F. J. Bohanon1, X. Wang1, B. M. Graham1, C. Ding2, Y. Ding2, C. Rastellini1, J. Zhou2, R. S. Radhakrishnan1 1University Of Texas Medical Branch,Surgery,Galveston, TX, USA 2University Of Texas Medical Branch,Pharmacology And Toxicology,Galveston, TX, USA
Introduction: Activated hepatic stellate cells (HSCs) are responsible for excess extracellular matrix (ECM) protein deposition in liver fibrosis. Our group reported previously that the natural compound oridonin induces apoptosis, inhibits cell proliferation, as well as down-regulates ECM proteins in activated HSCs. In this study, we investigate the anti-fibrosis effects of oridonin derivative CYD0682 on the activated human and rat HSC cell lines LX-2 and HSC-T6.
Methods: Cell proliferation was measured by Alamar Blue assay. Apoptosis was detected by cell death ELISA and staining of Yo-Pro-1/propidium iodide. Cell cycle was determined by flow cytometry. Immunoblot and Immunofluorescence staining were performed for cellular protein expression.
Results: CYD0682 treatment significantly inhibited LX-2 cells proliferation in a dose-and time-dependent manner with an IC50 value of ~0.6 μM for 48 hours, ~10–fold greater potency than oridonin. Similar results were observed in HSC-T6 cells. In contrast, 2.5 μM of CYD0682 treatment had no significant effects on proliferation of the human hepatocyte cell line C3A. CYD0682 treatment induced LX-2 cell apoptosis and S-phase cell cycle arrest, and was associated with activation of p53, p21, p16, and cleaved-caspase-3. The myofibroblast marker protein α-smooth muscle actin and major ECM proteins type I collagen (Fig. 1) and fibronectin were markedly suppressed in a time-and dose-dependent fashion by CYD0682. Furthermore, pre-treatment with CYD0682, blocked TGF-β-induced Smad2/3 phosphorylation, nuclear translocation and DNA binding, as well as TGF-β-induced type I collagen and fibronectin production in LX-2 cells.
Conclusion: In comparison with oridonin, its novel derivative CYD0682 may be a more potent anti-hepatic fibrosis agent.
