Abstract
Current anti-fibrotic effect of antioxidants in vivo is disappointing due probably to the fact that once liver fibrogenesis is established it is too advanced to be reversed by anti-oxidation mechanism. We consider antioxidant may only act on the early phase of fibrogenesis. Thus, we had previously established an early liver fibrosis animal model using an inducible expression vector (pPK9a), which contains TGF-β gene and was hydro-dynamically transferred into mice to induce a transient liver fibrosis. TGF-β1 has been well documented to up-regulate the expression of α2(1) collagen (Col 1A2) gene in the liver via the reactive oxygen species (ROS); the process triggers inflammation, leading to hepatic stellate cells (HSC) activation and liver fibrogenesis. Using our animal model and ROS, cyclooxygenase-2 (Cox-2) and Col 1A2 promoter assays as screening targets, we report here that a maleic acid derivative isolated from the Antrodia camphorata mycelium strongly decreases ROS production, promoter activity of Cox-2 and Col 1A2, intracellular calcium, expression of alpha-smooth muscle actin (α-SMA), Smad4-p-Smad2/3 co-localization in cell nucleus and the DNA binding activity of Sp1. Our results suggest that the maleic acid derivative prevents liver fibrosis at an early phase both in vitro and in vivo through the inhibition of ROS, inflammation and the activation of HSC.
Highlights
Advanced liver fibrosis represents a significant and severe healthcare problem and there are no effective drugs for therapy so far
The intracellular oxidation state of the cell was analyzed by flow cytometry, when reactive oxygen species (ROS) were induced by TGF-β1 (2 ng/ml) in the presence or absence of compound 2
Luciferase activities were assayed after cotransfection with pRL-TK and p353 Lux (Col 1A2-luciferase) or pxc918 (Cox2-luciferase) in hepatic stellate cells (HSC)-T6 cells, and cells were cultured with compound 2 and TGF-β1 (2 ng/ml)
Summary
Advanced liver fibrosis represents a significant and severe healthcare problem and there are no effective drugs for therapy so far. It has been documented that TGF-β1 up-regulates the expression of Col 1A2 gene in the liver via ROS generation and calcium influx [1, 2]. The method allows the achievement of a high expression level of exogenous gene in the liver [5, 6] Using this approach we have recently established a transient and early fibrosis animal model by using a pPk9a plasmid that contains TGF-β1 gene and was transferred via tail vein injection into a testing animal. Out of a panel of approximately 96 chemical compounds a small maleic acid derivative molecule isolated from the mycelia of Antrodia camphorata (designated here as compound 2) (Fig 1) was found to be a promising candidate molecule for inhibiting liver fibrogenesis.
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