Effect of aldosterone and its antagonist on the expression of PAI-1 and TGF-β1 in rat hepatic stellate cells.

Background: Aldosterone has been implicated in a variety of organ fibroses, but its role and mechanism in liver fibrosis remain unclear.
Methods: Rat primary hepatic stellate cells (HSCs) were isolated, cultured, and characterized. HSCs were incubated with aldosterone (10(-6) M) for 4 h, 8 h, 12 h, 24 h, and 48 h, after which TGF-β1 (transforming growth factor beta 1) expression was measured by real-time PCR. Rat HSCs were treated with different concentrations of aldosterone (10(-6) M, 10(-7) M, 10(-8) M, and 10(-9) M), and the expressions of PAI-1 (plasminogen activator inhibitor-1) and TGF-β1 were determined by measuring mRNA and protein. HSCs were incubated in groups containing aldosterone (10(-6) M), spironolactone (10(-5) M), both aldosterone and spironolactone, or neither aldosterone nor spironolactone (control), after which mRNA and protein expression of PAI-1 and TGF-β1 were measured. Collagen I expression was detected by immunohistochemical analysis of supernatants of the aldosterone (10(-6) M), TGF-β1, and aldosterone plus TGF-β1 groups. SMAD expression was detected in rat HSC control, HSC plus aldosterone (10(-6) M), HSC plus TGF-β1, and HSC plus aldosterone plus TGF-β1 groups.
Results: HSCs were incubated with aldosterone for 4 h, 8 h, 12 h, 24 h, and 48 h after which TGF-β1 expression was measured. We found that TGF-β1 expression increased in a time dependent manner and reached a peak at 24 h. The expression of TGF-β1 in groups treated with aldosterone for 4 h, 8 h, 12 h, and 24 h was significantly different from the control group (P < 0.01). No significant difference was seen in TGF-β1 expression between the groups treated with aldosterone for 24 h and 48 h (P > 0.05). Compared with the control group, TGF-β1 expression was significantly increased after incubation with different concentrations of aldosterone (10(-6) M, 10(-7) M, 10(-8) M, and 10(-9) M) (P < 0.01). There were significant differences in the expression of TGF-β1 between 10(-6) M and 10(-7) M aldosterone treatment groups (P < 0.01). Compared with the control group, the expression of PAI-1 was significantly increased after incubation with different concentrations of aldosterone (10(-6) M, 10(-7) M, 10(-8) M, and 10(-9) M) (P < 0.01). PAI-1 expression was increased in the aldosterone, spironolactone, and aldosterone plus spironolactone groups. The expression of PAI-1 was significantly enhanced in the aldosterone and aldosterone plus spironolactone groups compared with the control group (P < 0.01). There was a marked enhancement of collagen I expression in the aldosterone, TGF-β1, and aldosterone plus TGF-β1 groups (P < 0.05). Collagen I expressions in the aldosterone and TGF-β1 groups were significantly different from the aldosterone plus TGF-β1 group (P < 0.01). Compared with the control group, SMAD expression was markedly elevated in the aldosterone, TGF-β1, and aldosterone plus TGF-β1 groups (P < 0.05). The expression of SMAD was significantly increased in the aldosterone plus TGF-β1 group compared with the aldosterone group (P < 0.01).
Conclusion: This study demonstrated that aldosterone promoted HSC activation and the expression of TGF-β1, PAI-1, and collagen in hepatic fibrosis progression and that spironolactone administration partially reversed the effects. The aldosterone promotional effect on hepatic fibrosis was partially mediated by TGF-β1.