OBJECTIVETo investigate the expression and pathogenic relevance of angiotensin-converting enzyme 2 (ACE2) in liver fibrosis by using the rat model of CCl4-induced liver fibrosis.

METHODSThe liver fibrosis model was generated by delivering subcutaneous injections of CCl4 (dissolved in olive oil at a 2:3 ratio; injection dose: 3 ml/kg) every three days for six weeks into male Sprague-Dawley rats. Another group of rats that received simultaneous injections of olive oil alone (3 ml/kg) were used as controls. At week 0, 2, 4, or 6 after the first injection, a subset of rats from each group was sacrificed to obtain liver tissues and serum samples. Pathological analyses were carried out to detect the presence and extent of liver cell degeneration, necrosis, inflammatory cell infiltration, and collagen deposition. ACE2 and ACE gene and protein expressions were measured by real-time PCR and Western blotting, respectively. The significance of differential expression between groups and time points was assessed by t-test and one-way ANOVA or Kruskal-Wallis tests, and correlation with fibrosis was assessed by Spearman's rank correlation coefficient.

RESULTSCCl4 administration led to significantly up-regulated ACE2 mRNA levels at week 2 (3.055+/-1.034), 4 (3.545+/-1.947), and 6 (6.448+/-1.836) (vs. controls; H = 23.224, P less than 0.001). Similarly, hepatic ACE mRNA was significantly increased after the CCl4 injections (week 2: 3.055+/-1.034, week 4: 3.545+/-1.947, week 6: 6.448+/-1.836; vs. controls: F = 12.982, P less than 0.001). There was a significant correlation between the ACE and ACE2 gene expression levels (r = 0.750, P less than 0.001). Protein levels of ACE2 also showed an increasing trend following CCl4 administration (week 0: 0.034, week 2: 0.097, week 4: 0.356, week 6: 0.512). The hepatic ACE2 gene expression strongly correlated with levels of alanine aminotransferase (r = 0.669, P less than 0.0001) and aspartate aminotransferase (r = 0.815, P less than 0.0001), and with the Ishak fibrosis score (r = 0.850, P less than 0.001). Finally, there was a significant correlation between circulating ACE2 and the Ishak fibrosis score (r = 0.730, P less than 0.001).

CONCLUSIONA significant relationship exists between ACE2 gene expression and extent of liver fibrosis. ACE2 may play a crucial role in liver fibrogenesis.