Fuzhuan brick tea polysaccharides attenuate metabolic syndrome in

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Bioactive Constituents, Metabolites, and Functions

Fuzhuan brick tea polysaccharides attenuate metabolic syndrome in highfat diet induced mice in association with modulation in the gut microbiota Guijie Chen, Minhao Xie, Peng Wan, Dan Chen, Zhuqing Dai, Hong Ye, Bing Hu, Xiaoxiong Zeng, and Zhonghua Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00296 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Journal of Agricultural and Food Chemistry

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Fuzhuan brick tea polysaccharides attenuate metabolic syndrome in high-fat diet

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induced mice in association with modulation in the gut microbiota

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Guijie Chen,† Minhao Xie,† Peng Wan,† Dan Chen,† Zhuqing Dai,† Hong Ye,† Bing

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Hu,† Xiaoxiong Zeng,†,* Zhonghua Liu‡,§,*

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†

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210095, Jiangsu, China

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‡

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University, Changsha 410128, China

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§

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College of Food Science and Technology, Nanjing Agricultural University, Nanjing

Key Laboratory of Ministry of Education for Tea Science, Hunan Agricultural

National Research Center of Engineering Technology for Utilization of Botanical

Functional Ingredients, Changsha 410128, China

* To whom correspondence should be addressed. Tel & Fax: +86 25 84396791, E-mail: [email protected] (X. Zeng); +86 951 6886783; E-mail address: [email protected] (Z. Liu) 1

ACS Paragon Plus Environment


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ABSTRACT

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An increasing number of evidence suggests that the gut microbiota composition and

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structure contribute to the pathophysiology of metabolic syndrome (MS), which has

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been put forward as a new target in the treatment of diet-induced MS. In this work, we

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aimed to investigate effects of Fuzhuan brick tea polysaccharides (FBTPS) on MS and

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gut microbiota dysbiosis in high-fat diet (HFD)-fed mice and to further investigate

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whether its attenuation of MS being related to the modulation of gut microbiota. The

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results showed that FBTPS intervention could significantly attenuate metabolic

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syndrome in HFD-induced mice. Based on results of sequencing, FBTPS treatment

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could increase the phylogenetic diversity of HFD-induced microbiota. FBTPS

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intervention could significantly restore the HFD-induced increases in relative

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abundances

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Spearman’s correlation analysis showed that 44 key OTUs were negatively or

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positively associated with MS. Our results suggested that FBTPS could serve as a

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novel candidate for prevention of MS in association with the modulation of gut

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microbiota.

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Keywords: Fuzhuan brick tea; polysaccharides; metabolic syndrome; gut microbiota;

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modulation

of

Erysipelotrichaceae,

Coriobacteriaceae

2


and

Streptococcaceae.

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INTRODUCTION

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Metabolic syndrome (MS), a combination of medical disorders including obesity,

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hyperglycemia, dyslipidemia, hypertension and insulin resistance, can enhance the

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risk of developing cardiovascular disease (CVD) and type 2 diabetes (T2D).1-3 Due to

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the obesogenic shifts in nutritional composition, growing caloric intake and lack of

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exercise, humans are in the midst of worldwide epidemics of MS.1,4 Thus, prevention

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of MS has been a major challenge for modern society. A growing number of

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evidences suggests that the gut microbiota may be served as an important modulator

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of the crosstalk between diet and development of MS or related diseases through

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affecting the extraction of nutrients and energy from diets,5 energy homeostasis,6

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glucose metabolism,7 cholesterol metabolism,8 insulin resistance,3 non-alcoholic fatty

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liver9 and the chronic inflammatory state.10 Recently and more strikingly, some

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species or genera of beneficial gut microbiota, such as Akkermansia muciniphila,2,11

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Bifidobacterium12 and Lactobacillus,13 have been demonstrated to be negatively

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associated with the development of MS. On the contrary, increases in some

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pro-inflammatory/pathogenic

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Coriobacteriaceae and Streptococcaceae, are consistently associated with the

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development of obesity, adipose tissue, systemic inflammation and metabolic

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comorbidities in both humans and rodents.8,13,14 Thus, a potentially viable concept,

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modulating high-fat diet (HFD) induced gut microbiota dysbiosis as new therapeutic

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target for the prevention of MS and related diseases, has been presented.15,16

50

gut

microbiota,

such

as


A number of potential strategy or candidate based on modulation of gut

3



51

microbiota, including prebiotics, probiotics, antibiotics and natural products, has been

52

presented and evaluated for prevention of MS and its related diseases. For example,

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both Bifidobacterium and Lactobacillus could differentially attenuate HFD-induced

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obesity comorbidities partly due to the strain-specific effects on MS-related

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phylotypes of gut microbiota.13 The relative abundance of Akkermansia muciniphila

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was dramatically enhanced and the proportion of Firmicutes/Bacteroidetes (F/B) was

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significantly decreased after treatment of grape polyphenols, which led to reduction in

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intestinal and systemic inflammation and improvement in the protection against MS

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in HFD-induced mice.2 Likewise, both water extract and polysaccharides from

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Ganoderma lucidum could prevent obesity-related MS and gut dysbiosis.17 In addition,

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it has been reported that the host-indigestible, fermentable fibers and carbohydrates

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(regarded as prebiotics) could be definite assets for the potential modulation of gut

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microbiota by enhancing the growth of specific health-promoting bacteria.18 In

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particular, short-chain fatty acids (SCFAs) such as acetic, propionic and butyric acids

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are the main products of polysaccharides metabolized by gut microbiota, which not

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only contribute to gut health as a direct energy source for intestinal epithelial cells,

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but also enter the systemic circulation and directly regulate features of MS, such as

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insulin resistance and glucose metabolism.19,20 Therefore, the degradation and

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utilization of polysaccharides by gut microbiota and their health-promoting effects on

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human health and disease have been widely investigated and reported (reviewed in

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references18,21-23).

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Tea, made from the buds and leaves of Camellia sinensis L., is one of the most

4


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popular flavored and functional drinks consumed in the world due to its favorable

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flavor and health-promoting bioactivities.24 The dark tea is post-fermented tea

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produced by microbial fermentation of heat-processed green tea leaves.25 According

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to method of microbial fermentation, dark teas are further classified into Qingzhuan

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brick tea, Pu-erh tea and Fuzhuan brick tea (FBT), etc.26 Thereinto, FBT, one kind of

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dark tea produced in Hunan province of China with the help of fungus Eurotium

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cristatum, has been demonstrated to have preventive and therapeutic effects on

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MS.27,28 In our recent study, it was found that FBT could significantly attenuate

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features of the HFD-induced metabolic syndrome in mice, which might be related to

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the modulation of gut microbiota.29 However, the bioactive components contributing

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to the prevention of MS and the potential mechanisms responsible for regulating MS

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remain unclear. Tea polysaccharides, identified as one of the main active components

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in tea with a wide range of health-promoting bioactivities,30 have great potential

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applications as natural products. Our previous study in vitro demonstrated that

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polysaccharides from FBT (FBTPS) could pass through the digestive system without

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being degraded and reach the large intestine safely, where it could be broken down

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and utilized by gut microbiota.25 Furthermore, FBTPS could significantly reduce the

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ratio of F/B, which is expected to result in the decrease of the energy harvest thereby

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prevention of the risk of MS. Thus, it was speculated that FBTPS might be an

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important active component of FBT contributing to the prevention of MS and

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modulation of gut microbiota. The purpose of the present study, therefore, was to

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investigate whether the intervention of FBTPS to mice could prevent MS and

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modulate the gut microbiota dysbiosis in HFD-induced mice. Additionally, the gut

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microbe phylotypes responding to dietary HFD or FBTPS intervention as well as

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whether they were associated with MS in HFD-induced mice were analyzed, aiming

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to provide potential insights into mechanisms of the gut microbiota with prevention of

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MS.

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MATERIALS AND METHODS

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Preparation of FBTPS. FBTPS was prepared according to our reported method.25

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Briefly, the dried sample of FBT (provided by Hunan Yiyang Tea Processing Factory

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Co., Ltd., Yiyang, Hunan, China) was treated with aqueous ethanol solution (85%, v/v)

104

three times at 70 ℃ for 8 h to remove small molecule substances. The resulting

105

residue was dried and used for extraction of FBTPS by distilled water (10 mL/g, v/w)

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three times at 70 ℃ for 3 h. After filtration, the extracts were precipitated with triple

107

volumes of dehydrated ethanol and kept at 4 ℃ overnight. After centrifugation at

108

3040 g for 15 min, the precipitates were dissolved in distilled water, removed

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dissociative protein by Sevag method, dialyzed with distilled water and lyophilized,

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affording FBTPS. As reported, the average molecular weight of FBTPS was 828 kD.25

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The contents of natural carbohydrate, uronic acid and protein were 55.20 ± 3.07,

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37.78 ± 1.26 and 2.93 ± 0.06%, respectively, indicating that polysaccharide was the

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main composition of FBTPS. Moreover, FBTPS was composed of mannose, ribose,

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rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a

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molar ratio of 3.66:1.69:12.11:1.41:28.17:21.97:19.15:11.83.

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Animals and Experimental Design. All animal experiments were approved and 6


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carried out in accordance with the Guidelines of the Ethical Committee of

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Experimental Animal Center of Nanjing Agricultural University and the National

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Guidelines for Experimental Animal Welfare (MOST of People’s Republic of China,

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2006). Forty male C57BL/6 mice (6 weeks of age, animal number NO.201608240)

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were purchased from Comparative Medicine Centre of Yangzhou University

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(Yangzhou, Jiangsu, China) and housed in Animal Center of Nanjing Agricultural

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University (SYXK2011-0037) under controlled light conditions (12 h

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light-dark cycle) with ad libitum access to food and water. After one week

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acclimatization, the mice were randomly divided into 5 treatment groups (8 mice per

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group) and fed with (1) normal-chow diet (ND, 10% calories from fat, MD12031)

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with administration of water as healthy control, (2) HFD (45% calories from fat,

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MD12032) with administration of water as model control, (3) HFD plus daily

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administration of 200 mg/kg/day FBTPS (HFD-L group), (4) HFD plus daily

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administration of 400 mg/kg/day FBTPS (HFD-M group) or (5) HFD plus daily

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administration of 800 mg/kg/day FBTPS (HFD-H group) by intragastric gavage for 8

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weeks. The diets were obtained from Jiangsu Medicience Ltd. (Yangzhou, Jiangsu,

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China) and their compositions are showed in Table S1 (Supplemental material).

134

During the experiment, food intake and body weight were recorded weekly.

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Histology Analysis. Histology analysis was carried out according to our reported

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method.1 In brief, the liver and epididymal fat were immediately fixed in

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formaldehyde solution (12%) for 24 h after washing with saline. After dehydration

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with 30, 50, 70, 80, 90, 95 and 100% ethanol, respectively, all the samples were

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embedded in paraffin, cut into 5 µm thick microsections and stained with hematoxylin

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and eosin. Then, the sections were observed by light microscopy, and the size of

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epididymal adipocyte was evaluated by Image J software.

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Biochemical Analysis. The contents of triglyceride (TG), total cholesterol (TC),

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low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol

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(HDL-C) in serum were measured by commercially available kits (Nanjing Jiancheng

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Bioengineering Institute, Nanjing, China).

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RNA Preparation and qRT-PCR Analysis. The total RNA of liver was extracted

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by MiniBEST Universal RNA Extraction Kit (TaKaRa Bio. Inc., Dalian, China). The

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concentration and integrity of RNA were evaluated by Nanodrop 2000 (Thermo

149

Fisher Scientific Inc., USA). cDNA was prepared by reverse transcription of RNA by

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PrimeScript

151

reverse-transcription PCR (qRT-PCR) was performed with SYBR Green Master Mix

152

and QuantStudio 6 Flex (Thermo Fisher Scientific Inc.) according to the protocol of

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manufacturer. All samples were performed in duplicate in a single plate and relative

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quantification was calculated by the 2-∆∆Ct method. The primer sequences for the

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targeted mouse genes, including carnitine palmitoyl transterase-1 (CPT-1),

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peroxisome proliferator-activated receptor α (PPARα), fatty acid synthase (FAS),

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peroxisome

158

element-binding protein 1c (SREBP-1c) and liver X receptor (LXRα), are presented

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in Table S2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as

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housekeeping gene.

RT

Master

Mix

proliferator-activated

(TaKaRa

receptor

Bio.

γ

Inc.).

Quantitative

(PPARγ),

8


sterol

real-time

regulatory

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Gut microbiota Analysis. DNA of fecal sample was extracted by QiAamp DNA

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stool Mini Kit (NO.51504, Qiagen, Germany). The resulting DNA was amplified with

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barcoded specific bacterial primers targeting the variable region 4 (V4) of 16S rRNA

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gene using primers with the Primer F: Illumina adapter sequence 1 +

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AYTGGGYDTAAAGNG

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TACNVGGGTATCTAATCC.

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bioinformatics was carried out by Genesky Biotechnologies Inc. (Shanghai, China) on

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the Illumina MiSeq platform to generate 2 × 250 bp paired-end reads. The remaining

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unique reads were clustered into operational taxonomic units (OTUs) based on

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Ribosomal Database Project (RDP) by UPARSE with 97% similarity cutoff after the

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raw reads were quality filtered and merged by fastx, Cutadapt, Usearch and

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FLASH.31-33 Mothur was used to calculate rarefaction analysis, the community

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richness index and alpha diversities including Shannon and Simpson indexes.34

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Principal component analysis (PCA) and hierarchical cluster analysis by euclidean

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distance matrix and ward.D2 method were performed according to the abundances of

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OTUs by R package (V3.4.0).

and

Primer R: For

each

Illumina adapter sequence

fecal

sample,

the

sequencing

2

+ and

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Statistical analysis. All values are expressed as the mean ± standard deviation (SD)

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or box-and-whisker plots. Statistical significances between groups were evaluated by

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one-way ANOVA procedure followed by Tukey test using SPSS 22 software (IBM,

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USA). Differences in relative abundances of OTUs were calculated using Tukey's

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honest significant difference (HSD) test by R package (V3.4.0). Canoco for Windows

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4.5 (Microcomputer Power, USA) was used for redundancy analysis (RDA), which

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was assessed by Monte Carlo permutation procedure (MCPP) with 499 random

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permutations and P values of 0.002. The correlations between relative abundances of

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OTUs and host parameters of MS were analyzed by Spearman’s correlation (SPSS 22,

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IBM). Furthermore, adjusted p-values obtained based on false discovery rate (FDR)

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were used to evaluate differences in this work. A value at p < 0.05 was considered

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significant difference.

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RESULTS

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FBTPS Attenuated Features of HFD-induced MS. To investigate the effects of

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FBTPS on the development of MS after administration of HFD, the male C57BL/6

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mice (n = 8 per group) received intervention including HFD and HFD supplemented

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with 200, 400 or 800 mg/kg/day of FBTPS for 8 weeks. Compared with ND group,

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HFD treatment for 8 weeks led to significant increases in body weight gain, adipose

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tissue weight (epididymal and perirenal fat), epididymal adipocyte size, liver weight

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as well as hepatic lipid deposition (Figure 1a-1h). As expected, dietary

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supplementation with FBTPS could significantly prevent body weight gain,

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accumulation of fat, increase in epididymal adipocyte size and hepatic lipid deposition

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in a dose-dependent manner. Furthermore, the levels of TC and LDL-C in serum were

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significantly elevated after HFD intervention (Figure 2a and 2c) compared with those

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of ND group. As expected, FBTPS, especially high dosage of FBTPS, could

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significantly reduce these levels. However, the levels of serum TG and HDL-C were

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not significantly affected by treatment of FBTPS (Figure 2 b and 2d). The results

204

suggested that FBTPS could attenuate features of MS in HFD-fed mice. Food intake 10


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was not significantly different for any of the groups between HFD intervention groups

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(Figure S1), indicating that the prevention of MS by FBTPS was not due to the

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decrease in food consumption.17

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FBTPS Regulated Expression of Lipid Metabolism Related Genes. Previous

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studies have shown that hepatic fatty acid synthesis and oxidation might be regulated

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by genes expression after HFD treatment.35-37 Therefore, the effects of FBTPS on

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gene expression of fat metabolism-related genes in liver including CPT-1, PPARα,

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PPARγ, SREBP-1c, FAS and LXRα were investigated by qRT-PCR, and the results

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are shown in Figure 3. Compared with ND group, significantly higher expression of

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FAS, LXRα, PPARγ and SREBP-1c and while lower expression of CPT-1 and PPARα

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were observed in mice fed with HFD. Compared with HFD group, FBTPS treatment

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could lead to the down-regulation of LXRα, FAS, SREBP-1c and PPARγ and

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up-regulation of CPT-1. In particular, the expression levels of CPT-1, PPARγ,

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SREBP-1c, FAS and LXRα in treatment with high dosage of FBTPS exhibited no

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difference with those of ND group. However, FBTPS intervention had limited effect

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on expression of PPARα.

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Overall Structural Changes of Gut Microbiota in Response to HFD and FBTPS.

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An increasing number of works using germ-free animals and microbiota transplant

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shows that development of MS and associated diseases are strongly implicated in the

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gut microbiota.15,38,39 Therefore, the effects of HFD and FBTPS intervention on gut

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microbiota were investigated by pyrosequencing V4 of bacterial 16S rRNA genes.

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The average sequence number were all more than 44,397 to afford 3,208 distinct

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OTUs from 40 samples (n = 8 for each group) at a 97% similarity level. The results

228

from the Rarefaction and Shannon index curves suggested that most of the diversity

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and new phylotypes were covered (Figure S2). The changes of gut microbiota

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community diversity in response to the treatments with HFD and FBTPS were

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evaluated using Shannon and Simpson indexes. Similar to the previous findings,14 the

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diet was found to be a key factor in changing phylogenetic diversity. In this work, a

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significant increase in Simpson index and decrease in Shannon index, indicating the

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lower microbiota community diversity, were found in HFD group (Figure 4a).

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Nevertheless, the microbiota phylogenetic diversity was significantly restored by

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treatment with high dosage of FBTPS, which showed no difference with that of ND

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group.

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Beta-diversity analyses including PCA and hierarchical cluster analysis were

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carried out to further provide an overview of the effects of HFD and FBTPS

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treatments on the composition and structure of gut microbiota. PCA revealed a clear

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separation between the ND and HFD groups (Figure 4b). As expected, the

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composition of gut microbiota exhibited a obvious response to FBTPS intervention.

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Furthermore, PC1, which explained 59.45% of total variance, showed that FBTPS

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intervention shifted the gut microbiota composition of HFD group towards to that of

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ND group in dose-dependent manner. Notably, the HFD-H group had kept away from

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HFD group and was close to ND group as seen in PC1, indicating that FBTPS could

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reverse HFD-disrupted gut microbiota back to health status. On the other hand, the

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results of hierarchical cluster analysis also confirmed significant separation between

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HFD and ND groups (Figure 4c). As expected, shift from HFD group to ND group

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after FBTPS intervention was also observed. This is consistent with the result of PCA.

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Obviously, some mice samples from HFD-M and HFD-H groups got close to ND

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group, which demonstrated the similarity in gut microbiota composition between

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these mice from HFD-M and HFD-H groups with those of ND group. Taxonomic

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analysis suggested that the gut microbiota of mice at the phylum level was mainly

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dominated by the members of Firmicutes and Bacteroidetes, which is the same as

256

other reports.17,40 An increase in relative abundance of Firmicutes and a decrease in

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relative abundance of Bacteroidetes were noted for HFD treatment (Figure 4d and 4e),

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which led to a significant increase in the ratio of F/B compared to that of ND group.

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However, FBTPS intervention exhibited limited effects on the relative abundances of

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Firmicutes and Bacteroidetes and the ratio of F/B.

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After supplementation with HFD, significant decreased relative abundances of

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Porphyromonadaceae, Rikenellaceae, Lactobacillaceae and Bdellovibrionaceae and

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significant increased relative abundances of Erysipelotrichaceae, Coriobacteriaceae

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and Streptococcaceae were observed compared with ND group at family level (Figure

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5). FBTPS intervention could significantly reverse the HFD-induced increases in the

266

relative abundances of Erysipelotrichaceae, Coriobacteriaceae and Streptococcaceae.

267

However, FBTPS exhibited limited effect on HFD-induced decreased gut microbiota

268

such

269

Bdellovibrionaceae.

270

as

Porphyromonadaceae,

Rikenellaceae,

Lactobacillaceae

and

It is necessary to evaluate the changes of gut microbiota in OTUs levels after

13



271

FBTPS treatment, due to the fact that the different bacterial species in the same family

272

have different responses to HFD or FBTPS. Therefore, RDA was performed based on

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OTUs with more than 0.1% relative abundance at least in one group to identify the

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specific bacterial phylotypes of gut microbiota responding to HFD or FBTPS

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intervention. Mice treated with HFD exhibited changes in 122 OTUs (69 increased

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and 53 decreased) compared with those of the ND group (Figure S4a). As shown in

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Figure S4b-S4d, the treatments of HFD-L, HFD-M and HFD-H altered 58 (33

278

increased and 25 decreased), 50 (33 increased and 17 decreased) and 54 (31 increased

279

and 23 decreased) OTUs in comparison with HFD group, respectively. As a result, a

280

total of 95 distinct OTUs were significantly altered responding to FBTPS intervention

281

(Figure 6 and Supplementary Data 1). Thereinto, 27, 23 and 30 of the 95 OTUs that

282

altered by HFD treatment were reversed in HFD-L, HFD-M and HFD-H groups,

283

respectively, resulting in significant reversion of 46 OTUs in total.

284

The correlations between the relative abundances of dominant gut microbiota in

285

OTUs levels and parameters of MS were carried out by Spearman’s correlation

286

analysis to identify the OTUs that might contribute to attenuating MS in HFD-induced

287

mice. As shown in Figure 7, it was found that 44 of the 46 OTUs were negatively or

288

positively associated with at least one parameter of MS, including weight body gain,

289

liver weight, epididymal fat, perirenal fat, TG, TC, LDL-C and HDL-C. Thereinto,

290

OTU0021, OTU0345, OTU0316, OTU0197, OTU0493, OTU0363 and OTU0249

291

belonged to Erysipelotrichaceae, OTU0260 belonged to Coriobacteriaceae and

292

OTU0406 belonged to Streptococcaceae were significantly positively correlated with

14


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MS, which are in accord with the results mentioned above. Furthermore, 9 OTUs that

294

represented Lachnospiraceae, 8 OTUs that represented Porphyromonadaceae and 10

295

OTUs that represented Ruminococcaceae might also play an important role in

296

prevention of MS, supporting the prior findings.14,40,41 Notably, 26 of the 44 OTUs

297

which were positively correlated with parameters of MS were significantly reduced

298

by FBTPS treatment, including Acetatifactor (OTU0208), Anaerobacterium

299

(OTU0408), Clostridium_IV (OTU0267 and OTU0288), Coprobacter (OTU0009),

300

Flavonifractor (OTU0389), Leuconostoc

301

Oscillibacter (OTU0042 and OTU0300) and Streptococcus (OTU0406). On the

302

contrary, 18 of the 44 OTUs, negatively correlated with parameters of MS, were

303

significantly enhanced by FBTPS treatment, including Alistipes (OTU0054 and

304

OTU0218), Anaerobacterium (OTU0408), Bacteroides (OTU0391), Eisenbergiella

305

(OTU0029 and OTU0254), Erysipelotrichaceae_incertae_sedis (OTU0361) and

306

Odoribacter (OTU0008 and OTU0078). Taken all together, the results demonstrated

307

that FBTPS intervention could modulates the HFD-induced gut microbiota, resulting

308

in a healthy gut microbiota composition similar to that of ND group.

309

DISCUSSION

310

MS, characterized by central obesity, hypertension, hyperlipidemia, insulin resistance

311

and hyperglycemia, has been regarded as one of the urgent worldwide public health

312

problems of this century, which enhances the risk of developing CVD and T2D.2,13,42

313

In recent years, tea consumption as potentially viable nutritional strategy for the

314

prevention of MS has been demonstrated by using animal models and human studies,

(OTU0216), Olsenella (OTU0260),

15



315

thereby possible reducing the risk of T2D and CVD.35 Previous studies have showed

316

that both FBT and tea polysaccharides could attenuate MS,29,43 and the abundant

317

phenolic compounds in tea, specifically catechins, are considered to be the main

318

components contributing to the putative beneficial metabolic effects of tea.44 An

319

increasing number of evidences suggested that the human gut microbiota plays a

320

fundamental role in the metabolic capacity for processing nutrients and host health,45

321

and previous work showed that Fuzhuan brick, green, Oolong and black teas could

322

modulate the gut microbiota in HFD-induced obese mice or rat.27,44 Nevertheless,

323

there was no report about the effects of FBTPS on MS and gut microbiota and

324

possible mechanisms of FBTPS-mediated attenuation of MS. Thus, the effects of

325

FBTPS on the HFD-induced MS and gut microbiota dysbiosis were investigated in

326

this work. As expected, FBTPS could significantly suppress the HFD-induced body

327

weight gain, adipose tissue weight, adipocyte size, liver weight as well as hepatic lipid

328

deposition. Serum biochemical parameters, including TC, TG, LDL-C and HDL-C,

329

could also clearly reflect the status of lipid adsorption and metabolism.36 The present

330

results showed that FBTPS could reduce the levels of TC and LDL-C. However,

331

FBTPS exhibited limited effects on TG and HDL-C levels, which should be further

332

investigated. Wu et al. reported that tea polysaccharides from black tea could

333

significantly decrease the body weight, fat weight, fat cell size and Lee’s index, and

334

improve the biochemical profile in rats fed with HFD.46 Likewise, the effect of green

335

tea polysaccharides on HFD-induced obesity was investigated, and the significantly

336

suppressive effects on body weight gain and fat weight and improvement of blood

16


Page 16 of 46

Page 17 of 46


337

lipid and antioxidant levels were observed in HFD-induced rats.47 Interestingly, the

338

HFD-induced MS was improved by FBTPS in a dose-dependent manner from 200 to

339

800 mg/kg/day. The data suggested that FBTPS was effective for control of MS

340

parameters, making it a promising candidate for prevention of HFD-induced MS.

341

The liver is central to lipogenesis and lipids metabolism.48 It has been reported

342

that some anti-obesogenic candidates could regulate the gene expression levels related

343

to fatty acid synthesis and lipid metabolism in liver, which resulted in prevention of

344

obesity and serum lipid levels.37,49 LXRα, a member of the nuclear receptor family,

345

has a critical role in hepatic lipogenesis, which is mainly involved in the regulation of

346

cholesterol and lipid metabolism.36 Recent studies have shown that SREBP-1c, a

347

central transcription factor to dietary regulation of most hepatic lipogenic genes, is

348

related to the encoding enzymes catalyzing various steps in the synthetic pathways of

349

fatty acid and TG, such as FAS.48,50 PPARγ, which is another well-known nuclear

350

receptor, has been proven to play a major role in regulating the uptake of free fatty

351

acid and hepatic steatosis, and down-regulation of PPARγ expression might lead to

352

the suppression of fatty acid synthesis and stimulation of lipolysis in liver.37 In the

353

present study, the expression levels of LXRα, FAS, SREBP-1c and PPARγ related to

354

lipogenesis and accumulation of fat were significantly reduced by FBTPS.17,50

355

Furthermore, FBTPS treatment could significantly increase the expression level of

356

CPT-1, which is related to lipid catabolism.50 Up-regulation of CPT-1 expression

357

could result in the transfer of the acyl group of a long-chain fatty acyl-CoA from

358

coenzyme A to L-carnitine, which might stimulate the β-oxidation of fatty acids.51

17



359

Therefore, the results showed that alleviation of MS by FBTPS might be involved in

360

decrease in fat lipogenesis and accumulation as well as enhancement of fatty acid

361

catabolism and oxidation. Similarly, alleviation of HFD-induced insulin resistance

362

and nonalcoholic steatohepatitis by Pu-erh tea extract has been reported to be

363

involved in down-regulation of the mRNA expression levels related to lipogenesis and

364

accumulation of fat in the liver of HFD-fed mice.52

365

Accumulating evidences have suggested that gut microbiota might be a potential

366

target for treatment of MS.38,53 Furthermore, numerous reports indicated that the

367

prevention of MS by polysaccharides was related to the modulation of gut

368

microbiota.18,22 In the present work, HFD treatment led to profound alteration in gut

369

microbiota structure and composition based on results of PCA and hierarchical cluster

370

analysis, which is consistent with previous reports.14,54 Nevertheless, an obvious

371

amelioration of HFD-induced gut microbiota back to health status was observed for

372

FBTPS intervention, expecially for high dosage of FBTPS, suggesting superior gut

373

microbiota modulation by FBTPS. Low diversity has been associated with several

374

metabolic-disorders or immune-disorders.55 For example, it has been reported that gut

375

microbiota diversity in obese individuals or mice was lower than that of lean

376

hosts,8,41,56 and the measure of alpha diversity was negatively associated with

377

obesity-related phenotypes, specifically abdominal adiposity.40 Our results showed

378

that higher dosage of FBTPS could result in higher diversity of gut microbiota,

379

confirming the importance of gut microbiota diversity for prevention of MS. Based on

380

the results of alpha and beta-diversity analyses, FBTPS modulated gut microbiota in a

18


Page 18 of 46

Page 19 of 46


381

dose-dependent manner, which is in agreement with the dose-dependent manner of

382

MS amelioration, indicating a strong association between the modulation of gut

383

microbiota and syndrome amelioration alleviation.57 Besides decrease in gut

384

microbiota community diversity and alteration in the overall gut microbiota structure,

385

a significant increase in ratio of F/B was also observed after HFD treatment. An

386

increasing number of findings showed significant difference in the ratio of F/B

387

between the obesity and lean humans or mice.15,17 Furthermore, increase in the ratio

388

of F/B can result in enhancement of energy harvest, which is believed to be associated

389

with obesity.58,59 Thus, some researchers found that some potential candidates for

390

prevention of MS could reduce the ratio of F/B, which might play an important role in

391

amelioration of MS.2,17 However, it was found that supplementation with FBTPS had

392

limited effects on the relative abundances of Firmicutes and Bacteroides or the ratio

393

of F/B.

394

The early work showed that a bloom of Erysipelotrichaceae was observed in

395

diet-induced obese animals and individuals.60 It has also been reported that

396

Erysipelotrichaceae could lead to enhancement of energy extraction from the diet,61

397

which is associated with lipidemic profiles in the host.60 Martínez et al. found that the

398

abundances of several bacterial taxa from Coriobacteriaceae and Erysipelotrichaceae

399

displayed significantly high relationship with cholesterol metabolites in hamsters

400

model.8 In our study, the relative abundance of Erysipelotrichaceae increased from

401

1.65 ± 1.09% to 26.19 ± 10.63% after HFD treatment, and then decreased from 26.19

402

± 10.63% to 8.76 ± 4.39% after high dosage of FBTPS intervention. Furthermore, 7

19



Page 20 of 46

403

OTUs (OTU0021, OTU0316, OTU0345, OTU0197, OTU0493, OTU0363 and

404

OTU0249) within family of Erysipelotrichaceae in OTUs level all showed high

405

positive association with parameters of MS. Similarly, the enrichment of

406

Coriobacteriaceae and Streptococcaceae at family level has been reported in

407

HFD-induced MS in several studies.8,53,62,63 It has been demonstrated that the relative

408

abundance of Coriobacteriaceae had a high positive relation with plasma cholesterol

409

level in hamsters model (r = 0.84, P = 0.0002).64 Moreover, the hepatic glycogen,

410

glucose, TG and the metabolism of xenobiotics have been reported to have strong

411

association with the abundance of Coriobacteriaceae.65 Some strains within the family

412

of Streptococcaceae have been reported to induce mild inflammation in humans.57

413

Furthermore, high abundances of Streptococcaceae family in gut microbiota are

414

known to be related to the risks of metabolic disorders, diabetes and colon cancer.63 In

415

the current study, the HFD-induced increases in abundances of Coriobacteriaceae and

416

Streptococcaceae were significantly reversed to those of ND group. Both OTU0260

417

within the family of Coriobacteriaceae and OTU0406 within the family of

418

Streptococcaceae were also significantly positively correlated with MS. At the same

419

time, some researchers reported that decreases in relative abundance of

420

Erysipelotrichaceae, Coriobacteriaceae or Streptococcaceae by some potential

421

candidate or strategy for prevention of MS might play an important role in

422

amelioration of MS, which might be potentially therapeutic targets for prevention of

423

MS.8,61,66,67 Therefore, the alleviation of MS by FBTPS intervention might be also

424

owed

to

the

reversion

of

relative

abundances

20


of


Page 21 of 46


425

Coriobacteriaceae and Streptococcaceae.

426

Spearman’s correlation between the 46 key OTUs reversed by FBTPS and

427

parameters of MS was carried out to identify the potentially beneficial phylotypes of

428

gut microbiota. As results, 44 key OTUs, which were significantly associated with at

429

least one parameter of MS, mainly belonged to Erysipelotrichaceae (8 OTUs),

430

Lachnospiraceae (9 OTUs), Porphyromonadaceae (8 OTUs) and Ruminococcaceae

431

(10 OTUs). Besides Erysipelotrichaceae, Coriobacteriaceae and Streptococcaceae, the

432

families of Lachnospiraceae,44 Porphyromonadaceae68 and Ruminococcaceae54 had

433

also been reported to be closely related to MS in previous works. Notably, the bloom

434

of Erysipelotrichaceae in HFD group was mainly caused by the presence of OTU0021

435

whose abundance increased from 0.04 ± 0.04% to 23.27 ± 10.62%, and the relative

436

abundance decreased to 6.21 ± 4.21% after intervention with high dosage of FBTPS.

437

Furthermore, OTU0021 had significantly correlation with weight body gain, liver

438

weight, epididymal fat, perirenal fat, TC, LDL-C and HDL-C, indicating that

439

OTU0021 might play a central role in amelioration of MS by FBTPS. Gene

440

sequencing data showed that OTU0021 belonged to Faecalibacterium_rodentium,

441

however, limited information is available for it. Furthermore, FBTPS intervention

442

could significantly enhance the relative abundances of 18 OTUs which were

443

negatively correlated with MS. Thereinto, Alistipes,44 Bacteroides17 and Odoribacter69

444

have been reported to be negatively correlated with MS. On the other hand, FBTPS

445

intervention could significantly reduce the relative abundances of 26 OTUs which

446

were positively correlated with MS. Thereinto, Clostridium_IV,70 Flavonifractor,71

21



447

Leuconostoc,72 Oscillibacter10 and Streptococcus73 have been reported to be positively

448

correlated with MS. Although it is difficult to arrive at the cause-effect relationships

449

among these associations in this work, our data suggested that FBTPS-induced

450

modulation in the gut microbiota was highly associated with MS in obese mice and

451

these changes might be an important mechanism for FBTPS mediating its beneficial

452

metabolic effects. Therefore, the possible mechanism of MS prevention by FBTPS is

453

that indigestible FBTPS reach the large intestine and then modulate the gut

454

microbiota.

455

In conclusion, it was found that HFD-induced body weight gain, epididymal and

456

perirenal fat accumulation, epididymal adipocyte size and the levels of TC and

457

LDL-C in serum of mice were significantly restored by oral administration of FBTPS.

458

The effects were associated with alleviation of liver weight, hepatic lipid deposition

459

and expression of lipid metabolism-related genes. In addition, FBTPS intervention

460

could increase microbiota phylogenetic diversity and reduce the relative abundances

461

of Erysipelotrichaceae, Coriobacteriaceae and Streptococcaceae. The present results

462

further suggested that 44 key OTUs that were negatively or positively associated with

463

MS might play a key role in prevention of MS, leading us to propose that prevention

464

of MS by FBTPS may be significantly related to modulation of the gut microbiota.

465

ASSOCIATED CONTENT

466

Supporting information

467

Tables for compositions of experimental diets, target genes and primers sequence.

468

Figures for food intake of mice, alpha diversity analysis of sample, and biplot of the 22


Page 22 of 46

Page 23 of 46


469

RDA based on the OTUs abundance between HFD and (a) ND, (b) HFD-L, (c)

470

HFD-M and (d) HFD-H groups, respectively.

471

AUTHOR INFORMATION

472

Corresponding Author

473

*Phone: +86-25-84396791. E-mail: [email protected] (X Zeng).

474

ORCID

475

Xiaoxiong Zeng: 0000-0003-2954-3896

476

Funding

477

The study was supported by the National Key Research and Development Program of

478

China (2017YFD0400800), a project funded by the Priority Academic Program

479

Development of Jiangsu Higher Education Institutions and Postgraduate Research &

480

Practice Innovation Program of Jiangsu Province (KYCX17_0632).

481

Notes

482

The authors declare no competing financial interest.

483

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748

Figure Captions

749

Figure 1. Effects of FBTPS administration on (a) body weight, (b) body weight gain,

750

(c) epididymal fat, (d) perirenal fat, (e) epididymal adipocyte microsections, (f)

751

epididymal adipocyte size evaluated by Image J software, (g) liver weight and (h)

752

liver microsections stained with H&E, the arrows indicate small lipid droplets. Values

753

are means ± SD for n=8, and One-way ANOVA procedure followed by Tukey test was

754

used to evaluate the statistical significance. The different letters represent significant

755

differences between different groups (p < 0.05).

756

Figure 2. Effects of FBTPS on serum biochemical variables including TC (a), TG (b),

757

LDL-C (c) and HDL-C (d). Values are means ± SD for n=8, and One-way ANOVA

758

procedure followed by Tukey test was used to evaluate the statistical significance. The

759

different letters represent significant differences between different groups (p < 0.05).

760

Figure 3. Effects of FBTPS on the relative expression of CPT1 (a), LXRα (b), FAS

761

(c), SREBP-1c (d), PPARα (e) and PPARγ (f) in liver. Values are means ± SD for n=8,

762

and One-way ANOVA procedure followed by Tukey test was used to evaluate the

763

statistical significance. The different letters represent significant differences between

764

different groups (p < 0.05).

765

Figure 4. FBTPS modulate the HFD-disrupted gut microbiota composition. a,

766

comparison of alpha diversity accessed by Shannon and InvSimpson indexes; b, PCA

767

of gut microbiota based on OUT relative abundance; c, hierarchical cluster analysis by

768

euclidean distance matrix and ward.D2 method; d, bacterial taxonomic profiling at the

769

phylum level of gut microbiota; e, the relative abundances of Firmicutes,

36


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Page 37 of 46


770

Bacteroidetes, and the ratio of Firmicutes to Bacteroidetes. One-way ANOVA

771

procedure followed by Tukey test was used to evaluate the statistical significance. The

772

different letters represent significant differences between different groups (p < 0.05).

773

Figure 5. Bacterial taxonomic profiling at the family level of gut microbiota. a,

774

bubble chart of relative bacterial composition at the family level; b, comparative

775

analysis of 20 most relative abundance of gut microbiota at the family level. One-way

776

ANOVA procedure followed by Tukey test was used to evaluate the statistical

777

significance. The different letters represent significant differences between different

778

groups (p < 0.05).

779

Figure 6. The gut microbiota composition was altered by HFD or FBTPS intervention

780

according to RDA results. a, Heatmap shows the relative abundance (log10

781

transformed) of 95 OTUs, the color of the squares indicated the relative abundance of

782

the OUTs; b, the changing direction of OTUs induced by HFD or FBTPS intervention.

783

The circles (○) and dots (●) represent the less and more relative abundances of OTUs

784

in ND or FBTPS-fed groups compared with the HFD group, respectively. The asterisk

785

(*) indicates OTUs in ND group altered by HFD intervention were reversed by

786

FBTPS treatment; c, the OTUs represent bacterial taxa information (phylum, family,

787

genus and species) modulated by FBTPS. Differences in relative abundance of OTUs

788

were calculated using Tukey's HSD test. A value of p < 0.05 was considered

789

significant.

790

Figure 7. 46 OTUs reversed by FBTPS intervention were significantly correlated

791

with parameters of MS. a, the changing direction of OTUs induced by HFD and

37



792

FBTPS treatment. The circles (○) and dots (●) represent the less and more relative

793

abundances of OTUs in ND or FBTPS-fed groups compared with the HFD group,

794

respectively; b, the results of Spearman’s correlation between OTUs and parameters

795

of metabolic syndrome. Colors of squares represent R-value of Spearman’s

796

correlation. * and ** indicate the associations significant (p < 0.05 and p < 0.01,

797

respectively); c, the OTUs represent bacterial taxa information (phylum, family, genus

798

and species).

38


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Page 39 of 46


Figure 1.

799 39



800 801

Figure 2.

40


Page 40 of 46

Page 41 of 46


802 803

Figure 3.

41



804 805

Figure 4.

42


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Page 43 of 46


806 807

Figure 5.

43



808 809

Figure 6.

44


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810 811

Figure 7.

45



812

Graphic for table of contents

813

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Fuzhuan brick tea polysaccharides attenuate metabolic syndrome in

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