Gastroenterology

Gastroenterology

Volume 128, Issue 1, January 2005, Pages 108-120
Gastroenterology

Basic-liver, pancreas, and biliary tract
Inhibition of inhibitor of κB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis

https://doi.org/10.1053/j.gastro.2004.10.003Get rights and content

Background & Aims: Resolution of liver fibrosis is associated with clearance of hepatic myofibroblasts by apoptosis; development of strategies that promote this process in a selective way is therefore important. The aim of this study was to determine whether the inhibitor of κB kinase suppresser sulfasalazine stimulates hepatic myofibroblast apoptosis and recovery from fibrosis. Methods: Hepatic myofibroblasts were generated by culture activation of rat and human hepatic stellate cells. Fibrosis was established in rat livers by chronic injury with carbon tetrachloride followed by recovery with or without sulfasalazine (150 mg/kg) treatment. Results: Treatment of hepatic stellate cells with sulfasalazine (0.5–2.0 mmol/L) induced apoptosis of activated rat and human hepatic stellate cells. A single in vivo administration of sulfasalazine promoted accelerated recovery from fibrosis as assessed by improved fibrosis score, selective clearance of smooth muscle α-actin-positive myofibroblasts, reduced hepatic procollagen I and tissue inhibitor of metalloproteinase 1 messenger RNA expression, and increased matrix metalloproteinase 2 activity. Mechanistic studies showed that sulfasalazine selectively blocks nuclear factor-κB-dependent gene transcription, inhibits hepatic stellate cell expression of Gadd45β, stimulates phosphorylation of Jun N-terminal kinase 2, and promotes apoptosis by a mechanism that is prevented by the Jun N-terminal kinase inhibitor SP600125. As further evidence for a survival role for the inhibitor of κB kinase/nuclear factor-κB pathway in activated hepatic stellate cells, a highly selective cell-permeable peptide inhibitor of κB kinase activation also stimulated hepatic stellate cell apoptosis via a Jun N-terminal kinase-dependent mechanism. Conclusions: Inhibition of the inhibitor of κB kinase/nuclear factor-κB pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo apoptosis both in vitro and in vivo, and drugs that selectively target inhibitor of κB kinase have potential as antifibrotics.

Section snippets

Cell culture

HSC were isolated from normal livers of 350-g adult male Sprague-Dawley rats by sequential perfusion with collagenase and pronase, followed by discontinuous density centrifugation in 11.5% Optiprep (Life Technologies, UK). HSC were cultured on plastic in Dulbecco’s modified Eagle medium supplemented with penicillin 100 U/mL, streptomycin 100 μg/mL, l-glutamine 2 mmol/L, and 16% fetal calf serum and were maintained at 37°C in an atmosphere of 5% CO2. Activated HSC were generated by continuous

Sulfasalazine stimulates apoptosis of activated hepatic stellate cells and recovery from CCl4-induced fibrosis

Sulfasalazine induced a dose-dependent increase in HSC apoptosis as visualized by acridine orange staining (Figure 1). Apoptotic cells were identified by nuclear condensation/blebbing (Figure 1 A). Incubation of HSC with 0.5, 1, and 2 mmol/L sulfasalazine stimulated 28%, 43%, and 50% apoptosis, respectively, compared with dimethyl sulfoxide-treated HSC (Figure 1 B). Sulfasalazine treatment also induced a dose-dependent increase in caspase 3 activity (Figure 1 C). We also confirmed that

Discussion

There is now considerable interest in the discovery of compounds that selectively promote the apoptosis of activated HSC because proof-of-principle studies have shown that in vivo stimulation of HSC apoptosis will promote recovery from liver fibrosis. 7 In this study, we showed that a single administration of sulfasalazine to CCl4-injured rats promoted rapid clearance of α-SMA-positive myofibroblasts, reduced hepatic expression of procollagen I and TIMP1, increased hepatic MMP activity, and

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    • Cited by (0)

    Supported by the Wellcome Trust (050443/Z and 068524/Z/02/Z), the UK Medical Research Council (Co-operative Group Grant component Grant G9900279), and the Children’s Liver Disease Foundation. J.P.I. is an Medical Research Council senior clinician fellow.

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    Copyright © 2005 American Gastroenterological Association. Published by Elsevier Inc. All rights reserved.