TGF-β/p38 signaling axis regulates hepatocellular senescence and promotes Mallory-Denk body formation.

Mallory-Denk bodies (MDBs), a hallmark pathological feature of metabolic dysfunction-associated steatotic liver disease (MASLD), have been associated with hepatocellular senescence; however, the underlying regulatory mechanisms remain incompletely understood. We investigated whether the transforming growth factor-β (TGF-β)/p38 mitogen-activated protein kinase (p38 MAPK) signaling axis serves as a shared regulatory pathway driving both MDB formation and hepatocellular senescence. To address this, we established two MDB-positive mouse models: a metabolic dysfunction-associated steatohepatitis (MASH) model induced by a high-fat diet (HFD) combined with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), and a liver fibrosis model induced by carbon tetrachloride (CCl₄) plus DDC. These were complemented by an in vitro MDB-forming hepatocyte model stimulated with TNF-α/IFN-γ and TGF-β. In addition, three clinical hepatocellular carcinoma (HCC) specimens with abundant MDBs were analyzed. Multiplex immunofluorescence, RNA sequencing (RNA-seq), and functional in vitro and in vivo assays were employed. Both animal models consistently exhibited MDB formation, metabolic dysregulation, and liver fibrosis, accompanied by activation of p38 signaling, increased phosphorylation of keratin 8 at serine 73 (p-K8 S73), and features of hepatocellular senescence. RNA-seq analysis of the HFD+DDC-induced MASH model further implicated p38 signaling in MDB formation and cellular senescence at the transcriptomic level. Activation of p38 signaling was also confirmed in human HCC specimens. In vitro, pharmacological inhibition of p38 reduced p-K8 S73 levels, suppressed MDB formation, and attenuated the senescent phenotype. Collectively, these findings suggest the TGF-β/p38 signaling axis may be a common molecular mechanism underlying both MDB formation and hepatocellular senescence, providing new insights into the pathogenesis of MDB-associated liver diseases.