Deletion of Mixed Lineage Kinase Domain Like Pseudokinase Aggravates Chronic Alcohol-Induced Liver Injury via Increasing Apoptosis.

Abstract

he mixed lineage kinase domain like pseudokinase (MLKL) is known to play a protective role in non-alcoholic fatty liver disease (NAFLD) via inhibition of necroptosis pathway. However, the role of MLKL in alcoholic liver disease (ALD) is not yet clear. C57BL/6N wild-type (WT) and MLKL-knockout (KO) mice (8-10weeks old) were randomly divided into eight groups. To establish ALD model of different durations, ethanol (EtOH) was fed to WT and MLKL KO for 10days, 4weeks, and 8weeks. The control group was fed with Lieber-DeCarli control diet for 8weeks. Mortality, degree of hepatic inflammation, and steatosis were compared among the groups. Bulk mRNA transcriptome analysis was performed. Abundance of transcript and gene expressions were calculated based on read count or Transcript by Million (TPM) value. Survival rate of MLKL KO mice compared to WT was similar until 4weeks, but the survival of MLKL KO mice significantly decreased after 8weeks in ALD model. There was no difference in degree of inflammation, steatosis, and NAS scores between EtOH-fed MLKL KO and EtOH-fed WT mice at 10days. However, at 4weeks and 8weeks, the degree of hepatic steatosis, NAS, and inflammation were increased in MLKL KO mice. RNA transcriptome data showed that fatty acid synthesis, and lipogenesis, mitochondria, and apoptosis-related pathways were upregulated in EtOH-fed MLKL KO mice compared to EtOH-fed WT mice. Although hepatocyte apoptosis (BAX/BCL2 ratio, caspase-3, and TUNEL staining) increased after EtOH intake; however, apoptosis was more significantly increased in EtOH-fed MLKL KO mice compared to the WT group. At the same time, hepatic cFLIP was decreased in EtOH-fed MLKL KO mice compared to the WT group. MLKL deletion did not prevent chronic alcohol-induced liver damage independently of necroptosis and exacerbated hepatic steatosis by increasing hepatocyte apoptosis.