Background & Aims: Hepatocyte apoptosis, the hallmark of non‐alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis ofNASH, the final common step of apoptosis is executed by a family of cysteine‐proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan‐caspase inhibitor, ameliorates liver injury and fibrosis in a murine model ofNASH.Methods: C57/BL6J‐mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan.Results: Mice fed a HFD diet demonstrate a five‐fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5‐fold and 1.3‐fold increase in caspase‐3 and‐8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD‐Em). Likewise, liver injury and inflammation were reduced in mice fed HFD‐Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1‐β, TNF‐α, monocyte chemoattractant protein (MCP‐1) and C‐X‐C chemokine ligand‐2 (CXCL2) quantitative reverse‐transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR.Conclusion: In conclusion, these data demonstrate that in a murine model ofNASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy inNASH.

Liver cirrhosis is characterized by an excessive accumulation of extracellular matrix components, including hyaluronan (HA). In addition, cirrhosis is considered a pre-neoplastic disease for hepatocellular carcinoma (HCC). Altered HA biosynthesis is associated with cancer progression but its role in HCC is unknown. 4-Methylumbelliferone (4-MU), an orally available agent, is an HA synthesis inhibitor with anticancer properties. In this work, we used an orthotopic Hepa129 HCC model established in fibrotic livers induced by thioacetamide. We evaluated 4-MU effects on HCC cells and hepatic stellate cells (HSCs) in vitro by proliferation, apoptosis and cytotoxicity assays; tumor growth and fibrogenesis were also analyzed in vivo. Our results showed that treatment of HCC cells with 4-MU significantly reduced tumor cell proliferation and induced apoptosis, while primary cultured hepatocytes remained unaffected. 4-MU therapy reduced hepatic and systemic levels of HA. Tumors systemically treated with 4-MU showed the extensive areas of necrosis, inflammatory infiltrate and 2–3-fold reduced number of tumor satellites. No signs of toxicity were observed after 4-MU therapy. Animals treated with 4-MU developed a reduced fibrosis degree compared with controls (F1-2 vs F2-3, respectively). Importantly, 4-MU induced the apoptosis of HSCs in vitro and decreased the amount of activated HSCs in vivo. In conclusion, our results suggest a role for 4-MU as an anticancer agent for HCC associated with advanced fibrosis.

Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis.