Synergistic Protective Effect of Fermented Schizandrae Fructus Pomace and Hoveniae Semen cum Fructus Extracts Mixture in the Ethanol-Induced Hepatotoxicity

Abstract

Schizandrae Fructus (SF), fruits of Schisandra chinensis (Turcz.) Baill. and Hoveniae Semen cum Fructus (HSCF), the dried peduncle of Hovenia dulcis Thunb., have long been used for alcohol detoxification in the traditional medicine of Korea and China. In the current study, we aimed to evaluate the potential synergistic hepatoprotective effect of a combination mixture (MSH) comprising fermented SF pomace (fSFP) and HSCF hot water extracts at a 1:1 (w:w) ratio against ethanol-induced liver toxicity. Subacute ethanol-mediated hepatotoxicity was induced by the oral administration of ethanol (5 g/kg) in C57BL/6J mice once daily for 14 consecutive days. One hour after each ethanol administration, MSH (50, 100, and 200 mg/kg) was also orally administered daily. MSH administration significantly reduced the serum activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase. Histological observation indicated that MSH administration synergistically and significantly decreased the fatty changed region of hepatic parenchyma and the formation of lipid droplet in hepatocytes. Moreover, MSH significantly attenuated the hepatic triglyceride accumulation through reducing lipogenesis genes expression and increasing fatty acid oxidation genes expression. In addition, MSH significantly inhibited protein nitrosylation and lipid peroxidation by lowering cytochrome P450 2E1 enzyme activity and restoring the glutathione level, superoxide dismutase and catalase activity in liver. Furthermore, MSH synergistically decreased the mRNA level of tumor necrosis factor-α in the hepatic tissue. These findings indicate that MSH has potential for preventing alcoholic liver disease through inhibiting hepatic steatosis, oxidative stress, and inflammation.

Keywords: Hoveniae Semen cum Fructus; Schizandrae Fructus; alcoholic liver disease; anti-steatosis; antioxidant; ethanol.

Figure 1

Effect of fermented Schizandrae Fructus Pomace (fSFP) and Hoveniae Semen cum Fructus (HSCF) combination mixture (MSH) on body weight gain, liver weight, and serum activities of liver enzymes in ethanol intoxicated mice. (a) Body weight gain was calculated by subtracting the body weight of day 0 from those of day 14. (b) Relative liver weight. Absolute liver weight was divided by the body weight of each individual. (c) Enzymatic activity of liver enzymes in serum. Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyl transpeptidase (GGT) were measured in serum. All values were expressed as the mean ± standard deviation of 10 mice. Significant versus vehicle group, ** p < 0.01; versus EtOH group, ^## p < 0.01; versus fSFP-treated group, ^$ p < 0.05, ^$$ p < 0.01; versus HSCF-treated group, ^&& p < 0.01. N.S., not significant; EtOH, ethanol; Sily, silymarin.

Figure 2

Effect of MSH on the hepatic steatosis in ethanol-intoxicated mice. (a) Representative profile images of hematoxylin and eosin-stained (left) or oil red O-stained (right) liver tissues for histopathological observation. In the oil red O staining, nuclei were counterstained with hematoxylin solution. Scale bars indicate 200 μm. (b) Histopathological analysis. Percentage of fatty changed regions (upper), the number of fatty changed hepatocytes (middle), and mean hepatocyte diameter (lower) were observed using an automated image analyzer. Significant versus vehicle group, ** p < 0.01; versus EtOH group, ^## p < 0.01; versus fSFP-treated group, ^$$ p < 0.01; versus HSCF-treated group, ^&& p < 0.01. CT, central vein; PT, portal triad; EtOH, ethanol; Sily, silymarin.

Figure 3

Effect of MSH on triglycerides synthesis and fatty acid metabolism in the ethanol-intoxicated mice liver. (a) Triglyceride level in hepatic tissue (left) and serum (right). (b) Expression of genes related to fatty acid and triglyceride synthesis. Relative mRNA levels of SREBP-1c, PPARγ, SCD1, ACC1, FAS, and DGAT2 were measured. (c) Expression of genes related to fatty acid oxidation. Relative mRNA levels of PPARα, ACO and CPT1 were measured. mRNA level of each gene was measured using RT-qPCR and normalized with β-actin gene expression. Significant versus vehicle group, ** p < 0.01; versus EtOH group, ^## p < 0.01; versus fSFP-treated group, ^$$ p < 0.01; versus HSCF-treated group, ^&& p < 0.01. EtOH, ethanol; Sily, silymarin.

Figure 4

Effect of MSH on the ethanol-induced oxidative stress and inflammation. (a) Representative profiles of immunohistochemistry using anti-NT and 4-HNE antibodies. Scale bars indicate 200 μm. (b) The numbers of NT- (left) and 4-HNE- (right) positive cells. Cells showing more than 20% of immunoreactivity were counted. (c) MDA content and (d) tumor necrosis factor (TNF)-α in the hepatic tissue were quantified using liver homogenates. Significant versus vehicle group, ** p < 0.01; versus EtOH group, ^## p < 0.01; versus fSFP-treated group, ^$$ p < 0.01; versus HSCF-treated group, ^&& p < 0.01. CT, central vein; PT, portal triad; EtOH, ethanol; Sily, silymarin; NT, nitrotyrosine; 4-HNE, 4-hydroxynonenal; MDA, malondialdehyde.

Figure 5

Effect of MSH on the antioxidant system and CYP2E1 activity in ethanol-intoxicated mice liver. (a) Endogenous antioxidant capacities. The glutathione (GSH) level (left), superoxide dismutase (SOD) (middle), and catalase (CAT) (right) activities were measured in liver homogenates. (b) Relative level of Nrf2 mRNA. Nrf2 mRNA level was measured using RT-qPCR and normalized with β-actin mRNA expression. (c) CYP450 2E1 activity. Significant versus vehicle group, * p < 0.05, ** p < 0.01; versus EtOH group, ^## p < 0.01; versus fSFP-treated group, ^$$ p < 0.01; versus HSCF-treated group, ^&& p < 0.01. EtOH, ethanol; Sily, silymarin.