Cellular senescence exhibits context-dependent roles in liver pathophysiology, contributing to chronic disease progression while potentially facilitating acute injury repair. Although senescent hepatocytes are well-documented in chronic liver diseases, their role in acute liver injury (ALI) remains poorly understood. Exosomes have emerged as key mediators of intercellular communication during senescence, carrying biologically active molecules that can modulate recipient cell behavior. However, the specific functions of senescent hepatocyte-derived exosomes in ALI, particularly their effects on hepatic stellate cell (HSC) activation dynamics, have not been systematically investigated. The purpose of this study was to characterize the role and mechanisms of senescent hepatocyte exosomes in regulating HSC activation during ALI.

Acute liver injury (ALI) was induced in 8-week-old male C57BL/6 mice via intraperitoneal injection of LPS (30 μg/kg) and D-GalN (200 mg/kg). For intervention studies, mice received ATN-161 (50 mg/kg/day) for 3 days prior to injury induction. To induce cellular senescence, L02 cells were treated with 4 μM etoposide (HY-13629, MCE, USA) for 24 h. Subsequently, the medium was replaced with fresh, etoposide-free DMEM, and the cells were cultured for an additional 3 days. Exosomes were isolated from conditioned media of senescent (Sen) and non-senescent (Non-Sen) hepatocytes via differential ultracentrifugation and characterized by electron microscopy and western blotting.

In the LPS/D-GalN-induced acute liver injury (ALI) mouse model, we observed significant upregulation of hepatic senescence markers including γH2AX, P16, P21, and P53. Western blot analysis confirmed elevated expression of α-SMA and Col1A1, demonstrating that acute liver injury induces both hepatocyte senescence and hepatic stellate cell (HSC) activation. To establish an in vitro senescence model, hepatocytes were treated with etoposide (ETO). ETO-treated hepatocytes developed characteristic senescent morphology, appearing enlarged and flattened, with intense SA-β-galactosidase staining. These cells showed markedly increased protein levels of γH2AX, P16, P21, and P53, along with reduced expression of nuclear envelope protein Lamin B1, confirming successful induction of cellular senescence. Notably, etoposide-induced senescent hepatocytes demonstrated enhanced exosome secretion compared to non-senescent controls. Functional assays revealed that these senescent hepatocyte-derived exosomes significantly promoted HSC migration, proliferation and activation. Through comprehensive proteomic analysis, we identified integrin α5 (ITGA5) as a prominently upregulated component in senescent hepatocyte-derived exosomes. This finding was corroborated by elevated ITGA5 expression in both senescent hepatocytes and ALI mouse livers. Mechanistic investigation showed that mTORC1 inhibition by rapamycin substantially reduced ITGA5 expression in senescent hepatocytes, indicating mTORC1-dependent regulation of ITGA5 during senescence. To delineate ITGA5’s role in HSC activation, we performed siRNA-mediated ITGA5 knockdown in LX2 cells. This intervention led to significant downregulation of α-SMA and Col1A1, along with reduced Smad2 phosphorylation, suggesting ITGA5 mediates HSC activation primarily through the Smad2 pathway. Surprisingly, pharmacological ITGA5 inhibition with ATN-161 worsened liver injury and disrupted hepatic architecture, potentially due to compromised HSC activation. This paradoxical effect highlights the complex, context-dependent role of ITGA5 in liver pathophysiology.

Collectively, our study demonstrates that senescent hepatocytes secrete ITGA5-enriched exosomes that activate HSCs via Smad2 signaling, maintaining liver structure during ALI. These findings not only reveal a crucial protective mechanism in acute liver injury but also identify ITGA5 as a potential therapeutic target for liver repair.