Hepatic Nrf2 overexpression inhibits the deleterious effects induced by c-met deficiency in the progression of NASH

Background: We recently showed that hepatocyte-specific c-met deficiency accelerates the progression of non-alcoholic steatohepatitis in experimental murine models resulting in increased hepatic lipotoxicity, augmented production of reactive oxygen species and accelerated development of fibrosis. The aim of this study focuses on the elucidation of the underlying cellular mechanisms driven by Nrf2 over-activation in hepatocytes lacking c-met receptor characterized by a severe unbalance between pro-oxidant and anti-oxidant functions.

Methods: For this purpose, we generated double knockout mice lacking simultaneously c-met and keap1 genes employing the flox/cre technology under the control of the albumin promoter. Control mice (c-metfx/fx), single c-met knockouts (c-metΔhepa) and double c-met/keap1 knockouts (met/keap1Δhepa) were then fed a methionine-choline deficient (MCD) diet for 4 weeks in order to reproduce the features of NASH. Serum and liver samples were collected for biochemical, RNA and protein expression analyses.

Results: Double mutants displayed an increased liver mass as compared to the other experimental groups, but present less triglycerides accumulation. The marked increase of oxidative stress observed in c-metΔhepa was restored in the double mutants as assessed by 4-HNE immunostaining and by the expression of genes involved in the control of redox homeostasis, such as Cyp2e1 and NOX2. Accordingly, the number of TUNEL positive cells was also dramatically decreased in met/keap1Δhepa. Double ko mice also presented a reduced amount of liver infiltrating cells – neutrophils and resident macrophages (CD11b+/F4 – 80+) – as emerged by flow cytometry analysis of intrahepatic lymphocytes. These data were further supported by RT-PCR analyses indicating a decrease of the proinflammatory cytokine MCP1 in the double knockouts as compared to the other groups. Similarly, the worsening of fibrosis progression observed in c-metΔhepa livers was efficiently reduced at the levels of controls as indicated by Sirius Red staining and expression of pro-fibrotic mediators such as TGFb1 and Col1a1.

Conclusion: Genetic activation of the anti-oxidant transcription factor Nrf2 improves liver damage and repair in c-met deficient mice mainly through restoring a balance in the cellular redox homeostasis. This observation arouses further considerations for the development of novel therapeutic strategies.