Inhibition of P2X7RâNLRP3 Inflammasome Activation by Pleurotus

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Bioactive Constituents, Metabolites, and Functions

Inhibition of P2x7R-NLRP3 inflammasome activation by Pleurotus citrinopileatus: a possible protective role in alcoholic hepatosteatosis Xia Li, Quan Jin, Yu Zhang, Yan-Ling Wu, Cheng-Min Jin, Ben-Wen Cui, Ying Li, MingJi Jin, Yue Shang, Min Jiang, Hong-Xu Yang, Mei Wu, Jian Liu, Li-Hua Lian, and ji-xing nan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05756 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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Journal of Agricultural and Food Chemistry

Graphical abstract 56x58mm (300 x 300 DPI)

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Inhibition of P2x7R-NLRP3 inflammasome activation by Pleurotus citrinopileatus: a

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possible protective role in alcoholic hepatosteatosis

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Running title: Pleurotus citrinopileatus ameliorates hepatosteatosis

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Xia Li †,§, Quan Jin †,§, Yu Zhang †, Yan-Ling Wu †, Cheng-Min Jin⊥, Ben-Wen Cui †, Ying

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Li †, Ming-Ji Jin †, Yue Shang †, Min Jiang †, Hong-Xu Yang †, Mei Wu †, Jian Liu †, Li-Hua

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Lian*,†, Ji-Xing Nan*,†,‡

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†

Key Laboratory for Natural Resource of Changbai Mountain & Functional

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Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin

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Province 133002, China

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‡

Clinical Research Center, Yanbian University Hospital, Yanji, Jilin Province

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133002, China

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⊥ Analysis

center, Dt&CRO Inc., Yongin-si, Gyeonggi-do, 17042, Republic of Korea

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§ These

authors contributed equally to this work

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*Corresponding to

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Li-Hua Lian, Ji-Xing Nan, Key Laboratory for Natural Resource of Changbai Mountain &

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Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University,

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Yanji 133002 Jilin Province, China, Tel.: 86-433-2435061, fax: 86-433-2435072. E-mail

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address: [email protected] (L.-H. Lian), [email protected] (J.-X. Nan).

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Abbreviations: ALD, alcoholic liver disease; AH, alcoholic hepatitis; AMPK, AMP-

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activated kinase; ALT, alanine aminotransferase; AST, aspartate amino-transferase;

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GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-1β, Interleukin-1β; LPS,

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lipopolysaccharide; NLRP3, NOD-like receptor pyrin domains 3; P2x7R, purinergic

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receptor P2X ligand-gated ion channel 7; SREBP, Sterol regulatory element-binding

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protein; SIRT1, sirtuin 1; TLRs, Toll-like receptors.

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ABSTRACT

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Pleurotus citrinopileatus (golden oyster mushroom) is a widely used edible mushroom.

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We investigated the inhibitory effect of P. citrinopileatus aqueous extract against

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alcoholic steatohepatitis and its underlying mechanism. Acute and chronic ethanol

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feeding murine models were established by intragastrically administering ethanol or

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feeding ethanol-containing Lieber-DeCarli liquid diet to male C57BL/6 mice. In both

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models, P. citrinopileatus decreased serum alanine aminotransferase (ALT), aspartate

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transaminase (AST), triglyceride (TG) and hepatic TG levels. Hematoxylin and eosin (HE)

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and Oil red O staining confirmed that P. citrinopileatus ameliorated both of acute and

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chronic alcoholic hepatosteatosis, characterized by regulation of lipid metabolism-

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related protein, including sirtuin 1 (SIRT1), AMP-activated kinase (AMPK) and sterol

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regulatory

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inflammatory response via modulating purinergic receptor P2X ligand-gated ion

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channel 7 (P2x7R)-NOD-like receptor pyrin domains 3 (NLRP3) inflammasome. P.

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citrinopileatus restored the expression of those protein to normal level. In addition,

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HepG2 cells were incubated with P. citrinopileatus prior to ethanol stimulation. P.

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citrinopileatus reduced ethanol exposure-induced lipid deposition. Concomitantly P.

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citrinopileatus increased AMPK and SIRT1 expression, which were reduced by ethanol

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treatment. P. citrinopileatus ameliorated alcoholic hepatic steatosis and accompanied

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inflammatory response via regulating SIRT1-AMPK and P2x7R-NLRP3 inflammasome

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activation, highlighting a promising strategy and utility of P. citrinopileatus for

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alcoholic steatohepatitis as dietary health supplements.

element-binding

protein

(SREBP1).

P.

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citrinopileatus

reversed


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Keywords: Pleurotus citrinopileatus; alcoholic hepatosteatosis; AMPK; SIRT1, P2X7R

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INTRODUCTION

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Alcoholic liver disease (ALD) is caused by long-term alcohol consumption and can

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develop to liver fibrosis and cirrhosis 1. To date, the mainstay treatment for patients

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with all stage of ALD is alcohol abstinence 2, and adequate nutritional support is

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recommended in recent clinical guidelines 3, 4. However, few therapeutic option exists

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for severe ALD, and little changes has been made for medical treatment of ALD so far.

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Alcoholic steatosis, the earliest and most common response of liver to chronic

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alcohol exposure, is pathologically characterized by accumulation of lipid droplets in

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hepatocytes, mild inflammation but without hepatic fibrosis 5. Promoted fatty acid

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synthesis and impaired β-oxidation by excessive alcohol intake consequently result in

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hepatic lipid accumulation 2. AMP-activated kinase (AMPK), a vital lipid regulator, is

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dysregulated by alcohol during alcoholic hepatosteatosis. Downregulated AMPK by

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alcohol decreases its ability to promotes sterol regulatory element-binding protein-

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1 (SREBP1), which accelerates the progression of hepatic steatosis

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(SIRT1) plays a pivotal role in the regulation of hepatic fatty acid metabolism and

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inflammatory response by activating AMPK 9, 10. Therefore, targeting the SIRT1-AMPK

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partnership might be a breakthrough of ALD treatments.

6-8.

Sirtuin 1

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Bacterial translocation plays a key role in the progression of ALD. Alcohol abuse

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leads to an increased permeability of gut, which further results in an increasing level

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of lipopolysaccharide (LPS) in circulation 11. When translocated from gut lumen to liver,

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LPS is recognized by toll-like receptors (TLRs). Among TLRs, TLR4 is mainly responsible

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for LPS recognition, and activates nuclear factor-κB (NF-κB) signaling cascades, leading 6



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to Interleukin (IL)-1β synthesis. In addition, during the pathogenesis of ALD,

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extracellular adenosine triphosphate (ATP) is released from damaged hepatocytes and

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consequently aggravates hepatosteatosis 12. P2X ligand-gated ion channel 7 (P2x7R),

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an ATP-gated ion channel, initiates pro-inflammatory cascades via nucleotide-binding

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oligomerization domain (NOD)-like receptor pyrin domains 3 (NLRP3) inflammasome.

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NLRP3 inflammasome activates inflammatory caspases (i.e., caspase-1), which in turn

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catalyzes pro-inflammatory cytokines, including IL-1β 13.

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Pleurotus citrinopileatus, also known as “golden oyster mushroom”, is a popular

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edible mushroom, which is abundantly distributed in northeastern China, Japan and

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Korea

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effects, as anti-oxidation 15, 16, immunomodulation 17, 18, anti-tumor 17, anti-obesity 19

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and anti-inflammatory activities

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downregulated serum triglyceride and cholesterol levels. However, the effect of P.

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citrinopileatus on alcoholic liver disease remains elusive. Herein, we aimed to explore

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whether and how P. citrinopileatus would improve alcoholic hepatic steatosis using in

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vivo models of acute and chronic alcohol intake-induced hepatosteatosis and in vitro

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model of HepG2 cells with ethanol. The results showed that P. citrinopileatus aqueous

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extract alleivated both of acute and chronic mice alcoholic hepatosteatosis via P2x7R-

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NLRP3 inflammasome, suggesting the potential utility of P. citrinopileatus for ALD

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treatment.

14.

P. citrinopileatus is considered as “health food” with the pharmacological

20, 21.

Hu et al.

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reported that P. citrinopileatus

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MATERIALS AND METHODS

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Reagents. Anti-SIRT1, anti-P2X7R, anti-SREBP1, anti-AMPKα, anti-P-AMPKα and anti-

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GAPDH were purchased from Abcam (Cambridge, MA, USA). A438079 were from

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Abcam (Cambridge, MA, USA). A selective SIRT1 activator, SRT2104 was purchased

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from Selleck Chemicals (Houston, TX, USA). Metformin, a LKB1/AMPK activator was

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obtained from Beyotime Institute of Biotechnology (Haiman, Jiangsu, China).

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Preparation of P. citrinopileatus Extract. P. citrinopileatus were collected from the

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local farm (Wangqing, Jilin, China). Dried fruiting bodies of P. citrinopileatus (50 g) was

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grinded, extracted with 250 ml 80°C water three time for 2, 1 and 1 h respectively.

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And then combined crude aqueous extract was filtered with Whatman Grade 1 filter

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paper, concentrated with a rotary evaporator at 80°C to 100 ml, followed by fractional

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precipitation with 80 % (V/V) alcohol overnight and then finally lyophilized. These

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processes produced 0.65 g of 80 % (V/V) fraction. Compounds from P. citrinopileatus

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extract were analyzed using UltiMateTM 3000 Ultra Performance Liquid

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Chromatography system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The

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chromatographic separation was performed using ACQUITY UPLC BEH C18 column

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(2.1×100 mm I.D., 1.7 μm). The mobile phase consisted of water containing 0.1%

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formic acid (A) and acetonitrile (B). The gradient condition was 5-90% B at 1-25 min,

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100% B at 25.1-26 min, 100-5% B at 26-26.5 min, and 5% B at 26.5-30 min for

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equilibration. The flow rate was 0.3 mL/min. The separated substance was

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characterized by a TripleTOFTM 5600+ hybrid triple quadrupole time-of-flight mass

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spectrometer (SCIEX, Framingham, MA, USA). The following mass spectrometer (MS) 8



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conditions were used: ion spray voltage, 5.5 kV; decluttering potential (DP), 80V; the

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turbo spray temperature, 500 °C; nebulizer gas (Gas 1) of 50 psi; heater gas (Gas 2), 50

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psi; curtain gas, 25 psi. Nitrogen was kept as nebulizer and auxiliary gas. The time-of-

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flight mass spectrometry (TOF MS) scan was operated with the mass range of m/z 300-

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1800. Recalibration was carried by Calibrant delivery System before analysis.

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Animal experiments. Eight to ten weeks old male C57BL/6 mice (20-22 g) were

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purchased from Yisi Laboratory Animal Technology Co., Ltd (Changchun, Jilin, China)

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[SPF, SCXK (J) 2016-0003]. The mice were housed under constant temperature (22 ±

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2°C), relative humidity (50-60%), and light (12-h light-dark cycles) conditions with

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standard laboratory chow diet ad libitum. All mice were handled in compliance with

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“Guide for the Care and Use of Laboratory Animals” (8th edition, National Research

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Council, 2011) and all animal procedure were reviewed and approved by Animal

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Research Ethic Committee of Yanbian University, China. Murine model of acute and

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chronic alcoholic hepatosteatosis were carried out as follows: (1) Acute ethanol

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feeding (Fig. 1A): All mice were randomly divided into the following three groups:

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normal group, ethanol group, ethanol plus P. citrinopileatus group (100 mg/kg, body

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weight). Mice were treated three intragastric dose of ethanol (5 g/kg, body weight) or

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isocaloric/isovolumetric maltose dextrin every 12 h

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citrinopileatus group were gavaged with three doses of P. citrinopileatus extract every

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12 h at just before ethanol administration. P. citrinopileatus extract was diluted with

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saline. Four hours after the last dose of alcohol intake, mice were anesthetized with

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isoflurane and blood samples were collected by cardiac puncture. Liver tissues was

22, 23.

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Mice in ethanol plus P.

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removed and immediately snap-frozen in liquid nitrogen, and blood samples were

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collected. (2) Chronic ethanol feeding (Fig. 1B): C57BL/6 mice were randomly divided

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into the following four groups: pair-fed group, ethanol-fed group, and ethanol -fed

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mice treated with P. citrinopileatus groups (50 or 100 mg/kg, body weight). Ethanol

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group were fed with increasing concentration of ethanol in Lieber-DeCarli liquid diet.

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Ethanol concentrations were ramp-up from 1% (V/V) to 4% (V/V), every concentration

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lasted for 2 days, then followed by 5% ethanol for continuous 28 days. During these 4

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weeks, ethanol-fed plus P. citrinopileatus groups were daily gavaged 50 or 100 mg/kg

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of P. citrinopileatus extract. Pair-fed group was given pair-fed diets substituted with

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isocaloric maltose dextrin. Nine hours after last dosing, all mice were sacrificed under

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anesthesia, followed by the collection of blood and liver tissue samples.

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Serum biochemical parameters. Slanine aminotransferase (ALT) and aspartate amino-

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transferase (AST) levels of mice serum were detected by the dry chemistry blood

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analyzer SPOTCHEM™ SP-4410 (Arkray, Inc., Kyoto, Japan). Triglyceride concentration

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in mice serum and liver tissues were measured by an enzymic kit (Nanjing Jiancheng

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Bioengineering Institute Co., Ltd., Nanjing, Jiangsu, China) according to the

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manufacturer’s instructions.

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Enzyme-linked immunosorbent assay (ELISA). Levels of murine IL-1β protein were

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measured using Mouse IL-1β DuoSet ELISA (R&D Systems, Minneapolis, Minnesota,

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USA) according to the manufacturers’ protocols.

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Histopathological and immunohistochemistry examination. Liver tissues were fixed

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in 10% neutral-buffered formalin, embedded in paraffin, sectioned 5 μm-thick

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thickness and stained with hematoxylin-eosin (HE). Five μm-thick cryosections of mice

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liver were stained with Oil red O and counterstained with hematoxylin. In

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immunohistochemistry, after overnight incubated with primary antibodies (anti-SIRT1,

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anti-AMPKα, anti-SREBP1, anti-P2x7R, and anti-NLRP3 antibodies) in a humidified

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chamber overnight at 4°C, mouse tissue sections were incubated with for 30 min at

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room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody

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included in MaxVision™ HRP-Polymer anti-mouse/rabbit IHC kit (Fujian, Fuzhou,

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China), and incubated with diaminobenzidine (DAB) Chromogen, followed by

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counterstained with hematoxylin.

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Cell culture and Nile red staining. HepG2 cells were maintained in Dulbecco’s

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modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS),

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100 units/ml penicillin and 100 mg/ml streptomycin in a 37°C, 5% CO2 humidified

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incubator. HepG2 cells were fixed and stained with 100 ng/mL Nile red solution. Cells

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were washed with PBST for several times and mounted with UltraCruz aqueous

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mounting medium with DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

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Western Blot. The protein from the cells or livers were lysed by RIPA buffer. Nuclear

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and Cytoplasmic Protein Extraction Kit (Beyotime) was used to extract protein in

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nuclear and cytoplasmic fractions of liver cells. Equal amounts of protein were

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separated using 10% or 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and

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then transferred to the Amersham Hybond-P polyvinylidene fluoride (PVDF) 11


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membrane (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). After blocking, the

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membranes were washed briefly in PBS with 0.05% Tween 20 (PBST) and incubated

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with the specific primary antibodies overnight at 4°C with gentle agitation. The blots

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were washed with PBST and then incubated with the appropriate HRP-conjugated

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secondary antibodies. Membranes were finally developed with Clarity™ Western ECL

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Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the intensity of bands

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were quantified with Quantity One software (Bio-Rad, Hercules, CA, USA).

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

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statistical analysis was performed by one-way analysis of variance (ANOVA) and

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Tukey’s multiple comparison tests using the GraphPad Prism program (Graphpad

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Software, Inc, San Diego, CA, USA). A P value of less than 0.5 was regarded as

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statistically significant.

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RESULTS

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P. citrinopileatus reversed acute alcohol intake-induced hepatic steatosis. Profiling

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of compounds from P. citrinopileatus extract was analyzed by mass spectrometer (Fig.

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2). Then we utilized Formula FinderTM tool in PeakView ® software (SCIEX) and

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Chemspider database to search and identify compounds from P. citrinopileatus. As

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shown in table 1 and 2, a total of 24 compounds were identified from P. citrinopileatus.

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Serum ALT and AST levels were increased after mice gavaged three doses of

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alcohol (Fig. 3A). In addition, alcohol exposure caused the remarkable increase of

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hepatic and serum TG concentration (Fig. 3B). We also confirmed alcohol consumption

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leads to histological alterations of liver according to HE and Oil red O staining data (Fig.

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3C and 3D). P. citrinopileatus pretreatment reduced ethanol-induced elevatin level of

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serum aminotransferases, as well as serum and hepatic TG accumulation. Additionally

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hepatic lipid accumulation was notably diminished by P. citrinopileatus-pretreatment

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compared with those in ethanol group (Fig. 3D). These data indicated that acute

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alcohol intake induced a significant lipid accumulation in alcohol-gavaged mice, which

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could be attenuated by P. citrinopileatus administration. In addition, P. citrinopileatus

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didn’t affect ALT, AST and lipid accumulation within 4 weeks when gavaged to mice at

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100mg/kg dose (supplementary data F1).

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P. citrinopileatus reversed acute alcohol exposure-induced steatohepatitis through

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regulating lipid oxidation and synthesis. Growing evidences demonstrated that

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stimulation of hepatic SIRT1-AMPK signaling by nutritional or pharmacological 13


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intervention protects against the development of ALD in rodents 24. We hypothesized

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P. citrinopileatus could interfere the development of lipid accumulation by regulating

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SIRT1-AMPK-SREBP1 axis. Therefore, the expression of SIRT1, AMPK and SREBP1 were

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determined by immunohistochemistry in acute alcohol-exposed mice. P.

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citrinopileatus pretreatment increased the expression of SIRT1 and AMPK, which were

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obviously decreased after acute alcohol consumption (Fig. 4A and 4B). Simultaneously,

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P. citrinopileatus down-regulated the expression of SREBP1 evoked by alcohol intake

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(Fig. 4C), which was consistent with dynamically altered TG levels shown in Fig. 3B. We

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also employed Western blotting analysis to detect the protein expression of AMPK

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and nuclear active form of SREBP1. Alcohol consumption significantly suppressed

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AMPK phosphorylation, and P. citrinopileatus stimulated AMPK phosphorylation as

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expected (Fig. 4F and 4G). SREBP1 expression was increased in nucleus fractions after

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ethanol treatment, while PC administration suppressed the SREBP1 expression in

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nuclear ((Fig. 4F). These results hinted that P. citrinopileatus ameliorated hepatic lipid

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accumulation by regulating lipogenesis and lipolysis, and eventually attenuated

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impaired hepatic lipid metabolism balance via through regulation of SIRT1-AMPK. P.

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citrinopileatus inhibited P2x7R and NLRP3 inflammasome activation after ethanol

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exposure confirmed by immunohistochemistry staining (Fig. 4D and 4E). IL-1β level in

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serum was elevated after alcohol consumption, while P. citrinopileatus pretreatment

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declined IL-1β levels (Fig. 4H). Consistent with immunohistochemistry staining data,

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the protein expression of P2x7R was suppressed by P. citrinopileatus administration,

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which was increased by alcohol intake (Fig. 4F and 4G). 14



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P. citrinopileatus prevented lipid accumulation in chronic alcoholic hepatic steatosis.

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Considering the beneficial effect of P. citrinopileatus against acute alcohol binge-

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caused hepatic steatosis, we were intrigued whether P. citrinopileatus could reverse

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chronic ethanol feeding-induced hepatic steatosis and inflammation. After mice were

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fed with alcohol-containing Lieber-DeCarli liquid diet for 4 weeks, serum ALT and AST

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levels were significantly increased (Fig. 5A). The increasing of aminotransferases was

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abolished by pretreatment with P. citrinopileatus. Four weeks of alcohol consumption

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caused a significantly higher serum and hepatic TG contents, while P. citrinopileatus

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effectively altered TG contents in P. citrinopileatus treatment groups (Fig. 5B). As

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shown in Fig. 5C and Fig. 5D, chronic ethanol feeding induced an obvious lipid

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accumulation, which could be ameliorated by P. citrinopileatus administration. our

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data suggested that P. citrinopileatus also could effectively alleviate lipid accumulation

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in the liver caused by chronic alcohol exposure.

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Inhibition of P2x7R-NLRP3 activation by P. citrinopileatus contributed to lipid

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accumulation in chronic ethanol feeding-induced hepatic steatohepatitis. We

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determined the expression of lipid metabolism-related proteins such as SIRT1, AMPK,

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and SREBP1 by immunohistochemistry. As shown in Fig. 6A and Fig. 6B, positive

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staining area of SIRT1 and AMPK obviously reduced in ethanol-fed group, while P.

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citrinopileatus administration restored SIRT1 and AMPK activity. Protein expression of

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total and phosphorylated AMPK was reduced after chronic ethanol feeding and

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restored by P. citrinopileatus (Fig. 6F). As expected, chronic ethanol feeding increased

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the expression of SREBP1 confirmed by immunohistochemistry and western blot, 15


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which can be reversed by P. citrinopileatus (Fig. 6C and 6F). These data demonstrated

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that P. citrinopileatus prevented chronic alcoholic hepatic steatosis through SIRT1-

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AMPK-SREBP1 signaling. Long-term alcohol intake also promoted IL-1β release and up-

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regulated protein expression of P2x7R and NLRP3, meanwhile P. citrinopileatus

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abolished the alcohol-induced IL-1β release and the protein expression of P2x7R and

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NLRP3 (Fig. 6D, 6E, 6F and 6H).

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P. citrinopileatus regulated lipid accumulation in ethanol-induced steatotic

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hepatocytes. A human hepatoma cell line, HepG2 cells were incubated with ethanol

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at concentration of 50 mM to induce intracellular lipid accumulation. Nile red staining

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was employed to evaluate whether P. citrinopileatus could inhibit lipid accumulation

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in ethanol-exposed HepG2 cells. Metformin was used as a positive control, which

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could activate AMPK signaling. As shown in Fig. 7A, ethanol treatment increased the

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formation of lipid droplets in HepG2 cells, meanwhile P. citrinopileatus concentration-

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dependently alleviated lipid accumulation. P. citrinopileatus exhibited superior ability

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in controlling lipid accumulation compared with metformin. P. citrinopileatus also

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reversed alcohol-induced decreasing of total and phosphorylated AMPK expression

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(Fig. 7B and 7C). P. citrinopileatus increased the expression of SIRT1 in a concentration-

275

dependent manner. Especially P. citrinopileatus exhibited much superior ability on

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stimulating SIRT1 than SRT2104, a SIRT1 activator (Fig. 7D). P. citrinopileatus alone at

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concentration of 100 μM did not affect lipid synthesis.

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DISSUASION

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ALD is a major global public health problem, resulting in millions of deaths. So far,

281

ignificant efforts have been made to understand the molecular events and cellular

282

mechanisms in the development and progression of ALD. The classic therapies of ALD

283

include nutritional support, corticosteroids, tumor necrosis factor-α inhibitor

284

(pentoxifylline) and phosphodiesterase, unfortunately with serious side effects and

285

unsatisfactory outcome 25-27. Therefore, it is necessary to explore dietary supplements

286

from food targeted for ALD therapy. Recently edible mushrooms, such as P.

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citrinopileatus, received substantial investigative attention for the biofunction and

288

application as medicinal purposes and also as nutritional product, due to significant

289

benefits of low contamination and high economic value 16. We here reported that P.

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citrinopileatus alleviated hepatosteatosis induced by acute and chronic alcohol

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consumption, especially inhibited lipid accumulation through modulating SIRT1-AMPK

292

and inflammatory response via inhibiting P2x7R-NLRP3 in hepatocytes.

293

In order to confirm whether P. citrinopileatus could ameliorate alcoholic

294

hepatosteatosis, we established acute or chronic alcoholic hepatosteatosis models.

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Excessive alcohol intake amplifies inflammation in liver and further impairs

296

hepatocyte mitochondrial functions, eventually induces steatosis and inflammation in

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mice 16, 28-30. Alcohol intake simultaneously increases gut permeability and activates

298

LPS-TLR4 signaling. And according to preliminary experiments and published reports

299

19, 31, 32,

we selected 50 or 100 mg/kg as dose of P. citrinopileatus, especially at dose of 17


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100 mg/kg P. citrinopileatus didn’t affect any normal liver function. In addition, a

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human equivalent dose (HED) of 100 mg/kg P. citrinopileatus in mice

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converted to 480 mg daily for 60 kg man, which is equivalent to approximately 40 g

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dried P. citrinopileatus. According to our data, both of acute alcohol exposure and

304

chronic ethanol feeding caused the increase the level of serum ALT, AST and TG, which

305

were attenuated by P. citrinopileatus administration (Fig 3A, 3B and Fig. 5A, 5B). P.

306

citrinopileatus decreased the lipid accumulation in liver tissue significantly (Fig. 3C, 3D,

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5C and 5D), indicating the anti-steatotic effect of P. citrinopileatus. Furthermore, we

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established an in vitro model using human hepatoma cell line, HepG2 cells, to further

309

verify the inhibitory effect of P. citrinopileatus on lipid accumulation. P. citrinopileatus

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exhibited more stronger inhibitory effect on attenuating lipid accumulation than

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metformin in ethanol-induced steatotic hepatocytes (Fig. 7). Our results suggested

312

that P. citrinopileatus suppressed lipid accumulation in alcoholic hepatosteatosis, and

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this inhibitory capacity of P. citrinopileatus especially more focused to regulate lipid

314

metabolism in hepatocytes.

33

can be

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Hepatosteatosis is an initiation stage of ALD. Accumulation of triglyceride in

316

cellular lipid droplets leads to hepatic steatosis. Alcohol exposure promotes hepatic

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lipid accumulation by interfering the balance of lipogenesis and lipolysis, which was

318

directly or indirectly regulated by lipid metabolism-associated key transcription

319

factors, such as SIRT1, AMPK and SREBP1. We were intrigued whether the anti-

320

steatotic effect of P. citrinopileatus might be exerted through the modulation of SIRT1-

321

AMPK axis. In both murine models of acute and chronic alcoholic hepatosteatosis, P. 18



Page 20 of 39

322

citrinopileatus activated AMPK and SIRT1, and decreased SREBP1 expression,

323

indicating the elevated fatty acid β-oxidation and reduced lipogenesis. Our in vitro

324

data suggested that P. citrinopileatus abolished hepatic lipid accumulation through

325

regulating SIRT1-AMPK signaling in ethanol-exposed steatotic hepatocytes. AMPK and

326

SIRT both regulate each other, and AMPK can function as a SIRT1 activator

327

applied an AMPK activator, metformin and a SIRT1 activator, SRT2104 to verify the

328

stimulating effectiveness of SIRT1. Metformin and SRT2104 restored SIRT1 to a certain

329

extent, which was decreased by alcohol exposure, meanwhile P. citrinopileatus

330

completely restored SIRT1 to normal level (Fig. 7). Our data suggested that P.

331

citrinopileatus might activates hepatic SIRT1-AMPK axis, which leads to the

332

attenuation of lipid accumulation, thereby protecting liver from alcohol damage.

34.

We

333

Lipid accumulation induced by sustained alcohol consumption leads to

334

hepatocytes damages and inflammation, which will directly promote the progression

335

of alcoholic steatohepatitis. ATP released from damaged hepatocytes at sites of

336

inflammation will bind to P2x7R on neighboring cells. This binding results in the

337

activation of NLRP3 Inflammasome, which stimulates the cleavage of pro-IL-1β and

338

secretion of the mature IL-1β 35. We confirmed that upregulation of P2x7R by alcohol

339

intake is synchronized with activation of NLRP3 inflammasome, as well as its

340

downstream cytokines IL-1β (Fig. 4 and 6). Therefore, we detected whether P.

341

citrinopileatus could inhibit the expression of P2x7R and NLRP3 both in acute and

342

chronic alcoholic hepatosteatotic murine models. Alcohol treatment increased the

343

expression of P2x7R and NLRP3 in both models, while P. citrinopileatus completely 19


Page 21 of 39


344

suppressed P2x7R and NLRP3 expression, suggesting that the inhibitory ability of P.

345

citrinopileatus of alcohol-induced steatohepatitis might be linked with the blockade of

346

P2x7R-NLRP3 inflammasome activation.

347

Collectively, we firstly revealed the hepatoprotective effect of P. citrinopileatus

348

against alcoholic hepatosteatosis, and its capacity on the regulation of lipid

349

accumulation by activating SIRT1-AMPK signaling might be tightly linked with P2x7R-

350

NLRP3 inflammasome. Our data demonstrated the possibility of developing P.

351

citrinopileatus as potential dietary health supplements for preventing and treating

352

alcoholic steatohepatitis.

20



353

Author Contribution

354

Ms. Xia Li and Dr. Quan Jin is the primary investigator in this study. Ms. Yu Zhang,

355

Ms. Min Jiang, Mrs. Ben-Wen Cui and Ms. Ying Li participated in part of in vivo

356

experiments. Ms. Ming-Ji Jin, Ms. Ying Li, Ms. Yue Shang, Hong-Xu Yang, Mei Wu and

357

Jian Liu participated in part of in vitro experiments. Dr. Yan-Ling Wu participated in

358

part of statistical analysis. Cheng-Min Jin analyzed compounds from Pleurotus

359

citrinopileatus extract. Dr. Li-Hua Lian and Prof. Ji-Xing Nan designed the whole study

360

and wrote the manuscript.

361

Funding

362

This study was supported by a grant from the National Natural Science Foundation of

363

China (81560597, 81660689, 81460564 and 81860751), and partially by Science and

364

Technology Planning Projects from the Science and Technology Department of Jilin

365

Province (20160101205JC, 20180414048GH, 20180201065YY and 20180519010JH).

366

Conflict of Interest Statement

367

There is no potential conflict of interest.

368 369

21


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of

AMPK-Dependent

Lipogenesis

Mediated

476

477

26


by

P2X7R-NLRP3-


478

Figure legends

479

Figure 1. Animal experimental procedure. (A) Acute ethanol feeding model. C57BL

480

mice were intragastrically administrated with three dose of ethanol (5 g/kg, body

481

weight) every 12 h. P. citrinopileatus (100 mg/kg) were given by gavage three doses

482

just before ethanol administration. (B) Chronic ethanol feeding model. C57BL mice

483

were fed with gradually increased concentrations of ethanol containing Lieber-DeCarli

484

liquid diet from 1% to 4% every 2 days, then followed by 5% ethanol for 28 days. P.

485

citrinopileatus (50 or 100 mg/kg) were daily gavaged for 28 days.

486

Figure 2. Base peak chromatogram of P. citrinopileatus.

487

Figure 3. P. citrinopileatus attenuated acute ethanol intake-induced hepatic lipid

488

accumulation. Mice were gavaged with three dose of ethanol (5 g/kg, body weight)

489

every 12 h. P. citrinopileatus (100 mg/kg) was gavaged just before ethanol

490

administration. (A) ALT and AST level in serum. (B) TG contents in serum and liver. HE

491

(C, 200 × original magnification) and Oil red O staining (D, 400 × original magnification).

492

Black arrows mean lipid droplets in mice liver. Each value is expressed as mean ± SD

493

(n = 6). ### p< 0.001, significantly different when compared with normal group; *

494

p

Inhibition of P2X7RâNLRP3 Inflammasome Activation by Pleurotus

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