Glycyrrhizic acid attenuates CCl₄-induced hepatocyte apoptosis in rats via a p53-mediated pathway

Abstract

Aim: To investigate the effect of glycyrrhizic acid (GA) on carbon tetrachloride (CCl4)-induced hepatocyte apoptosis in rats via a p53-dependent mitochondrial pathway.

Methods: Forty-five male Sprague-Dawley rats were randomly and equally divided into three groups, the control group, the CCl4 group, and the GA treatment group. To induce liver fibrosis in this model, rats were given a subcutaneous injection of a 40% solution of CCl4 in olive oil at a dose of 0.3 mL/100 g body weight biweekly for 8 wk, while controls received the same isovolumetric dose of olive oil by hypodermic injection, with an initial double-dose injection. In the GA group, rats were also treated with a 40% solution of CCl4 plus 0.2% GA solution in double distilled water by the intraperitoneal injection of 3 mL per rat three times a week from the first week following previously published methods, with modifications. Controls were given the same isovolumetric dose of double distilled water. Liver function parameters, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Pathologic changes in the liver were detected by hematoxylin and eosin staining. Collagen fibers were evaluated by Sirius red staining. Hepatocyte apoptosis was investigated using the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay and the cleaved caspase-3 immunohistochemistry assay. The expression levels of p53 and apoptosis-related proteins were evaluated by immunohistochemistry or Western blotting analysis.

Results: After 8 wk of treatment, GA significantly reduced serum activity of ALT (from 526.7 ± 57.2 to 342 ± 44.8, P < 0.05) and AST (from 640 ± 33.7 to 462.8 ± 30.6, P < 0.05), attenuated the changes in liver histopathology and reduced the staging score (from 3.53 ± 0.74 to 3.00 ± 0.76, P < 0.05) in CCl4-treated rats. GA markedly reduced the positive area of Sirius red and the ratio of the hepatic fibrotic region (from 7.87% ± 0.66% to 3.68% ± 0.32%, P < 0.05) compared with the CCl4 group. GA also decreased the expression level of cleaved caspase-3 compared to the CCl4 group. TUNEL assay indicated that GA significantly diminished the number of TUNEL-positive cells compared with the CCl4 group (P < 0.05). GA treatment clearly decreased the level of p53 (P < 0.05) detected by immunohistochemistry and Western blotting analysis. Compared with the CCl4 group, we also found that GA reduced the Bax/Bcl-2 ratio (P < 0.05), the expression of cleaved caspase-3 (P < 0.05), cleaved caspase-9 (P < 0.05), and inhibited cytochrome C and second mitochondria-derived activator of caspases (Smac) release from mitochondria to cytoplasm, i.e., GA reduced the expression level of Smac, which inhibited c-IAP1 activity (P < 0.05), ultimately inhibiting the activity of caspase-3, according to Western blotting analysis. As a result, GA suppressed activation of the caspase cascades and prevented hepatocyte apoptosis.

Conclusion: GA can inhibit CCl4-induced hepatocyte apoptosis via a p53-dependent mitochondrial pathway to retard the progress of liver fibrosis in rats.

Keywords: Apoptosis; Glycyrrhizic acid; Liver fibrosis; Mitochondria; P53.

Figure 1

Histological examination of liver by hematoxylin and eosin and Sirius red staining. A: Histological examination. Rats were treated with carbon tetrachloride (CCl₄) and/or glycyrrhizic acid (GA). Liver tissue sections were stained with hematoxylin and eosin or Sirius red (original magnification, × 100); B: Quantitative analysis of liver fibrosis by Sirius red staining. Results are represented as fibrotic area (%), which signifies the proportion of area stained red/area of total area-vascular lumen. Values are mean ± SD. ^aP < 0.05 vs CCl₄.

Figure 2

Impact of glycyrrhizic acid treatment on hepatic apoptosis induced by carbon tetrachloride in rats. A: Liver tissue sections from the different groups were subjected to immunohistochemistry to determine the expression level of cleaved caspase-3 (original magnification, × 400); B: Fluorescence microscopy image showing terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) stain (dashed arrows), and the same tissue slices were respectively counterstained with 4’6-diamidino-2-phenylindole (DAPI) to localize the nuclei (arrows). Images of combined with DAPI, indicated TUNEL-positive cells (arrow heads) (original magnification, × 200). GA: Glycyrrhizic acid; CCl₄: Carbon tetrachloride.

Figure 3

Effect of glycyrrhizic acid treatment on the expression level of p53 in the livers of rats injured by carbon tetrachloride. A: Liver tissue slices from the different groups were subjected to immunohistochemistry (original magnification, × 400). B: Total protein fractions prepared from livers were analyzed by Western blotting to assess the expression level of p53 and GAPDH to confirm the same sample loading. The results of Western blotting analysis were similar in at least three replicate independent experiments. All values are presented as mean ± SD. Statistical significance was defined as follows: ^aP < 0.05 vs the carbon tetrachloride (CCl₄) group. GA: Glycyrrhizic acid.

Figure 4

Impact of glycyrrhizic acid on CCl₄-treated hepatocyte apoptosis signal cascades. Protein extracts from livers in the different groups were subjected to Western blotting. A: Expression levels of Bax and Bcl-2 in the mitochondria; B: Expression levels of cytochrome C (Cyt.c) in the cytoplasm and mitochondria; C: Expression level of caspase-9 in the total protein; D: Expression level of caspase-3 in the total protein; E: Expression level of Smac in the cytoplasm; F: Expression level of c-IAP1 the total protein. In all these experiments glyceraldehyde-3-phosphate dehydrogenase (GAPDH), COXIV were used to ensure equal sample loading. The Western blotting results represent three independent tests. The bar graph represents the value of in the different proteins via the density of bands from at least three independent tests. All values are presented as mean ± SD. Statistical significant was defined as follows: ^aP < 0.05 vs the CCl₄ group. GA: Glycyrrhizic acid; CCl_4: Carbon tetrachloride.

Figure 5

Schematic diagram of the effect of glycyrrhizic acid on the interruption of p53 signaling in carbon tetrachloride-induced hepatocyte apoptosis (blue arrows). Glycyrrhizic acid (GA) suppressed the activation of p53, decreased the expression level of Bax and increased the expression level of Bcl-2, which resulted in reduced cytochrome C release from the mitochondria into the cytoplasm, and inactivated caspase-9 and -3; GA also significantly inhibited Smac release from mitochondria into the cytoplasm and elevated the expression level of c-IAP1, resulting in inhibition of caspase-3 activity. Ultimately, GA suppressed the apoptosis of hepatocytes.