Enhanced anti-fibrogenic effects of novel oridonin derivative CYD0692 in hepatic stellate cells.

Oridonin, isolated from Rabdosia rubescens, has been proven to possess various anti-neoplastic and anti-inflammatory properties. Previously, we reported the anti-fibrogenic effects of oridonin for liver in vitro. In the present study, we investigated the effects of a newly designed analog CYD0692 in vitro. Cell viability was measured by Alamar Blue assay. Cell apoptosis was assessed by Cell Death ELISA and Yo-Pro-1 staining. Western blots were performed for cellular proteins. Flow cytometry was used to measure cell cycle regulation. CYD0692 significantly inhibited LX-2 cells proliferation in a dose- and time-dependent manner with an IC50 value of ~0.7 µM for 48 h, ~tenfold greater potency than oridonin. Similar results were observed in HSC-T6 cells. In contrast, on the human hepatocyte cell line C3A, only 12 % of the cell growth was inhibited with 5 µM of CYD0692 treatment for 48 h, while 30 % inhibited at 10 µM. After CYD0692 treatment on LX-2 cells, apoptosis and S-phase cell cycle arrest were induced; cleaved-PARP, p21, and p53 were activated while cyclin-B1 levels declined. In addition, α-smooth muscle actin, type I Collagen, and fibronectin (FN) were markedly down regulated. Transforming growth factor ß1 (TGF ß1) has been identified as a dominant stimulator for ECM production in HSC. Our results indicated that pretreatment with CYD0692 blocked TGF ß1-induced FN expression, thereby decreasing the downstream factors of TGF ß1 signaling, such as Phospho-Smad2/3 and phospho-ERK. In comparison with oridonin, its novel derivative CYD0692 has demonstrated to be a more potent and potentially safer anti-fibrogenic agent for the treatment of hepatic fibrosis.