(Lard) diet-induced non-alcoholic fatty liver disease in mice

3School of Aerospace Medicine, Fourth Military Medical University, Xi'an 710032,. 9 ... development of nonalcoholic fatty liver disease (NAFLD) 2. ...
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Bioactive Constituents, Metabolites, and Functions

Pectin alleviates high fat (Lard) diet-induced nonalcoholic fatty liver disease in mice: possible role of shortchain fatty acids and gut microbiota regulated by pectin Wenfeng Li, Kun Zhang, and Hongyan Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02979 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Journal of Agricultural and Food Chemistry

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Pectin alleviates high fat (Lard) diet-induced non-alcoholic fatty liver disease in

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mice: possible role of short-chain fatty acids and gut microbiota regulated by

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pectin

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Wenfeng Li1,*, Kun Zhang2, and Hongyan Yang3,*

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1

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408100, China

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2

School of Clinical Medicine, Jining Medical University, Jining 272067, China

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School of Aerospace Medicine, Fourth Military Medical University, Xi’an 710032,

School of Life Science and Biotechnology, Yangtze Normal University, Chongqing

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China

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*Corresponding authors:

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W.F. Li: [email protected]; +86 23 72792193 (Fax); No. 16, Juxian road, Fuling

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district, Chongqing, China.

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H.Y. Yang: [email protected]; +86 29 83246270 (Fax); No. 169, Changle-West

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road, Xi'an, Shaanxi, China.

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ABSTRACT: Consumption of pectin contributes to changes in the gut microbiota and

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the metabolism of short-chain fatty acids (SCFAs). We aimed to investigate the effects

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of and mechanism by which pectin prevented nonalcoholic fatty liver disease (NAFLD)

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in mice that were fed a high-fat diet containing 30% lard (HF). HF-fed mice that orally

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ingested pectin for eight weeks exhibited improvements in lipid metabolism, and

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decreased oxidative stress and inflammation through a mechanism regulated by the

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mitogen-activated protein kinase pathway. Pectin dose-dependently generated rise in

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acetic acid (from 566.4±26.6 to 694.6±35.9 μmol/mL, p 0.01

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was removed from the candidate pool of FA markers (Figure 4G). As shown in Figure

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4H and I, pectin intake significantly decrease the palmitic acid level in HF-fed mice,

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whereas oleic acid levels were not obviously reduced in mice in the HF-4% pectin

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group. Finally, the sPLS analysis was performed to reveal the correlation between the

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FA candidates and biochemical parameters (Figure 4J). A strong correlation was

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observed between palmitic acid levels and biochemical parameters, indicating that

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palmitic acid was a marker for the effects of pectin on HF-induced NAFLD.

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Pectin Affected the Short Chain Fatty Acid Profile. Pectin consumption is

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associated with changes in levels of short chain fatty acids, including acetic acid,

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propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid 9. As shown

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in the PCA score plot, the major modulator of SCFA metabolism is pectin consumption

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(Figure 5A). HF feeding prominently affected the cecal isovaleric acid content, whereas

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the levels of other SCFAs and the total SCFA content were unaffected (Figure 5B-H).

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Higher total SCFA, acetic acid and propionic acid levels were observed in the

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pectin-treated mice than in the HF-fed mice (Figure 5B-D). Conversely, pectin

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administration reduced the cecal isobutyric acid, isovaleric acid and valeric acid

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contents in HF-fed mice (Figure 5E, G, and H). The cecal butyric acid content was not

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affected by pectin consumption (Figure 5F). The sPLS analysis revealed a strong

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positive correlation between antioxidant and acetic acid levels and between antioxidant

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and propionic acid levels (Figure 5I). In addition, negative correlations were observed

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between parameters of de novo lipogenesis or oxidative stress and acetic acid or

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propionic acid levels, respectively (Figure 5I). Based on these findings, acetic acid and

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propionic acid played critical roles in preventing NAFLD by suppressing oxidative

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stress and fatty acid synthesis.

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Pectin Changes the Abundance of Specific Gut Bacteria. SCFAs are

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produced by different microbes 9, 22. Because we revealed a distinct change in the SCFA

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profile upon pectin intake, we further assessed whether this finding might be associated

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with changes in the gut microbiota composition. A Venn diagram displaying the

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overlapping OUT data from the four groups was developed to better understand their

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mutual microbes (Figure 6A). Four hundred fifty-one OTUs were shared by all samples.

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Moreover, 519 OTUs identified in the pectin-treated mice were not detected in the

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HF-fed mice. The nonmetric multidimensional scaling analysis (NMDS) showed an

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obvious difference in the composition of the gut microbiome between mice in the

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control, HF, HF-4% pectin and HF-8% pectin groups (Figure 6B). The first dimension

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separated control and HF mice from HF-4% pectin and HF-8% pectin mice, irrespective

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of the high fat diet, suggesting that changes may be induced by pectin intake (Figure

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6B). Additionally, the second dimension separated the control and HF-8% pectin groups

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from the HF and HF-4% pectin groups (Figure 6B). The most prevalent phyla in all

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samples were Firmicutes and Bacteroidetes (Figure S3A). Higher levels of Firmicutes

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were detected in the gut digesta from pectin-fed mice than in control- and HF-fed mice.

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At the class level (Figure S3B), Bacteroidia and Bacilli were influenced by pectin

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ingestion. In the colonic digesta, the most abundant order in all groups was

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Bacteroidales (Figure S3C). Long-term ingestion of HF decreased the proportion of

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Bacteroidales; however, pectin supplementation increased its proportion in the colonic

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contents

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Porphyromonadaceae and Ruminococcaceae were significantly increased in the colonic

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digesta of HF-fed mice (Figure S3D). Lower abundances of microbes in the families

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Lachnospiraceae and Lactobacillaceae were observed in the colonic digesta from

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pectin-fed mice than in HF-fed mice. Pectin consumption tended to increase the relative

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abundance of the genera Barnesiella, Anaerobacterium, Bacteroides, Parabacteroides

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and Clostridium IV, and decrease the relative abundance of the genera Lactobacillus,

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Helicobacter, Clostridium XIVa and Alistipes in HF-fed mice (Figure 6C).

of

HF-fed

mice.

The

abundance

of

microbes

in

the

families

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We cross-selected the results of the linear discriminant analysis effect size (LEfSe,

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Figure 6D) and the results of the sPLS analysis of SCFA and gut microbe datasets

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(Figure 6E) to explore which microbes were associated with the pectin-induced changes

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in SCFA metabolism in HF-fed mice. Although 16 genera were related to the regulation

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of SCFA metabolism in the gut of mice co-treated with HF and pectin (Figure 6E), 6

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also had substantial influences on microorganisms associated with the physiology of

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each group (Figure 6D; i.e., Bifidobacterium, Olsenella, Bacteroides, Parabacteroides,

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Lactobacillus, Marvinbryantia and Allobaculum). The relative abundance of

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Lactobacillus was increased by approximately 19-fold in response to HF feeding,

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whereas the abundance of other genera was not significantly altered (Figures 6F and S3).

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Strikingly, the pectin treatment dose-dependently reduced the HF-induced increase in

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the relative abundance of Lactobacillus. Moreover, the relative abundances of

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Bacteroides, Parabacteroides, Olsenella, and Bifidobacterium were significantly

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elevated by approximately 43-fold, 1379-fold, 466-fold and 74-fold, respectively,

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following the consumption of high-dose pectin (Figure 6F), and these effects were also

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dose-dependent. The administration of 4% pectin, but not the high dose of pectin, also

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affected the relative abundance of Allobaculum. Furthermore, the relative abundance of

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Marvinbryantia was not affected by the administration of pectin to HF-fed mice (Figure

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S3). Thus, pectin-induced changes in the abundances of Bifidobacterium, Olsenella,

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Bacteroides, Parabacteroides and Lactobacillus contributed to the mechanism

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regulating the short chain fatty acid profile in the gut of HF-fed mice.

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DISCUSSION

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Nonalcoholic fatty liver disease has emerged as a critical health issue in humans 3.

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The association between NAFLD and obesity led to the development of a high fat diet

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(HF) model that matches modern Western diets

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consistently been shown to cause NAFLD in mice, as evidenced by hepatic steatosis,

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increased serum transaminase levels, oxidative stress and abnormal proinflammatory

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cytokine production, and thus, HF has been applied to establish animal models of

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NAFLD as a reliable tool

23

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. Long-term intake of HF has

. In the current study, C57BL/6 mice fed a HF including

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30% lard for continuous 12 weeks exhibited NAFLD, which have the hallmark features

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observed in human patients with NAFLD, including obesity, hepatic steatosis and liver

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damage

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widely recognized to comprise a two-hit model 4. Triglyceride accumulation in

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hepatocytes is the first “hit” that occurs as a result of alterations in fatty acid uptake,

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synthesis, or oxidation, as well as defects in fatty acid transporters 4. Pectin ingestion

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markedly inhibited abnormal fat accumulation in the enterocoelia and liver of HF-fed

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mice. ChREBP was identified as a key regulator of lipogenic genes, including FAS and

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ACC, and shown to be associated with increased de novo lipogenesis in NAFLD

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Pectin significantly reduced hepatic ChREBP, FAS and ACC levels in the present study,

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and these decreases primarily affected palmitic acid metabolism in the liver of HF-fed

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mice. Thus, pectin likely ameliorates hepatic steatosis through pathways involving the

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down-regulation of key lipogenesis-related proteins. Pectin is not digested in the

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gastrointestinal tract

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substantial regulatory effect on fat metabolism in the bowel in vivo remains unclear. As

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shown in the study by Tian and co-workers, pectin supplementation modulates the

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composition of the microbiota and SCFA production 9. Among SCFAs, proionate was by

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far the most abundant of the intestinal SCFAs, and it was suggested to inhibit

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lipogenesis by acting on the transcription of several rate-limiting enzymes involved in

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de novo lipogenesis, namely ACC and FAS

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contents negatively correlated with hepatic ChREBP, FAS and ACC levels, which were

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positively correlated with the hepatic palmitic acid level. Consistent with findings from

23, 24

. NAFLD is a continuous spectrum of diseases

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23

, and its pathogenesis is

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.

. Accordingly, the mechanism by which pectin exerted a

11, 27

. The acetic acid and propionic acid

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a previous study

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propionic acid concentrations increased in the gut in the present study. Therefore,

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increased acetic acid and propionic acid contents in the gut have important roles in

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inhibiting hepatic triglyceride accumulation induced by pectin consumption.

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, the hepatic palmitic acid level decreased as acetic acid and

The “second hit” of NAFLD is postulated to be associated with lipid peroxidation 29

and oxidative stress

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plays a major role in fatty acid β-oxidation and the regulation of oxidative stress

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Pectin inhibited PPARα activation and the formation of oxidative markers (MDA) in the

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liver of HF-fed mice, suggesting that pectin prevented lipid peroxidation and oxidative

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stress. Nrf2 is a transcription factor with a central role in the protective molecular

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response to oxidative stress in most cells and is activated by ROS 33. In the current study,

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HF significantly increased the total Nrf2 level in the liver, suggesting an overproduction

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of ROS in HF-fed mice. The ROS-activated Nrf2 translocates to the nucleus and

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interacts with antioxidant response elements (AREs), which mediate the transcription of

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various antioxidants, including SOD and GSH-Px

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SOD activities in the liver in the present study, which correlates with Nrf2 activation.

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Importantly, Nrf2 is also modulated by some signaling kinases, such as ERK, JNK and

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p38 MAPK

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responsible for protective responses and stress-dependent apoptosis reactions

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Interestingly, pectin attenuated HF-induced JNK and ERK activation, implying that

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pectin might activate Nrf2 in a MAPK-independent manner and that these changes

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might contribute to the strengthening of antioxidant activity. Moreover, phosphorylated

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. Palmitic acid activates the nuclear receptor PPAR-α

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389

33

, which 31, 32

.

. Pectin increased the GSH-Px and

, which are key mediators of stress signals and seem to be mainly

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.

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MAPK activates NF-κB to regulate the production of proinflammatory cytokines such

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as TNF-α 35. HF consumption substantially increased hepatic NF-κB and TNF-α levels,

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which reflect liver inflammation. In contrast, the pectin treatment markedly reduced the

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levels of NF-κB and TNF-α in the liver of HF-fed mice, clearly indicating that pectin

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may effectively protect hepatocytes against the inflammatory effects of HF. SCFAs have

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been shown to inhibit oxidative stress and proinflammatory cytokine production

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According to Kobayashi and colleagues, short chain fatty acids, particularly propionate,

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decrease TNF-α-induced MCP-1 expression by suppressing the phosphorylation of p38

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and JNK 37. Moreover, butyrate directly inhibits the activation of the transcription factor

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NF-κB

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acid levels, propionic acid levels and MAPK or NF-κB activity, and, to a lesser extent,

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between butyric acid levels and MAPK or NF-κB activity. Although these findings must

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be verified in further investigations, pectin consumption substantially increased the

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bacterial generation of SCFAs in the gut, thus largely contributing to the prevention of

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oxidative stress and inflammation by regulating the MAPK pathway. Collectively,

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pectin inhibited the first and second “hits” of NAFLD was related to the regulation of

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the MAPK signaling pathway and SCFA metabolism in HF-fed mice. Additionally, the

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analyses of transaminase (AST and ALT) levels and histological observations confirmed

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that pectin effectively protected against HF-induced liver injury.

12, 38

36

.

. In our study, strong negative correlations were observed between acetic

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Consistent with the aforementioned discussion, SCFAs exerted a positive effect on

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preventing HF-induced NAFLD in pectin-treated mice. SCFAs are produced in the large

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intestine by the bacterial fermentation of plant-derived polysaccharides such as cellulose

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and pectin, which are not digested by host enzymes

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the acetate and propionate, whereas Firmicutes, which tend to utilize polysaccharides

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less efficiently, are believed to be the primary producers of butyrate 12. In present study,

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Bacteroides, a genus in the family Bacteroidetes, was related to increases in acetic acid

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and propionic acid levels in the gut, consistent with previous findings 39, 40. According to

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Henning and colleagues, the relative proportion of Parabacteroides significantly

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correlates with weight loss, fat accumulation and the intestinal formation of SCFA in

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mice

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Bacteroides, was significantly increased upon pectin feeding, thereby increasing the

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generation of acetic acid and propionic acid. Interestingly, this study is the first to reveal

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that the intestinal formation of acetic acid and propionic acid are associated with an

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increased abundance of Olsenella in pectin-fed mice. Although butyric acid levels were

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not affected significantly in this study, elevated levels of enteral butyric acid were

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slightly associated with the increase in the abundances of Firmicutes and Allobaculum

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in pectin-treated mice, which could improve host metabolism to resist HF-induced

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NAFLD 42. Conversely, pectin consumption decreased the abundance of Lactobacillus,

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suggesting that Lactobacillus was irrelevant to the prevention of NAFLD in pectin-fed

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mice. As shown in the study by Avila-Nava and co-workers, dietary fiber consumption

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is associated with an increased abundance of Bifidobacterium

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significantly increased the abundance of Bifidobacterium to a greater extent than the HF

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in the present study, indicating that pectin exerts prebiotic effects. Notably, most clinical

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investigations recommend the use of prebiotics/probiotics by patients with different

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. Bacteroidetes provide most of

. The abundance of another genus (Parabacteroides), which also belongs to

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. Pectin consumption

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stages of NAFLD as an adjunct to standard treatment regimens because they decrease

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manifestations of low-grade systemic inflammatory responses and improve liver

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aminotransferase levels in patients with NAFLD

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with a more balanced secretion of gastrointestinal hormones, namely, a low level of

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conversion of non-digestible carbohydrates (fibers) into short-chain fatty acids and

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thereby reducing the risk of NAFLD progression

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reports, these effects of probiotics increase insulin sensitivity and reduce autoimmune

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response

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flora of mice that were subjected to a high-fat diet, leads to an increase of

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glucose-stimulated insulin secretion, ghrelin secretion, hyperphagia and obesity

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seems contradictory that both HF as risk factor and pectin as a protective factor

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encouraged an increase in the acetic level. Lozupone and colleagues reported that the

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functional gene profiles are quite similar, despite the highly disparate compositions of

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gut microbiota in different individuals 47. Thus, the increase in the acetic level of HF-fed

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and pectin-treated mice might be due to different gut microorganisms, which displayed

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a converse health influence in vivo. Although the therapeutic use of prebiotics and

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probiotics for NAFLD is not supported by high-quality clinical studies

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the present study suggest that pectin is a potential prebiotic that might prevent

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HF-induced NAFLD by modulating the gut microbial composition and production of

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short-chain fatty acids in the gut.

44, 45

44

. These changes may be associated

44

. However, according to some

. For example, enhanced generation of acetic acid by modified intestinal

44

46

. It

, results from

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In conclusion, pectin prevents nonalcoholic fatty liver disease in mice fed a high

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fat diet enriched in lard. More importantly, mechanisms by which pectin regulates lipid

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metabolism, anti-oxidants, inflammation, the generation of short-chain fatty acids and

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the gut microbiota play important roles in protecting the liver. All these findings may

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support the hypothesis that non-digestible pectin displays hepatoprotective activity in

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vivo and provide a scientific basis for the application of pectin for beneficial effects on

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health.

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ASSOCIATED CONTENT

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Supporting Information

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Supplementary Figure 1: Fatty acid composition of lard. Supplementary Figure 2:

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The effects of pectin on body form (A), epididymal fat (B and C), liver weight (D and E)

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and levels of two key proteins (F and G) involved in lipogenesis. Supplementary Figure

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3: Bacterial community at the phylum (A), class (B), order (C) and family (D) levels in

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the colon of mice. Supplementary Figure 4: The effects of different diets on the relative

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abundance of Marvinbryantia in the mouse colon.

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Supplementary method: Materials. Short-chain fatty acid (SCFA) measurements.

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Histopathological observation.

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ACKNOWLEDGEMENTS

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This study was supported by the Plan for Supporting the Development of Youth Scientific Research Talent of Yangtze Normal University.

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Figure captions

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Figure 1 Effect of pectin on body weight and lipid metabolism in mice fed a high fat

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diet for consecutive 12 weeks. (A) Body weight evolution; (B) BMI = body weight (g) /

650

(body length (cm))2; (C) epididymal fat weight; (D) fat index = epididymal fat weight (g)

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/ body weight (g); and (E) food intake. (F) Serum total glycerin; (G) hepatic total

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glycerin; (H) serum total cholesterol; (I) hepatic total cholesterol; (J) serum low-density

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lipoprotein cholesterol; (K) serum high-density lipoprotein cholesterol; (L) hepatic

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non-esterified fatty acid; (M) hepatic acetyl-coenzyme A carboxylase; and (N) hepatic

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fatty acid synthase levels. (O) Representative histological images of liver Oil Red O

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staining (400×). Data in histograms are presented as the means ± SD. For ELISA results,

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each point in a group represents a value from a sample, and its average value is

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presented as a horizontal line. Mean values with different alphabetical letters indicate a

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statistically significant difference between the groups, as determined by ANOVA and

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Tukey’s post hoc test (p < 0.05).

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Figure 2 Pectin alleviated oxidative stress in the liver of mice fed a high fat diet. (A)

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Levels of peroxisome proliferator activated receptor-α, (B) malondialdehyde, and (C)

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hydroxyl radical scavenging activity. (D) Activities of glutathione peroxidase and (E)

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total activities of superoxide dismutase. (F) Levels of phosphorylated extracellular

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signal-regulated

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phosphorylated p38; (I-K) ERK, JNK and p38, respectively. (L-N) Ratios of

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pERK/ERK, pJNK/JNK and p-p38/p38, respectively. (O) Level of nuclear factor,

kinase;

(G)

phosphorylated

c-Jun

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kinase;

(H)

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erythroid 2-like 2. Data in histograms are presented as the means ± SD. For ELISA

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results, each point in a group represents a value from a sample, and its average value is

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presented as a horizontal line. Mean values with different alphabetical letters indicate

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statistically significant difference between groups, as determined by ANOVA and

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Tukey’s post hoc test (p < 0.05).

674 675

Figure 3 Pectin prevented inflammation and injury in the liver of mice fed a high fat

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diet. (A) Levels of nuclear factor-κB and (B) tumor necrosis factor-. (C) Glutamic

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oxaloacetic transaminase and (D) glutamic pyruvic transaminase activities. (E)

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Representative histological images of H&E staining in the liver, scale bar = 100 μm.

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Data in histograms are presented as the means ± SD. For ELISA results, each point in a

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group represents a value from a sample, and its average value is presented as a

681

horizontal line. Mean values with different alphabetical letters indicate statistically

682

significant differences between groups, as determined by ANOVA and Tukey’s post hoc

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test (p < 0.05).

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Figure 4 Pectin alleviated disorders in fatty acid metabolism in the liver of mice fed a

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high fat diet. (A) PCA score plots for hepatic fatty acids, (B) hepatic saturated fatty

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acids, (C) hepatic monounsaturated fatty acids, and (D) hepatic polyunsaturated fatty

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acids. (E) PLS-DA loading plot for hepatic fatty acids. (F) OPLS-DA s-plot for hepatic

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fatty acids. (G) Z-score and false positive rate analysis of the potential markers (H)

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hepatic palmitic acid levels and (I) hepatic linoleic acid levels. (J) Heat map showing

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the results of the correlation analysis. Mean values with different alphabetical letters

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indicate statistically significant differences between groups, as determined by ANOVA

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and Tukey’s post hoc test (p < 0.05).

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Figure 5 Pectin altered the cecal short-chain fatty acid metabolism in mice fed a high

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fat diet. (A) PCA score plots for short-chain fatty acids, (B) total contents of short-chain

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fatty acids in the cecum, (C) acetic acid, (D) propionic acid, (E) isobutyric acid, (F)

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butyric acid, (G) isovaleric acid, and (H) valeric acid. (I) Heat map showing the results

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of the correlation analysis. Mean values with different alphabetical letters indicate

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statistically significant differences between groups, as determined by ANOVA and

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Tukey’s post hoc test (p < 0.05).

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Figure 6 Pectin regulated the composition of the gut microbiota in mice fed a high fat

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diet. (A) Venn diagram illustrating the overlap of the OTUs identified in the gut

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microbiota of the four groups. (B) Nonmetric multidimensional scaling analysis of

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OTUs. (C) Linear relationships between samples and species. (D) Taxonomic

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cladogram obtained using linear discriminant analysis effect size of the 16s rRNA

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sequences. (E) sPLS analysis of the relationships between short-chain fatty acids and

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the gut microbiota. (F) Relative abundances of filtered gut bacteria analyzed by Tukey’s

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multiple comparisons test and the false discovery rate. Mean values with different

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alphabetical letters denote significant differences (p < 0.05).

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