title: Glycyrrhizic acid attenuates CCl₄-induced hepatocyte apoptosis in rats via a p53-mediated pathway.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  GA can inhibit CCl4-induced hepatocyte apoptosis via a p53-dependent mitochondrial pathway to retard the progress of liver fibrosis in rats.
authors.affiliation: Department of Gastroenterology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China.
authors.forename: Xiao-Ling
authors.initials: XL
authors.lastname: Guo
authors.forename: Bo
authors.initials: B
authors.lastname: Liang
authors.forename: Xue-Wei
authors.initials: XW
authors.lastname: Wang
authors.forename: Fu-Gang
authors.initials: FG
authors.lastname: Fan
authors.forename: Jing
authors.initials: J
authors.lastname: Jin
authors.forename: Rui
authors.initials: R
authors.lastname: Lan
authors.forename: Jing-Hui
authors.initials: JH
authors.lastname: Yang
authors.forename: Xiao-Chun
authors.initials: XC
authors.lastname: Wang
authors.forename: Lei
authors.initials: L
authors.lastname: Jin
authors.forename: Qin
authors.initials: Q
authors.lastname: Cao
mesh_terms: D000410:Alanine Transaminase / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001219:Aspartate Aminotransferases / Q000378:metabolism; D002251:Carbon Tetrachloride / Q000009:adverse effects / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D006034:Glycyrrhetinic Acid / Q000494:pharmacology / Q000627:therapeutic use*; D022781:Hepatocytes / Q000187:drug effects / Q000473:pathology* / Q000502:physiology; D008099:Liver / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000473:pathology / Q000517:prevention & control*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000502:physiology*
chemical_list: D016159:Tumor Suppressor Protein p53; D045304:Cytochromes c; D002251:Carbon Tetrachloride; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; D020169:Caspases; D006034:Glycyrrhetinic Acid
keywords: Apoptosis; Glycyrrhizic acid; Liver fibrosis; Mitochondria; P53