To explore the impact of high-salt diet (HSD) on the progression of high-fat diet (HFD)-induced metabolic dysfunction-associated steatohepatitis (MASH)-related liver fibrosis, as well as the underlying molecular mechanisms.

C57BL/6J mice were used to establish the following models: normal diet (Chow), HSD diet (Chow+8%salt), MASH diet (MASH), and MASH diet combined with HSD (MASH+8%salt). At the experience endpoint, the liver tissues were collected and subjected to HE staining, Sirius Red staining, immunohistochemistry (IHC) assay, and Oil Red O staining for histological assessment of steatosis, lipid deposition, and fibrosis. The levels of liver function indicators and serum lipids were measured and statistically analyzed, and the differences in liver weight, body weight, and their ratios were compared across the groups. RT-qPCR and Western blotting were applied to detect the mRNA and protein levels of hepatic fibrosis and inflammatory markers. Liver tissue samples were subjected to transcriptome sequencing, followed by bioinformatics analysis. Potential signaling pathways were validated through Western blotting to confirm the underlying mechanisms.

Oil Red O and HE staining confirmed the successful establishment of the mouse models of MASH, MASH+8%salt, and Chow+8%salt. Serum levels of aspartate transaminase (AST), alanine aminotransferase (ALT), total cholesterol (Tch), and triglyceride (TG) showed no significant differences between the Chow+8%salt and Chow groups (P>0.05). In contrast, the MASH+8%salt group exhibited significantly higher AST and ALT levels than the MASH group (P<0.01), indicating that HSD exacerbates liver injury in the context of MASH. Sirius Red and α-SMA IHC staining revealed that HSD aggravated MASH-related liver fibrosis (P<0.01). Similarly, the mRNA levels of fibrosis activation-related genes (Col1a1, α-SMA, Timp1, Pdgfrβ) were more significantly up-regulated in the MASH+8%salt group (P<0.01). Protein expression levels of α-SMA and Fibronectin were also markedly elevated. Furthermore, the mRNA levels of inflammation-related genes (TNF-α, IL-6, IL-1β, TGF-β1) were obviously up-regulated in the MASH+8%salt group (P<0.05), suggesting that HSD promotes hepatitis and liver fibrosis progression in MASH. Transcriptome sequencing indicated significant activation of the PI3K/AKT/mTOR signaling pathway and up-regulation of the TGF-β pathway. Heatmap analysis demonstrated marked up-regulation of fibrosis- and inflammation-related genes. Western blotting confirmed significant activation of the PI3K/AKT/mTOR signaling pathway in the MASH+8%salt group (P<0.01).

High-salt diet promotes the progression of hepatitis and liver fibrosis in metabolic dysfunction associated steatohepatitis by activating the PI3K/AKT/mTOR signaling pathway.