucan, can reduce blood cholesterol and the risk of cardiovascular diseases (Dong, Cai, Shen, & Liu, 2011; Tiwari & Cummins, 2011). The evidence for these beneficial effects of oat β-glucan has been accepted by the European Food Safety Authority (EFSA) and the Food and Drug Administration (FDA) (Grundy et al., 2017). However, the mechanisms of the lowering of blood cholesterol are still unclear. The beneficial effects of oats have been attributed not only to the presence of β-glucan but also to other bioactive compounds. Several studies have shown that oat oil and protein also significantly reduce blood cholesterol (Tong et al., 2014; Tong et al., 2016). Currently, increasing attention has been paid to the effects of investigations on the properties of individual oat constituents, and these effects would have impact on the extent of their
bioactivity (Grundy et al., 2017; Leão, Aquino, Dias, & Koifman, 2019). However, little attention has been paid to the impact of oat kernel processing. Different processes affect the characteristics of the oat matrix on a macroscale and the structural integrity (Albert Lihong, Nancie, Giovanni, & Michael, 2015). Furthermore, these characteristics impact the digestion and adsorption of oat products (Rasane et al., 2015). EROR is regarded as a whole grain product with good taste and special processing. However, little information is available about the health benefits of this whole grain food. Hence, investigations on its nutrition and function are crucial in determining its future development. The aim of this study was to investigate the healthy functions of EROR and its extract to gain a better understanding of how EROR affects serum and liver lipid levels. It has been reported that intestinal microbiota plays an important role in the regulation of lipid metabolism (Danneskiold-Samsøe et al., 2019; Li et al., 2019a), and previous studies have collectively suggested that cecal short chain fatty acids (SCFAs) effectively reduce plasma cholesterol concentrations (Gong et al., 2018; Koh et al., 2016). However, the effects of EROR on intestinal microbiota and cecal SCFAs and the regulatory functions in lipid synthesis remain insufficiently explained. In this study, the effects of EROR on the lipid profile, cecal SCFAs and intestinal microbiota in high-fat diet (HFD) fed rats were investigated. Additionally, the role of the ethanol extract of EROR on cell lipid accumulation in vitro was evaluated. 2. Materials and methods 2.1 Materials and reagents EROR was provided by Yan Gufang Co., Ltd. (Huhehaote, Inner Mongolia, China), and the compositions were measured by China Agricultural University (Supplementary Table 1). Acetic acid, propionic acid, and butyric acid were purchased from Aladdin Co., Ltd. (Shanghai, China). Oil Red O was purchased from Sigma-Aldrich (St. Louis, MO, USA) and other chemical reagents used in this study were of analytical grade. 2.2 Preparation of EROR extracts The dried EROR was ground into a powder using a pulverizer (HK-820, Xulang Machinery Equipment Co., Ltd., Guangzhou, Guangdong, China) at room temperature.
The lipid extract of EROR (OL) was obtained by extraction with n-hexane, and the ratio of grain flour to n-hexane was 1:4. Residue fractions were dried and mixed with deionized water (1:10 ratio) at 50 ℃ and then incubated for 2 h. The solution was centrifuged, and the supernatant was freeze-dried. The dried powder was regarded as the water extract of EROR (OW). The precipitate was dried and then dissolved in a 10fold volume of 80% ethanol for 2 h at 50 °C. The solution was centrifuged, and the supernatant was evaporated at 60 °C to remove the ethanol using a rotatory evaporator. The obtained fractions were freeze-dried, and the powder was regarded as the ethanol extract of EROR (OE). All the EROR extracts were stored at -20 °C and dissolved in DMSO for the following experiments. 2.3 EROR treatments on HepG2 cells HepG2 cells were cultured in 10% fetal bovine serum with Dulbecco’s minimal Eagle medium (DMEM) at 37 °C and 5% CO2. Cells were inoculated into 96-well plates (5 × 103 cells/well) and handled as follows: (1) control group: cultured in the stated medium without OA and extracts for 24 h; (2) model group: pretreated with 1 mM OA for 24 h and then cultured in the stated medium without extracts for 24 h; or (3) pretreated with 1 mM OA for 24 h and then cultured in the stated medium with extracts at low-dose level (100 and 200 µg/ml) or high-dose level (400 µg/ml) for 24 h. Cultured cells were fixed with 4% paraformaldehyde for 30 min and then stained with Oil Red O to measure lipid droplet accumulation with a MicroQ6 plate reader (BioRad, Hercules, CA, USA) at 510 nm. Stained cells were visualized using a microscope (Olympus, Tokyo, Japan) at a final magnification of 200×. Cell viability was determined using the MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime Biotech, Nanjing, Jiangsu, China) with various concentrations of EROR extract (0-400 µg/ml) for 24 h. The absorbance of formazan was measured at 570 nm. 2.4 Animal and sample collection A total of 24 male SD rats (weighing 180-220 g) were purchased from Shanghai Jie Si Jie Laboratory Animal Co., Ltd. (Shanghai, China). The rats were housed in stainless steel screen-bottomed cages. The room was illuminated with a dark/light cycle (8:00 am on - 8:00 pm off) at a constant temperature of 22 ± 2 °C and a humidity of 40-60%.
After adaptive feeding for a week, rats were discretionarily divided into four groups (n = 6 per group, 3 rats in the same group housed per cage): a normal diet group (ND), a high-fat diet group (HFD), a HFD supplemented with 10% EROR group (HFD-LO) and a HFD supplemented with 50% EROR group (HFD-HO). The composition of the experimental diet is presented in Supplementary Table 2. All rats were allowed free access to tap water and the corresponding diet mentioned above. Food intake and body weights were recorded weekly. At the end of 4 weeks, the rats were fasted for 12 h and then anesthetized with pentobarbital sodium (50 mg/kg). The rats were dissected, and blood samples were collected from the heart. Samples were centrifuged at 4000 rpm and 4 °C for 3 min, and the serum was stored at -80 °C until analysis. The livers were weighed and stored at -80 °C, and the liver index was calculated as liver weight (g)/body weight (g). The cecum of each rat was removed, and 2 g of fresh cecal contents were collected and stored at -80 °C. All animal experiments were conducted according to the guidelines of the Laboratory Animal Ethics Association of the University of Shanghai for Science and Technology (approval opinion: SCXK (SH) 20130006). 2.5 Biochemical analysis in the serum and liver The levels of TG, TC, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured with commercial reagent kits (Jiancheng Biotech, Naning, Jiangsu, China). The activities of serum alanine transaminase (ALT) and aspartate transaminase (AST) were assayed according to the manufacturer’s instructions for the colorimetric enzymatic kits (Jiancheng Biotech, Nanjing, Jiangsu, China). 2.6 Histological analysis of liver tissue A portion of the right liver lobe was fixed with 4% paraformaldehyde overnight. The fixed liver tissues were paraffin-embedded, cut into 5 μm sections and then stained with hematoxylin eosin (H&E) according to the standard method. The stained sections were observed and photographed with a microscope (Olympus, Tokyo, Japan) at a final magnification of 400×. 2.7 Gene expression analysis in the liver Liver RNA was extracted using the TRIzol Reagent Kit (Life Technologies, Carlsbad,
CA, USA). Reverse transcription reactions were conducted using the HiScript II Q RT SuperMix for qPCR Reagent Kit (Vazyme Biotech Co., Ltd, Nanjing, Jiangsu, China). The obtained product was regarded as the template for real-time PCR, which was performed with the ChamQ™ Universal SYBR qPCR Master Mix Regent Kit (Vazyme Biotech Co., Ltd, Nanjing, Jiangsu, China) and a CFX96 real-time PCR detection system (BioRad, Hercules, CA, USA). The primer sequences involved in our research are presented in Supplementary Table 3. The relative mRNA levels were normalized to the level of the β-actin gene in each sample and expressed as values of relative expression compared to that of the ND group. Relative levels of target mRNAs were determined using the 2-ΔΔCt method and normalization. 2.8 Cecal SCFA measurement The cecal SCFA concentrations were measured using a 7890 gas chromatograph (GC, Agilent Technologies, Palo Alto, CA, USA) with a DB-WAX capillary column (1227032, 30 m × 0.25 μm × 0.25 mm, Agilent Technologies) as in a previous report (Li et al., 2019a). Briefly, 50 mg of cecal contents were diluted with 500 μl of distilled water and then centrifuged at 1000 rpm for 5 min. The obtained supernatant was mixed with 25% metaphosphoric acid solution (9:1), incubated overnight at 4 °C, and then centrifuged at 10000 rpm for 5 min. The prepared sample (1 μl) was injected into the GC, and the procedures were as follows: 80 °C for 30 s, raised to 130 °C at a rate of 5 °C/min and held for 2 min, raised to 240 °C at a rate of 20 °C/min and held for 1 min. 2.9 Analysis of gut microbiota Bacterial genomic DNA was extracted from 300 mg of cecal contents from each rat with the DNA Isolation Kit (Takara Co., Dalian, Liaoning, China). The 16S rDNA V3V4
region
was
amplified
ACTCCTACGGGAGGCAGCAG-3′)
using
the and
primers 806R
338F
(5′(5′-
GGACTACHVGGGTWTCTAAT-3′). DNA concentration was quantified using a QuantiFluor™-ST fluorescent quantitative system (Promega Co., Madison, WI, USA). High-throughput sequencing was performed on an Illumina MiSeq platform (Illumina Co., San Diego, CA, USA) according to the standard proposed by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw sequence data were deposited into the
NCBI sequence read archive database (https://www.ncbi.nlm.nih.gov/sra) with accession No. SRP171421. 2.10 Bioinformatics and statistical analysis The composition and richness of the intestinal microbiota were analyzed using the online platform provided by the Majorbio I-Sanger Cloud Platform (www.isanger.com). Operational taxonomic units (OTUs) were clustered with 97% identity by UPARSE (http://drive5.com/uparse/). Alpha diversity parameters such as ACE and Shannon index were analyzed using mothur software (http://mothur.org/). A heat map at the genus level was generated using the R package heat map followed by relative abundance calculation at the genus level by the R software package (https://www.rproject.org/). The relationships between the intestinal microbiota and biochemical indicators were all preformed using the heat map package by Spearman’s rank correlation. Data were analyzed with GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) by one-way ANOVA. Significant differences were evaluated by Tukey’s post hoc test. The values are expressed as the mean ± SEM. The significance level was set at P < 0.05. 3. Results 3.1 Effects of EROR extracts on lipid accumulation in HepG2 cells Different EROR extracts were prepared with a sequential extraction strategy. A total of 6.7 g of OL, 20.5 g of OW and 3.5 g of OE were extracted from 100 g of EROR. As shown in Fig. 1a, compared with the ND group, OA treatment significantly induced lipid accumulation. OE treatment inhibited lipid accumulation (up to 20%) in a dosedependent manner. However, the OL and OW treatments showed no significant difference at any concentration level (data not shown). As shown in Fig. 1b-e, MTT analysis showed that the OE did not have cytotoxic effects at concentrations ranging from 0 to 400 μg/ml. Furthermore, OE treatment significantly decreased the intracellular TG and TC levels in OA-induced HepG2 cells. 3.2 Effect of EROR supplementation on body weight, liver index, and hepatic lipid accumulation As shown in Fig. 2a, compared with the HFD group, the intake of EROR did not affect
the body weights of the rats. However, EROR supplementation decreased the liver index (Fig. 2b). As shown in Fig. 2c, compared with the ND group, the hepatocytes were swollen and their volume increased significantly in the HFD group, and the cytoplasm was filled with fat vacuoles. Moreover, the cell boundary was not clear, and the hepatocytes showed severe steatosis. Compared with the HFD group, the HFD-LO and HFD-HO groups had smaller fat vacuoles and clearer cell boundaries. 3.3 Rat lipid profile analysis As shown in Fig. 3a-d, compared with the ND group, the serum TG, TC, and LDL-C levels in the HFD, HFD-LO and HFD-HO groups were significantly higher, while the HDL-C levels were lower. Compared with the HFD group, the intake of EROR significantly decreased the serum TG, TC and LDL-C levels and increased the HDL-C levels. The liver lipid distributions are shown in Fig. 3e-f, which suggests that the rat model of non-alcoholic fatty liver was successfully established. Compared with the HFD group, the HFD-HO group showed significantly lower hepatic TG and TC levels, while the HFD-LO group only showed a significantly lower TC level but no significant difference in the TG level. 3.4 Analysis of hepatic dysfunction markers and gene expression in liver The effects of EROR supplementation on liver enzymes were investigated. As shown in Fig. 4a-b, the activities of ALT and AST in the HFD group were significantly higher than those in the ND group. Compared with the HFD group, supplementation with EROR significantly decreased the activities of ALT and AST. To assess the mechanism of lipid metabolism in the liver, the expression levels of the genes SREBP-1C, FAS, and HMGCR were determined. As shown in Fig. 4c-e, compared with the ND group, the FAS gene in the HFD group was significantly upregulated, and the other two genes were upregulated slightly. Compared with the HFD group, the expression levels of all hepatic lipid metabolism-, fatty acid- and cholesterol synthesis-related genes were significantly downregulated in the HFD-LO and HFD-HO groups. 3.5 SCFA analysis The generated SCFAs (the concentrations of total SCFAs, acetate, propionate and
butyrate) in the cecal contents of each rat were determined. As shown in Fig. 5, compared with the ND group, there were significant decreases in the contents of total SCFAs, acetate and butyrate in the HFD group. The concentrations of total SCFAs, acetate and propionate in the HFD-HO group were significantly higher than those in the HFD group. Interestingly, the concentration of propionate in the HFD-HO group was significantly higher than that in the ND group. Acetate, propionate and butyrate showed no significant difference in the HFD-LO group compared with the HFD group. 3.6 Effect of EROR on the composition of intestinal microbiota To determine the specific changes in intestinal microbiota, the OTUs were obtained by high-throughput sequencing. A total of 16 rat samples from the four groups were evaluated. After the sequence optimization, a total of 1,072,565 reads were generated, corresponding to an average of 268,414 reads per group. After quality filtering, the average length of each read was more than 430 bp. Sequences were clustered into a total of 848 OTUs at a 97% similarity level (data not shown). The results of the ACE and Shannon index showed that the intake of EROR did not affect the intestinal microbiota diversity (Fig. 6a-b). As shown in Fig. 6c-f, compared with the ND group, a HFD mainly led to a higher abundance of Firmicutes (93.0%, phylum level), Bacilli and Clostridia (29.6% and 58.3%, respectively, class level), Clostridiales and Lactobacillales (58.3% and 27.2%, respectively, order level), and Lachnospiraceae and Lactobacillaceae (41.3% and 26.5%, respectively, family level). Compared with the HFD group, supplementation of EROR mainly increased the abundance of Actinobacteria (34.0-34.6%, phylum level), Coriobacteriales, Bacillales and Verrucomicrobiales (26.1-32.3%, 5.7-6.7% and 2.3-4.7%, respectively, order level) and Bifidobacteriaceae, Staphylococcaceae, and Coriobacteriaceae (0.5-2.1%, 5.7-6.7% and 26.1-32.3%, respectively, family level), and decreased the abundance of Firmicutes (60.1-62.7%), Bacilli and Clostridia (11.2-18.5% and 40.9-47.7%, respectively), Clostridiales and Lactobacillales (40.9-47.7% and 5.4-11.6%, respectively) and Lachnospiraceae and Lactobacillaceae (24.9-30.0% and 4.5-10.5% respectively). Furthermore, as shown in Fig. 7, the abundances of 20 key bacteria with significant changes at each level were investigated. At the genus level, intake of EROR decreased
the abundance of Desulfovibrio, Holdemanella, Fusicatenibacter, Faecalitalea, Turicibacter and Bifidobacterium and increased the abundance of Akkermansia, Ruminococcus ssp, Faecalibaculum and Roseburia. 3.7 Correlation between gut microbiota and lipid metabolism related indices The correlations between gut microbiota (the relative abundance of 50 key species at the genus level) and lipid metabolism-related indices were analyzed using Spearman correlation analysis (Fig. 8). The serum TC, TG, LDL-C, hepatic TC, TG and genes SREBP-1C, FAS, and HMGCR were positively correlated with Fusicatenibacter, Faecalitalea,
Blautia,
Eubacterium_hallii_group,
Akkermansia,
Eubacterium_nodatum_group and unclassified Lachnospiraceae but negatively correlated
with
Bifidobacterium,
Lachnospiraceae_UCG006,
norank
Feacalibacterium, Coriobacteriaceae,
Enterohabdus, Romboutsia
and
Staphylococcus. However, the correlation between the serum HDL-C and gut microbiota showed the opposite trend, which was positively correlated with Feacalibacterium, Enterohabdus, norank Coriobacteriaceae, Staphylococcus and Romboutsia
and
negatively
correlated
with
Faecalitalea,
Blautia
and
Eubacterium_hallii_group. In addition, the liver TC also showed a negative correlation with
Tuicibacter
and
a
positive
correlation
with
Roseburia
and
Ruminococcus_torques_group. 4. Discussion Recent studies have reported that the health benefits of whole grains are related to dietary fiber (Xia et al., 2017; Chakraborty et al., 2019). OR is an excellent source of dietary fiber, which usually accounts for 10.2-12.1% of OR (Manthey, Hareland, & Huseby, 1999). Partial oat bran removal leads to decreases in dietary fiber and some other constituents. In this study, the total dietary fiber of EROR was only 6.36%, and nearly 7.04% of the dietary fiber was removed (Supplementary Table 1). However, EROR presents the advantages of cooking properties. Furthermore, we found that EROR alleviated lipid disorder in HFD fed rats in the present study. In addition to dietary fiber, constituent phytochemicals also play important roles in health benefits, such as β-glucan, oil, protein and polyphenol (Grundy et al., 2017; Tong et al., 2014;
Tong et al., 2016). All these constitutes mainly remained in the lipid, water and ethanol extracts. Numerous studies have claimed that natural plant ethanol extracts modulate lipid metabolism in vivo and in vitro, and these functions are mainly attributed to the phenolic compounds, such as ethanol extracts from millet, black adzuki bean, mulberry, Cynara scolymus, and Schisandra chinensis berry (Chung et al., 2017; Kim, Kim, & Cha, 2017; Li et al., 2019a; Safat, Sheibani, Mohammadi, Hasanabadi, & Sakhaee, 2017). Similar to these reports, OE treatment effectively inhibited lipid accumulation and decreased the levels of TC and TG in OA-induced HepG2 cells. Thus, it has been proposed that the effect of OE on lipid modulation is also related to the presence of oat phenolic compounds. Further investigation of oat phenolic compounds on lipid disorder is currently being investigated in our laboratory. A HFD results in an increased risk of hyperlipidemia, which is accompanied by a significant increase in serum TC, TG and LDL-C and a simultaneous decrease in HDLC (Scicchitano et al., 2014). Consumptions of whole grain foods, such as sorghum, purple rice, rhubarb rice, qingke and millet, have been reported to reduce serum lipid concentrations (Arbex et al., 2018; De Sousa et al., 2018; Li et al., 2019a; Shen et al., 2017; Xia et al., 2017). Similar to these reports, EROR supplementation improved the serum lipid profile in HFD fed rats. Disease research has indicated that LDL-C and TG, as the key risk indicators of heart and cerebrovascular diseases, play important roles in the process of intervention and treatment (Li et al., 2019b). The changes in serum biochemical indicators were associated with healthy morphological alterations in rat livers. A HFD promotes the accumulation of fat droplets and then causes fatty liver (Scicchitano et al., 2014). Histopathological analysis showed that EROR could regulate the structure of liver tissue and that the number of lipid droplets were significantly reduced. Furthermore, EROR improved AST and ALT levels, which indicated that EROR ameliorated fatty liver in HFD fed rats. The serine kinase AMPK plays an important role in glucose and lipid metabolism. Lipid metabolism disorder is usually regulated by downstream genes in the AMPK signaling pathway, such as SREBP-1C, HMGCR and FAS (Brahma Naidu et al., 2016). SREBP1C, as a transcription factor that maintains lipid homeostasis, regulates the expression
of genes (FAS and HMGCR) and fat synthesis in the liver (Moreira et al., 2018). The intake of a HFD increased the expression of FAS, which induced the production of C16:0 (Dziadek, Kopeć, & Piątkowska, 2019). Downregulation of FAS showed the regulation of fatty acids in HFD fed rats. HMGCR, the rate-limiting enzyme of cholesterol biosynthesis that transforms HMG-CoA into mevalonic acid, is regulated through a negative feedback mechanism and increases the decomposition rate of plasma LDL (Chen et al., 2019). The results showed that compared with the HFD group, EROR treatment significantly lowered the expression levels of SREBP-1C, FAS and HMGCR. These results suggested that EROR alleviated lipid metabolic disorder by regulating the related genes through the AMPK signaling pathway, which could provide a theoretical basis for the development of EROR. Undigested carbohydrates can be rapidly consumed by the intestinal microbiota to produce SCFAs, which are mainly composed of acetate, propionate and butyrate (Xia et al., 2017). Acetate is the most abundant SCFA, which serves as a substrate for fatty acid synthesis, enhances ileal motility, and downregulates lipogenesis-related gene expression, such as FAS and ACC (Choque Delgado & Tamashiro, 2018). Propionate can lower lipogenesis and serum cholesterol and is regarded as a good precursor for gluconeogenesis and protein synthesis, which decreases food intake via glucagon-like peptide-1- and peptide YY (PYY)-driven mechanisms (Koh et al., 2016). Butyrate is a good energy source for colonocytes and acts as an antitumor or anti-inflammatory agent (Morrison & Preston, 2016; Xia et al., 2017). Treatment of obese rodents with SCFA has been proven to reduce body weight and improve the plasma lipid profile (Lu et al., 2016). The HFD decreased the concentrations of SCFAs; however, the intake of EROR significantly improved the SCFA profile in HFD fed rats. Similarly, many studies have reported that the consumption of whole grains increased the content of the SCFAs (Gong et al., 2018; Li et al., 2019b; Xia et al., 2017). Most of these studies showed that higher SCFA production mainly resulted from dietary fiber (Koh et al., 2016; Maurer et al., 2019). However, some other components, such as tannins and phenolic compounds, also promote SCFA production (Li et al., 2019a; Maurer et al., 2019; Rocchetti et al., 2017). This is a potential reason for EROR having obvious stimulation
of SCFA production in HFD fed rats, even though its dietary fiber was partially removed. The intestinal microbiota is directly associated with lipid metabolic disorder, liver steatosis and obesity (Danneskiold-Samsøe et al., 2019; Li et al., 2019b; Monk et al., 2019). A HFD induces intestinal dysbiosis, which is usually accompanied by an increase in Firmicutes and Verrucomicrobia at the phylum level and an increase in Clostridia and Bacilli at the class level (Gao et al., 2019; Xia et al., 2017). A higher abundance of Firmicutes and a lower abundance of Bacteroidetes indicate an acceleration of energy harvest from food and increased energy storage in adipose tissue (Arbex et al., 2018). This acceleration and storage further suppresses the production of fasting-induced adipose factors and leads to the higher storage of triacylglycerols (Luo et al., 2018). Intake of EROR did not affect the intestinal microbiota diversity, although it decreased the abundance of Firmicutes, Clostridia and Bacilli and increased the abundance of Bacteroidetes (though its proportion was low in each group). At the family level, the intake of EROR increased the abundance of Porphyromonadaceae, Enterobacteriaceae, and Coriobacteriaceae. These bacteria are positively correlated with serum HDL-C and negatively correlated with obesity (Kong, Gao, Yan, Huang, & Qin, 2019). At the genus level, Spearman correlation analysis showed that the levels of TC, TG and LDL-C in the serum and liver and the expression of the related genes were positively correlated with Fusicatenibacter, Faecalitalea, Blautia, Akkermansia, Roseburia and unclassified Lachnospiraceae, while negatively correlated with Bifidobacterium, Feacalibacterium, Enterohabdus, Romboutsia, Tuicibacter and Staphylococcus. Similar to our results, studies have shown that Lachnospiraceae and Blautia contribute to the development of obesity and lipid metabolic disorders in obese mice (Chen et al., 2019; Ju et al., 2019; Lv, Guo, Li, Yu, & Liu, 2019). A HFD leads to a decrease in beneficial bacteria, such as Bifidobacterium and Feacalibacterium (Dziadek et al., 2019; Xia et al., 2017). Turicibacter has a negative impact on the healthy functions of the gut and maintaining the serum metabolic index (Luo et al., 2018; Monk et al., 2019). Furthermore, it has been reported that Rombutsia, Fusicatenibacter and Holdemanella are negatively correlated with serum HDL-C and
positively correlated with TG, TC and LDL-C (Arbex et al., 2018; Chen et al., 2019; Shen et al., 2017). The concentrations of SCFAs are related to the intestinal microbiota community richness, and a growing number of recent studies have shown that more attention should be focused on the lower classification levels of SCFA-producing bacteria (Morrison & Preston, 2016). It has been reported that the increased abundance of Akkermansia, Bifidobacterium, Blautia, Roseburia and Lachnospiraceae promoted the production of SCFAs (De Sousa et al., 2018; Koh et al., 2016; Xia et al., 2017). Interestingly, the intake of EROR increased the abundance of the bacteria mentioned above. The experimental results suggest that EROR may regulate the intestinal microbiota, promote SCFA production and alleviate lipid metabolic disorders against a HFD. In addition to these functions, intestinal microbiota regulation also plays therapeutic roles in other metabolism functions. EROR improved the abundance of Ruminococcus ssp and Faecalibacterium and decreased the abundance of Desulfovibrio. Ruminococcus ssp and Faecalibacterium have been reported as beneficial bacteria related to improving malnutrition and promoting health recovery (Kong, Gao, Yan, Huang, & Qin, 2019; Liang et al., 2018). Desulfovibrio, as an H2S-producing bacterium, is involved in blood pressure and plasmatic insulin levels (Prieto et al., 2018). Conclusions The present study confirmed that the ethanol extract of EROR decreased lipid accumulation in HepG2 cells. Furthermore, the intake of EROR, especially at a high dose, mediated the lipid disorder in HFD fed rats with an improvement in the lipid parameters in the serum and liver, modulated the related gene expression levels in liver, ameliorated cecal SCFAs, and beneficially regulated the intestinal microbiota. The current results contribute to the development of EROR as a potential functional food for dietary strategies to prevent lipid metabolic disorder. Funding This work was supported by the National Key Research and Development Program of China (2017YFD0401202), Yangtze River Delta Cooperation Program (18395810200) and Youth Scientific Talent Program of National Grain Industry (LQ2018204). Conflict of interest
The authors have no conflict of interests Authorship KH, SL, WWY, XG and JL designed the study and had primary responsibility for the content of the paper; WWY, DDL and RQD performed the experiments; HK, WWY analyzed the data and wrote the manuscript. All authors approved the manuscript. Reference Albert Lihong, Z., Nancie, H., Giovanni, R., & Michael, L. (2015). Whole grain oats improve insulin sensitivity and plasma cholesterol profile and modify gut microbiota composition in C57BL/6J mice. Journal of Nutrition, 145(2), 222-230. Arbex, P. M., Moreira, M. E. D. C., Toledo, R. C. L., Cardoso, L. D. M., Pinheiro-Sant'Ana, H. M., Benjamin, L. D. A., . . . Martino, H. S. D. (2018). Extruded sorghum flour (Sorghum bicolor L.) modulate adiposity and inflammation in high fat diet-induced obese rats. Journal of Functional Foods, 42, 346-355. Brahma Naidu, P., Uddandrao, V. V. S., Ravindar Naik, R., Suresh, P., Meriga, B., Begum, M. S., . . . Saravanan, G. (2016). Ameliorative potential of gingerol: Promising modulation of inflammatory factors and lipid marker enzymes expressions in HFD induced obesity in rats. Molecular and Cellular Endocrinology, 419, 139-147. Chakraborty, P., Witt, T., Harris, D., Ashton, J., Stokes, J. R., & Smyth, H. E. (2019). Texture and mouthfeel perceptions of a model beverage system containing soluble and insoluble oat bran fibres. Food Research International, 120, 62-72. Chen, H., Zeng, F., Li, S., Liu, Y., Gong, S., Lv, X., . . . Liu, B. (2019). Spirulina active substance mediated gut microbes improve lipid metabolism in high-fat diet fed rats. Journal of Functional Foods, 59, 215-222. Choque Delgado, G. T., & Tamashiro, W. M. d. S. C. (2018). Role of prebiotics in regulation of microbiota and prevention of obesity. Food Research International, 113, 183-188. Chung, M. Y., Shin, E. J., Choi, H. K., Kim, S. H., Sung, M. J., Park, J. H., & Hwang, J. T. (2017). Schisandra chinensis berry extract protects against steatosis by inhibiting histone acetylation in oleic acid-treated HepG2 cells and in the livers of diet-induced obese mice. Nutrition Research, 46, 1-10. Danneskiold-Samsøe, N. B., Dias de Freitas Queiroz Barros, H., Santos, R., Bicas, J. L., Cazarin, C. B. B., Madsen, L., . . . Maróstica Júnior, M. R. (2019). Interplay between food and gut microbiota in health and disease. Food Research International, 115, 23-31. De Sousa, A. R., de Castro Moreira, M. E., Toledo, R. C. L., dos Anjos Benjamin, L., Queiroz, V. A. V., Veloso, M. P., . . . Martino, H. S. D. (2018). Extruded sorghum (Sorghum bicolor L.) reduces metabolic risk of hepatic steatosis in obese rats consuming a high fat diet. Food Research International, 112, 48-55. Dong, J., Cai, F., Shen, R., & Liu, Y. (2011). Hypoglycaemic effects and inhibitory effect on intestinal disaccharidases of oat beta-glucan in streptozotocin-induced diabetic mice. Food Chemistry, 129(3), 1066-1071. Dziadek, K., Kopeć, A., & Piątkowska, E. (2019). Intake of fruit and leaves of sweet cherry beneficially affects lipid metabolism, oxidative stress and inflammation in Wistar rats fed with high fat-
cholesterol diet. Journal of Functional Foods, 57, 31-39. Gao, J., Ding, G., Li, Q., Gong, L., Huang, J., & Sang, Y. (2019). Tibet kefir milk decreases fat deposition by regulating the gut microbiota and gene expression of Lpl and Angptl4 in high fat diet-fed rats. Food Research International, 121, 278-287. Gong, L., Cao, W., Chi, H., Wang, J., Zhang, H., Liu, J., & Sun, B. (2018). Whole cereal grains and potential health effects: Involvement of the gut microbiota. Food Research International, 103, 84-102. Grundy, M. M. L., Quint, J., Rieder, A., Ballance, S., Dreiss, C. A., Cross, K. L., . . . Ellis, P. R. (2017). The impact of oat structure and β-glucan on in vitro lipid digestion. Journal of Functional Foods, 38(Pt A), 378-388. Ju, M., Liu, Y., Li, M., Cheng, M., Zhang, Y., Deng, G., . . . Liu, H. (2019). Baicalin improves intestinal microecology and abnormal metabolism induced by high-fat diet. European Journal of Pharmacology, 857, 172457. Koh, A., Filipe D. V., Petia K. D., & Fredrik B. (2016). From dietary fiber to host physiology: shortchain fatty acids as key bacterial metabolites. Cell, 165(6), 1332-1345. Kong, C., Gao, R., Yan, X., Huang, L., & Qin, H. (2019). Probiotics improve gut microbiota dysbiosis in obese mice fed a high-fat or high-sucrose diet. Nutrition, 60, 175-184. Leão, L. S. C. d. S., Aquino, L. A. d., Dias, J. F., & Koifman, R. J. (2019). Addition of oat bran reduces HDL-C and does not potentialize effect of a low-calorie diet on remission of metabolic syndrome: A pragmatic, randomized, controlled, open-label nutritional trial. Nutrition, 65, 126130. Li, S., Yu, W., Guan, X., Huang, K., Liu, J., Liu, D., & Duan, R. (2019a). Effects of millet whole grain supplementation on the lipid profile and gut bacteria in rats fed with high-fat diet. Journal of Functional Foods, 59, 49-59. Li, T.-T., Tong, A.-J., Liu, Y.-Y., Huang, Z.-R., Wan, X.-Z., Pan, Y.-Y., . . . Zhao, C. (2019b). Polyunsaturated fatty acids from microalgae Spirulina platensis modulates lipid metabolism disorders and gut microbiota in high-fat diet rats. Food and Chemical Toxicology, 131, 110558. Liang, Y., Liang, S., Zhang, Y., Deng, Y., He, Y., Chen, Y., . . . Yang, Q. (2018). Oral administration of compound probiotics ameliorates HFD-induced gut microbe dysbiosis and chronic metabolic inflammation via the G protein-coupled receptor 43 in non-alcoholic fatty liver disease rats. Probiotics Antimicrob Proteins, 1-11. Lu, Y., Fan, C., Li, P., Lu, Y., Chang, X., & Qi, K. (2016). Short chain fatty acids prevent high-fat-dietinduced obesity in mice by regulating G protein-coupled receptors and gut microbiota. Scientific Reports, 6, 37589. Luo, J., Yuetong, L., Yunshi, M., Lijuan, G., Shiyi, O., Yong, W., . . . Xichun, P. (2018). Flaxseed gum reduces body weight by regulating gut microbiota. Journal of Functional Foods, 47, 136-142. Lv, X. C., Guo, W. L., Li, L., Yu, X. D., & Liu, B. (2019). Polysaccharide peptides from Ganoderma lucidum ameliorate lipid metabolic disorders and gut microbiota dysbiosis in high-fat diet-fed rats. Journal of Functional Foods, 57, 48-58. Manthey, F. A., Hareland, G. A., & Huseby, D. J. (1999). Soluble and insoluble dietary fiber content and composition in oat. Cereal Chemistry, 76(3), 417-420. Maurer, L. H., Cazarin, C. B. B., Quatrin, A., Minuzzi, N. M., Costa, E. L., Morari, J., . . . Emanuelli, T. (2019). Grape peel powder promotes intestinal barrier homeostasis in acute TNBS-colitis: A major role for dietary fiber and fiber-bound polyphenols. Food Research International, 123,
425-439. Miller, S. S., & Fulcher, R. G. (2011). CHAPTER 5 – Microstructure and chemistry of the oat kernel 1. Oats, 77-94. Monk, J. M., Wu, W., Lepp, D., Wellings, H. R., Hutchinson, A. L., Liddle, D. M., . . . Power, K. A. (2019). Navy bean supplemented high-fat diet improves intestinal health, epithelial barrier integrity and critical aspects of the obese inflammatory phenotype. The Journal of Nutritional Biochemistry, 70, 91-104. Moreira, M. E. d. C., de Oliveira Araújo, F., de Sousa, A. R., Toledo, R. C. L., dos Anjos Benjamin, L., Veloso, M. P., . . . Martino, H. S. D. (2018). Bacupari peel extracts (Garcinia brasiliensis) reduces the biometry, lipogenesis and hepatic steatosis in obese rats. Food Research International, 114, 169-177. Morrison, D. J., & Preston, T. (2016). Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes, 7(3), 189-200. Prieto, I., Hidalgo, M., Segarra, A. B., Martã-Nez-Rodrã-Guez, A. M., Cobo, A., Ramã-Rez, M., . . . Martã-Nez-Caã-Amero, M. (2018). Influence of a diet enriched with virgin olive oil or butter on mouse gut microbiota and its correlation to physiological and biochemical parameters related to metabolic syndrome. Plos One, 13(1), e0190368. Rasane, P., Jha, A., Sabikhi, L., Kumar, A., & Unnikrishnan, V. S. (2015). Nutritional advantages of oats and opportunities for its processing as value added foods - a review. Journal of Food Science & Technology, 52(2), 662-675. Rocchetti, G., Lucini, L., Chiodelli, G., Giuberti, G., Gallo, A., Masoero, F., & Trevisan, M. (2017). Phenolic profile and fermentation patterns of different commercial gluten-free pasta during in vitro large intestine fermentation. Food Research International, 97, 78-86. Safat, A. A., Sheibani, H., Mohammadi, P., Hasanabadi, N., & Sakhaee, E. (2017). Evaluation of lipidlowering effect of Cynara scolymus extract-loaded mesoporous silica nanoparticles on ultralipid-fed mice. Comparative Clinical Pathology, 27(6), 1-6. Scicchitano, P., Cameli, M., Maiello, M., Modesti, P. A., Muiesan, M. L., Novo, S., . . . Ciccone, M. M. (2014). Nutraceuticals and dyslipidaemia: Beyond the common therapeutics. Journal of Functional Foods, 6, 11-32. Shen, Y., Song, X., Chen, Y., Li, L., Sun, J., Huang, C., . . . Zhang, H. (2017). Effects of sorghum, purple rice and rhubarb rice on lipids status and antioxidant capacity in mice fed a high-fat diet. Journal of Functional Foods, 39, 103-111. Tong, L. T., Zhong, K., Liu, L., Guo, L., Cao, L., & Zhou, S. (2014). Oat oil lowers the plasma and liver cholesterol concentrations by promoting the excretion of faecal lipids in hypercholesterolemic rats. Food Chemistry, 142, 129-134. Tong, L. T., Guo, L., Zhou, X., Qiu, J., Liu, L., Zhong, K., & Zhou, S. (2016). Effects of dietary oat proteins on cholesterol metabolism of hypercholesterolemic hamsters: Cholesterol-lowering effects of oat proteins. Journal of the Science of Food & Agriculture, 96(4), 1396-1401. Wood, P. J. (2010). Oat Bran. Journal of Food Biochemistry, 16(6), 401-401. Xia, X., Li, G., Ding, Y., Ren, T., Zheng, J., & Kan, J. (2017). Effect of whole grain qingke (Tibetan Hordeum vulgare L. Zangqing 320) on the serum lipid levels and intestinal microbiota of rats under high-fat Diet. Journal of Agricultural & Food Chemistry, 65(13), 2686-2693.
Figure legends Fig. 1 Effects of OE on lipid accumulation and viability of HepG2 cells. HepG2cells were treated
for 24 h with OE in the presence or absence of OA, and then lipid accumulation was measured by Oil-Red O staining (a, b). The effects of OE on intracellular TG and TC levels were determined in OA induced HepG2 cells (c, d). Cytotoxicity was measured by the MTT assay (e). All results are expressed as the mean ± SD. #p < 0.05,
##p<0.01, ###p<0.001
vs. Control; *p < 0.05, **p<0.01,
***p<0.001 vs. OA Fig. 2 Effect of EROR supplementation on body weight (a), liver index (b), liver fat accumulation (c). Data are expressed as the means ± SD. #P<0.05, ##P<0.01, ###P<0.001 compared to the ND group; *P<0.05, **P<0.01, ***P<0.001 compared to the HFD group (n=4 per group). Fig. 3 Effect of EROR supplementation on Lipid profile in serum and liver. Levels of TG, TC, HDL-C, and LDL-C in serum (a-d), and levels of TG and TC in liver (e-f). Data are expressed as the means ± SD. #P<0.05,
##P<0.01, ###P<0.001
compared to the ND group; *P<0.05, **P<0.01,
***P<0.01 compared to the HFD group (n=4 per group). Fig. 4 Effect of EROR supplementation on the activity of ALT (a) and AST (b), and the expression of SREBP-1C (c), FAS (d), HMGCR (e). One-way ANOVA was used for statistical analysis. The expression of hepatic lipid metabolism genes in liver was determined by real-time PCR analysis. Multiple comparison of two-way ANOVA was employed for statistical analysis (n=4 per group). Fig. 5 Effect of EROR supplementation on cecal SCFAs. Total SCFA (a), Acetate (b), Propionate (c) and Butyrate (d). Values are presented as the means ± SD. #P<0.05,
##P<0.01, ###P<0.001
compared to the ND group; *P<0.05, **P<0.01, ***P<0.001 compared to the HFD group (n=4 per group). Fig. 6 Effect of EROR supplementation on microbial community richness and Diversity. The α-diversity index of Ace and Shannon (a-b), and relative the abundance at the phylum, class, order and family level (c-f). Fig. 7 Detailed proportions for the specific bacteria at each classification levels. Firmicutes and Bacteroidetes (at the phylum level), Clostridia and Bacilli (at the class level), Porphyromonadaceae, Enterobacteriaceae, Lachnospiraceae and Coriobacteriaceae (at the family level), Akkermansia, Bifidobacterium,
Blautia,
Roseburia,
Fusicatenibacter,
Desulfovibrio,
Turicibacter,
Faecalibaculum, Holdemanella, Faecalitalea and Ruminococcus ssp (at the genus level). The ND group (), the HFD group (), the HFD-LO group () and the HFD-HO group () were
represented with different colors. Fig. 8 Heatmap of Spearman’s correlation between gut microbiota (the relative abundances of 50 key phylotypes at genus level) and lipid metabolism related indices. The colors range from blue (negative correlation) to red (positive correlation). Significant correlations are noted by *P<0.05, **P<0.01, ***P<0.001.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Conflict of Interest form
The authors have no conflict of interest/financial disclosure.
Highlights: 1. EROR is popular in China for its good taste, welcomed appearance and short cooking time 2. Ethanol extract of EROR could alleviate the lipid accumulation in OA induced HepG2 cells 3. EROR could dramatically modulate the lipid profile in serum and liver in HFD fed rats 4. Supplementation of EROR could significantly increase total SCFAs, acetate and propionate 5. EROR is beneficial for the regulation of intestinal microbiota dysbiosis against HFD