H2O2-mediated autophagy during ethanol metabolism

Background Alcoholic liver disease (ALD) is the most common liver disease worldwide and its underlying molecular mechanisms are still poorly understood. Moreover, conflicting data have been reported on potentially protective autophagy, the exact role of ethanol-metabolizing enzymes, and ROS.

Methods Expression of LC3B, CYP2E1, and NOX4 was studied in a mouse model of acute ethanol exposure. Autophagy was further studied in primary mouse hepatocytes and huh7 cells in response to ethanol and acetaldehyde. Experiments were carried out in cells overexpressing CYP2E1 and NOX4 silencing. The response to external H2O2 was studied by using the GOX/CAT system. Autophagic flux was monitored using the mRFP-GFP-LC3 plasmid, while rapamycin and chloroquine served as positive and negative controls.

Results Acute ethanol exposure of mice significantly induced LC3B expression but also induced the ROS-generating CYP2E1 and NOX4 enzymes. Notably, ethanol but not its downstream metabolite acetaldehyde induced autophagy in primary mouse hepatocytes. In contrast, autophagy could only be induced in CYP2E1 overexpressed huh7 cells. In addition, overexpression of NOX4 also significantly increased autophagy, which could be blocked by siRNA silencing. The antioxidant N-acetylcysteine (NAC) also efficiently blocked CYP2E1- and NOX4-mediated induction of autophagy. Finally, specific and non-toxic production of H2O2 by the GOX/CAT system as evidenced by elevated peroxiredoxin (Prx-2) also induced LC3B which was efficiently blocked by NAC. H2O2 strongly increased the autophagic flux as measured by mRFP-GFP-LC3 plasmid

Conclusion We here provide evidence that short-term ethanol exposure induces autophagy in hepatocytes both in vivo and in vitro through the generation of ROS.

 

Scroll to top

Publikationsverlauf

Artikel online veröffentlicht:
26. Januar 2022