Lychee (Litchi chinensis Sonn.) Pulp Phenolic Extract Provides

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Lychee (Litchi chinensis Sonn.) pulp phenolic extract provides protection against alcoholic liver injury in mice by alleviating intestinal microbiota dysbiosis, intestinal barrier dysfunction and liver inflammation Juan Xiao, Ruifen Zhang, Qiuyun Zhou, Lei Liu, Fei Huang, Yuanyuan Deng, Yongxuan Ma, Zhencheng Wei, Xiaojun Tang, and Mingwei Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03791 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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Journal of Agricultural and Food Chemistry

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Lychee (Litchi chinensis Sonn.) pulp phenolic extract

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provides protection against alcoholic liver injury in mice by

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alleviating intestinal microbiota dysbiosis, intestinal barrier

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dysfunction and liver inflammation

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Juan Xiao1, Ruifen Zhang1, Qiuyun Zhou2, Lei Liu1, Fei Huang1, Yuanyuan Deng1,

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Yongxuan Ma1, Zhencheng Wei1, Xiaojun Tang1, Mingwei Zhang1*

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1

Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural

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Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture/Guangdong

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Key laboratory of Agricultural Products Processing, Guangzhou 510610, China

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2

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Guangzhou 510631, China

Institute for Brain Research and Rehabilitation, South China Normal University,

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*

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Tel: +86-20-8723 7865; Fax: +86-20-8723 6354; E-mail: [email protected]

Corresponding author: Mingwei Zhang

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The authors declare no competing financial interest.

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Abstract

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Liver injury is the most common consequence of alcohol abuse, which is promoted by

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the inflammatory response triggered by gut-derived endotoxins produced as a

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consequence of intestinal microbiota dysbiosis and barrier dysfunction. The aim of

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this study was to investigate whether modulation of intestinal microbiota and barrier

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function, and liver inflammation contributes to the hepatoprotective effect of lychee

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pulp phenolic extract (LPPE) in alcohol-fed mice. Mice were treated with an ethanol-

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containing liquid diet alone or in combination with LPPE for 8 weeks. LPPE

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supplementation alleviated ethanol-induced liver injury and downregulated key

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markers of inflammation. Moreover, LPPE supplementation reversed the ethanol-

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induced alteration of intestinal microbiota composition and increased the expression

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of intestinal tight junction proteins, mucus protecting proteins and antimicrobial

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proteins. Furthermore, in addition to decreasing serum endotoxin level, LPPE

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supplementation suppressed CD14 and toll-like receptor 4 expression, and repressed

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the activation of nuclear factor-κB p65 in the liver. These data suggest that intestinal

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microbiota dysbiosis, intestinal barrier dysfunction and liver inflammation are

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improved by LPPE, and therefore the intake of LPPE or Litchi pulp may be an

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effective strategy to alleviate the susceptibility to alcohol-induced hepatic diseases.

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Keywords: Alcoholic liver injury; Intestinal barrier; Inflammation; Lychee pulp;

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Microbiota; Phenolics;

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Introduction

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Alcohol abuse is an alarming public health concern. Alcohol-induced risks to health

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in modern society bring an important financial burden for the health care system.1,2

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Chronic alcohol consumption is associated with the development of various medical

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disorders such as alcoholic liver disease, pancreatitis, and brain injury.2,3 Liver injury

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is the most common consequence of alcohol abuse, which can progress to fatty liver,

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steatohepatitis, fibrosis, cirrhosis and even liver cancer.1,3 According to the global

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status report on alcohol and health by WHO, alcohol consumption causes

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approximately 3.3 million deaths every year, which contributes to 5.9% of all

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deaths.2The pathogenesis of liver injury induced by long-term alcohol consumption

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appears to be complex and multifactorial. The inflammatory response induced by the

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imbalance of the gut-liver axis plays an important role in the initiation and

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development of alcoholic liver injury.4-6 Accumulating studies have revealed that

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intestinal microbiota dysbiosis and barrier dysfunction are common in patients and

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animals with alcohol-induced liver injury.5-8 Chronic alcohol consumption causes

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intestinal microbiota dysbiosis, which results in the dysfunction of the intestinal

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barrier and the translocation of bacteria and bacterial products.4-7 As bacterial

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products, endotoxins are a critical trigger of liver inflammation.5,6 Endotoxins interact

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with the endotoxin receptor CD14 and other receptors in the liver, followed by

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cooperation with toll-like receptor 4 (TLR4), and finally induce the activation of

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nuclear factor-κB (NF-κB).5,6 NF-κB regulates the expression of inflammatory 3 ACS Paragon Plus Environment


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factors, such as tumor necrosis factor-ߙ (TNF-ߙ), which in turn triggers liver

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inflammation.5,10 Therefore, the endotoxin-TLR4-NF-κB pathway is a bridge linking

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intestinal microbiota, intestinal barrier and liver inflammation. The prevention of

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intestinal dysbiosis and liver inflammation effectively slows the progression of

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alcoholic liver injury in animals.9-12

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Lychee (Litchi chinensis Sonn.) is a subtropical fruit that is cultivated throughout

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Southeast Asia and has an attractive appearance, delicious taste and good nutritional

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value.13 Recent studies have revealed that lychee pulp is rich in phenolic

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compounds.14-17 Our previous studies have found that lychee pulp phenolic extract

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(LPPE) confers a hepatoprotective effect on alcohol-induced liver injury,18,19 prevents

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intestinal microbiota dysbiosis, and decreases serum endotoxin level,18 indicating that

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LPPE may exert a hepatoprotective effect by reversing the imbalance of the gut-liver

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axis in alcohol-fed animials. However, the protective effect of LPPE on the

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inflammatory response triggered by gut-derived endotoxins produced as a

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consequence of intestinal microbiota dysbiosis and barrier dysfunction remains

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unknown. Intriguingly, procyanidin B2, rutin and (-)-epicatechin, three major

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phenolic compositions in LPPE,18-20 have been demonstrated to exert anti-

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inflammatory activity in animals.21-23 Therefore, the present study investigated the

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mechanisms underlying the hepatoprotective effect of LPPE in C57BL/6 mice fed an

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ethanol-containing liquid diet, focusing on the link between intestinal microbiota,

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intestinal barrier, and hepatic inflammation. 4 ACS Paragon Plus Environment

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Materials and Methods

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Preparation and analysis of LPPE. Fresh Lychee (cv. Feizixiao) was purchased

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from a local fruit market in Guangzhou, Guangdong, China. LPPE was prepared

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following our previous method.18,19 In our previous studies, the phenolic compositions

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of LPPE were identified using HPLC-MS, and their contents were determined by

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HPLC-DAD.19,20 Briefly, the major phenolic components in LPPE were procyanidin

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B2 (104.98 ± 3.11 mg/g), (-)-epicatechin (34.91 ± 1.20 mg/g), quercetin 3-O-

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rutinoside-7-O-a-L-rhamnosidase (260.49 ± 9.21 mg/g), rutin (54.06 ± 1.52 mg/g), and

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isorhamnetin-3-O-rutinoside (16.49 ± 0.50 mg/g).19 The content of A-type

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procyanidin trimer, B-type procyanidin trimer and B-type procyanidin dimer

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calculated as epicatechin equivalent (mg EE/g) using the standard curve of

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epicatechin were 33.96 ± 0.96, 6.36 ± 0.21, and 4.56 ± 0.13 mg EE/g, respectively.19

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The content of kaempferol rhamnosyl-rutinoside, rhamnetin rhamnosyl-rutinoside and

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isorhamnetin rhamnosyl-rutinoside calculated as rutin equivalent (mg RE/g) using the

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standard curve of rutin were 22.79 ± 0.45, 16.04 ± 0.56, and 26.76 ± 0.43 mg RE/g,

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respectively.19

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Animals and experimental design. The experimental procedures involving the use of

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animals were approved by the Animal Ethical and Welfare Committee of Sun Yat-Sen

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University (approval no. IACUC-DB-16-0302) and followed the Guiding Principles

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in the Care and Use of Animals. Ten-week-old specific-pathogen-free male C57BL/6

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mice (26 ± 2 g) were purchased from the Center of Laboratory Animal Science 5 ACS Paragon Plus Environment


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Research of Sun Yat-Sen University (Guangzhou, Guangdong, China). Animals were

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housed in a specific-pathogen-free and environmentally controlled room with constant

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temperature (22 ± 1 ºC), humidity (55-60%) and a 12-hour light-dark cycle.

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During the one-week acclimation period, the mice were fed a rodent chow diet and

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water ad libitum. The mice were then randomly divided into four groups (n = 10 per

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group) and supplied a control liquid diet (control group, CON), a 4% (w/v) ethanol-

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containing liquid diet (ethanol group, EtOH), a 4% (w/v) ethanol-containing liquid

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diet supplemented with 0.2 g/L LPPE (low-dose LPPE-supplemented group,

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EtOH+L-LPPE), or a 4% (w/v) ethanol-containing liquid diet supplemented with 0.4

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g/L LPPE (high-dose LPPE-supplemented group, EtOH+H-LPPE) for 8 weeks. The

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mice were housed two per cage. The liquid diet (TROPHIC Animal Feed High-Tech

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Co. Ltd., Nantong, Jiangsu, China) provides 1 kcal/mL based on the Lieber–DeCarli

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formulation, and 35% of the calories are provided from fat, 19% from carbohydrate,

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18% from protein, and 28% from isocaloric maltose dextrin (control liquid diet) or

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ethanol (4% (w/v) ethanol-containing liquid diet). The CON group was pair-fed with

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the EtOH group, and the other groups were fed ad libitum. The diets were freshly

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prepared from powder and provided daily at 5:00 p.m. Body weight and caloric intake

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were recorded weekly and daily, respectively.

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The animals were euthanized with ether inhalation after fasting for 12 h. Blood

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samples were collected and centrifuged at 3000g for 10 min at 4 ºC to obtain serum.

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After the mice were sacrificed, the livers were immediately removed, washed, 6 ACS Paragon Plus Environment

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weighed and cut into several portions. One portion was fixed in 4% paraformaldehyde

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immediately for histopathological analysis. The others were flash frozen in liquid

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nitrogen. Faeces samples were immediately collected from the ileum and rectum, and

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frozen in liquid nitrogen. The small intestines were immediately removed, washed

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with chilled PBS, and frozen in liquid nitrogen. All samples were stored at −80°C.

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Measurement of serum biomarkers of hepatic function. The activities of alanine

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aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were

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measured using the respective commercial kits (Nanjing Jiancheng Bioengineering

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Institute, Nanjing, Jiangsu, China). All biochemical indices were determined using an

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Infinite® M200 PRO plate reader (Tecan Austria GmbH, Grödig, Salzburg-

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Umgebung, Austria).

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Measurement of lipid profiles in the serum and liver. The levels of triglyceride

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(TG) and total cholesterol (TC) in the serum were measured using the respective

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commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu,

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China). For measurement of hepatic TG and TC levels, total lipids were extracted

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from liver homogenates using a chloroform/methanol mixture (2:1, v/v),24 and TG and

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TC levels in total lipids were measured using commercial kits (Nanjing Jiancheng

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Bioengineering Institute, Nanjing, Jiangsu, China).

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Measurement of endotoxin and inflammatory factors levels in the serum. The

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endotoxin level was determined using the Limulus Amebocyte Lysate test kit

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(Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). Murine TNF7 ACS Paragon Plus Environment


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ߙ, interleukin-1ߚ (IL-1ߚ), interleukin-6 (IL-6), interferon-ߛ (IFN-ߛ) and monocyte

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chemotactic protein-1 (MCP-1) were detected in the serum using a Bio-Plex Mouse

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Cytokine 5-Plex Assay (Bio-Rad, Hercules, CA, USA).

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Liver histopathology. Liver histopathology was assessed via hematoxylin and eosin

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(H&E) staining following a standard procedure. Briefly, paraffin sections (5 µm-

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thick) were cut, deparaffinized in xylene, rehydrated in an alcohol gradient, and

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stained with H&E. Stained sections were observed using a light microscope (Leica

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DMI 4000B, Heidelberger, Baden-Württemberg, Germany).

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Quantitative real-time PCR (qRT-PCR). Total RNA was extracted from the frozen

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liver samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and reverse-

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transcribed to synthesize cDNA on a B960 real-time thermocycler (Hangzhou Jingle

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Scientific Instruments Co., Ltd., Hangzhou, Zhejiang, China) using a Reverse

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Transcriptase M-MLV (RNase H-) (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu,

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China). qRT-PCR was performed on an ABI ViiA 7 Detection System (Applied

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Biosystems, Foster City, CA,USA) using an AceQ® qPCR SYBR® Green Master Mix

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(Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China). Each sample was assessed in

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triplicate, and normalized to GAPDH or 18S. The relative gene expression levels were

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calculated using the 2-△△CT method as previously described, and presented as a ratio

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of the treatment group to the CON group.25 The primer sequences used (Sangon

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Biotech (Shanghai) Co., Ltd., Shanghai, China) are listed in Table 1.


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Western blot analysis. Cytoplasmic protein extract, nuclear protein extract and

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membrane protein extract from frozen liver were obtained by homogenizing the liver

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in ice-cold extraction buffer for cytoplasmic protein, nuclear protein and membrane

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protein, respectively, using the respective commercial kits (Beyotime Biotechnology,

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Shanghai, China). Protein concentrations were determined by the BCA assay. Western

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blot analysis was conducted as previously described.19 Briefly, the protein extract was

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loaded onto a 10% SDS-polyacrylamide gel, transferred onto a polyvinylidene

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difluoride membrane (0.45 µm, Merck Millipore, Darmstadt, Hesse-Darmstadt,

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Germany), and blocked with 5% skimmed milk, followed by immunostaining with

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primary antibodies against CD14, TLR4, inhibitor kappa Bα (IκBα), phosphorylated-

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IκBα (p-IκBα) or NF-κB p65 (1:1000, Cell Signaling Technology, Danvers, MA,

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USA). After incubation with a horseradish peroxidase-conjugated secondary antibody

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(1:10000, Tianjin Sungene Biotech, Tianjin, China), immunoreactive proteins were

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stained with ECL substrate from the Fast Western Blot Kit (Pierce, Rockford, IL,

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USA) and then exposed to a film. The film was scanned on a Plustek SW500 scanner

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(Plustek, Taiwan, China), and the band intensities were subsequently recorded using

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Quantity One 1-D analysis software (Bio-Rad, Hercules, CA, USA). β-actin, Histone

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and ATP1B2 (Tianjin Sungene Biotech, Tianjin, China) were detected as the loading

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controls for the cytoplasmic protein, nuclear protein and membrane protein extracts,

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respectively.



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Measurement of faecal microbial composition by high-throughput sequencing

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analysis. Total bacterial DNA was isolated from faeces using the PowerFecal™ DNA

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Isolation kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the

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manufacturer's instructions. Sequencing and sequence analysis were conducted at

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BGI-Shenzhen (Shenzhen, China) according to previously published methods.26

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Briefly, the isolated DNA was subjected to Illumina MiSeq sequencing of the V4

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hypervariable region of the 16S rRNA gene, followed by analysis of faecal microbiota

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using QIIME 1.8.0.

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Statistical analysis. Data were expressed as the means ± standard deviation (SD) and

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analyzed by one-way ANOVA followed by a Duncan post hoc test using SPSS 16.0

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software. For intestinal microbial composition analysis, data were expressed as the

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means ± standard error (SE) and compared using Metastats. p < 0.05 was regarded as

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statistical significance.

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Results

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Effects of LPPE on general parameters. As shown in Table 2, there were no

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significant differences in the initial body weight, final body weight or total caloric

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intake among the four groups (p > 0.05). Ethanol feeding resulted in a significant

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increase in the liver-to-body weight ratio compared with the CON group (p < 0.05).

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Both L-LPPE and H-LPPE supplementation significantly decreased the liver-to-body

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weight ratio compared with the EtOH group, indicating that LPPE supplementation

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alleviated ethanol-induced liver swelling. 10 ACS Paragon Plus Environment

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Effects of LPPE on liver histopathology. Ethanol feeding resulted in liver damage

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— characterized by an irregular arrangement of hepatocytes, extensive fat droplets

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and inflammatory infiltration in the hepatocytes — that was not seen in the CON

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group (Fig. 1). The vacuoles in H&E-stained sections reflected the fat droplets in the

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hepatocytes. Fewer and smaller fat droplets, and less inflammatory infiltration in the

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hepatocytes were observed in the LPPE-supplemented groups than in the EtOH group.

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The results indicated that LPPE supplementation alleviated ethanol-induced hepatic

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steatosis and inflammation.

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Effects of LPPE on serum biomarkers of hepatic function. As shown in Fig. 1,

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ethanol intake led to significant increases in serum AST and ALT activities compared

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with those in the CON group (1.37-fold and 1.36-fold, respectively, p < 0.05).

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However, compared with the EtOH group, L-LPPE and H-LPPE supplementation

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decreased serum AST activity by 27.23% and 36.24%, respectively, and decreased

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serum ALT activity by 15.80% and 26.90%, respectively (p < 0.05). The results

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indicated that LPPE supplementation alleviated ethanol-induced liver injury.

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Effects of LPPE on serum and liver lipid profiles. Ethanol feeding led to significant

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increases in serum and hepatic TG levels (1.26-fold and 1.46-fold, respectively, p

Lychee (Litchi chinensis Sonn.) Pulp Phenolic Extract Provides

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