Liver fibrosis is a critical pathological stage in chronic liver diseases, characterized by excessive extracellular matrix deposition and progressive loss of functional liver units, ultimately leading to hepatocellular carcinoma. Macrophages play a key role in liver fibrosis, while cellular senescence is an irreversible cell cycle arrest that alters the tissue microenvironment via the senescence-associated secretory phenotype (SASP). Although macrophages can undergo senescence, their specific role in liver fibrosis remains unclear.

Liver fibrosis was induced in young (8-week) and aged (12-month) mice by intraperitoneal injection of 25% carbon tetrachloride (CCl₄). Hepatic function and immune microenvironment were assessed by histopathology, serum biochemistry, Western blot, RT-qPCR, immunohistochemistry, and immunofluorescence. In vitro, a senescent macrophage model was established in RAW264.7 cells using 200 μmol/L H₂O₂, confirmed by senescence-associated β-galactosidase (SA-β-Gal) staining. Conditioned medium (CM) from senescent macrophages was applied to LX-2 cells to evaluate hepatic stellate cell (HSC) activation. Transcriptome sequencing was performed on the senescent RAW264.7 cells to identify potential mechanisms. Finally, to investigate the functional role of fatty acid synthase (FASN) in macrophage senescence, cycloheximide (CHX) was used to assess FASN protein half-life and stability, while combined treatment with MG132 and chloroquine (CQ) was employed to preliminarily explore the degradation mechanism of FASN during macrophage senescence.

In vivo, aged mice treated with CCl₄ exhibited higher serum ALT and AST levels than young mice, along with more severe hepatic collagen deposition, fibrosis, and senescence. SA-β-Gal positive cells were mainly distributed along fibrotic scars. Expression of senescence and SASP markers (γH2AX, p21, p53, TNF-α) was significantly elevated in aged mice. Immunohistochemistry and immunofluorescence double staining further demonstrated the presence of marked cellular senescence in macrophages within the livers of CCl₄-induced fibrotic mice. Human liver biopsies from fibrotic patients also showed increased CD68 and p21 double-positive cells compared with hepatitis patients. In vitro, senescent macrophages showed upregulated expression of SASP-related cytokines and DNA damage markers, and their conditioned medium promoted LX-2 cell activation, as evidenced by increased α-SMA and COL1A1 expression. Transcriptome sequencing of senescent RAW264.7 cells revealed upregulation of FASN and p38, alongside downregulation of fatty acid β-oxidation enzymes (CPT1A, CPT2) and regulators (CD36, PPAR γ, ACACA). FASN protein was degraded over time after CHX treatment, and its degradation was partially rescued by MG132 or CQ, indicating involvement of both ubiquitin-proteasome and autophagy pathways. Moreover, treatment with FASN inhibitors reduced the expression of p21, p53, and γH2AX, alleviating the DNA damage response in senescent macrophages.

Macrophages in liver fibrosis exhibit cellular senescence. Senescent macrophages, through FASN-mediated enhancement of fatty acid synthesis and inhibition of fatty acid β-oxidation, lead to lipid metabolic disorder and accumulation, thereby sustaining the secretion of SASP inflammatory factors that promote hepatic stellate cell activation and contribute to the progression of liver fibrosis.