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Antifibrotic effects of ZK14, a novel nitric oxide-donating biphenyldicarboxylate derivative, on rat HSC-T6 cells and CCl4-induced hepatic fibrosis.

ABSTRACT: AIM: To study the pharmacologic effect of ZK(14), a novel nitric oxide-donating biphenyldicarboxylate (DDB) derivative, on HSC-T6 cells and on CCl(4)-induced hepatic fibrosis. METHODS: Inhibition of HSC-T6 cell growth by ZK(14) was evaluated by MTT assay. The effect of ZK(14) 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% CCl(4). Rats with hepatic fibrosis were randomly divided into four groups: model control, ZK(14) (20 mg/kg), ZK(14) (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 ZK(14) on HSC-T6 cell apoptosis was concentration- and time-dependent, with up to 50% of cells becoming apoptotic when exposed to 100 mumol/L ZK(14) for 18 h. ZK(14) treatment resulted in mitochondrial membrane depolarization and activation of caspases 3 and 9. At a dose of 20 mg/kg, ZK(14) significantly decreased serum transaminase (AST, ALT) activities and fibrotic index (HA, PCIII) levels and significantly inhibited fibrogenesis. CONCLUSION: These data indicate that ZK(14), a novel NO-donating DDB derivative, promotes HSC-T6 apoptosis in vitro through a signaling mechanism involving mitochondria and caspase activation and it inhibits CCl(4)-induced hepatic fibrosis in vivo. The results suggest that ZK(14) has potential therapeutic value in the treatment of hepatic fibrosis.

SUBMITTER: Dai L

PROVIDER: S-EPMC4002687 | biostudies-other | 2010 Jan

REPOSITORIES: biostudies-other

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Project description:Resolvin D1 (RvD1) was previously reported to relieve inflammation and liver damage in several liver diseases, but its potential role in liver fibrosis remains elusive. The aim of our study was to investigate the effects and underlying mechanisms of RvD1 in hepatic autophagy in liver fibrosis. In vivo, male C57BL/6 mice were intraperitoneally injected with 20% carbon tetrachloride (CCl4, 5 ml/kg) twice weekly for 6 weeks to establish liver fibrosis model. RvD1 (100 ng or 300 ng/mouse) was added daily in the last 2 weeks of the modeling period. In vitro, lipopolysaccharide (LPS)-activated LX-2 cells were co-treated with increasing concentrations (2.5-10 nM) of RvD1. The degree of liver injury was measured by detecting serum AST and ALT contents and H&E staining. Hepatic fibrosis was assessed by masson's trichrome staining and metavir scoring. The qRT-PCR, western blot, immunohistochemistry, and immunofluorescence were applied to liver tissues or LPS-activated LX-2 cells to explore the protective effects of RvD1 in liver fibrosis. Our findings reported that RvD1 significantly attenuated CCl4 induced liver injury and fibrosis by decreasing plasma AST and ALT levels, reducing collagen I and α-SMA accumulation and other pro-fibrotic genes (CTGF, TIMP-1 and Vimentin) expressions in mouse liver, restoring damaged histological architecture and improving hepatic fibrosis scores. In vitro, RvD1 also repressed the LPS induced LX-2 cells activation and proliferation. These significant improvements mainly attributed to the inhibiting effect of RvD1 on autophagy in the process of hepatic stellate cell (HSC) activation, as demonstrated by decreased ratio of LC3-II/I and elevated p62 after RvD1 treatment. In addition, using AZD5363 (an AKT inhibitor that activates autophagy) and AZD8055 (an mTOR inhibitor, another autophagy activator), we further verified that RvD1 suppressed autophagy-mediated HSC activation and alleviated CCl4 induced liver fibrosis partly through AKT/mTOR pathway. Overall, these results demonstrate that RvD1 treatment is expected to become a novel therapeutic strategy against liver fibrosis.

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Antifibrotic effects of ZK14, a novel nitric oxide-donating biphenyldicarboxylate derivative, on rat HSC-T6 cells and CCl4-induced hepatic fibrosis.

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