Short interfering RNA targetting NF-kappa B induces apoptosis of hepatic stellate cells and attenuates extracellular matrix production

Background: Pharmacological inhibition of the NF-kappa B activity enhances hepatic stellate cell apoptosis and reverses experimental fibrosis. However, there is no report on the effects of NF-kappa B knockdown on apoptosis and extracellular matrix secretion in hepatic stellate cells. The aim of the present study is to explore the effects of siRNA targetting NF-kappa B on the apoptosis and extracellular matrix production in hepatic stellate cells. Methods: The immortalised hepatic stellate cell line HSC-T6 was transfected with siRNA; 72 h later, cells were stimulated by LPS for 1 h; these cells were collected for further use. Hepatic stellate cell apoptosis was determined by fluorescence activated cell sorter analysis, TUNEL assay and caspase-3 activity measurement. Matrix metalloproteinase 2 activity was evaluated with Gelatin zymography. The quantities of mRNA transcriptions of NF-kappa B p65, type I collagen, tissue inhibitor of metalloproteinases-1, alpha-smooth muscle actin and transforming growth factor beta 1 and anti-apoptotic protein Al were evaluated with quantitative reverse transcriptase real-time polymerase chain reaction. Results: siRNA targetting NF-kappa B p65 effectively abrogated the expression of NF-kappa B p65 in hepatic stellate cells; decreased anti-apoptotic protein Bcl-2 and the mRNA transcription of hepatic type I collagen, alpha-smooth muscle actin, transforming growth factor beta 1, Al and tissue inhibitor of metalloproteinases-1; increased matrix metalloproteinase 2 activity and promoted hepatic stellate cell apoptosis. Conclusion: NF-kappa B knockdown enhances hepatic stellate cell apoptosis and attenuates extracellular matrix production. (C) 2010 Editrice Gastroenterologica ltaliana S.r.l. Published by Elsevier Ltd. All rights reserved.