Mistletoe alkaloid fractions alleviates carbon tetrachloride-induced liver fibrosis through inhibition of hepatic stellate cell activation via TGF-β/Smad interference

Ethnopharmacological relevance: Mistletoe (Viscum coloratum (Kom.) Nakai) has long been categorized as a traditional herbal medicine in Asia. In addition to its application in cancer therapy, mistletoe has also been used in the treatment of chronic hepatic disorders in China. In the present study, we investigated the antifibrotic effect and mechanisms of action of mistletoe extracts in a rat model of carbon tetrachloride (CCl4)-induced hepatotoxicity. Materials and methods: An experimental model of hepatic fibrosis was established by intraperitoneal injection of rats with CCl4 for 8 weeks. Rats were subsequently treated with a mistletoe alkaloid fraction preparation via oral administration (120 mg/kg daily for 8 weeks) or with distilled water as a control. Histopathological changes were observed by hematoxylin and eosin staining and Masson's trichrome staining. The expression of markers relevant to hepatic stellate cell (HSC) activation in the liver was assessed by real-time reverse transcription-polymerase chain reaction, immunohistochemistry and western blotting. The anti-fibrosis activity and mechanisms of action of mistletoe alkaloid fractions were further investigated in the HSC-T6 HSC line, following treatment with mistletoe alkaloid fractions (12 mg/ml) for 48 h. Results: Hepatic fibrosis decreased markedly in CCl4-treated animals following treatment with mistletoe alkaloid fractions, compared to controls. The mRNA levels of transforming growth factor-beta 1 (TGF-beta 1), procollagen I and tissue inhibitors of metalloproteinases (TIMPs) were significantly downregulated, by about 40%, 40% and 45%, respectively, in liver tissues from rats treated with mistletoe alkaloid fractions. Furthermore, significant downregulation of TGF-beta 1, TGF-beta 1 receptor, phosphorylated Smad 2 and alpha smooth muscle actin (alpha-SMA) proteins, by about 45%, 30% and 40%, respectively, was also observed in liver tissues from mistletoe alkaloid fractions-treated rats. In contrast, Smad 7 levels were significantly increased by about 30% in mistletoe alkaloid fractions-treated rats. Treatment of HSC-T6 cells with mistletoe alkaloid fractions significantly induced Smad 7 expression and inhibited the expression of alpha-SMA, TGF beta 1, TGF-beta 1 receptor, Smad 2 and TIMP-1, in vitro. Conclusion: We demonstrate that mistletoe alkaloid fractions decrease extracellular matrix accumulation by inhibiting HSC activation. Mechanistically, this may occur via inhibition of TGF-beta 1/Smad 2 and Smad 7 signal transduction, thereby blocking the synthesis of procollagen I and TIMP-1. These findings suggest that mistletoe alkaloid fractions may be a potential therapeutic agent for the treatment of hepatic fibrosis. (C) 2014 Elsevier Ireland Ltd. All rights reserved.