Betulin and betulinic acid attenuate ethanol-induced liver stellate cell activation by inhibiting reactive oxygen species (ROS), cytokine (TNF-α, TGF-β) production and by influencing intracellular signaling

Abstract

Background/aims Liver fibrosis has been reported to be inhibited in vivo by oleanolic and ursolic acids. However, the mechanisms of the action of those triterpenoids are poorly understood. In this study, we aimed to determine the antifibrotic potential of other triterpenes, betulin and betulinic acid, and to characterize their influence on the signal transduction pathways involved in ethanol-activated hepatic stellate cells (HSCs). Methods Investigated was the influence of preincubation of rat HSCs with betulin and betulinic acid, at non-toxic concentrations, on ethanol-induced toxicity, migration, and several markers of HSC activation such as smooth muscle α-actin (α-SMA) and procollagen I expression, release of reactive oxygen species (ROS) and cytokines: tumor necrosis factor-α (TNF-α) and tumor growth factor-β1 (TGF-β1), and production of metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). To assess the mechanism of the action of those triterpenes, intracellular signals such as nuclear factor-κB (NFκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol were examined. Results In vitro, betulin, but not betulinic acid, protected HSCs against ethanol toxicity. However, both betulin and betulinic acid inhibited the production of ROS by HSCs treated with ethanol and inhibited their migration as well as ethanol-induced TNF-α, and TGF-β1, production. Betulin and betulinic acid down-regulated ethanol-induced production of TIMP-1 and TIMP-2. Betulin and betulinic acid, also decreased ethanol-induced activity of MMP-2. In ethanol-induced HSCs, betulin inhibited the activation of the p38 MAPK and the JNK transduction pathways, while betulinic acid inhibited the JNK transduction pathway only. They also significantly inhibited phosphorylation of IκB and Smad 3 and attenuated the activation of TGF-β1 and NFκB/IκB transduction signaling. Conclusion The results indicated that betulin and betulinic acid inhibited ethanol-induced activation of HSCs on different levels, acting as antioxidants, inhibitors of cytokine production, and inhibitors of TGF-β, and NFκB/IκB transduction signaling. Betulin was also inhibitor of both JNK and p38 MAPK signal transduction, while betulinic acid inhibited only JNK. The remarkable inhibition of several markers of HCS activation makes triterpenes, especially betulin, promising agents for anti-fibrotic combination therapies.