C. B. Cummins1, X. Wang1, J. Xu2, Y. Ding2, H. Chen2, J. Zhou2, R. Radhakrishnan1 1University Of Texas Medical Branch,Department Of Surgery,Galveston, TX, USA 2University Of Texas Medical Branch,Department Of Pharmacology And Toxicology,Galveston, TX, USA
Introduction:
Liver fibrosis is characterized as excessive deposition of the extracellular matrix (ECM) proteins, especially collagen type I. Activated hepatic stellate cells (HSCs) are the primary cell type responsible for ECM deposition, and NF-κB signal has been reported as one of major mediators of HSC activation. Previously, our team reported oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, exhibited anti-hepatic fibrogenetic activity In vitro. In the present study, we examined the effects of its novel derivative CYD0618 on HSC viability, apoptosis and NF-κB signaling.
Methods:
The proliferation effects of CYD0618 treatment on activated human and rat HSC cell lines LX-2 and HSC-T6 were measured by Alamar Blue Assay. Apoptosis was measured by Cell Death ELISA. Cellular proteins were determined by Western blots and immunofluorescence.
Results:
CYD0618 significantly inhibited LX-2 cells proliferation in a dose-dependent manner with an IC50 value of ~0.45 μM after 48 hours treatment, this was ~15 fold more potent than the parent compound oridonin. Similar effects were seen in HSC-T6 cells with an IC50 of ~0.75 μM. Cell apoptosis was induced by CYD0618 in both cell lines. CYD0618 treatment increased cell cycle inhibitory protein p21, p27, and induced apoptosis marker cleaved poly (ADP-ribose) polymerase (c-PARP), while significantly suppressing the expression of collagen type I. Notably, CYD0618 blocked lipopolysaccharides(LPS)-induced NF-κB p65 nuclear translocation and DNA binding activity, in addition to preventing LPS-induced NF-κB inhibitory protein IκBα phosphorylation and degradation. LPS-stimulated NF-κB downstream target cytokines IL-6, MCP-1 were also attenuated by CYD0618. It has been reported that phosphorylation of the NF-κB p65 on the serine 536 residue affects its nuclear translocation and transcription of target genes. Our data showed that endogenous NF-κB p65 S536 phosphorylation was inhibited by CYD0618 treatment in a time-dependent fashion. Importantly, NF-κB specific chemical inhibitor Bay-11-0781 was found to inhibit the proliferation, and promote apoptosis in both LX-2 and HSC-T6 cells.
Conclusion:
The potent anti-hepatic fibrogenetic effect of CYD0618 may be mediated via suppression of the NF-κB pathway.