Background and aim: The role of epigenetic changes in liver diseases has been described through abundant experimental evidence, highlighting the de-acetylation in residue lysine 9 of histone H3 (H3K9) regulated by SIRT1 (NAD-dependent deacetylase), and methylation of promoters of genes involved in fibrogenic response regulated by DNA-Methyl transferase 1 (DNMT1). Our research group characterized the regulation of pirfenidone (PFD) on PPARalpha/gamma-SIRT1 axis in two experimental animal models: (1) NASH and (2) hepatocarcinoma. The objective of this work was to characterize the changes in the acetylation/de-acetylation of H3K9 induced by PFD treatment in hepatic stellate cells (HSCs), in addition to analyzing the global methylation patterns
Material and methods: HSCs were treated with PFD (500μM), SIRT172 activator (20μM) and inhibitor EX527 (80μM) of SIRT1 for 24hrs. Subsequently, changes in the expression of SIRT1 and DNMT1 were analyzed by western blot. Immunofluorescence was carried out using markers that detect H3K9 acetylation and global methylation (5MeC; 5Metyl-Cytosine), images were captured with a confocal microscope in order to visualize the cellular co-location of proteins.
Results: The western blot shows that the treatments with PFD and SIRT1720 treatments increase the expression of SIRT1 and DNMT1 proteins, while EX527 reduces them. Immunofluorescence demonstrated that PFD and SIRT1720 decrease the acetylation of H3K9, but increase overall methylation; contrarily, treatment with EX527 induces an increase in acetylation of H3K9 but decrease overall methylation.
Conclusions: The results of our work indicate for the first time that PFD can regulate epigenetic marks possibly through modulation of the PPARγ-SIRT1-DNMT1 axis. Acetylation in H3K9 decreases with PFD treatment, however overall methylation increases. The perspectives of this work will be to analyze the methylation of specific genes (PPARalpha, IL-6, TNFalpha) involved in the development of liver diseases.
This work was partially subsidized by CONACyT basic science 259096 CB-2015-01, CONACyT No. 658152
Conflicts of interest: The authors have no conflicts of interest to declare.
