Styrylpyrones from Phellinus linteus Mycelia Alleviate Non-Alcoholic Fatty Liver by Modulating Lipid and Glucose Metabolic Homeostasis in High-Fat and High-Fructose Diet-Fed Mice

Abstract

Phellinus linteus (PL), an edible and medicinal mushroom containing a diversity of styrylpyrone-type polyphenols, has been shown to have a broad spectrum of bioactivities. In this study, the submerged liquid culture in a 1600-L working volume of fermentor was used for the large-scale production of PL mycelia. Whether PL mycelia extract is effective against nonalcoholic fatty liver disease (NAFLD) is still unclear. In the high fat/high fructose diet (HFD)-induced NAFLD C57BL/6 mice study, the dietary supplementation of ethyl acetate fraction from PL mycelia (PL-EA) for four weeks significantly attenuated an increase in body weight, hepatic lipid accumulation and fasting glucose levels. Mechanistically, PL-EA markedly upregulated the pgc-1α, sirt1 genes and adiponectin, downregulated gck and srebp-1c; upregulated proteins PPARγ, pAMPK, and PGC-1α, and downregulated SREBP-1 and NF-κB in the liver of HFD-fed mice. Furthermore, the major purified compounds of hispidin and hypholomine B in PL-EA significantly reduced the level of oleic and palmitic acids (O/P)-induced lipid accumulation through the inhibition of up-regulated lipogenesis and the energy-metabolism related genes, ampk and pgc-1α, in the HepG2 cells. Consequently, these findings suggest that the application of PL-EA is deserving of further investigation for treating NAFLD.

Keywords: NAFLD; Phellinus linteus; centrifugal partition chromatography (CPC); dyslipidemia; hepatoprotection; hispidin; hypholomine B; mice; styrylpyrone polyphenolics.

Figure 1

Experimental procedures of solvent extraction, partition and purification of extract prepared using the Phelinus linteus mycelia extract and HPLC analysis of extracts obtained in each procedure. (A) 75% methanol. (B) ethyl acetate. (C) hispidin. (D) hypholomine B and hypholomine B isomer. Peaks 1: hispidin; 2: hypholomine B, and 3: hypholomine B isomer.

Figure 2

Radical scavenging capabilities of different preparations fractionated from P. linteus mycelia. (a) for DPPH radicals. (b) for ABTS⁺ radicals. PL: 75% methanol crude extract. PL-EA: ethyl acetate fraction. PL-H: CPC isolated hispidin. PL-HB: CPC isolated hypholomine B and hypholomine B isomer. Data are expressed as mean ± SD from triplicate experiments. Different letters in lower case on each curve indicate significantly different from each other (p < 0.05).

Figure 3

PL-EA inhibits high-fat high-fructose (HFD)-induced obese in mice. HFD: high-fat/high-fructose diet. PL-EA-H: HDF + high dose PL-EA (70 mg/kg). PL-EA-L: HDF + low dose PL-EA (35 mg/kg). Data are expressed as mean ± SE (n = 9). ## p <0.01; ### p < 0.001 vs. the HFD group; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. the control.

Figure 4

Effects of ethyl acetate fraction of P. linteus mycelia on the lipid profile. (a) total plasma cholesterol content, (b) content of plasma high density lipoprotein cholesterol, (c) content of plasma low density lipoprotein cholesterol, (d) the ratio LDL-C/HDL-C and (e) the plasma TG content. HFD: high-fat/high-fructose diet (HFD). PL-EA-L: HDF + low dose PL-EA (35 mg/kg). PL-EA-H: HDF + high dose PL-EA (70 mg/kg). Data are expressed as mean ± SE (n = 9). ## p < 0.01; ### p < 0.001 vs. the HFD group; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. the control. ^& p < 0.05 significant differences between the groups.

Figure 5

Effects of ethyl acetate fraction from P. linteus mycelia on the activities of (a) plasma aspartate aminotransferase and (b) plasma alanine aminotransferase. HFD: high-fat/high-fructose diet (HFD). PL-EA-L: HDF + low dose PL-EA (35 mg/kg). PL-EA-H: HDF + high dose PL-EA (70 mg/kg). Data are expressed as mean ± SE (n = 9). ## p <0.01; ### p < 0.001 vs. the HFD group; * p < 0.05; *** p < 0.001 vs. the control.

Figure 6

Effects of ethyl acetate fraction from P. linteus mycelia on the OGTT at week 9 (a) and at week 14 (b). HFD: high-fat/high-fructose diet (HFD). PL-EA-L: HDF + low dose PL-EA (35 mg/kg). PL-EA-H: HDF + high dose PL-EA (70 mg/kg). Data are expressed as mean ± SE (n = 9). ## p < 0.01; vs. the HFD group; ** p < 0.01; *** p < 0.001 vs. the control.

Figure 7

Effects of PL-EA on the hepatic lipogenesis in NAFLD mouse model. Representative photographs of hematoxylin-eosin (H&E) (A) and Oil Red O staining (B) of mice liver tissues for histological examination. (a-1,a-2) control. (B) (b-1,b-2) HFD, high-fat/high-fructose diet. (c-1,c-2) PL-EA-L: HDF + low dose PL-EA (35 mg/kg). (d-1,d-2) PL-EA-H: HDF + high dose PL-EA (70 mg/kg). Magnification, 200×.

Figure 8

Effects of purified compounds from PL-EA on Oil Red O staining and lipid. accumulation in HepG2 cells. Lipid droplets in HepG2 cells were photographed by phase contrast microscopy (original magnification ×200) (A). The lipid content from Oil Red O stained cells was quantified by spectrophotometric analysis at 500 nm (B). Bars represent mean ± SE (n = 3) *** p < 0.001 vs. the control; ### p < 0.001 vs. the O/P group; ^&&& p < 0.001. Significant differences between groups were determined using one-way ANOVA followed by Tukey’s procedure.

Figure 9

Effects of ethyl acetate fraction from P. linteus mycelia on the relative gene expression of mice livers. HFD: high-fat/high-fructose diet (HFD). PL-EA-L: HDF + low dose PL-EA (35 mg/kg). PL-EA-H: HDF + high dose PL-EA (70 mg/kg). Data are expressed as mean ± SE (n = 9). * and ***, p < 0.05 and 0.001, respectively vs. the control; ^#, ^## and ^###, p < 0.05, 0.01 and 0.001, respectively vs. the O/P group. ^&& and ^&&&, p < 0.01 and 0.001, respectively, significant differences between the groups. PGC1-α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Sirt-1: NAD-dependent deacetylase sirtuin-1; SREBP-1c: sterol regulatory element-binding protein 1c.

Figure 10

Effects of hispidin and hypholomine B isolated from the ethyl acetate fraction of P. linteus mycelia on the relative gene expression of HepG2 cell. Data are expressed as mean ± SE (n = 3). *, **, and ***, p < 0.05, 0.01 and 0.001, respectively vs. the control; ^#, ^## and ^###, p < 0.05, 0.01 and 0.001, respectively vs. the O/P group. PGC1-α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Sirt-1: NAD-dependent deacetylase sirtuin-1; SREBP-1c: sterol regulatory element-binding protein 1c.

Figure 11

Effects of ethyl acetate fraction from P. linteus mycelia on the relative proteins expression of mice livers. Representative Western blots (left panel) and quantified bar graphs relative to each control (right panel) showing alterations among groups. HFD: high-fat/high-fructose diet (HFD). PL-EA-L: HDF + low dose PL-EA (35 mg/kg). PL-EA-H: HDF + high dose PL-EA (70 mg/kg). Data are expressed as mean ± SE (n = 9). # p < 0.05; ## p < 0.01; ### p < 0.001 vs. the HFD group; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. PPARγ: peroxisome proliferator-activated receptor gamma. AMPK: AMP activated protein kinase. p-AMPK: phosphorylated AMPK. PGC1-α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha. SREBP1: sterol regulatory element-binding protein 1. NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells.

Figure 12

Mechanism of action related to the alleviative effect of NAFLD in mice with the ethyl acetate partition from Phellinus linteus (a) and that of in vitro hypolipidemic effect of hispidin and hypolomine B in HepG2 cell model (b). In Figure 12b, the items highlighted in pink are not shown in the HepG2 cell model but were found in the in vivo mice model. The purified active components from PL-EA used in the in vitro experiment are hispidin (HPD) and hypholomine B and isomers (HLM). ALT: alanine aminotransferase (GPT). AMPK: AMP-activated protein kinase. AST: aspartic aminotransferase (GOT). FAS: fatty acid synthase. gck: glucokinase gene. HFD: high fat diet. HDL-C: high density lipoprotein–cholesterol. IR: insulin resistance. LDL-C: low density lipoprotein–cholesterol. NAFLD: non-alcoholic fatty liver disease. NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells. pAMPK: phosphorylated AMPK. sirt-1: NAD⁺-dependent deacetylase sirtuin-1 gene. PGC-1α: peroxisome proliferator-activated receptor-gamma coactivator-1α. pgc-1α: peroxisome proliferator-activated receptor-gamma coactivator-1α gene. PPAR-γ: peroxisome proliferator- activated receptor gamma (PPAR-γ). ROS: reactive oxygen species. SREBP-1c: sterol regulatory element-binding protein-1c. srebp-1c: sterol regulatory element-binding protein-1c gene. TC: plasma total cholesterol. TG: triglycerides. PL-EA: the ethyl acetate partition from Phellinus linteus.

Figure 12

Mechanism of action related to the alleviative effect of NAFLD in mice with the ethyl acetate partition from Phellinus linteus (a) and that of in vitro hypolipidemic effect of hispidin and hypolomine B in HepG2 cell model (b). In Figure 12b, the items highlighted in pink are not shown in the HepG2 cell model but were found in the in vivo mice model. The purified active components from PL-EA used in the in vitro experiment are hispidin (HPD) and hypholomine B and isomers (HLM). ALT: alanine aminotransferase (GPT). AMPK: AMP-activated protein kinase. AST: aspartic aminotransferase (GOT). FAS: fatty acid synthase. gck: glucokinase gene. HFD: high fat diet. HDL-C: high density lipoprotein–cholesterol. IR: insulin resistance. LDL-C: low density lipoprotein–cholesterol. NAFLD: non-alcoholic fatty liver disease. NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells. pAMPK: phosphorylated AMPK. sirt-1: NAD⁺-dependent deacetylase sirtuin-1 gene. PGC-1α: peroxisome proliferator-activated receptor-gamma coactivator-1α. pgc-1α: peroxisome proliferator-activated receptor-gamma coactivator-1α gene. PPAR-γ: peroxisome proliferator- activated receptor gamma (PPAR-γ). ROS: reactive oxygen species. SREBP-1c: sterol regulatory element-binding protein-1c. srebp-1c: sterol regulatory element-binding protein-1c gene. TC: plasma total cholesterol. TG: triglycerides. PL-EA: the ethyl acetate partition from Phellinus linteus.