Lipophagy and epigenetic alterations are related to metabolic dysfunction-associated steatotic liver disease progression in an experimental model

Abstract

Background: Genetic and epigenetic alterations are related to metabolic dysfunction-associated steatotic liver disease (MASLD) pathogenesis.

Aim: To evaluate micro (mi)RNAs and lipophagy markers in an experimental model of metabolic dysfunction-associated steatohepatitis (MASH).

Methods: Adult male Sprague Dawley rats were randomized into two groups: Control group (n = 10) fed a standard diet; and intervention group (n = 10) fed a high-fat-choline-deficient diet for 16 weeks. Molecular evaluation of lipophagy markers in liver tissue [sirtuin-1, p62/sequestosome-1, transcription factor-EB, perilipin-2 (Plin2), Plin3, Plin5, lysosome-associated membrane proteins-2, rubicon, and Cd36], and serum miRNAs were performed.

Results: Animals in the intervention group developed MASH and showed a significant decrease in sirtuin-1 (P = 0.020) and p62/sequestosome-1 (P < 0.001); the opposite was reported for transcription factor-EB (P = 0.020), Plin2 (P = 0.003), Plin3 (P = 0.031), and Plin5 (P = 0.005) compared to the control group. There was no significant difference between groups for lysosome-associated membrane proteins-2 (P = 0.715), rubicon (P = 0.166), and Cd36 (P = 0.312). The intervention group showed a significant increase in miR-34a (P = 0.005) and miR-21 (P = 0.043) compared to the control. There was no significant difference between groups for miR-375 (P = 0.905), miR-26b (P = 0.698), and miR-155 (P = 0.688).

Conclusion: Animals with MASH presented expression changes in markers related to lysosomal stress and autophagy as well as in miRNAs related to inflammation and fibrogenesis, processes that promote MASLD progression.

Keywords: Animal model; Epigenetic; Lipophagy; Metabolic dysfunction-associated steatohepatitis; Metabolic dysfunction-associated steatotic liver disease; MicroRNAs.

Figure 1

Study design. Animals received a standard or high-fat and choline-deficient diet, together with water (ad libitum) during the 16 weeks of the experiment. All animals were euthanized at the end. HFCD: High-fat and choline-deficient.

Figure 2

Gene expression of circulating microRNAs. A: Micro (mi)R-34a; B: MiR-21; C: MiR-375; D: MiR-26b; E: MiR-155 (P = 0.688). Mann-Whitney U test, ^aP < 0.05 considered significant. Variables are described as median (25^th-75^th percentiles).

Figure 3

Hepatic gene expression of lipophagy markers. A: Sirtuin-1; B: Lysosome-associated membrane protein-2; C: P62/sequestosome-1; D: Transcription factor EB; E: Rubicon; F: Cd36; G: Perilipin 2 (Plin2); H: Plin3; I: Plin5. ^aP < 0.05 considered significant, Mann-Whitney U test. Variables are described as median (25^th-75^th percentiles). Lamp2: Lysosome-associated membrane protein-2; Plin: Perilipin; p62/Sqstm1: P62/sequestosome-1; Sirt1: Sirtuin-1; Tfeb: Transcription factor EB.

Figure 4

Inflammation, fibrosis, and metabolic dysfunction-associated fatty liver disease status. A: Results after dividing the intervention group in two halves according to their inflammation status; B: Results after dividing the intervention group in two halves according to their fibrosis status; C: Results after dividing the intervention group in two halves according to their metabolic dysfunction-associated fatty liver disease status. Plin: Perilipin; Tfeb: Transcription factor EB; NAFLD: Non-alcoholic fatty liver disease.

Figure 5

Correlations between circulating microRNAs and gene expression. A: Correlation between Sirtuin-1 (Sirt1) and p62/sequestosome-1; B: Correlation between Sirt1 and rubicon; C: Correlation between micro (mi)R26b and miR-21. P62/Sqstm1: Sequestosome-1; Sirt1: Sirtuin-1.