Theerut Luangmonkong

Human precision-cut liver slices as a model to test antifibrotic drugs in the early onset of liver fibrosis.

Liver fibrosis is the progressive accumulation of connective tissue ultimately resulting in loss of organ function. Currently, no effective antifibrotics are available due to a lack of reliable human models. Here we investigated the fibrotic process in human precision-cut liver slices (PCLS) and studied the efficacy of multiple putative antifibrotic compounds. Our results demonstrated that human PCLS remained viable for 48h and the early onset of fibrosis was observed during culture, as demonstrated by an increased gene expression of Heat Shock Protein 47 (HSP47) and Pro-Collagen 1A1 (PCOL1A1) as well as increased collagen 1 protein levels. SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK) showed a marked decrease in HSP47 and PCOL1A1 gene expression, whereas specific inhibitors of Smad 3 and Rac-1 showed no or only minor effects. Regarding the studied antifibrotics, gene levels of HSP47 and PCOL1A1 could be down-regulated with sunitinib and valproic acid, while PCOL1A1 expression was reduced following treatment with rosmarinic acid, tetrandrine and pirfenidone. These results are in contrast with prior data obtained in rat PCLS, indicating that antifibrotic drug efficacy is clearly species-specific. Thus, human PCLS is a promising model for liver fibrosis. Moreover, MAPK signaling plays an important role in the onset of fibrosis in this model and transforming growth factor beta pathway inhibitors appear to be more effective than platelet-derived growth factor pathway inhibitors in halting fibrogenesis in PCLS.

Organ- and species-specific biological activity of rosmarinic acid

Rosmarinic acid (RA), a compound found in several plant species, has beneficial properties, including anti-inflammatory and antibacterial effects. We investigated the toxicity, anti-inflammatory, and antifibrotic effects of RA using precision-cut liver slices (PCLS) and precision-cut intestinal slices (PCIS) prepared from human, mouse, and rat tissue. PCLS and PCIS were cultured up to 48 h in the absence or presence of RA. Gene expression of the inflammatory markers: IL-6, IL-8/CXCL1/KC, and IL-1β, as well as the fibrosis markers: pro-collagen 1a1, heat shock protein 47, α-smooth muscle actin, fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1) were evaluated by qPCR. RA was only toxic in murine PCIS. RA failed to mitigate the inflammatory response in most models, while it clearly reduced IL-6 and CXCL1/KC gene expression in murine PCIS at non-toxic concentrations. With regard to fibrosis, RA decreased the gene levels of Fn2 and PAI-1 in murine PCLS, and Fn2 in murine PCIS. Yet, no effect was observed on the gene expression of fibrosis markers in human and rat PCIS. In conclusion, we observed clear organ- and species-specific effects of RA. RA had little influence on inflammation. However, our study further establishes RA as a potential candidate for the treatment of liver fibrosis.

Evaluating the antifibrotic potency of galunisertib in a human ex vivo model of liver fibrosis

Liver fibrosis is a major cause of liver‐related mortality and, so far, no effective antifibrotic drug is available. Galunisertib, a TGF‐β receptor type I kinase inhibitor, is a potential candidate for the treatment of liver fibrosis. Here, we evaluated the potency of galunisertib in a human ex vivo model of liver fibrosis.

Targeting Oxidative Stress for the Treatment of Liver Fibrosis

Oxidative stress is a reflection of the imbalance between the production of reactive oxygen species (ROS) and the scavenging capacity of the antioxidant system. Excessive ROS, generated from various endogenous oxidative biochemical enzymes, interferes with the normal function of liver-specific cells and presumably plays a role in the pathogenesis of liver fibrosis. Once exposed to harmful stimuli, Kupffer cells (KC) are the main effectors responsible for the generation of ROS, which consequently affect hepatic stellate cells (HSC) and hepatocytes. ROS-activated HSC undergo a phenotypic switch and deposit an excessive amount of extracellular matrix that alters the normal liver architecture and negatively affects liver function. Additionally, ROS stimulate necrosis and apoptosis of hepatocytes, which causes liver injury and leads to the progression of end-stage liver disease. In this review, we overview the role of ROS in liver fibrosis and discuss the promising therapeutic interventions related to oxidative stress. Most importantly, novel drugs that directly target the molecular pathways responsible for ROS generation, namely, mitochondrial dysfunction inhibitors, endoplasmic reticulum stress inhibitors, NADPH oxidase (NOX) inhibitors, and Toll-like receptor (TLR)-affecting agents, are reviewed in detail. In addition, challenges for targeting oxidative stress in the management of liver fibrosis are discussed.

In vitro and ex vivo anti-fibrotic effects of LY2109761, a small molecule inhibitor against TGF-β

ABSTRACT Fibrosis is a pathophysiological state characterized by the excessive formation/deposition of fibrous extracellular matrix. Transforming growth factor‐beta (TGF‐&bgr;) is a central profibrotic mediator, and targeting TGF‐&bgr; is a promising strategy in the development of drugs for the treatment of fibrosis. Therefore, the effect of LY2109761, a small molecule inhibitor against TGF‐&bgr; with targets beyond TGF‐&bgr; signaling, on fibrogenesis was elucidated in vitro (HepG2 cells and LX‐2 cells) and ex vivo (human and rat precision‐cut liver slices). Our results displayed an anti‐fibrotic effect of LY2109761, as it markedly down‐regulated gene and protein expression of collagen type 1, as well as gene expression of the inhibitor of metalloproteinases 1. This effect on fibrosis markers was partially mediated by targeting TGF‐&bgr; signaling, seeing that LY2109761 inhibited TGF‐&bgr;1 gene expression and SMAD2 protein phosphorylation. Interestingly, particularly at a high concentration, LY2109761 decreased SMAD1 protein phosphorylation and gene expression of the inhibitor of DNA binding 1, which appeared to be TGF‐&bgr;‐independent effects. In conclusion, LY2109761 exhibited preclinical anti‐fibrotic effects via both TGF‐&bgr;‐dependent and ‐independent pathways. These results illustrate that small molecule inhibitors directed against TGF‐&bgr; could possibly influence numerous signaling pathways and thereby mitigate fibrogenesis. HIGHLIGHTSLY2109761 elicits anti‐fibrotic effects both in vitro and ex vivo.At low concentrations, LY2109761 mainly affects the TGF‐&bgr; pathway.LY2109761 has targets beyond TGF‐&bgr; signaling, increasing its anti‐fibrotic efficacy.SMAD1 and ID1 are novel and interesting therapeutic targets for drug development.