Modulation of hepatocyte-specific c-Jun N-terminal kinase 2 (JNK2) during chronic liver disease using gene silencing

Background:

The prevalence of non-alcoholic fatty liver disease (NAFLD), including the more aggressive non-alcoholic steatohepatitis (NASH) is continuously increasing in developed countries, associated with major complications due to cirrhosis and end-stage hepatocellular carcinoma (HCC). The c-Jun N-terminal kinases (JNKs) play a crucial role in liver physiology and disease pathogenesis. In the present study, we aimed to investigate the relevance of hepatocyte-specific Jnk2 inhibition in an experimental model of chronic liver disease (CLD) -the NEMOΔhepamice.

Methods:

Jnk2 inhibition specifically in hepatocytes using siRNA (siJnk2) was first performed in vitro in Hepa 1 – 6 cells and validated in vivo in NEMOΔhepamice. The effects of siJnk2 administration were investigated at different stages of CLD. Furthermore, fluorescence molecular and microcomputed tomography (FMT, µCT) were included to analyze the phenotype. In addition, validation of the results was performed in hepatocyte-specific Jnk2 mice crossed with NEMOΔhepa (DKO).

Results:

siJnk2 treatment in an early phase of CLD in NemoΔhepamice caused significant elevated serum levels, which was in line with increased immune cellinfiltration in the liver parenchyma normally visible at this age. Furthermore, siJnk2 treatment caused increased hepatocellular apoptosis and compensatory proliferation. In addition, elevated hepatic stellate cell activation/matrix deposition markers caused more pronounced hepatic fibrogenesis progression. In contrast, siJnk2 administration in aged NEMOΔhepamice (44 – 52 weeks) resulted in improved serum values and a significant reduction in liver immune cell infiltration. Compensatory hepatocyte proliferation (PCNA) was significantly decreased in livers alongside with the absence of MAPK9 signaling. Sirius red collagen staining revealed significantly improved hepatic fibrogenesis also displayed by a reduction of hepatic stellate cell activation/matrix deposition markers. In addition, fluorescence molecular tomography and microcomputed tomography (FMT/µCT) analysis revealed reduced hepatocellular apoptosis in these mice. Moreover, chronic siJnk2 treatment dramatically reduced HCC formation in NEMOΔhepamice at the age of 52 weeks. This was shown by a reduced presence of premalignant and malignant liver tumors corresponding to dysplastic nodules and differentiated adenomas, respectively. In line with the siRNA treatment, DKO (NEMOΔhepa/JNK2Δhepa) mice resemble the phenotype observed after Jnk2 knockdown. Concomitantly, HCC progression was diminished in DKO mice compared to control mice. Interestingly, DKO and siJnk2 treated mice display significantly reduced tumor numbers but increased in total diameter. In addition, the expression of HCC markers such as TNF-α, AFP, VEGF-B and c-MYC were reduced.

Conclusions:

siJnk2 treatment successfully depleted Jnk2 levels both in vitro and in vivo. Interestingly, siJnk2 treatment and DKO mice show stage-dependent liver disease progression. In particular, siRNA-formulated delivery to hepatocytes in the late phase of CLD and DKO display an amelioration of NASH and HCC progression. Thus, Jnk2 downregulation in hepatocytes might be a novel suitable therapeutic option to target a specific stage of CLD.