AFM-based nanomechanical evaluation of human umbilical cord mesenchymal stem cells for alcohol-associated liver injury

Abstract

Alcohol-associated liver injury is a major consequence of chronic or excessive alcohol consumption. Mesenchymal stem cells (MSCs) have shown considerable therapeutic potential. However, previous studies have focused mainly on molecular regulation, while the mechanical alterations of injured hepatocytes remain poorly understood. In this study, we established an in vitro model of alcohol-associated liver injury by exposing the human liver epithelial (THLE-2) cells to ethanol. We then evaluated the therapeutic effects of human umbilical cord mesenchymal stem cells (UCSCs). Using inverted microscopy, confocal microscopy and atomic force microscopy (AFM), we assessed cell morphology, intracellular reactive oxygen levels (ROS), F-actin cytoskeletal remodeling, cell surface ultrastructure, and nanomechanical properties at the single-cell level. Treatment with 200 mM ethanol significantly reduced THLE-2 cell viability and increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, confirming the successful model establishment. At the nanoscale, ethanol-injured cells showed markedly increased surface roughness, adhesion and elastic modulus, indicating pronounced cell stiffening and the disruption of membrane mechanical homeostasis. After the UCSC treatment, the morphology of injured cells was clearly improved. ROS levels were reduced and the F-actin cytoskeleton was restored. These changes were accompanied by the normalization of membrane nanomechanical properties, including surface roughness, stiffness and adhesion. Overall, these findings suggest that UCSCs alleviate the alcohol-associated liver injury by promoting cytoskeletal remodeling and restoring mechanical homeostasis at the cell membrane interface. This study also highlights the unique value of AFM in characterizing dynamic cellular nanomechanical properties. It provides a new way for evaluating stem cell therapy from a nanomechanical perspective.