Lonicera caerulea Extract Attenuates Non-Alcoholic Fatty Liver Disease in Free Fatty Acid-Induced HepG2 Hepatocytes and in High Fat Diet-Fed Mice

Abstract

Honeyberry (Lonicera caerulea) has been used for medicinal purposes for thousands of years. Its predominant anthocyanin, cyanidin-3-O-glucoside (C3G), possesses antioxidant and many other potent biological activities. We aimed to investigate the effects of honeyberry extract (HBE) supplementation on HepG2 cellular steatosis induced by free fatty acids (FFA) and in diet-induced obese mice. HepG2 cells were incubated with 1 mM FFA to induce lipid accumulation with or without HBE. Obesity in mice was induced by a 45% high fat diet (HFD) for 6 weeks and subsequent supplementation of 0.5% HBE (LH) and 1% HBE (MH) for 6 weeks. HBE suppressed fatty acid synthesis and ameliorated lipid accumulation in HepG2 cells induced by FFA. Moreover, HBE also decreased lipid accumulation in the liver in the supplemented HBE group (LH, 0.5% or MH, 1%) compared with the control group. The expressions of adipogenic genes involved in hepatic lipid metabolism of sterol regulatory element-binding protein-1 (SREBP-1c), CCAAT/enhancer-binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), and fatty acid synthase (FAS) were decreased both in the HepG2 cells and in the livers of HBE-supplemented mice. In addition, HBE increased mRNA and protein levels of carnitine palmitoyltransferase (CPT-1) and peroxisome proliferator-activated receptor α (PPARα), which are involved in fatty acid oxidation. Furthermore, HBE treatment increased the phosphorylation of AMP-activated protein kinase (AMPK) and Acetyl CoA Carboxylase (ACC). Honeyberry effectively reduced triglyceride accumulation through down-regulation of hepatic lipid metabolic gene expression and up-regulation of the activation of AMPK and ACC signaling in both the HepG2 cells as well as in livers of diet-induced obese mice. These results suggest that HBE may actively ameliorate non-alcoholic fatty liver disease.

Keywords: HepG2 cells; Honeyberry; free fatty acids; high fat diet; nonalcoholic fatty liver disease.

Figure 1

High-Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD) chromatogram of main component in honeyberry (Lonicera caerulea). HPLC-DAD traces at 517 nm, Cyanidin 3-O-glucoside (C3G), was detected at 5.91 mg/mL in 25% ethanolic extract of honeyberry using high-performance liquid chromatography (HPLC) system.

Figure 2

Effect of Honeyberry extract (HBE) on the viability of HepG2 cells. HepG2 cells were incubated in the various concentration of HBE with 1 mM free fatty acids (FFA) for 24 h. All experiments were repeated at least three times and data represent means ± SD.

Figure 3

Effect of honeyberry extract (HBE) on Oil Red O staining and lipid accumulation in HepG2 cells. Lipid droplets in HepG2 cells were dyed red (magnification 200X). (A) Oil Red O staining images of HepG2 cells treated with 1 mM free fatty acids (FFA) and exposed to various concentration of HBE with 1mM FFA for 24 h. Control (Con) cells were incubated with 1% fat-free bovine serum albumin. (B) Quantitative lipid accumulation of Oil Red O contents at 500 nm. (C) Total intracellular triglyceride in HepG2 cells treated with HBE and 1 mM FFA. Data represent means ± SD. ### p < 0.001 vs. Con; ** p < 0.01, *** p < 0.001 vs. FFA.

Figure 4

Effects of HBE on the expression of genes associated with lipogenesis in HepG2 cells. (A–D) The expressions of SREBP-1c (A), C/EBPα (B), PPARγ (C), and fatty acid synthase (FAS) (D) were quantified by real-time PCR and normalized by β-actin as an internal control. (E) SREBP-1c, C/EBPα, PPARγ, and FAS protein levels were monitored by Western blot analysis. (F) Protein density of SREBP-1c, C/EBPα, PPARγ, and FAS. Equal loading of protein was verified by probing β-actin. Data represent means ± SD. # p < 0.5, ## p < 0.01, ### p < 0.001 vs. Con; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. free fatty acids (FFA).

Figure 5

Effects of HBE on CPT1 and PPARα expression and AMPK, ACC signaling in FFA-treated HepG2 cells, with and without HBE supplementation. The mRNA expression of genes associated with fatty acid oxidation factors CPT1 (A) and PPARα (B) were quantified by real-time PCR and normalized by β-actin as an internal control. Western blot analysis of p-AMPK/AMPK (C) and p-ACC/ACC (D) in HepG2 cells. AMPK and ACC were used as a protein loading control of phosphorylated AMPK (p-AMPK) and phosphorylated ACC (p-ACC), respectively. (E) CPT-1, PPARα, phosphorylated AMPK and phosphorylated ACC proteins level by immunoblot analysis. The results from three independent experiments are expressed as the mean ± SD. (F) Protein density of CPT-1 and PPARα. Equal loading of protein was verified by probing β-actin. The results from three independent experiments are expressed as the mean ± SD. ### p < 0.001 vs. Con; ∗ p < 0.05, ** p < 0.01, *** p < 0.001 vs. FFA.

Figure 6

Effect of HBE treatment on hepatic steatosis in normal or HFD-fed mice. (A) Liver tissue histology (400×). (B) Adipocyte sizes of ND, HFD, LH, and MH. (C) The accumulation of liver TG. (D) Hepatic MDA levels. ND, normal diet control; HFD, high-fat diet; LH, HFD-supplemented 0.5% HBE; MH, HFD-supplemented 1% HBE. The results from three independent experiments are expressed as the mean ± SD. ### p < 0.001 vs. ND; ** p < 0.01, *** p < 0.001 vs. HFD.

Figure 7

Effects of HBE on the expression of genes associated with lipogenesis in HFD-fed mice. (A–D) The expression of SREBP-1c (A), C/EBPα (B), PPARγ (C), and FAS (D) were quantified by real-time PCR and normalized by β-actin as an internal control. (E) C/EBPα, PPARγ, and FAS protein levels by Immunoblot analysis. (F) Protein density of C/EBPα, PPARγ, and FAS. Equal loading of protein was verified by probing β-actin. ND, normal diet control; HFD, high-fat diet; LH, HFD-supplemented 0.5% HBE; MH, HFD-supplemented 1% HBE. Data represent means ± SD. # p < 0.5, ## p < 0.01, ### p < 0.001 vs. ND; * p < 0.5, ** p < 0.01, *** p < 0.001 vs. HFD.

Figure 8

Effects of HBE on CPT-1 and PPARα expression and AMPK, ACC signaling in HFD-fed mice. The expression of CPT-1 (A) and PPARα (B) were quantified by real-time PCR and normalized by β-actin as an internal control. (C–D) Western blot analysis of p-AMPK/AMPK and p-ACC/ACC protein in the livers of mice fed an ND, HFD, or HFD with supplemented HBE (LH, 0.5% or MH, 1%). β-actin was used as a protein loading control. AMPK and ACC were used as protein loading controls of phosphorylated AMPK (p-AMPK) and phosphorylated ACC (p-ACC), respectively. (E) CPT-1, PPARα, phosphorylated AMPK, and phosphorylated ACC protein levels by immunoblot analysis. (F) Protein density of CPT-1, PPARα. Equal loading of protein was verified by probing β-actin. Data represent means ± SD. * p < 0.5, ** p < 0.01, *** p < 0.001 vs. HFD.