Background: ASK1 is a redox-sensitive kinase that is activated by pathological stimuli including oxidative and ER stress. ASK1 promotes activation of p38 and JNK MAP kinases to induce hepatocyte apoptosis, hepatic inflammation and myofibroblast activation. Here we characterized ASK1 pathway activity in the livers of patients with NASH and evaluated the therapeutic efficacy and mechanism of a selective ASK1 inhibitor in two rodent models of NASH.
Methods: Liver biopsies were obtained from NAFLD patients with fibrosis staged according to the NASH CRN classification. p-p38 immunohistochemistry (IHC) was used as a marker of ASK1 activity. Male wistar rats were fed a choline-deficient high-fat diet (CDHFD) for 14 weeks and administered an ASK1 inhibitor (GS-444217) from week 4-14 (n=8/group). Male C57BL/6 mice were fed a fast-food diet (FFD) for 330 days and treated with GS-444217 from day 240-330 (n=15/group). Endpoints for both rodent models of NASH included hepatic fibrosis (quantified by morphometric image analysis of Picosirius red), analysis of p-p38 and p-JNK (by western blot and IHC), and pro-fibrotic and inflammatory gene expression (by RT-PCR).
Results: Hepatic p-p38 was elevated in NASH liver biopsies and increased with fibrosis stage (Spearman ρ=0.38; ###i
/i###=0.003). In patients with F1-F3 fibrosis, p-p38 was detected in the nuclei of steatotic hepatocytes, inflammatory cells and fibroblasts. In patients with F4 fibrosis (cirrhosis), p-p38 staining was found in subsets of inflammatory cells, fibroblasts and reactive ductal epithelial cells in fibrotic septa. In both the CDHFD rat and the FFD mouse models of NASH, ASK1 inhibition significantly reduced hepatic p38 and JNK phosphorylation (p < 0.05), reduced hepatic fibrosis (p < 0.05), and reduced the expression of profibrotic genes (Col1a1 and TIMP1) and proinflammatory genes (TNF-a and MIP1-a) (p < 0.05).
Conclusions: These data demonstrate that the ASK1 pathway is activated in human NASH and correlates with fibrosis stage. Pharmacologic inhibition of ASK1 reduced hepatic fibrosis in rodent NASH models. These data support the ongoing clinical evaluation of the ASK1 inhibitor selonsertib as a therapy to reduce fibrosis in patients with NASH.
