Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

Abstract

Qushi Huayu Decoction (QHD), a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD) in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK) in vivo and in vitro. Nonalcoholic fatty liver (NAFL) model was duplicated with high-fat diet in rats and with free fatty acid (FFA) in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC) and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1) and carbohydrate-responsive element-binding protein (ChREBP) in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro.

Figure 1

The HPLC chromatogram of QHD. (a) QHD. (b) Standard compounds. Compounds were labeled as internal standard, chlorogenic acid (2.457 min, Peak 1), polygonin (10.213 min, Peak 2), resveratrol (14.792 min, Peak 3), and jasminoidin (39.104 min, Peak 4).

Figure 2

Effect of QHD on hepatic morphology and pathological changes. (A) The liver samples of ND, HDF, and HFD + QHD rats. (B) Histological micrograph of liver specimen from ND, HDF, and HFD + QHD rats (H&E and oil red O staining, magnification ×200). (a) The ND group; (b) the HFD group; (c) the HFD + QHD group. The major histopathological change induced by HFD in rat liver was hepatocyte steatosis (filled arrow) with inflammation (arrowhead) and ballooning (open arrow).

Figure 3

Effects of QHD on AMPK and ACCα protein expression and activation in hepatic tissues of HFD-fed rats. (a) Western blot. (b) Gray-level score. **P < 0.01, versus the ND group; *P < 0.05, versus the ND group; ^## P < 0.01, versus the HFD group.

Figure 4

Effects of QHD on SREBP-1 and ChREBP in total and nuclear protein expressions in hepatic tissuesof HFD-fed rats. (a) Western blot. (b) Gray-level score. **P < 0.01, versus the ND group; *P < 0.05, versus the ND group; ^## P < 0.01, versus the HFD group.

Figure 5

Effects of QHD-containing serum on cellular steatosis in L02 cells stimulated with FFA. (A) Intracellular TG content in L02 cells incubated with different concentrations of FFA for different times. The concentrations of low, middle, and high dose of FFA were 0.25 mM oleate/0.125 mM palmitate, 0.5 mM oleate/0.25 mM palmitate, and 1 mM oleate/0.5 mM palmitate, respectively. **P < 0.01, versus the control group at the same time point. (B) Effects of QHD-containing serum on cell viability of L02 cells. Cell viability was determined by the alamarBlue assay. (C) Effects of different concentrations of QHD-containing serum on intracellular TG contents in L02 cells incubated with FFA. **P < 0.01, versus the 5% vehicle control group; ^&& P < 0.01, versus the 10% vehicle control group; ^## P < 0.01, versus the FFA + 10% vehicle control group. (D) Effect of QHD-containing serum on cellular lipid droplets in L02 cells stimulated with FFA (oil red O staining, magnification ×100). (a) Control group; (b) FFA group; (c) FFA + QHD group.

Figure 6

Effects of QHD on SREBP-1 and ChREBP in total and nuclear protein expressions in L02 cells stimulated with FFA. (a) Western blot. (b) Gray-level score. **P < 0.01, versus the control group; ^## P < 0.01, versus the FFA group; ^# P < 0.05, versus the FFA group.

Figure 7

Effects of QHD on AMPK and ACCα protein expression and activation in L02 cells stimulated with FFA. (a) Western blot. (b) Gray-level score. **P < 0.01, versus the control group; *P < 0.05, versus the control group; ^## P < 0.01, versus the FFA group; ^# P < 0.05, versus the FFA group.

Figure 8

QHD inhibits hepatic lipid accumulation by activating AMPK. QHD significantly stimulates AMPK phosphorylation, decreases nuclear SREBP-1 and ChREBP protein contents, down-regulates ACCα expression and up-regulates phosphorylation of ACCα, and then decreases hepatic de novo lipogenesis and accumulation.