Objective: To investigate the inhibitory potential of aldosterone antagonist on NOX4 protein expression in hepatic fibrosis by using a rat model of carbon tetrachloride (CCl4)-induced hepatotoxicity.
Methods: Twenty-four male Wistar rats were randomly divided into three equal groups: fibrosis model group (receiving three subcutaneous injections per week of 2.5 ml/kg 40% CCl4); spironolactone (Sp)-treated fibrosis model group (receiving CCl4 regimen plus three injections per day of 20 mg/kg Sp in olive oil); negative-treatment fibrosis model group (receiving CCl4 regimen plus three injections per day of olive oil alone). Unmanipulated rats (receiving no CCl4 and no supplemental treatments) served as normal controls. After 4 weeks, liver histology was carried out to assess cytotoxicity (by hematoxylin-eosin staining), fibrosis (by Masson staining and METAVIR scoring), and NOX4 protein expression (by immunohistochemistry). In addition, in vitro analyses of immortalized rat hepatic stellate cells, HSC-T6, were performed to evaluate dose-response (10-9, 10-7 and 10-5 mol/L) and time-response (6, 12 and 24 h) of aldosterone agonist (Ald) and an aldosterone antagonist, eplerenone (EPLE). Effects on NOX4 protein expression were evaluated by western blotting.
Results: The fibrosis model group showed significantly more fibrosis than the normal control group (16.060 +/- 0.300 vs. 2.471 +/- 0.160, P = 0.000]; however, the Sp-treated fibrosis model group showed significantly less CCl4-induced fibrosis (5.761 +/- 0.152 vs. model: 16.060 +/- 0.300, P = 0.000). The fibrosis model group also showed significantly higher NOX4 protein expression in liver tissues than the normal control group (7.231 +/- 0.211 vs. 1.350 +/- 0.252, P = 0.000), and the Sp-treated fibrosis model tissues showed significantly less CCl4-induced up-regulated NOX4 protein expression (4.270 +/- 0.242 vs. model: 7.231 +/- 0.211, P = 0.000]. Ald induced up-regulated NOX4 protein expression in HSC-T6 cells in dose- and concentration-dependent manners, with the peak expression being induced by the 10-5 mol/L concentration and 24 h exposure. The Ald-treated cells expressed significantly more NOX4 protein than the untreated control cells (0.710 +/- 0.011 vs. 0.316 +/- 0.015, P = 0.000]. and the EPLE-treated cells showed significantly less Ald-induced up-regulated NOX4 expression (0.615 +/- 0.014 vs. 0.710 +/- 0.011, P = 0.000].
Conclusion: Aldosterone antagonists inhibit the fibrosis-induced NOX4 protein expression in rat hepatic cells.