Herbal SGR Formula Prevents Acute Ethanol-Induced Liver Steatosis via Inhibition of Lipogenesis and Enhancement Fatty Acid Oxidation in Mice

Abstract

Our previous study indicated that herbal SGR formula partially attenuates ethanol-induced fatty liver, but the underlying mechanisms remain unclear. In the present study, mice were pretreated with SGR (100 and 200 mg/kg/d bw) for 30 d before being exposed to ethanol (4.8 g/kg bw). The biochemical indices and histopathological changes were examined to evaluate the protective effects and to explore potential mechanisms by investigating the adiponectin, tumor necrosis factor-α (TNF-α), peroxisome proliferators-activated receptor-α (PPAR-α), sterol regulatory element binding protein-1c (SREBP-1c), adenosine monophosphate-activated protein kinase (AMPK), and so forth. Results showed that SGR pretreatment markedly inhibited acute ethanol-induced liver steatosis, significantly reduced serum and hepatic triglyceride (TG) level, and improved classic histopathological changes. SGR suppressed the protein expression of hepatic SREBP-1c and TNF-α and increased adiponectin, PPAR-α, and AMPK phosphorylation in the liver. Meanwhile, acute toxicity tests showed that no death or toxic side effects within 14 days were observed upon oral administration of the extracts at a dose of 16 g/kg body wt. These results demonstrate that SGR could protect against acute alcohol-induced liver steatosis without any toxic side effects. Therefore, our studies provide novel molecular insights into the hepatoprotective effect of SGR formula, which may be exploited as a therapeutic agent for ethanol-induced hepatosteatosis.

Figure 1

(a) Preparation of SGR, Semen Hoveniae extract (SHE) power mixed properly with Ginkgo biloba extract (GBE) and Rosa roxburghii Tratt extract (RRTE) power at the ratio of 8 : 1 : 1 in this study. (b) and (c) Flavonoids of herbal formula SGR were analysed by HPLC. ①: dihydromyricetin, ②: dihydroquercetin, and ③: quercetin.

Figure 2

Effects of SGR on the body weight and liver index in acutely inebriated mice (n = 12). ^Δ P < 0.05, ^ΔΔ P < 0.01, compared with normal control group; ^∗ P < 0.05, ^∗∗ P < 0.01, compared with ethanol group.

Figure 3

Effect of SGR pretreatment on the TG levels in serum and hepatic, pathological changes in the liver with acute alcoholism (×100). (a) and (b) Supplement with SGR for 30 d significantly inhibited the increase of the TG levels in serum and in liver, respectively. (c) H&E (hematoxylin and eosin) staining of liver tissue. The red arrow indicated the fat droplets in the liver sections. (A) normal; (B) ethanol; (C) SGR (100 mg/kg); (D) SGR (200 mg/kg). ^Δ P < 0.05, ^ΔΔ P < 0.01, compared with normal control group; ^∗ P < 0.05, ^∗∗ P < 0.01, compared with ethanol group.

Figure 4

Effects of SGR on the hepatic TNF-α protein expression, serum adiponectin level in mice with acute ethanol exposure. (a) and (b) TNF-α protein levels were quantified with Tublin as an internal control and expressed as the relative content of the control value. These data were representative of three independent experiments. (c) Serum adiponectin levels were quantified with ELISA (n = 12). ^Δ P < 0.05, ^ΔΔ P < 0.01, compared with normal control group; ^∗ P < 0.05, ^∗∗ P < 0.01, compared with ethanol group.

Figure 5

Effects of SGR on the hepatic AMPK, P-AMPKβ, PPAR-α, and SREBP-1c protein expression in mice with acute ethanol exposure, respectively. (a) AMPK, P-AMPKβ, PPAR-α, and SREBP-1c were quantified with Tublin as an internal control. (b) Hepatic AMPK levels were quantified with ELISA (n = 12). (c, e, and f) AMPK, PPAR-α, and SREBP-1c protein levels of them were expressed as the relative content of the control value. (d) P-AMPKβ/AMPK expressed as the degree of phosphorylated AMPK. These data were representative of three independent experiments. ^Δ P < 0.05, ^ΔΔ P < 0.01, compared with normal control group; ^∗ P < 0.05, ^∗∗ P < 0.01, compared with ethanol group.

Figure 6

The effects of SGR supplementation on PPAR-α immunofluorescence staining of acute ethanol-induced liver injury in mice. The graph showed the average number of fluorescence dots of images from each treatment group (×400). (a) (A) normal; (B) ethanol; (C) SGR (100 mg/kg); (D) SGR (200 mg/kg). (b) Changes in the cumulative value of optical density in cytoplasm immunofluorescence staining of PPAR-α, expressed as the relative content of the ethanol control value. These data were representative of three independent experiments. ^Δ P < 0.05, ^ΔΔ P < 0.01, compared with normal control group; ^∗ P < 0.05, ^∗∗ P < 0.01, compared with ethanol group.

Figure 7

Excessive consumption of alcohol can upregulate the expression of SREBP-1c and downregulate the expression of PPAR-α. Meanwhile, alcohol exposure also inhibits AMPK and subsequently increases ACC activity but decreases carnitine palmitoyltransferase 1 (CPT-1) activity. All of them lead to an increase in fatty acid synthesis and a decrease in fatty acid β-oxidation. Furthermore, alcohol exposure stimulates adipose tissue adipokine imbalance, such as adiponectin and TNF-α. Moreover, alcohol-induced also stimulates adipose tissue lipolysis, the triglycerides reverse transported and deposited in the liver. Moreover, herbal formula SGR contains abundant flavonoids, including dihydromyricetin, dihydroquercetin, and quercetin which inhibit lipogenesis to prevent ethanol-induced liver steatosis.