Integrative Behavioral, Histological and Proteomics Profiling Identifies Cerebellar Drivers of Motor Dysfunction in a Rat Model of Hepatic Encephalopathy

Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome resulting from liver dysfunction or portosystemic shunting, characterized by cognitive and motor impairments. Although often considered reversible after liver transplantation, emerging evidence suggests HE may cause lasting neurological damage, underscoring the need to explore its molecular basis. The cerebellum, critical for motor coordination and cognitive functions, is increasingly recognized for its role in HE pathogenesis. This study employed proteomic analyses to investigate molecular alterations in the cerebellum of a thioacetamide-induced moderate-grade HE (MoHE) rat model. Motor deficits were confirmed through rotarod and gait test, showing impaired balance, reduced endurance, and disrupted coordination. Histological analyses revealed degeneration of Purkinje neurons and hypertrophy of Bergmann glial fibers. Label-free quantitative mass spectrometry identified 2,002 proteins, with 65 differentially expressed (35 upregulated, 30 downregulated). Bioinformatic analyses indicated dysregulation of pathways involved in calcium signaling, GTPase cycles, endocytosis, and apoptosis. Protein-protein interaction networks highlighted the interconnected roles of these proteins. Overexpression of RheB, MPPCB, and fetuin-A was validated by western blotting. Immunofluorescence further confirmed the overexpression and cerebellar localization of these proteins, along with lactadherin-a novel protein implicated in HE-in the Granular and Purkinje layers. This study offers a comprehensive molecular profile of cerebellar changes in HE, identifying key proteins and pathways that may contribute to neurological impairments. These findings provide a foundation for developing targeted therapies aimed at mitigating HE-related brain dysfunction and improving clinical outcomes.