Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 46
Journal of Agricultural and Food Chemistry
1
Fuzhuan brick tea polysaccharides attenuate metabolic syndrome in high-fat diet
2
induced mice in association with modulation in the gut microbiota
3
Guijie Chen,† Minhao Xie,† Peng Wan,† Dan Chen,† Zhuqing Dai,† Hong Ye,† Bing
4
Hu,† Xiaoxiong Zeng,†,* Zhonghua Liu‡,§,*
5
†
6
210095, Jiangsu, China
7
‡
8
University, Changsha 410128, China
9
§
10
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
Journal of Agricultural and Food Chemistry
Page 2 of 46
11
ABSTRACT
12
An increasing number of evidence suggests that the gut microbiota composition and
13
structure contribute to the pathophysiology of metabolic syndrome (MS), which has
14
been put forward as a new target in the treatment of diet-induced MS. In this work, we
15
aimed to investigate effects of Fuzhuan brick tea polysaccharides (FBTPS) on MS and
16
gut microbiota dysbiosis in high-fat diet (HFD)-fed mice and to further investigate
17
whether its attenuation of MS being related to the modulation of gut microbiota. The
18
results showed that FBTPS intervention could significantly attenuate metabolic
19
syndrome in HFD-induced mice. Based on results of sequencing, FBTPS treatment
20
could increase the phylogenetic diversity of HFD-induced microbiota. FBTPS
21
intervention could significantly restore the HFD-induced increases in relative
22
abundances
23
Spearman’s correlation analysis showed that 44 key OTUs were negatively or
24
positively associated with MS. Our results suggested that FBTPS could serve as a
25
novel candidate for prevention of MS in association with the modulation of gut
26
microbiota.
27
Keywords: Fuzhuan brick tea; polysaccharides; metabolic syndrome; gut microbiota;
28
modulation
of
Erysipelotrichaceae,
Coriobacteriaceae
2
ACS Paragon Plus Environment
and
Streptococcaceae.
Page 3 of 46
Journal of Agricultural and Food Chemistry
29
INTRODUCTION
30
Metabolic syndrome (MS), a combination of medical disorders including obesity,
31
hyperglycemia, dyslipidemia, hypertension and insulin resistance, can enhance the
32
risk of developing cardiovascular disease (CVD) and type 2 diabetes (T2D).1-3 Due to
33
the obesogenic shifts in nutritional composition, growing caloric intake and lack of
34
exercise, humans are in the midst of worldwide epidemics of MS.1,4 Thus, prevention
35
of MS has been a major challenge for modern society. A growing number of
36
evidences suggests that the gut microbiota may be served as an important modulator
37
of the crosstalk between diet and development of MS or related diseases through
38
affecting the extraction of nutrients and energy from diets,5 energy homeostasis,6
39
glucose metabolism,7 cholesterol metabolism,8 insulin resistance,3 non-alcoholic fatty
40
liver9 and the chronic inflammatory state.10 Recently and more strikingly, some
41
species or genera of beneficial gut microbiota, such as Akkermansia muciniphila,2,11
42
Bifidobacterium12 and Lactobacillus,13 have been demonstrated to be negatively
43
associated with the development of MS. On the contrary, increases in some
44
pro-inflammatory/pathogenic
45
Coriobacteriaceae and Streptococcaceae, are consistently associated with the
46
development of obesity, adipose tissue, systemic inflammation and metabolic
47
comorbidities in both humans and rodents.8,13,14 Thus, a potentially viable concept,
48
modulating high-fat diet (HFD) induced gut microbiota dysbiosis as new therapeutic
49
target for the prevention of MS and related diseases, has been presented.15,16
50
gut
microbiota,
such
as
Erysipelotrichaceae,
A number of potential strategy or candidate based on modulation of gut
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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,
53
both Bifidobacterium and Lactobacillus could differentially attenuate HFD-induced
54
obesity comorbidities partly due to the strain-specific effects on MS-related
55
phylotypes of gut microbiota.13 The relative abundance of Akkermansia muciniphila
56
was dramatically enhanced and the proportion of Firmicutes/Bacteroidetes (F/B) was
57
significantly decreased after treatment of grape polyphenols, which led to reduction in
58
intestinal and systemic inflammation and improvement in the protection against MS
59
in HFD-induced mice.2 Likewise, both water extract and polysaccharides from
60
Ganoderma lucidum could prevent obesity-related MS and gut dysbiosis.17 In addition,
61
it has been reported that the host-indigestible, fermentable fibers and carbohydrates
62
(regarded as prebiotics) could be definite assets for the potential modulation of gut
63
microbiota by enhancing the growth of specific health-promoting bacteria.18 In
64
particular, short-chain fatty acids (SCFAs) such as acetic, propionic and butyric acids
65
are the main products of polysaccharides metabolized by gut microbiota, which not
66
only contribute to gut health as a direct energy source for intestinal epithelial cells,
67
but also enter the systemic circulation and directly regulate features of MS, such as
68
insulin resistance and glucose metabolism.19,20 Therefore, the degradation and
69
utilization of polysaccharides by gut microbiota and their health-promoting effects on
70
human health and disease have been widely investigated and reported (reviewed in
71
references18,21-23).
72
Tea, made from the buds and leaves of Camellia sinensis L., is one of the most
4
ACS Paragon Plus Environment
Page 4 of 46
Page 5 of 46
Journal of Agricultural and Food Chemistry
73
popular flavored and functional drinks consumed in the world due to its favorable
74
flavor and health-promoting bioactivities.24 The dark tea is post-fermented tea
75
produced by microbial fermentation of heat-processed green tea leaves.25 According
76
to method of microbial fermentation, dark teas are further classified into Qingzhuan
77
brick tea, Pu-erh tea and Fuzhuan brick tea (FBT), etc.26 Thereinto, FBT, one kind of
78
dark tea produced in Hunan province of China with the help of fungus Eurotium
79
cristatum, has been demonstrated to have preventive and therapeutic effects on
80
MS.27,28 In our recent study, it was found that FBT could significantly attenuate
81
features of the HFD-induced metabolic syndrome in mice, which might be related to
82
the modulation of gut microbiota.29 However, the bioactive components contributing
83
to the prevention of MS and the potential mechanisms responsible for regulating MS
84
remain unclear. Tea polysaccharides, identified as one of the main active components
85
in tea with a wide range of health-promoting bioactivities,30 have great potential
86
applications as natural products. Our previous study in vitro demonstrated that
87
polysaccharides from FBT (FBTPS) could pass through the digestive system without
88
being degraded and reach the large intestine safely, where it could be broken down
89
and utilized by gut microbiota.25 Furthermore, FBTPS could significantly reduce the
90
ratio of F/B, which is expected to result in the decrease of the energy harvest thereby
91
prevention of the risk of MS. Thus, it was speculated that FBTPS might be an
92
important active component of FBT contributing to the prevention of MS and
93
modulation of gut microbiota. The purpose of the present study, therefore, was to
94
investigate whether the intervention of FBTPS to mice could prevent MS and
5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
95
modulate the gut microbiota dysbiosis in HFD-induced mice. Additionally, the gut
96
microbe phylotypes responding to dietary HFD or FBTPS intervention as well as
97
whether they were associated with MS in HFD-induced mice were analyzed, aiming
98
to provide potential insights into mechanisms of the gut microbiota with prevention of
99
MS.
100
MATERIALS AND METHODS
101
Preparation of FBTPS. FBTPS was prepared according to our reported method.25
102
Briefly, the dried sample of FBT (provided by Hunan Yiyang Tea Processing Factory
103
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)
106
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
109
dissociative protein by Sevag method, dialyzed with distilled water and lyophilized,
110
affording FBTPS. As reported, the average molecular weight of FBTPS was 828 kD.25
111
The contents of natural carbohydrate, uronic acid and protein were 55.20 ± 3.07,
112
37.78 ± 1.26 and 2.93 ± 0.06%, respectively, indicating that polysaccharide was the
113
main composition of FBTPS. Moreover, FBTPS was composed of mannose, ribose,
114
rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a
115
molar ratio of 3.66:1.69:12.11:1.41:28.17:21.97:19.15:11.83.
116
Animals and Experimental Design. All animal experiments were approved and 6
ACS Paragon Plus Environment
Page 6 of 46
Page 7 of 46
Journal of Agricultural and Food Chemistry
117
carried out in accordance with the Guidelines of the Ethical Committee of
118
Experimental Animal Center of Nanjing Agricultural University and the National
119
Guidelines for Experimental Animal Welfare (MOST of People’s Republic of China,
120
2006). Forty male C57BL/6 mice (6 weeks of age, animal number NO.201608240)
121
were purchased from Comparative Medicine Centre of Yangzhou University
122
(Yangzhou, Jiangsu, China) and housed in Animal Center of Nanjing Agricultural
123
University (SYXK2011-0037) under controlled light conditions (12 h
124
light-dark cycle) with ad libitum access to food and water. After one week
125
acclimatization, the mice were randomly divided into 5 treatment groups (8 mice per
126
group) and fed with (1) normal-chow diet (ND, 10% calories from fat, MD12031)
127
with administration of water as healthy control, (2) HFD (45% calories from fat,
128
MD12032) with administration of water as model control, (3) HFD plus daily
129
administration of 200 mg/kg/day FBTPS (HFD-L group), (4) HFD plus daily
130
administration of 400 mg/kg/day FBTPS (HFD-M group) or (5) HFD plus daily
131
administration of 800 mg/kg/day FBTPS (HFD-H group) by intragastric gavage for 8
132
weeks. The diets were obtained from Jiangsu Medicience Ltd. (Yangzhou, Jiangsu,
133
China) and their compositions are showed in Table S1 (Supplemental material).
134
During the experiment, food intake and body weight were recorded weekly.
135
Histology Analysis. Histology analysis was carried out according to our reported
136
method.1 In brief, the liver and epididymal fat were immediately fixed in
137
formaldehyde solution (12%) for 24 h after washing with saline. After dehydration
138
with 30, 50, 70, 80, 90, 95 and 100% ethanol, respectively, all the samples were
7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 46
139
embedded in paraffin, cut into 5 µm thick microsections and stained with hematoxylin
140
and eosin. Then, the sections were observed by light microscopy, and the size of
141
epididymal adipocyte was evaluated by Image J software.
142
Biochemical Analysis. The contents of triglyceride (TG), total cholesterol (TC),
143
low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol
144
(HDL-C) in serum were measured by commercially available kits (Nanjing Jiancheng
145
Bioengineering Institute, Nanjing, China).
146
RNA Preparation and qRT-PCR Analysis. The total RNA of liver was extracted
147
by MiniBEST Universal RNA Extraction Kit (TaKaRa Bio. Inc., Dalian, China). The
148
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
150
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
153
manufacturer. All samples were performed in duplicate in a single plate and relative
154
quantification was calculated by the 2-∆∆Ct method. The primer sequences for the
155
targeted mouse genes, including carnitine palmitoyl transterase-1 (CPT-1),
156
peroxisome proliferator-activated receptor α (PPARα), fatty acid synthase (FAS),
157
peroxisome
158
element-binding protein 1c (SREBP-1c) and liver X receptor (LXRα), are presented
159
in Table S2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as
160
housekeeping gene.
RT
Master
Mix
proliferator-activated
(TaKaRa
receptor
Bio.
γ
Inc.).
Quantitative
(PPARγ),
8
ACS Paragon Plus Environment
sterol
real-time
regulatory
Page 9 of 46
Journal of Agricultural and Food Chemistry
161
Gut microbiota Analysis. DNA of fecal sample was extracted by QiAamp DNA
162
stool Mini Kit (NO.51504, Qiagen, Germany). The resulting DNA was amplified with
163
barcoded specific bacterial primers targeting the variable region 4 (V4) of 16S rRNA
164
gene using primers with the Primer F: Illumina adapter sequence 1 +
165
AYTGGGYDTAAAGNG
166
TACNVGGGTATCTAATCC.
167
bioinformatics was carried out by Genesky Biotechnologies Inc. (Shanghai, China) on
168
the Illumina MiSeq platform to generate 2 × 250 bp paired-end reads. The remaining
169
unique reads were clustered into operational taxonomic units (OTUs) based on
170
Ribosomal Database Project (RDP) by UPARSE with 97% similarity cutoff after the
171
raw reads were quality filtered and merged by fastx, Cutadapt, Usearch and
172
FLASH.31-33 Mothur was used to calculate rarefaction analysis, the community
173
richness index and alpha diversities including Shannon and Simpson indexes.34
174
Principal component analysis (PCA) and hierarchical cluster analysis by euclidean
175
distance matrix and ward.D2 method were performed according to the abundances of
176
OTUs by R package (V3.4.0).
and
Primer R: For
each
Illumina adapter sequence
fecal
sample,
the
sequencing
2
+ and
177
Statistical analysis. All values are expressed as the mean ± standard deviation (SD)
178
or box-and-whisker plots. Statistical significances between groups were evaluated by
179
one-way ANOVA procedure followed by Tukey test using SPSS 22 software (IBM,
180
USA). Differences in relative abundances of OTUs were calculated using Tukey's
181
honest significant difference (HSD) test by R package (V3.4.0). Canoco for Windows
182
4.5 (Microcomputer Power, USA) was used for redundancy analysis (RDA), which
9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
183
was assessed by Monte Carlo permutation procedure (MCPP) with 499 random
184
permutations and P values of 0.002. The correlations between relative abundances of
185
OTUs and host parameters of MS were analyzed by Spearman’s correlation (SPSS 22,
186
IBM). Furthermore, adjusted p-values obtained based on false discovery rate (FDR)
187
were used to evaluate differences in this work. A value at p < 0.05 was considered
188
significant difference.
189
RESULTS
190
FBTPS Attenuated Features of HFD-induced MS. To investigate the effects of
191
FBTPS on the development of MS after administration of HFD, the male C57BL/6
192
mice (n = 8 per group) received intervention including HFD and HFD supplemented
193
with 200, 400 or 800 mg/kg/day of FBTPS for 8 weeks. Compared with ND group,
194
HFD treatment for 8 weeks led to significant increases in body weight gain, adipose
195
tissue weight (epididymal and perirenal fat), epididymal adipocyte size, liver weight
196
as well as hepatic lipid deposition (Figure 1a-1h). As expected, dietary
197
supplementation with FBTPS could significantly prevent body weight gain,
198
accumulation of fat, increase in epididymal adipocyte size and hepatic lipid deposition
199
in a dose-dependent manner. Furthermore, the levels of TC and LDL-C in serum were
200
significantly elevated after HFD intervention (Figure 2a and 2c) compared with those
201
of ND group. As expected, FBTPS, especially high dosage of FBTPS, could
202
significantly reduce these levels. However, the levels of serum TG and HDL-C were
203
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
ACS Paragon Plus Environment
Page 10 of 46
Page 11 of 46
Journal of Agricultural and Food Chemistry
205
was not significantly different for any of the groups between HFD intervention groups
206
(Figure S1), indicating that the prevention of MS by FBTPS was not due to the
207
decrease in food consumption.17
208
FBTPS Regulated Expression of Lipid Metabolism Related Genes. Previous
209
studies have shown that hepatic fatty acid synthesis and oxidation might be regulated
210
by genes expression after HFD treatment.35-37 Therefore, the effects of FBTPS on
211
gene expression of fat metabolism-related genes in liver including CPT-1, PPARα,
212
PPARγ, SREBP-1c, FAS and LXRα were investigated by qRT-PCR, and the results
213
are shown in Figure 3. Compared with ND group, significantly higher expression of
214
FAS, LXRα, PPARγ and SREBP-1c and while lower expression of CPT-1 and PPARα
215
were observed in mice fed with HFD. Compared with HFD group, FBTPS treatment
216
could lead to the down-regulation of LXRα, FAS, SREBP-1c and PPARγ and
217
up-regulation of CPT-1. In particular, the expression levels of CPT-1, PPARγ,
218
SREBP-1c, FAS and LXRα in treatment with high dosage of FBTPS exhibited no
219
difference with those of ND group. However, FBTPS intervention had limited effect
220
on expression of PPARα.
221
Overall Structural Changes of Gut Microbiota in Response to HFD and FBTPS.
222
An increasing number of works using germ-free animals and microbiota transplant
223
shows that development of MS and associated diseases are strongly implicated in the
224
gut microbiota.15,38,39 Therefore, the effects of HFD and FBTPS intervention on gut
225
microbiota were investigated by pyrosequencing V4 of bacterial 16S rRNA genes.
226
The average sequence number were all more than 44,397 to afford 3,208 distinct
11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
227
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
229
and new phylotypes were covered (Figure S2). The changes of gut microbiota
230
community diversity in response to the treatments with HFD and FBTPS were
231
evaluated using Shannon and Simpson indexes. Similar to the previous findings,14 the
232
diet was found to be a key factor in changing phylogenetic diversity. In this work, a
233
significant increase in Simpson index and decrease in Shannon index, indicating the
234
lower microbiota community diversity, were found in HFD group (Figure 4a).
235
Nevertheless, the microbiota phylogenetic diversity was significantly restored by
236
treatment with high dosage of FBTPS, which showed no difference with that of ND
237
group.
238
Beta-diversity analyses including PCA and hierarchical cluster analysis were
239
carried out to further provide an overview of the effects of HFD and FBTPS
240
treatments on the composition and structure of gut microbiota. PCA revealed a clear
241
separation between the ND and HFD groups (Figure 4b). As expected, the
242
composition of gut microbiota exhibited a obvious response to FBTPS intervention.
243
Furthermore, PC1, which explained 59.45% of total variance, showed that FBTPS
244
intervention shifted the gut microbiota composition of HFD group towards to that of
245
ND group in dose-dependent manner. Notably, the HFD-H group had kept away from
246
HFD group and was close to ND group as seen in PC1, indicating that FBTPS could
247
reverse HFD-disrupted gut microbiota back to health status. On the other hand, the
248
results of hierarchical cluster analysis also confirmed significant separation between
12
ACS Paragon Plus Environment
Page 12 of 46
Page 13 of 46
Journal of Agricultural and Food Chemistry
249
HFD and ND groups (Figure 4c). As expected, shift from HFD group to ND group
250
after FBTPS intervention was also observed. This is consistent with the result of PCA.
251
Obviously, some mice samples from HFD-M and HFD-H groups got close to ND
252
group, which demonstrated the similarity in gut microbiota composition between
253
these mice from HFD-M and HFD-H groups with those of ND group. Taxonomic
254
analysis suggested that the gut microbiota of mice at the phylum level was mainly
255
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
257
relative abundance of Bacteroidetes were noted for HFD treatment (Figure 4d and 4e),
258
which led to a significant increase in the ratio of F/B compared to that of ND group.
259
However, FBTPS intervention exhibited limited effects on the relative abundances of
260
Firmicutes and Bacteroidetes and the ratio of F/B.
261
After supplementation with HFD, significant decreased relative abundances of
262
Porphyromonadaceae, Rikenellaceae, Lactobacillaceae and Bdellovibrionaceae and
263
significant increased relative abundances of Erysipelotrichaceae, Coriobacteriaceae
264
and Streptococcaceae were observed compared with ND group at family level (Figure
265
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
273
OTUs with more than 0.1% relative abundance at least in one group to identify the
274
specific bacterial phylotypes of gut microbiota responding to HFD or FBTPS
275
intervention. Mice treated with HFD exhibited changes in 122 OTUs (69 increased
276
and 53 decreased) compared with those of the ND group (Figure S4a). As shown in
277
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
ACS Paragon Plus Environment
Page 14 of 46
Page 15 of 46
Journal of Agricultural and Food Chemistry
293
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 16 of 46
Page 17 of 46
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 18 of 46
Page 19 of 46
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
of
Erysipelotrichaceae,
Page 21 of 46
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 22 of 46
Page 23 of 46
Journal of Agricultural and Food Chemistry
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
REFERENCES
484
1. Dai, Z.; Lyu, W.; Xie, M.; Yuan, Q.; Ye, H.; Hu, B.; Zhou, L.; Zeng, X. X. Effects
485
of α-galactooligosaccharides from chickpeas on high-fat-diet-induced metabolic
486
syndrome in mice. J. Agric. Food Chem. 2017, 65, 3160-3166.
487
2. Roopchand, D. E.; Carmody, R. N.; Kuhn, P.; Moskal, K.; Rojas-Silva, P.;
488
Turnbaugh, P. J.; Raskin, I. Dietary polyphenols promote growth of the gut
489
bacterium Akkermansia muciniphila and attenuate high-fat diet-induced
23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
490
metabolic syndrome. Diabetes 2015, 64, 2847-2858.
491
3. Ussar, S.; Griffin, N. W.; Bezy, O.; Fujisaka, S.; Vienberg, S.; Softic, S.; Deng, L.;
492
Bry, L.; Gordon, J. I.; Kahn, C. R. Interactions between gut microbiota, host
493
genetics and diet modulate the predisposition to obesity and metabolic syndrome.
494
Cell Metab. 2015, 22, 516-530.
495
4. Zhang, J.; Wang, O.; Guo, Y.; Wang, T.; Wang, S.; Li, G.; Ji, B.; Deng, Q. Effect
496
of increasing doses of linoleic and α-linolenic acids on high-fructose and
497
high-fat diet induced metabolic syndrome in rats. J. Agric. Food Chem. 2016, 64,
498
762-772.
499
5. Lozupone, C. A.; Stombaugh, J. I.; Gordon, J. I.; Jansson, J. K.; Knight, R.
500
Diversity, stability and resilience of the human gut microbiota. Nature 2012,
501
489, 220-230.
502 503
6. Rosenbaum, M.; Knight, R.; Leibel, R. L. The gut microbiota in human energy homeostasis and obesity. Trends Endocrinol. Metab. 2015, 26, 493-501.
504
7. Sung, M. M.; Kim, T. T.; Denou, E.; Soltys, C. L. M.; Hamza, S. M.; Byrne, N. J.;
505
Masson, G.; Park, H.; Wishart, D. S.; Madsen, K. L.; Schertzer, J. D.; Dyck, J. R.
506
B. Improved glucose homeostasis in obese mice treated with resveratrol is
507
associated with alterations in the gut microbiome. Diabetes 2016, 66, 418-425.
508
8. Martínez, I.; Perdicaro, D. J.; Brown, A. W.; Hammons, S.; Carden, T. J.; Carr, T.;
509
Carrb, T. P.; Eskridgec, K. M.; Waltera, J. Diet-induced alterations of host
510
cholesterol metabolism are likely to affect the gut microbiota composition in
511
hamsters. Appl. Environ. Microbiol. 2013, 79,516-524.
24
ACS Paragon Plus Environment
Page 24 of 46
Page 25 of 46
Journal of Agricultural and Food Chemistry
512 513
9. Bashiardes, S.; Shapiro, H.; Rozin, S.; Shibolet, O.; Elinav, E. Non-alcoholic fatty liver and the gut microbiota. Mol. Metab. 2016, 5, 782-794.
514
10. Lam, Y. Y.; Ha, C. W.; Campbell, C. R.; Mitchell, A. J.; Dinudom, A.; Oscarsson,
515
J.; Cook, D. I.; Hunt, N. H.; Caterson, I. D.; Holmes, A. J.; Storlien, L. H.
516
Increased gut permeability and microbiota change associate with mesenteric fat
517
inflammation and metabolic dysfunction in diet-induced obese mice. PLoS One
518
2011, 7, e34233 (DOI: 10.1371/journal.pone.0034233).
519
11. Dao, M. C.; Everard, A.; Aron-Wisnewsky, J.; Sokolovska, N.; Prifti, E.; Verger,
520
E. O.; Kayser, B. D.; Levenez, F.; Chilloux, J.; Hoyles, L.; MICRO-Obes
521
Consortium; Dumas, M. E.; Rizkalla, S. W.; Doré, J.; Cani, P. D.; Clément, K.
522
Akkermansia muciniphila and improved metabolic health during a dietary
523
intervention in obesity: relationship with gut microbiome richness and ecology.
524
Gut 2016, 65, 426-436.
525
12. Cani, P. D.; Neyrinck, A. M.; Fava, F.; Knauf, C.; Burcelin, R. G.; Tuohy, K. M.;
526
Gibson, G. R.; Delzenne, N. M. Selective increases of bifidobacteria in gut
527
microflora improve high-fat-diet-induced diabetes in mice through a mechanism
528
associated with endotoxaemia. Diabetologia 2007, 50, 2374-2383.
529
13. Wang, J.; Tang, H.; Zhang, C.; Zhao, Y.; Derrien, M.; Rocher, E.; van-Hylckama
530
Vlieg, J. E. T.; Strissel, K.; Zhao, L.; Obin, M.; Shen, J. Modulation of gut
531
microbiota during probiotic-mediated attenuation of metabolic syndrome in high
532
fat diet-fed mice. ISME J. 2015, 9, 1-15.
533
14. Ziętak, M.; Kovatcheva-Datchary, P.; Markiewicz, L. H.; Ståhlman, M.; Kozak,
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
534
L. P.; Bäckhed, F. Altered microbiota contributes to reduced diet-induced obesity
535
upon cold exposure. Cell Metab. 2016, 23, 1216-1223.
536 537 538 539
15. Zhao, L. The gut microbiota and obesity: from correlation to causality. Nat. Rev. Microbiol. 2013, 11, 639-647. 16. Lynch, S. V.; Pedersen, O. The human intestinal microbiome in health and disease. N. Engl. J. Med. 2016, 375, 2369-2379.
540
17. Chang, C. J.; Lin, C. S.; Lu, C. C.; Martel, J.; Ko, Y. F.; Ojcius, D. M.; Tseng, S.
541
F.; Wu, T. R.; Chen, Y. Y. M.; Young, J. D.; Lai, H. C. Ganoderma lucidum
542
reduces obesity in mice by modulating the composition of the gut microbiota.
543
Nat. Commun. 2015, 6, 7489 (DOI: 10.1038/ncomms8489).
544
18. Koh, A.; De Vadder, F.; Kovatcheva-Datchary, P.; Bäckhed, F. From dietary fiber
545
to host physiology: Short-chain fatty acids as key bacterial metabolites. Cell
546
2016, 165, 1332-1345.
547
19. Canfora, E. E.; Jocken, J. W.; Blaak, E. E. Short-chain fatty acids in control of
548
body weight and insulin sensitivity. Nat. Rev. Endocrinol. 2015, 11, 577-591.
549
20. Brooks, L.; Viardot, A.; Tsakmaki, A.; Stolarczyk, E.; Howard, J. K.; Cani, P. D.;
550
Everard, A.; Sleeth, M. L.; Psichas, A.; Anastasovskaj, J.; Bell, J. D.;
551
Bell-Anderson, K.; Mackay, C. R.; Ghatei, M. A.; Bloom, S. R.; Frost, G.;
552
Bewick, G. Fermentable carbohydrate stimulates FFAR2-dependent colonic
553
PYY cell expansion to increase satiety. Mol. Metab. 2017, 6, 48-60.
554
21. Flint, H. J.; Bayer, E. A.; Rincon, M. T.; Lamed, R.; White, B. A. Polysaccharide
555
utilization by gut bacteria: potential for new insights from genomic analysis. Nat.
26
ACS Paragon Plus Environment
Page 26 of 46
Page 27 of 46
Journal of Agricultural and Food Chemistry
556 557 558
Rev. Microbiol., 2008, 6, 121-131. 22. Xu, X.; Xu, P.; Ma, C.; Jian, T.; Zhang, X. Gut microbiota, host health, and polysaccharides. Biotechnol. Adv. 2013, 31, 318-337.
559
23. Cockburn, D. W.; Koropatkin, N. M. Polysaccharide degradation by the intestinal
560
microbiota and its influence on human health and disease. J. Mol. Biol. 2016,
561
428, 3230-3252.
562
24. Peng, C. Y.; Cai, H. M.; Zhu, X. H.; Li, D. X.; Yang, Y. Q.; Hou, R. Y.; Wan, X.
563
C. Analysis of naturally occurring fluoride in commercial teas and estimation of
564
its daily intake through tea consumption. J. Food Sci. 2016, 81, 235-239.
565
25. Chen, G.; Xie, M.; Wan, P.; Chen, D.; Ye, H.; Chen, L.; Zeng, X. X.; Liu, Z.
566
Digestion under saliva, simulated gastric and small intestinal conditions and
567
fermentation in vitro by human intestinal microbiota of polysaccharides from
568
Fuzhuan brick tea. Food Chem. 2018, 244, 331-339.
569
26. Tian, Y. Z.; Liu, X.; Liu, W.; Wang, W. Y.; Long, Y. H.; Zhang, L.; Xu, Y.; Bao,
570
G. H.; Wan, X. C.; Ling, T. J. A new anti-proliferative acylated flavonol
571
glycoside from Fuzhuan brick-tea. Nat. Prod. Res. 2016, 30, 2637-2641.
572
27. Foster, M. T.; Gentile, C. L.; Cox-York, K.; Wei, Y.; Wang, D.; Estrada, A. L.;
573
Reese, L.; Miller, T.; Pagliassotti, M. J.; Weir, T. L. Fuzhuan tea consumption
574
imparts hepatoprotective effects and alters intestinal microbiota in high saturated
575
fat diet-fed rats. Mol. Nutr. Food Res. 2016, 60, 1213-1220.
576
28. Fu, D.; Ryan, E. P.; Huang, J.; Liu, Z.; Weir, T. L.; Snook, R. L.; Ryan, T. P.
577
Fermented Camellia sinensis, Fu Zhuan Tea, regulates hyperlipidemia and
27
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 28 of 46
578
transcription factors involved in lipid catabolism. Food Res. Int. 2011, 44,
579
2999-3005.
580
29. Chen, G.; Xie, M.; Dai, Z.; Wan, P.; Ye, H.; Zeng, X. X.; Sun, Y. Kudingcha and
581
Fuzhuan brick tea prevent obesity and modulate gut microbiota in high-fat diet
582
fed
583
10.1002/mnfr.201700485).
mice.
Mol.
Nutr.
Food
Res.
2018,
62,
1700485
(DOI:
584
30. Chen, G.; Yuan, Q.; Saeeduddin, M.; Ou, S.; Zeng, X.; Ye, H. Recent advances in
585
tea polysaccharides: Extraction, purification, physicochemical characterization
586
and bioactivities. Carbohydr. Polym. 2016, 153, 663-678.
587 588 589 590
31. Magoč, T.; Salzberg, S. L. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011, 27, 2957-2963. 32. Edgar, R. C. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods, 2013, 10, 996-998.
591
33. Cole, J. R.; Wang, Q.; Cardenas, E.; Fish, J.; Chai, B.; Farris, R. J.;
592
Kulam-Syed-Mohideen, A. S.; McGarrell, D. M.; Marsh, T.; Garrity, G. M.;
593
Tiedje, J. M. The Ribosomal Database Project: improved alignments and new
594
tools for rRNA analysis. Nucleic Acids Res. 2009, 37, 141-145.
595
34. Schloss, P. D.; Westcott, S. L.; Ryabin, T.; Hall, J. R.; Hartmann, M.; Hollister, E.
596
B.; Lesniewski, R. A.; Oakley, B. B.; Parks, D. H.; Robinson, C. J.; Sahl, J. W.;
597
Stres, B.; Thallinger, G. G.; Van Horn, D. J.; Webe, C. F. Introducing mothur:
598
open-source,
599
describing and comparing microbial communities. Appl. Environ. Microbiol.
platform-independent,
community-supported
28
ACS Paragon Plus Environment
software
for
Page 29 of 46
Journal of Agricultural and Food Chemistry
600
2009, 75, 7537-7541.
601
35. Yang, C. S.; Zhang, J.; Zhang, L.; Huang, J.; Wang, Y. Mechanisms of body
602
weight reduction and metabolic syndrome alleviation by tea. Mol. Nutr. Food
603
Res. 2016, 60, 160-174.
604
36. Huang, J.; Zhang, Y.; Zhou, Y.; Zhang, Z.; Xie, Z.; Zhang, J.; Wan. X. Green tea
605
polyphenols alleviate obesity in broiler chickens through the regulation of
606
lipid-metabolism-related genes and transcription factor expression. J. Agric.
607
Food Chem. 2013, 61, 8565-8572.
608
37. den Besten, G.; Bleeker, A.; Gerding, A.; van Eunen, K.; Havinga, R.; van Dijk,
609
T. H.; Oosterveer, M. H.; Jonker, J. W.; Groen, A. K.; Reijngoud, D. J.; Bakker,
610
B. M. Short-chain fatty acids protect against high-fat diet-induced obesity via a
611
PPARγ-dependent switch from lipogenesis to fat oxidation. Diabetes 2015, 64,
612
2398-2408.
613 614
38. Tremaroli, V.; Bäckhed, F. Functional interactions between the gut microbiota and host metabolism. Nature 2012, 489, 242-249.
615
39. Gérard, P. Gut microbiota and obesity. Cell. Mol. Life Sci. 2016, 73, 147-162.
616
40. Beaumont, M.; Goodrich, J. K.; Jackson, M. A.; Yet, I.; Davenport, E. R.;
617
Vieira-Silva, S.; Debelius, J.; Pallister, T.; Mangino, M.; Raes, J.; Knight, R.;
618
Clark, A. G.; Ley, R. E.; Spector, T. D.; Bell, J. T. Heritable components of the
619
human fecal microbiome are associated with visceral fat. Genome Biol. 2016, 17,
620
189 (DOI: 10.1186/s13059-016-1052-7).
621
41. Yang, J.; Bindels, L. B.; Munoz, R. R. S.; Martínez, I.; Walter, J.; Ramer-Tait, A.
29
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 30 of 46
622
E.; Rose, D. J. Disparate metabolic responses in mice fed a high-fat diet
623
supplemented
624
polysaccharides are linked to changes in the gut microbiota. PLoS One 2016, 11,
625
e0146144 (DOI: 10.1371/journal.pone.0146144).
626 627
with
maize-derived
non-digestible
feruloylated
oligo-and
42. O'neill, S.; O'driscoll, L. Metabolic syndrome: a closer look at the growing epidemic and its associated pathologies. Obes. Rev. 2015, 16, 1-12.
628
43. Zheng, W. J.; Wan, X. C.; Bao, G. H. Brick dark tea: a review of the manufacture,
629
chemical constituents and bioconversion of the major chemical components
630
during fermentation. Phytochem. Rev. 2015, 14, 499-523.
631
44. Liu, Z.; Chen, Z.; Guo, H.; He, D.; Zhao, H.; Wang, Z.; Zhang, W.; Liao, L.;
632
Zhang, C.; Ni, L. The modulatory effect of infusions of green tea, oolong tea,
633
and black tea on gut microbiota in high-fat-induced obese mice. Food Funct.
634
2016, 7, 4869-4879.
635
45. Clemente, J. C.; Ursell, L. K.; Parfrey, L. W.; Knight, R. The impact of the gut
636
microbiota on human health: an integrative view. Cell 2012, 148, 1258-1270.
637
46. Wu, T.; Guo, Y.; Liu, R.; Wang, K.; Zhang, M. Black tea polyphenols and
638
polysaccharides improve body composition, increase fecal fatty acid, and
639
regulate fat metabolism in high-fat diet-induced obese rats. Food Funct. 2016, 7,
640
2469-2478.
641
47. Xu, Y.; Zhang, M.; Wu, T.; Dai, S.; Xu, J.; Zhou, Z. The anti-obesity effect of
642
green tea polysaccharides, polyphenols and caffeine in rats fed with a high-fat
643
diet. Food Funct. 2015, 6, 297-304.
30
ACS Paragon Plus Environment
Page 31 of 46
Journal of Agricultural and Food Chemistry
644
48. Li, D.; Cheng, M.; Niu, Y.; Chi, X.; Liu, X.; Fan, J.; Chang, Y. S.; Yang, W.
645
Identification of a novel human long non-coding RNA that regulates hepatic
646
lipid metabolism by inhibiting SREBP-1c. Int. J. Biol. Sci. 2017, 13, 349-357.
647
49. Suk, S.; Kwon, G. T.; Lee, E.; Jang, W. J.; Yang, H.; Kim, J. H.; Thimmegowda,
648
N. R.; Chung, M. Y.; Kwon, J. Y.; Yang, S. H.; Kim, J. K.; Park, J. H. Y.; Lee,
649
K. W. Gingerenone A, a polyphenol present in ginger, suppresses obesity and
650
adipose tissue inflammation in high-fat diet-fed mice. Mol. Nutr. Food Res.
651
2017, 61, 1700139 (DOI: 10.1002/mnfr.201700139).
652
50. Ann, J. Y.; Eo, H.; Lim, Y. Mulberry leaves (Morus alba L.) ameliorate
653
obesity-induced hepatic lipogenesis, fibrosis, and oxidative stress in high-fat
654
diet-fed mice. Genes Nutr. 2015, 10, 46 (DOI: 10.1007/s12263-015-0495-x).
655
51. Kim, D. H.; Jeong, D.; Kang, I. B.; Kim, H.; Song, K. Y.; Seo, K. H. Dual
656
function of Lactobacillus kefiri DH5 in preventing high-fat-diet-induced obesity:
657
direct reduction of cholesterol and upregulation of PPARα in adipose tissue.
658
Mol. Nutr. Food Res. 2017, 61, 1700252 (DOI: 10.1002/mnfr.201700252).
659
52. Cai, X.; Fang, C.; Hayashi, S.; Hao, S.; Zhao, M.; Tsutsui, H.; Nishiguchi, S.;
660
Sheng, J. Pu-erh tea extract ameliorates high-fat diet-induced nonalcoholic
661
steatohepatitis and insulin resistance by modulating hepatic IL-6/STAT3
662
signaling in mice. J. Gastroenterol. 2016, 51, 819-829.
663
53. Murphy, E. F.; Cotter, P. D.; Hogan, A.; O'sullivan, O.; Joyce, A.; Fouhy, F.;
664
Clarke, S. F.; Marques, T. M.; O'Toole, P. W.; Stanton, C.; Quigley, E. M. M.;
665
Daly, C.; Ross, P. R.; O'Doherty, R. M.; Shanahan, F. Divergent metabolic
31
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 32 of 46
666
outcomes arising from targeted manipulation of the gut microbiota in
667
diet-induced obesity. Gut 2013, 62, 220-226
668
54. Daniel, H.; Gholami, A. M.; Berry, D.; Desmarchelier, C.; Hahne, H.; Loh, G.;
669
Mondot, S.; Lepage, P.; Rothballer, M.; Walker, A.; Böhm, C.; Wenning, M.;
670
Wagner, M.; Blaut, M.; Schmitt-Kopplin, P.; Kuster, B.; Haller, D.; Clavel, T.
671
High-fat diet alters gut microbiota physiology in mice. ISME J. 2014, 8,
672
295-308.
673 674
55. Doré, J.; Blottiere, H. The influence of diet on the gut microbiota and its consequences for health. Curr. Opin. Biotechnol. 2015, 32, 195-199.
675
56. Li, J.; Zhao, F.; Wang, Y.; Chen, J.; Tao, J.; Tian, G.; Wu, S.; Liu, W.; Cui, Q.;
676
Geng, B.; Zhang, W.; Weldon, R.; Auguste, K.; Yang, L.; Liu, X.; Chen, L.;
677
Yang, X.; Zhu, B.; Cai, J. Gut microbiota dysbiosis contributes to the
678
development
679
10.1186/s40168-016-0222-x).
of
hypertension.
Microbiome
2017,
5,
14
(DOI:
680
57. Xu, J.; Lian, F.; Zhao, L.; Zhao, Y.; Chen, X.; Zhang, X.; Guo, Y.; Zhang, C.;
681
Zhou, Q.; Xue, Z.; Pang, X.; Zhao, L.; Tong, X. Structural modulation of gut
682
microbiota during alleviation of type 2 diabetes with a Chinese herbal formula.
683
ISME J. 2015, 9, 552-562.
684
58. Turnbaugh, P. J.; Magrini, V.; Gordon, J. I.; Mardis, E. R.; Ley, R. E.; Mahowald,
685
M. A. An obesity-associated gut microbiome with increased capacity for energy
686
harvest. Nature 2006, 444, 1027-1031.
687
59. Turnbaugh, P. J.; Hamady, M.; Yatsunenko, T.; Cantarel, B. L.; Duncan, A.; Ley,
32
ACS Paragon Plus Environment
Page 33 of 46
Journal of Agricultural and Food Chemistry
688
R. E.; Sogin, M. L.; Jones,W. J.; Roe, B. A.; Affourtit, J. P.; Egholm, M.;
689
Henrissat, B.; Heath, A. C.; Knight, R.; Gordon, J. I. A core microbiome in obese
690
and lean twins. Nature 2008, 457, 480-484.
691
60. Kaakoush, N. O. Insights into the role of Erysipelotrichaceae in the human host.
692
Front. Cell. Infect. Microbiol. 2015, 5, 84 (DOI: 10.3389/fcimb.2015.00084).
693
61. Etxeberria, U.; Arias, N.; Boqué, N.; Macarulla, M. T.; Portillo, M. P.; Martínez,
694
J. A.; Milagro, F. I. Reshaping faecal gut microbiota composition by the intake
695
of trans-resveratrol and quercetin in high-fat sucrose diet-fed rats. J. Nutr.
696
Biochem. 2015, 26, 651-660.
697
62. Zhang, H.; DiBaise, J. K.; Zuccolo, A.; Kudrna, D.; Braidotti, M.; Yu, Y.;
698
Parameswaran,
699
Krajmalnik-Brown, R. Human gut microbiota in obesity and after gastric bypass.
700
Proc. Natl. Acad. Sci. USA. 2009, 106, 2365-2370.
701
P.;
Crowell,
M.
D.;
Wing,
R.;
Rittmann,
B.
E.;
63. Zeng, H.; Ishaq, S. L.; Zhao, F. Q.; Wright, A. D. G. Colonic inflammation
702
accompanies
an
increase
of
β-catenin
signaling
and
703
Lachnospiraceae/Streptococcaceae bacteria in the hind gut of high-fat diet-fed
704
mice. J. Nutr. Biochem. 2016, 35, 30-36.
705
64. Martínez, I.; Wallace, G.; Zhang, C.; Legge, R.; Benson, A. K.; Carr, T. P.;
706
Moriyama, E. N.; Walter, J. Diet-induced metabolic improvements in a hamster
707
model of hypercholesterolemia are strongly linked to alterations of the gut
708
microbiota. Appl. Environ. Microbiol. 2009, 75, 4175-4184.
709
65. Claus, S. P.; Ellero, S. L.; Berger, B.; Krause, L.; Bruttin, A.; Molina, J.; Paris, A.;
33
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
710
Want, E. J.; de Waziers, I.; Cloarec, O.; Richards, S. E.; Wang, Y.; Dumas, M.
711
E.; Ross, A.; Rezzi, S.; Kochhar, S.; Bladeren, P. V.; Lindon, J. C.; Holmes, E.;
712
Nicholson, J. K. Colonization-induced host-gut microbial metabolic interaction.
713
MBio 2011, 2, e00271-10 (DOI:10.1128/mBio.00271-10).
714
66. Neyrinck, A. M.; Van Hée, V. F.; Bindels, L. B.; De Backer, F.; Cani, P. D.;
715
Delzenne, N. M. Polyphenol-rich extract of pomegranate peel alleviates tissue
716
inflammation and hypercholesterolaemia in high-fat diet-induced obese mice:
717
potential implication of the gut microbiota. Br. J. Nutr. 2013, 109, 802-809.
718
67. Zhang, C.; Li, S.; Yang, L.; Huang, P., Li, W.; Wang, S.; Zhao, G.; Zhang, M.;
719
Pang, X.; Yan, Z.; Liu, Y.; Zhao, L. Structural modulation of gut microbiota in
720
life-long calorie-restricted mice. Nat. Commun. 2013, 16, 2163 (DOI:
721
10.1038/ncomms3163).
722
68. Le Roy, T.; Llopis, M.; Lepage, P.; Bruneau, A.; Rabot, S.; Bevilacqua, C.;
723
Martin, P.; Philippe, C.; Walker, F.; Bado, A.; Perlemuter, G.; Cassard-Doulcier,
724
A. M.; Gérard, P. Intestinal microbiota determines development of non-alcoholic
725
fatty liver disease in mice. Gut 2013, 62, 1787-1794.
726
69. Cani, P. D.; Bibiloni, R.; Knauf, C.; Waget, A.; Neyrinck, A. M.; Delzenne, N.
727
M.; Burcelin, R. Pterostilbene-induced changes in gut microbiota composition in
728
relation to obesity. Mol. Nutr. Food Res. 2017, 61, 1500906 (DOI:
729
10.1002/mnfr.201500906).
730
70. Arnoldussen, I.; Wiesmann, M.; Pelgrim, C.; Wielemaker, E.; van Duyvenvoorde,
731
W.; Amaral-Santos, P.; Verschuren, L.; Keijser, B. J. F.; Heerschap, A.;
34
ACS Paragon Plus Environment
Page 34 of 46
Page 35 of 46
Journal of Agricultural and Food Chemistry
732
Kleemann, R.; Wielinga, P. Y.; Kiliaan, A. J. Butyrate restores HFD-induced
733
adaptations in brain function and metabolism in mid-adult obese mice. Int. J.
734
Obes. 2017, 41, 935-944.
735
71. Ruiz, A.; Cerdó, T.; Jáuregui, R.; Pieper, D. H.; Marcos, A.; Clemente, A.; García,
736
F.; Margolles, A.; Ferrer, M.; Campoy, C.; Suárez, A. One-year calorie
737
restriction impacts gut microbial composition but not its metabolic performance
738
in obese adolescents. Environ. Microbiol. 2017, 19, 1536-1551.
739
72. Selma, M. V.; Romo-Vaquero, M.; García-Villalba, R.; González-Sarrías, A.;
740
Tomás-Barberán, F. A.; Espín, J. C. The human gut microbial ecology associated
741
with overweight and obesity determines ellagic acid metabolism. Food Funct.
742
2016, 7, 1769-1774.
743
73. Langley, G.; Hao, Y.; Pondo, T.; Miller, L.; Petit, S.; Thomas, A.; Lindegren, M.
744
L.; Farley, M. M.; Dumyati, G.; Como-Sabetti, K.; Harrison, L. H.; Baumbach,
745
J.; Watt, J.; Beneden, C. V. The impact of obesity and diabetes on the risk of
746
disease and death due to invasive group A Streptococcus infections in adults.
747
Clin. Infect. Dis. 2016, 62, 845-852.
35
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 36 of 46
Page 37 of 46
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 38 of 46
Page 39 of 46
Journal of Agricultural and Food Chemistry
Figure 1.
799 39
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
800 801
Figure 2.
40
ACS Paragon Plus Environment
Page 40 of 46
Page 41 of 46
Journal of Agricultural and Food Chemistry
802 803
Figure 3.
41
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
804 805
Figure 4.
42
ACS Paragon Plus Environment
Page 42 of 46
Page 43 of 46
Journal of Agricultural and Food Chemistry
806 807
Figure 5.
43
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
808 809
Figure 6.
44
ACS Paragon Plus Environment
Page 44 of 46
Page 45 of 46
Journal of Agricultural and Food Chemistry
810 811
Figure 7.
45
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
812
Graphic for table of contents
813
46
ACS Paragon Plus Environment
Page 46 of 46
