Ursolic acid suppresses TGF-β1-induced quiescent HSC activation and transformation by inhibiting NADPH oxidase expression and Hedgehog signaling

Activation of quiescent hepatic stellate cells (q-HSCs) and their transformation to myofibroblasts (MFBs) is a key event in liver fibrosis. Hedgehog (Hh) signaling stimulates q-HSCs to differentiate into MFBs, and NADPH oxidase (NOX) may be involved in regulating Hh signaling. The author's preliminary study demonstrated that ursolic acid (UA) selectively induces apoptosis in activated HSCs and inhibits their proliferation in vitro via negative regulation of NOX activity and expression. However, the effect of UA on q-HSCs remains to be elucidated. The present study aimed to investigate the effect of UA on q-HSC activation and HSC transformation and to observe alterations in the NOX and Hh signaling pathways during q-HSC activation. q-HSC were isolated from adult male Sprague-Dawley rats. Following culture for 3 days, the cells were treated with or without transforming growth factor-beta 1 (TGF-beta 1; 5 mu g/l); intervention groups were pretreated with UA (40 mu M) or diphenyleneiodonium chloride (DPI; 10 mu M) for 30 min prior to addition of TGF-beta 1. mRNA and protein expression of NOX and Hh signaling components and markers of q-HSC activation were examined by western blotting and reverse transcription-polymerase chain reaction. TGF-beta 1 induced activation of q-HSCs, with increased expression of a-smooth muscle actin alpha-SMA) and type I collagen. In addition, expression of NOX subunits (gp91(phox), p67(phox), p22(phox), and Rac1) and Hh signaling components, including sonic Hh, sterol-4-alpha-methyl oxidase, and Gli family zinc finger 2, were upregulated in activated HSCs. Pretreatment of q-HSCs with UA or DPI prior to TGF-beta 1 significantly downregulated expression of NOX subunits and Hh signaling components and additionally inhibited expression of alpha-SMA and type I collagen, thereby preventing transformation to MFBs. UA inhibited TGF-beta 1-induced activation of q-HSCs and their transformation by inhibiting expression of NOX subunits and the downstream Hh pathway.