Hepatoprotective activity of chrysin is mediated through TNF-α in chemically-induced acute liver damage: An in vivo study and molecular modeling

Abstract

Chrysin (5,7-dihydroxyflavone) is a naturally occurring flavonoid present at high levels in honey, propolis and numerous plant extracts. Chrysin is known to have hepatoprotective activity, however, the mechanisms by which it exerts this effect remain unclear. In the present study, the effects of chrysin in carbon tetrachloride (CCl₄)-induced acute liver damage were investigated and the results used to infer a possible mechanism behind chrysin's hepatoprotective activity. Prior to an intraperitoneal injection of CCl4 (1 ml/kg) to induce acute liver damage, chrysin (50 mg/kg) was administered orally to mice for 7 days. The positive control group was given 50 mg/kg standardized silymarin, a well-studied hepatoprotective flavonoid. Twenty-four h following CCl4 administration, an increase in the activity levels of serum aspartate-amino-transferase and alanine-amino-transferase was found. This was accompanied by extended centrilobular necrosis, steatosis and an altered hepatocyte ultrastructure. In addition, CCl4-induced acute hepatotoxicity was associated with an increase in hepatic tumor necrosis factor-α (TNF-α) and α-smooth muscle actin (α-SMA) protein expression, which was significantly decreased in the livers of mice pre-treated with chrysin (P<0.001), similar to the results of the silymarin pre-treated group (P<0.001). Treatment with chrysin prior to CCl4 exposure significantly reduced the activity of enzymes used as biochemical markers of poor liver function compared with the group which did not receive pre-treatment (P<0.001). In addition, the results of histopathological and electron microscopy liver examination showed chrysin pre-treatment reduced the effects of CCl4 treatment. Molecular modeling results demonstrated that the hepatoprotective activity of chrysin is mediated through TNF-α, as it reduces soluble TNF-α generation via blocking TNF-α-converting enzyme activity. In conclusion, the results of the present study suggest that inflammatory pathways are activated in CCl4-induced acute liver damage, which are ameliorated by chrysin pre-treatment. This indicates that chrysin is a potent hepatoprotective agent, similarly to silymarin at the same dose, which has the potential to be a viable alternative to conventional hepatoprotective treatments.

Keywords: carbon tetrachloride; chrysin; hepatoprotection; liver; molecular modeling; silymarin; tumor necrosis factor-α; α-smooth muscle actin.

Figure 1.

Effect of chrysin on the activities of serum (A) AST and (B) ALT following CCl₄ treatment. Chrysin pre-treatment reduced on serum AST and ALT activities. Results are represented as the mean ± standard deviation (n=8). ***P<0.001 vs. the control group; ^###P<0.001 vs. the CCl₄ group. AST, aspartate transaminase; ALT, alanine transaminase; CCl₄, carbon tetrachloride; CHR, chrysin; Sy, silymarin.

Figure 2.

Effect of chrysin on histological changes in the liver of CCl₄-treated mice. Representative images of hematoxylin and eosin stained liver sections of the (A) control group, (B) CHR group, (C) CCl₄ group, (D) CHR+CCl₄ group and (E) Sy+CCl₄ group. Scale bar, 50 µm. Necrosis areas are indicated with asterisks, lymphocyte infiltration are indicated by arrows. (F) The percentage of necrotic area in mice liver samples. Results are represented as the mean ± standard deviation (n=5). Scale bar, 50 µm. ***P<0.001 vs. the control group; ^###P<0.001 vs. the CCl₄ group. CCl₄, carbon tetrachloride; CHR, chrysin; Sy, silymarin.

Figure 3.

Effect of chrysin on lipid accumulation in the liver of CCl₄-treated mice. Representative images of Oil Red O stained liver sections of the (A) control group, (B) CHR group, (C) CCl₄ group, (D) CHR+CCl₄ group and (E) Sy+CCl₄ group. Lipid drops, indicated in red. Scale bar, 50 µm. (F) The percentage of lipids in mice liver samples. Results are represented as the mean ± standard deviation (n=5). Scale bar, 50 µm ***P<0.001 vs. control; ^###P<0.001 vs. the CCl₄ group. CCl₄, carbon tetrachloride; CHR, chrysin; Sy, silymarin.

Figure 4.

Effect of chrysin on the expression and distribution of TNF-α protein in the liver of CCl₄-treated mice. (A) Control group, (B) CHR group, (C) CCl₄ group, (D) CHR+CCl₄ group and (E) Sy+CCl₄ group. Scale bar, 200 µm. (F) Quantification of TNF-α staining intensity relative to control and chrysin groups. Results are represented as the mean ± standard deviation (n=5 mice per group). ***P<0.001 vs. the control group; ^###P<0.001 vs. the CCl₄ group. TNF-α, tumor necrosis factor-α; CCl₄, carbon tetrachloride; CHR, chrysin; Sy, silymarin.

Figure 5.

Effect of chrysin on the expression and distribution of α-SMA protein in the liver of CCl₄-treated mice. (A) Control group, (B) CHR group, (C) CCl₄ group, (D) CHR+CCl₄ group and (E) Sy+CCl₄ group. Scale bar, 200 µm. (F) Quantification of α-SMA staining intensity. Results are represented as the mean ± standard deviation (n=5). ***P<0.001 vs. the control group; ^###P<0.001 vs. the CCl₄ group. α-SMA, α-smooth muscle actin; CCl₄, carbon tetrachloride; CHR, chrysin; Sy, silymarin.

Figure 6.

Effect of chrysin on hepatocyte ultrastructure in CCl₄-treated mice. (A) The control group showed normal N and L. (B) CHR group. (C) The CCl₄ group showed an edematous cytoplasm matrix with sER proliferation, and an increased quantity and size of L. (D) The CHR+CCl₄ group showed a reduction in the quantity and size of L, and no sER proliferation. (E) The Sy+CCl₄ group showed a reduction in the quantity and size of L, and no sER proliferation. Transmission electron miscopy images representative of the group. Scale bar, 50 µm. N, nucleus; L, lipid drop; sER, smooth endoplasmic reticulum.

Figure 7.

Validation of the FlexX docking method through re-docking of the inhibitor IK682 into the crystal structure (PDB ID: 2FV5) of tumor necrosis factor-α converting enzyme (TACE). (A) Pharmacophoric features of TACE interaction with inhibitor IK682, obtained using the FlexX docking method. (B) Pharmacophoric interaction of TACE with inhibitor IK682, as observed in the crystal structure. Top right, structure of the inhibitor IK682, obtained from docking with the orientation observed in the crystal structure. Amino acid names are abbreviated using standard International Union of Pure and Applied Chemistry convention. Green lines represent hydrophobic interactions, while dashed lines represent hydrogen bonds and metal interactions.

Figure 8.

Structure of the lowest energy docking solution of the tumor necrosis factor-α converting enzyme (TACE)-chrysin complex obtained from molecular modeling. (A) Interactions of docked chrysin within the TACE binding site. Chrysin is rendered as a stick representation, TACE is rendered as a ribbon representation and hydrogen bonds indicated by dashed lines. (B) Orientation of docked chrysin within the active site of TACE. The active site is represented in surface mode and colored according to B-factor value (flexibility), with blue to green to red signifying increasing flexibility. (C) The essential pharmacophoric interactions of TACE inhibition by chrysin. Hydrogen bonds are indicated by dashed lines. Amino acid names are abbreviated using standard International Union of Pure and Applied Chemistry convention. Green lines represent hydrophobic interactions, while dashed lines represent hydrogen bonds.