Inhibition of P2X7R–NLRP3 Inflammasome Activation by Pleurotus

Article Cite This: J. Agric. Food Chem. 2018, 66, 13183−13190

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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,‡ Ming-Ji Jin,‡ Yue Shang,‡ Min Jiang,‡ Hong-Xu Yang,‡ Mei Wu,‡ Jian Liu,‡ Li-Hua Lian,*,‡ and Ji-Xing Nan*,‡,∥

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‡

Key Laboratory for Natural Resource of Changbai Mountain and Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin 133002, People’s Republic of China ∥ Clinical Research Center, Yanbian University Hospital, Yanji, Jilin 133002, People’s Republic of China § Analysis Center, Dt&CRO, Incorporated, Yongin-si, Gyeonggi-do 17042, Republic of Korea S Supporting Information *

ABSTRACT: Pleurotus citrinopileatus (golden oyster mushroom) is a widely used edible mushroom. We investigated the inhibitory effect of P. citrinopileatus aqueous extract against alcoholic steatohepatitis and its underlying mechanism. Acute and chronic ethanol-feeding murine models were established by intragastrically administering ethanol or feeding an ethanolcontaining Lieber−DeCarli liquid diet to male C57BL/6 mice. In both models, P. citrinopileatus decreased serum alanine aminotransferase (ALT), aspartate transaminase (AST), triglyceride (TG), and hepatic TG levels. Hematoxylin and eosin (HE) and Oil Red O staining confirmed that P. citrinopileatus ameliorated both acute and chronic alcoholic hepatosteatosis, characterized by regulation of lipid-metabolism-related proteins, including sirtuin 1 (SIRT1), AMP-activated kinase (AMPK), and sterol regulatory element-binding protein (SREBP1). P. citrinopileatus reversed inflammatory response via modulating purinergic receptor P2X ligand-gated ion channel 7 (P2X7R)-NOD-like receptor pyrin domain 3 (NLRP3) inflammasome. P. citrinopileatus restored the expressions of those proteins to a normal level. In addition, HepG2 cells were incubated with P. citrinopileatus prior to ethanol stimulation. P. citrinopileatus reduced ethanol exposure-induced lipid deposition. Concomitantly, P. citrinopileatus increased AMPK and SIRT1 expressions, which were reduced by ethanol treatment. P. citrinopileatus ameliorated alcoholic hepatic steatosis and accompanied inflammatory response via regulating SIRT1−AMPK and P2X7R− NLRP3 inflammasome activation, highlighting a promising strategy and utility of P. citrinopileatus for alcoholic steatohepatitis as dietary health supplements. KEYWORDS: Pleurotus citrinopileatus, alcoholic hepatosteatosis, AMPK, SIRT1, P2X7R

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INTRODUCTION

SIRT1−AMPK partnership might be a breakthrough of ALD treatments. Bacterial translocation plays a key role in the progression of ALD. Alcohol abuse leads to an increased permeability of the gut, which further results in an increasing level of lipopolysaccharide (LPS) in circulation.11 When translocated from the gut lumen to liver, LPS is recognized by toll-like receptors (TLRs). Among TLRs, TLR4 is mainly responsible for LPS recognition and activates nuclear factor-κB (NF-κB) signaling cascades, leading to interleukin 1β (IL-1β) synthesis. In addition, during the pathogenesis of ALD, extracellular adenosine triphosphate (ATP) is released from damaged hepatocytes and, consequently, aggravates hepatosteatosis.12 P2X ligand-gated ion channel 7 (P2X7R), an ATP-gated ion channel, initiates pro-inflammatory cascades via nucleotidebinding oligomerization domain (NOD)-like receptor pyrin domain 3 (NLRP3) inflammasome. NLRP3 inflammasome

Alcoholic liver disease (ALD) is caused by long-term alcohol consumption and can develop to liver fibrosis and cirrhosis.1 To date, the mainstay treatment for patients with all stages of ALD is alcohol abstinence,2 and adequate nutritional support is recommended in recent clinical guidelines.3,4 However, few therapeutic options exist for severe ALD, and little changes has been made for medical treatment of ALD thus far. Alcoholic steatosis, the earliest and most common response of liver to chronic alcohol exposure, is pathologically characterized by accumulation of lipid droplets in hepatocytes, mild inflammation, but without hepatic fibrosis.5 Promoted fatty acid synthesis and impaired β-oxidation by excessive alcohol intake consequently result in hepatic lipid accumulation.2 AMP-activated kinase (AMPK), a vital lipid regulator, is dysregulated by alcohol during alcoholic hepatosteatosis. Downregulated AMPK by alcohol decreases its ability to promote sterol regulatory element-binding protein 1 (SREBP1), which accelerates the progression of hepatic steatosis.6−8 Sirtuin 1 (SIRT1) plays a pivotal role in the regulation of hepatic fatty acid metabolism and inflammatory response by activating AMPK.9,10 Therefore, targeting the © 2018 American Chemical Society

Received: Revised: Accepted: Published: 13183

October 23, 2018 November 28, 2018 November 29, 2018 November 29, 2018 DOI: 10.1021/acs.jafc.8b05756 J. Agric. Food Chem. 2018, 66, 13183−13190

Article

Journal of Agricultural and Food Chemistry activates inflammatory caspases (i.e., caspase-1), which, in turn, catalyzes pro-inflammatory cytokines, including IL-1β.13 Pleurotus citrinopileatus, also known as “golden oyster mushroom”, is a popular edible mushroom, which is abundantly distributed in northeastern China, Japan, and Korea.14 P. citrinopileatus is considered as a “health food” with pharmacological effects, such as antioxidation,15,16 immunomodulation,17,18 antitumor,17 antiobesity,19 and anti-inflammatory activities.20,21 Hu et al.16 reported that P. citrinopileatus downregulated serum triglyceride (TG) and cholesterol levels. However, the effect of P. citrinopileatus on ALD remains elusive. Herein, we aimed to explore whether and how P. citrinopileatus would improve alcoholic hepatic steatosis using in vivo models of acute and chronic alcohol intake-induced hepatosteatosis and an in vitro model of HepG2 cells with ethanol. The results showed that P. citrinopileatus aqueous extract alleivated both acute and chronic mice alcoholic hepatosteatosis via P2X7R−NLRP3 inflammasome, suggesting the potential utility of P. citrinopileatus for ALD treatment.

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Figure 1. Animal experimental procedure. (A) Acute ethanol feeding model. C57BL mice were intragastrically administrated with three doses of ethanol (5 g/kg of body weight) every 12 h. P. citrinopileatus (100 mg/kg) was given by gavage with three doses just before ethanol administration. (B) Chronic ethanol feeding model. C57BL mice were fed with gradually increased concentrations of ethanol containing a Lieber−DeCarli liquid diet from 1 to 4% every 2 days and then followed by 5% ethanol for 28 days. P. citrinopileatus (50 or 100 mg/kg) was gavaged daily for 28 days.

MATERIALS AND METHODS

Reagents. Anti-SIRT1, anti-P2X7R, anti-SREBP1, anti-AMPKα, anti-P-AMPKα, and anti-GAPDH were purchased from Abcam (Cambridge, MA, U.S.A.). A438079 were from Abcam (Cambridge, MA, U.S.A.). A selective SIRT1 activator, SRT2104, was purchased from Selleck Chemicals (Houston, TX, U.S.A.). Metformin, a LKB1/ AMPK activator, was obtained from Beyotime Institute of Biotechnology (Haiman, Jiangsu, China). Preparation of the P. citrinopileatus Extract. P. citrinopileatus was collected from a local farm (Wangqing, Jilin, China). Dried fruiting bodies of P. citrinopileatus (50 g) was ground and extracted with 250 mL of 80 °C water 3 times for 2, 1, and 1 h. Then, combined crude aqueous extract was filtered with Whatman grade 1 filter paper, concentrated with a rotary evaporator at 80 °C to 100 mL, followed by fractional precipitation with 80% (v/v) alcohol overnight, and then finally lyophilized. These processes produced 0.65 g of 80% (v/v) fraction. Compounds from P. citrinopileatus extract were analyzed using an UltiMate 3000 ultra performance liquid chromatography system (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). The chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (2.1 × 100 mm inner diameter, 1.7 μm). The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B). The gradient condition was 5−90% B at 1−25 min, 100% B at 25.1−26 min, 100−5% B at 26−26.5 min, and 5% B at 26.5−30 min for equilibration. The flow rate was 0.3 mL/min. The separated substance was characterized by a TripleTOF 5600+ hybrid triple quadrupole time-of-flight mass spectrometer (SCIEX, Framingham, MA, U.S.A.). The following mass spectrometer (MS) conditions were used: ion spray voltage, 5.5 kV; decluttering potential (DP), 80 V; the turbo spray temperature, 500 °C; nebulizer gas (gas 1) of 50 psi; heater gas (gas 2), 50 psi; and curtain gas, 25 psi. Nitrogen was kept as a nebulizer and auxiliary gas. The time-of-flight mass spectrometry (TOF MS) scan was operated with the mass range of m/z 300−1800. Recalibration was carried out by a Calibrant delivery system before analysis. Animal Experiments. The 8−10 week old male C57BL/6 mice (20−22 g) were purchased from Yisi Laboratory Animal Technology Co., Ltd. (Changchun, Jilin, China) [SPF, SCXK (J) 2016-0003]. The mice were housed under a constant temperature (22 ± 2 °C), relative humidity (50−60%), and light (12 h light−dark cycles) conditions with a standard laboratory chow diet ad libitum. All mice were handled in compliance with the Guide for the Care and Use of Laboratory Animals (eighth edition, National Research Council), and all animal procedures were reviewed and approved by the Animal Research Ethic Committee of Yanbian University, China. Murine models of acute and chronic alcoholic hepatosteatosis were carried out as follows: (1) For acute ethanol feeding (Figure 1A), all mice

were randomly divided into the following three groups: normal group, ethanol group, and ethanol plus P. citrinopileatus group (100 mg/kg of body weight). Mice were treated with three intragastric doses of ethanol (5 g/kg of body weight) or isocaloric/isovolumetric maltose dextrin every 12 h.22,23 Mice in the ethanol plus P. citrinopileatus group were gavaged with three doses of P. citrinopileatus extract every 12 h at just before ethanol administration. The P. citrinopileatus extract was diluted with saline. At 4 h after the last dose of alcohol intake, mice were anesthetized with isoflurane and blood samples were collected by cardiac puncture. Liver tissues was removed and immediately snap-frozen in liquid nitrogen, and blood samples were collected. (2) For chronic ethanol feeding (Figure 1B), C57BL/6 mice were randomly divided into the following four groups: pair-fed group, ethanol-fed group, and ethanol-fed mice treated with P. citrinopileatus groups (50 or 100 mg/kg of body weight). The ethanol group was fed with an increasing concentration of ethanol in a Lieber−DeCarli liquid diet. Ethanol concentrations were ramped up from 1 to 4% (v/v), with every concentration lasting for 2 days, and then followed by 5% ethanol for a continuous 28 days. During these 4 weeks, ethanol-fed plus P. citrinopileatus groups were gavaged daily with 50 or 100 mg/kg of P. citrinopileatus extract. The pair-fed group was given pair-fed diets substituted with isocaloric maltose dextrin. At 9 h after the last dosing, all mice were sacrificed under anesthesia, followed by the collection of blood and liver tissue samples. Serum Biochemical Parameters. Slanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of mice serum were detected by the dry chemistry blood analyzer SPOTCHEM SP4410 (Arkray, Inc., Kyoto, Japan). TG concentrations in mice serum and liver tissues were measured by an enzymic kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, Jiangsu, China) according to the instructions of the manufacturer. Enzyme-Linked Immunosorbent Assay (ELISA). Levels of murine IL-1β protein were measured using Mouse IL-1β DuoSet ELISA (R&D Systems, Minneapolis, MN, U.S.A.) according to the protocols of the manufacturer. Histopathological and Immunohistochemistry Examination. Liver tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned 5 μm thick, and stained with 13184

DOI: 10.1021/acs.jafc.8b05756 J. Agric. Food Chem. 2018, 66, 13183−13190

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Journal of Agricultural and Food Chemistry hematoxylin and eosin (HE). The 5 μm thick cryosections of mice liver were stained with Oil Red O and counterstained with hematoxylin. In immunohistochemistry, after incubation overnight with primary antibodies (anti-SIRT1, anti-AMPKα, anti-SREBP1, anti-P2X7R, and anti-NLRP3 antibodies) in a humidified chamber at 4 °C, mouse tissue sections were incubated for 30 min at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody included in a MaxVision HRP-Polymer anti-Mouse/ Rabbit Immunohistochemistry (IHC) Kit (Fujian, Fuzhou, China) and incubated with diaminobenzidine (DAB) chromogen, followed by counterstaining with hematoxylin. Cell Culture and Nile Red Staining. HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin in a 37 °C and 5% CO2 humidified incubator. HepG2 cells were fixed and stained with 100 ng/mL Nile Red solution. Cells were washed with phosphate-buffered saline with 0.05% Tween 20 (PBST) several times and mounted with UltraCruz aqueous mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). Western Blot. The protein from the cells or livers were lysed by radioimmunoprecipitation assay (RIPA) buffer. The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) was used to extract protein in nuclear and cytoplasmic fractions of liver cells. Equal amounts of protein were separated using 10 or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE) and then transferred to the Amersham Hybond P polyvinylidene fluoride (PVDF) membrane (GE Healthcare Bio-Sciences, Pittsburgh, PA, U.S.A.). After blocking, the membranes were washed briefly in PBST and incubated with the specific primary antibodies overnight at 4 °C with gentle agitation. The blots were washed with PBST and then incubated with the appropriate HRP-conjugated secondary antibodies. Membranes were finally developed with Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.), and the intensity of bands was quantified with Quantity One software (BioRad Laboratories, Inc., Hercules, CA, U.S.A.). Statistical Analysis. All values are expressed as the mean ± standard deviation (SD). The statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests using the GraphPad Prism program (GraphPad Software, Inc., San Diego, CA, U.S.A.). A p value of less than 0.5 was regarded as statistically significant.

alcohol exposure caused the remarkable increase of hepatic and serum TG concentrations (Figure 3B). We also confirmed that alcohol consumption leads to histological alterations of liver according to HE and Oil Red O staining data (panels C and D of Figure 3). P. citrinopileatus pretreatment reduced the ethanol-induced elevation level of serum aminotransferases as well as serum and hepatic TG accumulation. Additionally, hepatic lipid accumulation was notably diminished by P. citrinopileatus pretreatment compared to those in the ethanol group (Figure 3D). These data indicated that acute alcohol intake induced a significant lipid accumulation in alcoholgavaged mice, which could be attenuated by P. citrinopileatus administration. In addition, P. citrinopileatus did not affect ALT, AST, and lipid accumulation within 4 weeks when gavaged to mice at 100 mg/kg dose (Supplementary data F1 of the Supporting Information). P. citrinopileatus Reversed Acute Alcohol ExposureInduced Steatohepatitis through Regulating Lipid Oxidation and Synthesis. Growing evidence demonstrated that stimulation of hepatic SIRT1−AMPK signaling by nutritional or pharmacological intervention protects against the development of ALD in rodents.24 We hypothesized that P. citrinopileatus could interfere with the development of lipid accumulation by regulating the SIRT1−AMPK−SREBP1 axis. Therefore, the expressions of SIRT1, AMPK, and SREBP1 were determined by immunohistochemistry in acute alcoholexposed mice. P. citrinopileatus pretreatment increased the expressions of SIRT1 and AMPK, which were obviously decreased after acute alcohol consumption (panels A and B of Figure 4). Simultaneously, P. citrinopileatus downregulated the expression of SREBP1 evoked by alcohol intake (Figure 4C), which was consistent with dynamically altered TG levels shown in Figure 3B. We also employed western blotting analysis to detect the protein expressions of AMPK and the nuclear active form of SREBP1 (nSREBP1). Alcohol consumption significantly suppressed AMPK phosphorylation, and P. citrinopileatus stimulated AMPK phosphorylation as expected (panels F and G of Figure 4). SREBP1 expression was increased in nucleus fractions after ethanol treatment, while pyruvate carboxylase (PC) administration suppressed the SREBP1 expression in the nucleus (Figure 4F). These results hinted that P. citrinopileatus ameliorated hepatic lipid accumulation by regulating lipogenesis and lipolysis and eventually attenuated impaired the hepatic lipid metabolism balance through regulation of SIRT1−AMPK. P. citrinopileatus inhibited P2X7R and NLRP3 inflammasome activation after ethanol exposure, confirmed by immunohistochemistry staining (panels D and E of Figure 4). The IL-1β level in serum was elevated after alcohol consumption, while P. citrinopileatus pretreatment declined IL-1β levels (Figure 4H). Consistent with immunohistochemistry staining data, the protein expression of P2X7R was suppressed by P. citrinopileatus administration, which was increased by alcohol intake (panels F and G of Figure 4). P. citrinopileatus Prevented Lipid Accumulation in Chronic Alcoholic Hepatic Steatosis. Considering the beneficial effect of P. citrinopileatus against acute alcohol bingecaused hepatic steatosis, we were intrigued whether P. citrinopileatus could reverse chronic ethanol feeding-induced hepatic steatosis and inflammation. After mice were fed with an alcohol-containing Lieber−DeCarli liquid diet for 4 weeks, serum ALT and AST levels were significantly increased (Figure 5A). The increase of aminotransferases was abolished by

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RESULTS P. citrinopileatus Reversed Acute Alcohol IntakeInduced Hepatic Steatosis. Profiling of compounds from P. citrinopileatus extract was analyzed by a mass spectrometer (Figure 2). Then, we utilized the Formula Finder tool in

Figure 2. Base peak chromatogram of P. citrinopileatus.

PeakView software (SCIEX) and ChemSpider database to search and identify compounds from P. citrinopileatus. As shown in Tables 1 and 2, a total of 24 compounds were identified from P. citrinopileatus. Serum ALT and AST levels were increased after mice were gavaged with three doses of alcohol (Figure 3A). In addition, 13185

DOI: 10.1021/acs.jafc.8b05756 J. Agric. Food Chem. 2018, 66, 13183−13190

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Journal of Agricultural and Food Chemistry Table 1. Identity of Amino Acids Based on Accurate Mass and Empirical Formulas of the P. citrinopileatus Extract number

name

formula

mass (Da)

found at mass (Da)

adduct

found at RT (min)

intensity

area

error (ppm)

error (mDa)

1 2 3 4 5 6 7 8 9 10 11 12 13

arginine asparagine aspartic acid glutamic acid histidine isoleucine/leucine lysine phenylalanine proline serine tryptophan tyrosine valine

C6H14N4O2 C4H8N2O3 C4H7NO4 C5H9NO4 C6H9N3O2 C6H13NO2 C6H14N2O2 C9H11NO2 C5H9NO2 C3H7NO3 C11H12N2O2 C9H11NO3 C5H11NO2

174.11 132.05 133.04 147.05 155.07 131.09 146.11 165.08 115.06 105.04 204.09 181.07 117.08

175.12 133.06 134.04 148.06 156.08 132.1 147.11 166.09 116.07 106.05 205.1 182.08 118.09

+H +H +H +H +H +H +H +H +H +H +H +H +H

1.19 1.31 1.32 1.31 1.19 2.09 1.18 3.08 1.33 1.31 5.09 1.75 1.4

175844.7 10644 5703 82472.14 20722.24 912034.1 87489.26 1348427 198231.4 8320 260289.3 606607.3 207618.9

11418.75 569.68 542.21 4990.74 1129.72 87836.14 4562.18 156869.37 10870.70 417.99 19385.62 36550.80 18244.96

0.55 0.76 0.96 −0.68 0.07 0.91 0.85 −0.02 −0.19 −1.01 0.62 0.27 0.52

0.10 0.10 0.13 −0.10 0.01 0.12 0.13 0.00 −0.02 −0.11 0.13 0.05 0.06

citrinopileatus increased the expression of SIRT1 in a concentration-dependent manner. P. citrinopileatus especially exhibited much superior ability upon stimulating SIRT1 compared to SRT2104, a SIRT1 activator (Figure 7D). P. citrinopileatus alone at a concentration of 100 μM did not affect lipid synthesis.

pretreatment with P. citrinopileatus. Four weeks of alcohol consumption caused significantly higher serum and hepatic TG contents, while P. citrinopileatus effectively altered TG contents in P. citrinopileatus treatment groups (Figure 5B). As shown in panels C and D of Figure 5, chronic ethanol feeding induced an obvious lipid accumulation, which could be ameliorated by P. citrinopileatus administration. Our data suggested that P. citrinopileatus could also effectively alleviate lipid accumulation in the liver caused by chronic alcohol exposure. Inhibition of P2X7R−NLRP3 Activation by P. citrinopileatus Contributed to Lipid Accumulation in Chronic Ethanol Feeding-Induced Hepatic Steatohepatitis. We determined the expressions of lipid-metabolism-related proteins, such as SIRT1, AMPK, and SREBP1, by immunohistochemistry. As shown in panels A and B of Figure 6, the positive staining area of SIRT1 and AMPK obviously reduced in the ethanol-fed group, while P. citrinopileatus administration restored SIRT1 and AMPK activities. Protein expressions of total and phosphorylated AMPK were reduced after chronic ethanol feeding and restored by P. citrinopileatus (Figure 6F). As expected, chronic ethanol feeding increased the expression of SREBP1 confirmed by immunohistochemistry and western blot, which can be reversed by P. citrinopileatus (panels C and F of Figure 6). These data demonstrated that P. citrinopileatus prevented chronic alcoholic hepatic steatosis through SIRT1− AMPK−SREBP1 signaling. Long-term alcohol intake also promoted IL-1β release and upregulated protein expressions of P2X7R and NLRP3; meanwhile, P. citrinopileatus abolished the alcohol-induced IL-1β release and the protein expressions of P2X7R and NLRP3 (panels D−F and H of Figure 6). P. citrinopileatus Regulated Lipid Accumulation in Ethanol-Induced Steatotic Hepatocytes. As a human hepatoma cell line, HepG2 cells were incubated with ethanol at a concentration of 50 mM to induce intracellular lipid accumulation. Nile Red staining was employed to evaluate whether P. citrinopileatus could inhibit lipid accumulation in ethanol-exposed HepG2 cells. Metformin was used as a positive control, which could activate AMPK signaling. As shown in Figure 7A, ethanol treatment increased the formation of lipid droplets in HepG2 cells; meanwhile, P. citrinopileatus concentration-dependently alleviated lipid accumulation. P. citrinopileatus exhibited superior ability in controlling lipid accumulation compared to metformin. P. citrinopileatus also reversed alcohol-induced decreasing of total and phosphorylated AMPK expressions (panels B and C of Figure 7). P.

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DISCUSSION ALD is a major global public health problem, resulting in millions of deaths. Thus far, significant efforts have been made to understand the molecular events and cellular mechanisms in the development and progression of ALD. The classic therapies of ALD include nutritional support, corticosteroids, tumor necrosis factor α inhibitor (pentoxifylline), and phosphodiesterase, unfortunately with serious side effects and unsatisfactory outcomes.25−27 Therefore, it is necessary to explore dietary supplements from food targeted for ALD therapy. Recently edible mushrooms, such as P. citrinopileatus, received substantial investigative attention for the biofunction and application as medicinal purposes and also as nutritional products as a result of the significant benefits of low contamination and high economic value.16 We here reported that P. citrinopileatus alleviated hepatosteatosis induced by acute and chronic alcohol consumption, especially inhibiting lipid accumulation through modulating SIRT1−AMPK and the inflammatory response via inhibiting P2X7R−NLRP3 in hepatocytes. To confirm whether P. citrinopileatus could ameliorate alcoholic hepatosteatosis, we established acute or chronic alcoholic hepatosteatosis models. Excessive alcohol intake amplifies inflammation in liver and further impairs hepatocyte mitochondrial functions, eventually inducing steatosis and inflammation in mice.16,28−30 Alcohol intake simultaneously increases gut permeability and activates LPS−TLR4 signaling. According to preliminary experiments and published reports,19,31,32 we selected 50 or 100 mg/kg as the dose of P. citrinopileatus. Especially at a dose of 100 mg/kg, P. citrinopileatus did not affect any normal liver function. In addition, a human equivalent dose (HED) of 100 mg/kg P. citrinopileatus in mice33 can be converted to 480 mg daily for a 60 kg man, which is equivalent to approximately 40 g of dried P. citrinopileatus. According to our data, both acute alcohol exposure and chronic ethanol feeding caused the increase in the levels of serum ALT, AST, and TG, which were attenuated by P. citrinopileatus administration (panels A and B of Figures 3 13186

DOI: 10.1021/acs.jafc.8b05756 J. Agric. Food Chem. 2018, 66, 13183−13190

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0.56 0.25 −0.04 0.14 0.50 0.86 1.37 −0.82 0.12 −0.11 0.13 2.64 1.07 −0.18 0.73 2.19 3.48 4.86 −2.03 0.91 −0.68 0.62 8280.43 9380.77 104260.03 48059.82 54629.84 10144.47 11653.48 60477.64 87836.14 16341.59 7588.01 157248.06 165791.46 1489484.70 653094.72 814146.00 177992.51 175577.84 853393.59 912034.13 300284.25 92126.59 8.16 5.68 1.40 5.09 5.79 6.58 26.46 11.57 2.09 1.75 1.32 +H +H +H +H +H +H +H +H +H +H +Na 210.14 230.16 201.17 187.064 226.17 245.16 280.24 400.26 131.09 164.05 182.08 2-methyl-2-propanyl 4-cyano-1-piperidinecarboxylate carboxylate 11-aminoundecylic acid N-benzylmaleimide tert-butyl 2,7-diazaspiro[4.4]nonane-2-carboxylate 7-(boc-amino)heptanoic acid linoleic acid actinopyrone a aminocaproic acid coumaric acid mannitol 1 2 3 4 5 6 7 8 9 10 11

C11H18N2O2 C11H22N2O3 C11H23NO2 C11H9NO2 C12H22N2O2 C12H23NO4 C18H32O2 C25H36O4 C6H13NO2 C9H8O3 C6H14O6

211.14 231.17 202.18 188.07 227.18 246.17 281.25 401.27 132.10 165.06 205.07

intensity found at RT (min) adduct found at mass (Da) mass (Da) formula name number

Table 2. Identity of Other Compounds Based on Accurate Mass and Empirical Formulas of the P. citrinopileatus Extract

area

error (ppm)

error (mDa)

Journal of Agricultural and Food Chemistry

Figure 3. P. citrinopileatus attenuated acute ethanol intake-induced hepatic lipid accumulation. Mice were gavaged with three doses of ethanol (5 g/kg of body weight) every 12 h. P. citrinopileatus (100 mg/kg) was gavaged just before ethanol administration. (A) ALT and AST levels in serum. (B) TG contents in serum and liver. HE staining (C, 200 × original magnification) and Oil Red O staining (D, 400 × original magnification). Black arrows indicate mean lipid droplets in mice liver. Each value is expressed as the mean ± SD (n = 6). (###) p < 0.001, significantly different when compared to the normal group. (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001, significantly different when compared to the ethanol group.

and 5). P. citrinopileatus decreased the lipid accumulation in liver tissue significantly (panels C and D of Figures 3 and 5), indicating the antisteatotic effect of P. citrinopileatus. Furthermore, we established an in vitro model using human hepatoma cell line HepG2 cells to further verify the inhibitory effect of P. citrinopileatus on lipid accumulation. P. citrinopileatus exhibited a stronger inhibitory effect on attenuating lipid accumulation than metformin in ethanolinduced steatotic hepatocytes (Figure 7). Our results suggested that P. citrinopileatus suppressed lipid accumulation in alcoholic hepatosteatosis, and this inhibitory capacity of P. citrinopileatus was especially more focused to regulate lipid metabolism in hepatocytes. Hepatosteatosis is an initiation stage of ALD. Accumulation of TG in cellular lipid droplets leads to hepatic steatosis. Alcohol exposure promotes hepatic lipid accumulation by interfering with the balance of lipogenesis and lipolysis, which was directly or indirectly regulated by lipid-metabolismassociated key transcription factors, such as SIRT1, AMPK, and SREBP1. We were intrigued whether the antisteatotic effect of P. citrinopileatus might be exerted through the modulation of the SIRT1−AMPK axis. In both murine models of acute and chronic alcoholic hepatosteatosis, P. citrinopileatus activated AMPK and SIRT1 and decreased SREBP1 expression, indicating the elevated fatty acid β-oxidation and reduced lipogenesis. Our in vitro data suggested that P. citrinopileatus abolished hepatic lipid accumulation through regulating SIRT1−AMPK signaling in ethanol-exposed steatotic hepatocytes. AMPK and SIRT both regulate each other, and AMPK can function as a SIRT1 activator.34 We applied an AMPK activator, metformin, and a SIRT1 activator, SRT2104, 13187

DOI: 10.1021/acs.jafc.8b05756 J. Agric. Food Chem. 2018, 66, 13183−13190

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Journal of Agricultural and Food Chemistry

Figure 4. P. citrinopileatus suppressed lipid metabolism and inflammation in acute alcohol hepatosteatosis. Immunohistochemistry staining for (A) SIRT1, (B) AMPKα, (C) SREBP1, (D) P2X7R, and (E) NLRP3 (200 × original magnification). Black arrows indicate positive staining in mice liver. (F) Protein expressions of AMPKα, phosphorylated AMPKα, P2X7R, and nSREBP1 were detected by western blotting. (G) Each protein density was normalized to GAPDH or TOPO1. (H) Serum IL-1β protein level was measured by ELISA. (##) p < 0.01 and (###) p < 0.001, significantly different when compared to the normal group. (∗∗∗) p < 0.001, significantly different when compared to the ethanol group.

Figure 5. P. citrinopileatus attenuated chronic ethanol feeding-induced hepatosteatosis. C57BL/6 mice were fed with an ethanol-containing Lieber−DeCarli liquid diet. (A) ALT and AST levels in serum. (B) TG contents in serum and liver. (C and D) HE staining (200 × original magnification) and Oil Red O staining (200 × original magnification). Each value is expressed as the mean ± SD (n = 6). (#) p < 0.05 and (##) p < 0.01, significantly different from the pair-fed group. (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001, significantly different when compared to the ethanol-fed group.

Figure 6. P. citrinopileatus inhibited lipid metabolism in chronic ethanol feeding-induced steatohepatitis. Immunohistochemistry staining for (A) SIRT1, (B) AMPKα, (C) SREBP1, (D) P2X7R, and (E) NLRP3 (200 × original magnification). Black arrows indicate positive staining in mice liver. (F) Protein expressions of AMPKα, phosphoAMPKα, P2X7R, and nSREBP1 were detected by western blotting. (G) Each protein density was normalized to GAPDH or TOPO1. (H) Serum IL-1β protein level was measured by ELISA. (##) p < 0.01 and (###) p < 0.001, significantly different when compared to the pair-fed group. (∗) p < 0.05 and (∗∗∗) p < 0.001, significantly different when compared to the ethanol-fed group.

to verify the stimulating effectiveness of SIRT1. Metformin and SRT2104 restored SIRT1 to a certain extent, which was decreased by alcohol exposure; meanwhile, P. citrinopileatus completely restored SIRT1 to a normal level (Figure 7). Our 13188

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supplements for preventing and treating alcoholic steatohepatitis.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b05756. Effect of P. citrinopileatus on liver function (Supplementary data F1) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: 86-433-2435061. Fax: 86-433-2435072. E-mail: [email protected]. *Telephone: 86-433-2435061. Fax: 86-433-2435072. E-mail: [email protected]. ORCID

Ji-Xing Nan: 0000-0002-6221-4309 Author Contributions †

Xia Li and Quan Jin contributed equally to this work.

Author Contributions

Xia Li and Quan Jin are the primary investigators in this study. Yu Zhang, Min Jiang, Ben-Wen Cui, and Ying Li participated in part of the in vivo experiments. Ming-Ji Jin, Ying Li, Yue Shang, Hong-Xu Yang, Mei Wu, and Jian Liu participated in part of the in vitro experiments. Yan-Ling Wu participated in part of the statistical analysis. Cheng-Min Jin analyzed compounds from the P. citrinopileatus extract. Li-Hua Lian and Ji-Xing Nan designed the whole study and wrote the manuscript.

Figure 7. P. citrinopileatus regulated lipid accumulation in ethanolexposed HepG2 cells. HepG2 cells were pretreated with the P. citrinopileatus extract, metformin, or SRT2104 at 1 h prior to ethanol stimulation (50 mM) and then continuously incubated for 24 h. (A) Lipid deposition in HepG2 cells was assessed by immunofluorescence staining for Nile Red (200 × original magnification). (B) Total and phosphorylated AMPKα protein expressions of HepG2 cells were evaluated by western blotting. Each protein density was normalized to GAPDH. (###) p < 0.001, significantly different from untreated cells. (∗∗∗) p < 0.001, significantly different from ethanol-treated cells. (D) Immunofluorescence staining for SIRT1 of HepG2 cells (200 × original magnification).

Funding

This study was supported by grants from the National Natural Science Foundation of China (81560597, 81660689, 81460564, and 81860751) and partially supported by the Science and Technology Planning Projects from the Science and Technology Department of Jilin Province (20160101205JC, 20180414048GH, 20180201065YY, and 20180519010JH).

data suggested that P. citrinopileatus might activate the hepatic SIRT1−AMPK axis, which leads to the attenuation of lipid accumulation, thereby protecting liver from alcohol damage. Lipid accumulation induced by sustained alcohol consumption leads to hepatocyte damage and inflammation, which will directly promote the progression of alcoholic steatohepatitis. ATP released from damaged hepatocytes at sites of inflammation will bind to P2X7R on neighboring cells. This binding results in the activation of NLRP3 inflammasome, which stimulates the cleavage of pro-IL-1β and secretion of mature IL-1β.35 We confirmed that upregulation of P2X7R by alcohol intake is synchronized with activation of NLRP3 inflammasome as well as its downstream cytokine IL-1β (Figures 4 and 6). Therefore, we detected whether P. citrinopileatus could inhibit the expressions of P2X7R and NLRP3 in both acute and chronic alcoholic hepatosteatotic murine models. Alcohol treatment increased the expressions of P2X7R and NLRP3 in both models, while P. citrinopileatus completely suppressed P2X7R and NLRP3 expressions, suggesting that the inhibitory ability of P. citrinopileatus of alcohol-induced steatohepatitis might be linked to the blockade of P2X7R−NLRP3 inflammasome activation. Collectively, we first revealed the hepatoprotective effect of P. citrinopileatus against alcoholic hepatosteatosis, and its capacity on the regulation of lipid accumulation by activating SIRT1−AMPK signaling might be tightly linked with P2X7R− NLRP3 inflammasome. Our data demonstrated the possibility of developing P. citrinopileatus as potential dietary health

Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ALD, alcoholic liver disease; AH, alcoholic hepatitis; AMPK, AMP-activated kinase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-1β, interleukin 1β; LPS, lipopolysaccharide; NLRP3, NOD-like receptor pyrin domain 3; P2X7R, purinergic receptor P2X ligand-gated ion channel 7; SREBP, sterol regulatory element-binding protein; SIRT1, sirtuin 1; TLR, toll-like receptor

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