The role of MAPK/mTOR pathway in regulating ULK1 mediated autophagy in hyperammonemia induced liver injury

Hyperammonemia is frequently encountered in cirrhosis; nevertheless, its direct hepatotoxic effects remain poorly characterized. In this study, we aimed to delineate the hepatotoxic effects of chronic hyperammonemia and and explore the related mechanisms of liver injury. Thirty male C57BL/6 mice were randomly divided into three groups (n = 10 per group): a control group, a hyperammonemia group, and a rifaximin-treated group. Starting on day 0, the hyperammonemia and rifaximin-treated groups were fed a diet supplemented with an amino acid (AA) mixture at a 1:2 ratio to normal powdered (NP) diet for 14 consecutive days to establish chronic hyperammonemia, while the control group received NP diet. From day 8 onward, mice in the rifaximin-treated group received rifaximin by gavage at 100 mg/kg once daily for 7 days; the control and hyperammonemia groups received an equivalent volume of 0.9% saline. Twenty-four hours after the final administration, blood was collected; serum was separated by centrifugation and analyzed on an automatic biochemical analyzer for blood ammonia, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Immediately after blood sampling, mice were euthanized; whole brain and liver were rapidly excised, blotted dry, weighed, and used to calculate brain weight and liver index values. Tissue specimens were fixed for hematoxylin-eosin (H&E) staining to evaluate histopathological damage. Commercial kits were used to measure superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, urea content and glutamine synthetase (GS) activity in the liver tissue. Take paraffin sections of liver tissue and use Terminal Deoxynucleotide Transferase dUTP Nick End Labeling (TUNEL) method to detect the apoptosis level of liver cells. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) were performed on liver samples to measure the mRNA and protein expression levels, respectively. Hyperammonemia significantly increased brain weight index, liver index, blood ammonia, serum ALT and AST (p < 0.001); rifaximin reversed these changes (p < 0.001). Liver urea content was significantly lower in the hyperammonemia group (0.245 ± 0.008 mmol/g, p < 0.01) than in the control group (0.281 ± 0.004 mmol/g), and rifaximin obviously rescued this reduction and raised hepatic urea to 0.300 ± 0.014 mmol/g (p < 0.01). Hepatic GS activity declined remarkably in the hyperammonemia group (8.37 ± 0.50, p < 0.05) compared with the control group (8.87 ± 0.41), and rifaximin treatment effectively increased GS activity (9.42 ± 0.27, p < 0.05). Histologically, H&E-stained brain sections from hyperammonemia mice displayed pronounced cytotoxic edema, while liver sections showed disordered hepatocellular cords, and marked inflammatory infiltration, both of which were mitigated by rifaximin. Compared with the control group, the liver tissue SOD activity in the hyperammonemia model group was significantly reduced, and the MDA content was significantly increased (p < 0.01); After intervention with rifaximin, SOD activity increased and MDA content decreased (p < 0.01). TUNEL staining further showed that the apoptosis level of liver cells in the model group mice was significantly increased compared to the control group, while treatment with rifaximin significantly reduced the number of apoptotic cells (p < 0.0001). Mechanically, hyperammonemia-induced hepatic injury are underpinned by its ability to upregulate MAPK/mTOR signaling and downregulate ULK1 activity, thereby impairing autophagic flux. Hyperammonemia may negatively regulate ULK1 activity through the MAPK/mTOR pathway, impair autophagic flux, promote liver oxidative stress and hepatocyte apoptosis, and lead to liver injury.