Prevention of TGF-β-induced early liver fibrosis by a maleic acid derivative anti-oxidant through suppression of ROS, inflammation and hepatic stellate cells activation

<div><p>Current anti-fibrotic effect of antioxidants <i>in vivo</i> 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 <i>Antrodia camphorata</i> 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 <i>in vitro</i> and <i>in vivo</i> through the inhibition of ROS, inflammation and the activation of HSC.</p></div

Mutation identification and amino acid sequence alignment.

<p><b>a</b> Chromatograms of direct cDNA sequencing of R767H, S885N and R1441H. <b>b</b> Restriction enzyme RFLP or ARMS analysis of R767H, S885N, R1441H mutations. On agarose gel, R767H results restriction by <i>Bsp</i>HI and leads to additional 419 and 154 bp bands, whereas R1441H prevents restriction by <i>Bst</i>UI and leads to an additional 715 bp band. <b>c</b> Evolutionary conservation of the regions of LRRK2 R767H, S885N and R1441H using the program Vector NTI.</p