Regulatory Effects of Ammonium Chloride in an In Vitro Model of Hepatic Encephalopathy: Mechanisms Involving ACTA2‐Mediated Cytoskeletal Remodeling

ABSTRACT Background Hyperammonemia is a central feature of hepatic encephalopathy (HE) and contributes to astrocytic dysfunction; however, the mechanisms underlying ammonia‐induced cytoskeletal remodeling remain unclear. This study aimed to identify key molecular mediators of ammonia‐induced astrocyte injury and to elucidate the role of ACTA2 in this process. Methods We established an in vitro HE model using the human astrocyte line SVG p12 treated with either ammonium chloride (NH4Cl) (5–20 mM) or serum from patients with HE. Cellular responses were evaluated by cell counting kit‐8 assay and flow cytometry. RNA sequencing was performed to identify key regulatory genes, followed by molecular and functional validation of the lead candidate, ACTA2, which encodes alpha‐smooth muscle actin (α‐SMA), using knockdown and overexpression strategies. Filamentous actin organization was assessed by phalloidin staining and fluorescence microscopy. The expressions of water channel (aquaporin‐4) and junction‐associated proteins (zonula occludens‐1, occludin, claudin‐5) were measured to characterize astrocytic junctional protein alterations. Results NH4Cl exposure effectively recapitulated the cytotoxic effects of serum from patients with HE, revealing a distinct biphasic response: low‐dose ammonia (5 mM) transiently boosted metabolic activity, whereas higher doses (10–20 mM) reduced viability, promoted apoptosis, and caused G2/M phase arrest. RNA‐seq, cytoskeletal analysis, and biochemical validation identified ACTA2 downregulation as a sentinel event in ammonia‐induced injury. Functional assays demonstrated that ACTA2 deficiency phenocopied NH4Cl‐induced cytotoxicity, while its restoration significantly mitigated apoptosis and cell cycle arrest. Ammonia treatment led to upregulation of aquaporin‐4 alongside altered expression of junctional proteins, marked by diminished zonula occludens‐1 and claudin‐5 expression and elevated occludin levels. Mechanistically, NH4Cl reduced the phosphorylation of FAK (Tyr397) and proto‐oncogene tyrosine‐protein kinase Src (c‐Src) (Tyr416), whereas ACTA2 overexpression in NH4Cl‐treated cells restored their activation, implicating suppression of the α‐SMA‐FAK/c‐Src signaling axis in ammonia‐induced astrocytic injury. Conclusion This study demonstrates that NH4Cl disrupts cell cytoskeletal stability by dose‐dependently downregulating ACTA2 and suppressing the downstream FAK/c‐Src pathway, thereby affecting the viability and fate of SVG p12 cells and inducing molecular alterations associated with HE. This provides new mechanistic insights into understanding ammonia‐induced astrocytic dysfunction.