ABSTRACT
BACKGROUND: Intestinal fibrosis is a hallmark of Crohn's disease. Here, we investigated the impact of several putative antifibrotic compounds on the expression of fibrosis markers using murine precision-cut intestinal slices. METHODS: Murine precision-cut intestinal slices were cultured for 48 hours in the presence of profibrotic and/or antifibrotic compounds. The fibrotic process was studied on gene and protein level using procollagen 1a1 (Col1α1), heat shock protein 47 (Hsp47), fibronectin (Fn2), and plasminogen activator inhibitor-1 (Pai-1). The effects of potential antifibrotic drugs mainly inhibiting the transforming growth factor ß (TGF-ß) pathway (eg, valproic acid, tetrandrine, pirfenidone, SB203580, and LY2109761) and compounds mainly acting on the platelet-derived growth factor (PDGF) pathway (eg, imatinib, sorafenib, and sunitinib) were assessed in the model at nontoxic concentrations. RESULTS: Murine precision-cut intestinal slices remained viable for 48 hours, and an increased expression of fibrosis markers was observed during culture, including Hsp47, Fn2, and Pai-1. Furthermore, TGF-ß1 stimulated fibrogenesis, whereas PDGF did not have an effect. Regarding the tested antifibrotics, pirfenidone, LY2109761, and sunitinib had the most pronounced impact on the expression of fibrosis markers, both in the absence and presence of profibrotic factors, as illustrated by reduced levels of Col1α1, Hsp47, Fn2, and Pai-1 after treatment. Moreover, sunitinib significantly reduced Hsp47 and Fn2 protein expression and the excretion of procollagen 1. CONCLUSIONS: Precision-cut intestinal slices can successfully be used as a potential preclinical screening tool for antifibrotic drugs. We demonstrated that sunitinib reduced the expression of several fibrosis markers, warranting further evaluation of this compound for the treatment of intestinal fibrosis.
Subject(s)
Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Gastrointestinal Agents/pharmacology , Intestines/drug effects , Signal Transduction/drug effects , Animals , Collagen Type I/drug effects , Collagen Type I, alpha 1 Chain , Crohn Disease/drug therapy , Crohn Disease/pathology , Fibronectins/drug effects , Fibrosis/drug therapy , Fibrosis/pathology , HSP47 Heat-Shock Proteins/drug effects , Intestines/pathology , Mice , Platelet-Derived Growth Factor/drug effects , Serpin E2/drug effects , Sunitinib/pharmacology , Transforming Growth Factor beta/antagonists & inhibitorsABSTRACT
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, characterized by excess fat accumulation (steatosis). Nonalcoholic steatohepatitis (NASH) develops in 15-20% of NAFLD patients and frequently progresses to liver fibrosis and cirrhosis. We aimed to develop an ex vivo model of inflammation and fibrosis in steatotic murine precision-cut liver slices (PCLS). NASH was induced in C57Bl/6 mice on an amylin and choline-deficient l-amino acid-defined (CDAA) diet. PCLS were prepared from steatohepatitic (sPCLS) and control (cPCLS) livers and cultured for 48 h with LPS, TGFß1, or elafibranor. Additionally, C57Bl/6 mice were placed on CDAA diet for 12 wk to receive elafibranor or vehicle from weeks 7 to 12. Effects were assessed by transcriptome analysis and procollagen Iα1 protein production. The diets induced features of human NASH. Upon culture, all PCLS showed an increased gene expression of fibrosis- and inflammation-related markers but decreased lipid metabolism markers. LPS and TGFß1 affected sPCLS more pronouncedly than cPCLS. TGFß1 increased procollagen Iα1 solely in cPCLS. Elafibranor ameliorated fibrosis and inflammation in vivo but not ex vivo, where it only increased the expression of genes modulated by PPARα. sPCLS culture induced inflammation-, fibrosis-, and lipid metabolism-related transcripts, explained by spontaneous activation. sPCLS remained responsive to proinflammatory and profibrotic stimuli on gene expression. We consider that PCLS represent a useful tool to reproducibly study NASH progression. sPCLS can be used to evaluate potential treatments for NASH, as demonstrated in our elafibranor study, and serves as a model to bridge results from rodent studies to the human system.NEW & NOTEWORTHY This study showed that nonalcoholic steatohepatitis can be studied ex vivo in precision-cut liver slices obtained from murine diet-induced fatty livers. Liver slices develop a spontaneous inflammatory and fibrogenic response during culture that can be augmented with specific modulators. Additionally, the model can be used to test the efficacy of pharmaceutical compounds (as shown in this investigation with elafibranor) and could be a tool for preclinical assessment of potential therapies.
