To explore the protective effects and potential mechanism of bifendate against methionine-choline-deficient (MCD) diet-induced metabolic dysfunction-associated steatotic liver disease (MASLD) in mice.

A total of 24 male C57BL/6J mice were randomly divided into control group, model group, and experimental group. The model group was fed with MCD diet to establish the MASLD model, while the experimental group received bifendate (300 mg·kg^-1) in addition to the MCD diet. After 6 weeks, liver pathology was assessed using Hematoxylin-Eosin (H&E) and Oil Red O staining. Hepatic triglyceride (TG) content, serum lipopolysaccharide (LPS) and serum alanine aminotransferase (ALT) levels were measured using assay kits. Gut microbiota composition and diversity were analyzed by high-throughput sequencing. The relative expression levels of the intestinal tight junction protein ZO-1 and Occludin were detected by Western blot.

Oil Red O staining revealed that the number and degree of hepatocellular steatosis were significantly reduced in the experimental group compared to the model group. The MCD diet successfully induced steatohepatitis in mice, characterized by significantly increased hepatic TG, serum ALT, and marked hepatocellular steatosis. Hepatic TG contents were (66.43±14.58), (231.29±20.26), and (190.39±29.33) μmol·g^-1 for the control, model and experimental groups, respectively; serum ALT levels were (52.04±16.80), (219.74±123.37) and (31.68±10.70) IU·L^-1, respectively; serum LPS levels were (0.89±0.57), (4.01±0.65) and (0.52±0.25) EU·mL^-1, respectively. The relative expression levels of ZO-1 were 1.00±0.94, 0.06±0.03 and 0.25±0.27, respectively, and for Occludin were 1.00±0.57, 0.06±0.03 and 0.15±0.15, respectively. Compared with the model group, the experimental group showed statistically significant differences in all the aforementioned indicators (P<0.05, P<0.001). Bifendate intervention significantly reduced serum ALT and hepatic TG levels and markedly alleviated the extent and severity of hepatocellular steatosis. Regarding the gut-liver axis, bifendate significantly improved the diversity and richness of the gut microbiota in model mice, particularly increasing the relative abundance of Bilophila and Desulfovibrio, genera closely related to bile acid metabolism. Furthermore, bifendate intervention significantly up-regulated the expression of intestinal tight junction proteins and decreased serum LPS levels.

Bifendate can effectively ameliorate MCD diet-induced steatohepatitis in mice. Its protective mechanism is closely related to the regulation of the gut-liver axis, including remodeling the intestinal flora structure (increasing the relative abundance of Bilophila and Desulfovibrio), enhancing intestinal barrier function (upregulating tight junction proteins, reducing serum LPS), and potentially affecting bile acid metabolism.