Ethyl acetate fraction of Terminalia bellirica fruit inhibits rat hepatic stellate cell proliferation and induces apoptosis

Fibrosis results from excessive accumulation of extracellular matrix (ECM), and hepatic stellate cell (HSC) plays a central role in hepatic fibrosis. Thus, removal of excess ECM and promotion of apoptosis in HSC are two antifibrotic approaches. Ethyl acetate fraction (EF) of Terminalia bellirica fruit has long been used in liver diseases, but little scientific evidence exists for EF mechanisms in such illnesses. EF has demonstrated both antiproliferative and proapoptotic activities in HSC-T6. The present study investigated the effects of EF on cellular proliferation, ECM removal, cellular signaling pathways, and apoptosis of a rat HSC line (HSC-T6). HSC-T6 cells were incubated with EF, and their proliferation was assessed by MU assay. Expression of Smad2, platelet-derived growth factor receptor (PDGFR), alpha-smooth muscle actin (alpha-SMA), matrix metalloproteinase-2 (MMP-2) was determined by real-time polymerase chain reaction (PCR) and western blot analysis. Expression of tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 mRNAs was determined by real-time PCR. Collagen I, collagen III, TGF-beta 1, and hydroxyproline levels were assessed by ELISA. Apoptosis was measured and confirmed by Annexin V-PI staining using fluorescence microscopy and flow cytometry. Apoptotic pathways involving Fas/FasL expression and Bcl-2/Bax family were investigated by real-time PCR. Results showed that 31.25-250 mu g/mL EF exhibited cytotoxic and antiproliferative effects on HSC-T6 cells. EF at 50 mu g/mL significantly decreased the levels of collagen I, collagen III, TGF-beta 1, and hydroxyproline. EF suppressed the gene expression of Smad2, PDGFR, alpha-SMA, TIMP-1, and TIMP-2 but elevated that of MMP-2. These events consequently facilitated ECM removal and fibrosis resolution. The highest potency of EF promoting HSC-T6 apoptosis was detected at 50 mu g/mL concentration. This finding was associated with upregulation of Fas/FasL and Bax, as well as downregulation of Bcl-2 in HSC-T6. These results indicate that EF demonstrates antifibrotic activity, and its mechanism of action can be ascribed to inhibition of collagen synthesis, cytokine secretion, and TGF-beta 1/Smad pathway. Furthermore, EF facilitates apoptosis in HSCs. (C) 2015 Elsevier B.V. All rights reserved.