Inhibitory Effect of Styrylpyrone Extract of Phellinus linteus on Hepatic Steatosis in HepG2 Cells

Abstract

The prevalence of nonalcoholic fatty liver disease (NAFLD) is estimated to be approximately about 25.24% of the population worldwide. NAFLD is a complex syndrome and is characterized by a simple benign hepatocyte steatosis to more severe steatohepatitis in the liver pathology. Phellinus linteus (PL) is traditionally used as a hepatoprotective supplement. Styrylpyrone-enriched extract (SPEE) obtained from the PL mycelia has been shown to have potential inhibition effects on high-fat- and high-fructose-diet-induced NAFLD. In the continuous study, we aimed to explore the inhibitory effects of SPEE on free fatty acid mixture O/P [oleic acid (OA): palmitic acid (PA); 2:1, molar ratio]-induced lipid accumulation in HepG2 cells. Results showed that SPEE presented the highest free radical scavenging ability on DPPH and ABTS, and reducing power on ferric ions, better than that of partitions obtained from n-hexane, n-butanol and distilled water. In free-fatty-acid-induced lipid accumulation in HepG2 cells, SPEE showed an inhibition effect on O/P-induced lipid accumulation of 27% at a dosage of 500 μg/mL. As compared to the O/P induction group, the antioxidant activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced by 73%, 67% and 35%, respectively, in the SPEE group. In addition, the inflammatory factors (TNF-α, IL-6 and IL-1β) were significantly down-regulated by the SPEE treatment. The expressions of anti-adipogenic genes involved in hepatic lipid metabolism of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were enhanced in the SPEE supplemented HepG2 cells. In the protein expression study, p-AMPK, SIRT1 and PGC1-α were significantly increased to 121, 72 and 62%, respectively, after the treatment of SPEE. Conclusively, the styrylpyrone-enriched extract SPEE can ameliorate lipid accumulation and decrease inflammation and oxidative stress through the activation of SIRT1/AMPK/PGC1-α pathways.

Keywords: HepG2 cells; NAFLD; Phellinus linteus; SIRT1/AMPK/PGC1-α pathway; hepatic steatosis; oil droplet accumulation.

Figure 1

The antioxidant activities of 75% methanol crude extract and its different solvent partitions including n-hexane, ethyl acetate, n-butanol and water from freeze-dried powder of Phellinus linteus mycelia. Each value represents the mean ± standard deviation (SD, n = 3) SD of triplicate experiments. (A) DPPH radical scavenging activity, (B) ABTS radical scavenging activity and (C) reducing power on ferric ions. Means with different letters are significantly different (p < 0.05).

Figure 2

Representative HPLC profile of main constituents present in the SPEE. The diode-array detector was utilized at the wavelength of 210–600 nm in the HPLC analysis. Peak 1: hispidin; 2: MW. 490; 3: hypholomine B; 4: hypholomine B isomer; 5: unidentified compound. Chemical structures of 1 and 3 from PubChem are cited in the figure.

Figure 3

The effects of O/P SPEE and their combined treatments on HepG2 cell proliferation at 24 hr. (A) The viability of HepG2 cells. (B) The cytotoxicity of SPEE on HepG2 cells. (C) The rescue of HepG2 cells treated with O/P 0.7 mM by SPEE within a dose range of 100 to 500 μg/mL. Data are expressed as mean ± SD from triplicate experiments (n = 3). The Student’s t-test was used to compare the means between two groups. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group. ^# p < 0.05 vs. the O/P group.

Figure 4

The effects SPEE on O/P-induced intracellular lipid accumulation analyzed by Oil Red O staining and Nile red staining in HepG2 cell. HepG2 cells were co-treated with 0.3 mM O/P and SPEE at 100, 150 and 500 μg/mL, respectively, for 24 h. Silibinin (25 μM) was used as the positive control. Oil-Red-stained cells were observed under inverted microscope and Nile-red-stained cells were assessed by phase-contrast fluorescence microscopy (magnification, ×200). (A) Control, (B) O/P 0.3 mM, (C) O/P 0.3 mM + Silibinin 25 µM, (D) O/P 0.3 mM + SPEE 100 μg/mL, (E) O/P 0.3 mM + SPEE 250 μg/mL and (F) O/P 0.3 mM + SPEE 500 μg/mL. Values are expressed as the mean ± SD (n = 3). * p < 0.05 and *** p < 0.001 vs. the control group. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs. the O/P group. ^&& p < 0.01 and ^&&& p < 0.001 vs. the O/P 0.3 mM + SPEE 100 μg/mL group.

Figure 5

The effects SPEE on O/P-induced intracellular reactive oxygen species (ROS). HepG2 cells were co-treated with 0.3 mM O/P and SPEE at 100, 150 and 500 μg/mL, respectively, for 24 h. Silibinin (25 μM) was used as the positive control. Intracellular ROS were assessed using DCFH-DA fluorescence staining and were observed by phase-contrast fluorescence microscopy (left panel, original magnification ×100). (A) Control, (B) O/P 0.3 mM, (C) O/P 0.3 mM + Silibinin 25 µM, (D) O/P 0.3 mM + SPEE 100 μg/mL, (E) O/P 0.3 mM + SPEE 250 μg/mL and (F) O/P 0.3 mM + SPEE 500 μg/mL. Values are expressed as the mean ± SD (n = 3, right panel). * p < 0.05 and ** p < 0.01 vs. the control group. ^# p < 0.05 and ^### p < 0.001 vs. O/P group. ^&& p < 0.01 and ^&&& p < 0.001 vs. O/P 0.3 mM + SPEE 100 μg/mL group.

Figure 6

The effects of SPEE on O/P-inhibited antioxidant activities in HepG2 cells. HepG2 cells were co-treated with 0.3 mM O/P and SPEE at 100, 250 and 500 μg/mL for 24 h. SPEE inhibited the O/P-induced MDA production (A), and increased the antioxidant enzyme activities of SOD (B), catalase (C) and GPx (D). Silibinin (25 μM) was used as the positive control. Values are expressed as mean ± SD from triplicated experiments (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the control group. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs. the O/P group. ^&& p < 0.01 and ^&&& p < 0.001 vs. the O/P 0.3 mM + SPEE 100 μg/mL group.

Figure 7

The effects of SPEE on O/P-induced intracellular inflammatory factors in HepG2 cells. HepG2 cell lines were co-treated with 0.3 mM O/P and SPEE (100, 250 and 500 μg/mL, respectively) for 24 h. The expressions of pro-inflammatory factors IL-1β (A), IL-6 (B) and TNF-α (C) were analyzed as described in Materials and Methods. Silibinin was used as the positive control. Data are expressed as mean ± SD from triplicate experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the control group. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs. the O/P group. ^&& p < 0.01 and ^&&& p < 0.001 vs. the O/P 0.3 mM + SPEE 100 μg/mL group.

Figure 8

The effects of SPEE on O/P-induced m-RNA (A) and protein expressions (B,C) in HepG2 cells. HepG2 cell lines were co-treated with 0.3 mM O/P and SPEE (100, 250 and 500 μg/mL, respectively) for 24 h. Silibinin was used as the positive control. Data are expressed as mean ± SD from triplicate experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the control group. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs. the O/P group. ^& p < 0.05, ^&& p < 0.01 and ^&&& p < 0.001 vs. the O/P 0.3 mM + SPEE 100 μg/mL group.

Figure 9

O/P insults HepG2 cell to induce ROS signaling via ER stress associated with oil droplet deposition, which enhances energy stress and upregulates AMPK, Sirt1 and, subsequently, PGC1-α. SPEE further upregulates AMPK, Sirt1 and PGC1-α. Simultaneously, O/P stimulates macrophage to produce IL-1β, which activates NFκB to upregulate antioxidant and pro-inflammatory cytokines TNF-α, IL-6 and IL-1β. On the other hand, O/P may upregulate PERK which in turn upregulates the pro-inflammatory genes and UPR target genes. SPEE downregulated pro-inflammatory cytokines; conversely, it upregulated the antioxidant enzymes’ activities. Finally, the upregulated PGC1-α improved the mitochondrial function (Mito function) and fatty acid oxidation (FAO). Items with dotted lines and dotted boxes are depicted from the literature and were not included in this experiment.