Delineating the heterogeneity of senescence-induced-functional alterations in hepatocytes

Background and Aim: Cellular senescence in hepatocytes is characterized by permanent cell cycle arrest, impaired bioenergetics, resistance to apoptosis, and the secretion of pro-inflammatory cytokines. This “zombie-like” state drives tissue dysfunction and may contribute to the progression of liver diseases. Importantly, the features of senescence vary depending on both the type of stressor and the target cell. Therefore, this study aims to investigate stressor-specific changes in senescence phenotypes, their molecular profiles, and cellular bioenergetics in primary mouse hepatocytes (PMH) and hepatocyte-derived liver organoids (HepOrgs).

Methods: PMH were isolated from collagenase-perfused livers of 18–23-week-old C57B6/J mice and cultured overnight in William’s E-medium with 2% FBS, L-glutamine, and hepatocyte growth supplements. HepOrgs were generated by culturing hepatocytes in a 3D matrix for two weeks. Senescence was induced using various stressors: DNA damage (doxorubicin, cisplatin, etoposide), oxidative stress (H₂O₂, ethanol), telomere inhibition (BIBR-1532), p53 activation (nutlin-3a), DNA methyltransferase inhibition (5-azacitidine), and metabolism inhibitors (galactosamine, hydroxyurea). Senescence markers were evaluated through SA-β-galactosidase activity, immunofluorescence, immunoblotting, SASP profiling, and bioenergetic assessments.

Results: Each senescence inducer triggered distinct senescence markers BIBR 1532 in hepatocytes. All stressors, except hydroxyurea and ethanol, increased SA-β-galactosidase activity, a hallmark of senescence. Among SASP factors, CCL2 and IL-10 were consistently upregulated, while plasminogen activator inhibitor-1 (PAI-1) was globally downregulated. DNA damage inducers activated the DNA damage response (DDR), and cell cycle arrest was most pronounced with doxorubicin, cisplatin, and galactosamine. Additionally, DNA damage-induced senescence caused a metabolic shift from glycolysis to oxidative phosphorylation. In HepOrgs, exposure to senescence inducers led to higher levels of γH2A.X, p53, and p21. Notably, SASP gene expression was markedly elevated in HepOrgs compared to PMH, showing several-fold increases.

Conclusion: Our findings reveal that each stressor induces a unique senescence signature in PMH, with doxorubicin showing the strongest effect, followed by cisplatin and H₂O₂, without triggering apoptosis. All inducers activated DDR and mitochondrial dysfunction, independent of the MAPK/AKT pathway. These results highlight the importance of stressor-specific responses in hepatocyte senescence and their potential implications for liver disease progression.