Rat HSCs cultured on plastic dish spontaneously undergo myofibroblastic transdifferentiation (“activation”) from day 2 to 3 and become fully activated by day 5 to 7. Upon treatment of day 3 activating or day 7 fully activated HSCs with the YGW extract for 2 days, activation of HSC is morphologically attenuated as compared to the cells treated with the solvent control or no treatment (Fig. 1A). YGW decreases the expression of SMA, the bona fide see more marker for the HSC activation as detected by immunohistochemistry (Fig. 1B), and increases oil red O staining upon addition of retinol and palmitic acid, the parameter for vitamin A storage and the unique

feature of quiescent HSCs (Fig. 1C). In addition, the YGW treatment Sotrastaurin markedly suppresses messenger RNA (mRNA) expression of markers for HSC activation such as α1(I) procollagen, SMA, and TGF-β1 while up-regulating the HSC quiescence marker PPARγ (Fig. 1D). As restored expression of PPARγ reverses activated HSCs to quiescent

cells,8, 9 the observed YGW effect to prevent or reverse culture-activation of HSCs is most likely mediated by way of PPARγ induction. Our recent study revealed the epigenetic mechanisms of Pparγ repression in HSC activation involving up-regulation and recruitment of the DNA methyl-CpG binding protein MeCP2 to the Pparγ promoter, resulting in the recruitment of the HP-1α corepressor.17 That study also demonstrated MeCP2-dependent up-regulation of EZH2, the histone H3 lysine 27 (H3K27) methyltransferase of polychrome repressor complex 2 (PRC2), increasing H3K27 di- and trimethylation in the Pparγ exons with consequent formation of a repressive chromatic structure.17 this website Thus, we tested whether YGW’s inductive effect on Pparγ is associated with epigenetic effects on this gene. First, we examined the recruitment of elongating RNA polymerase

II (Ser2-p RNAPoly II) to the Pparγ gene. As previously shown, culture-activated HSCs at day 7 have a markedly reduced recruitment of the Ser2-p RNAPoly II as compared with day 1 quiescent HSCs, and this suppression is attenuated by YGW treatment (Fig. 2A). MeCP2 enrichment to the Pparγ promoter is increased in day 7 culture-activated HSCs but reduced by the YGW treatment to the level seen in day 1 HSCs (Fig. 2B). This reduction is associated with abrogation of MeCP2 protein induction seen in day 5 HSCs subsequently incubated with the YGW extract for 24 or 48 hours (Fig. 2C). Increased H3K27 dimethylation (H3K27me2) noted at the exon 2 of Pparγ in culture-activated HSCs17 with or without the solvent is also normalized by the YGW extract (Fig. 2D), most likely attributable to suppressed expression of PRC2 components, EZH2, Suz12, and EED (Fig. 2E).