Acta Pharmacologica Sinica (2010) 31: 27–34; doi: 10.1038/aps.2009.170; published online 7 December 2009

Aim: To study the pharmacologic effect of ZK₁₄, a novel nitric oxide-donating biphenyldicarboxylate (DDB) derivative, on HSC-T6 cells and on CCl₄-induced hepatic fibrosis. 

Methods: Inhibition of HSC-T6 cell growth by ZK₁₄ was evaluated by MTT assay.  The effect of ZK₁₄ on the percentage of HSC-T6 cells undergoing apoptosis was measured using Annexin-V/PI double-staining and TUNEL assay.  Mitochondrial membrane potential (MMP) and caspase activities were tested.  Hepatic fibrosis was induced in Sprague-Dawley rats by intraperitoneal injection with 14% CCl₄.  Rats with hepatic fibrosis were randomly divided into four groups: model control, ZK₁₄ (20 mg/kg), ZK₁₄ (10 mg/kg) and DDB (5
mg/kg).  Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), hyaluronic acid (HA), type III collagen (PCIII), and nitric oxide (NO) were assessed, and liver samples were stained with hematoxylin-eosin.  The NO level in cells treated with ZK ₁₄ in vitro was also measured.

Results: The effect of ZK₁₄ on HSC-T6 cell apoptosis was concentration- and time-dependent, with up to 50% of cells becoming apoptotic when exposed to 100 μmol/L ZK₁₄ for 18 h.  ZK₁₄ treatment resulted in mitochondrial membrane depolarization and activation of caspases 3 and 9.  At a dose of 20 mg/kg, ZK₁₄ significantly decreased serum transaminase (AST, ALT) activities and fibrotic index (HA, PCIII) levels and significantly inhibited fibrogenesis.

Conclusion: These data indicate that ZK₁₄, a novel NO-donating DDB derivative, promotes HSC-T6 apoptosis in vitro through a signaling mechanism involving mitochondria and caspase activation and it inhibits CCl₄-induced hepatic fibrosis in vivo.  The results suggest that ZK₁₄ has potential therapeutic value in the treatment of hepatic fibrosis.

Keywords: nitric oxide; apoptosis; hepatic fibrosis; HSC-T6; CCl₄; biphenyldicarboxylate; ZK₁₄