Hyperlipidemia, a major cause of cardiovascular disease, is a significant health concern. Bile acid (BA) synthesis and excretion in the liver are closely related to hyperlipidemia. Restoring blood lipids, particularly cholesterol and BA metabolism, is crucial for treatment. Two pathways convert cholesterol to BAs: the classical neutral pathway and the alternative acidic pathway. BA metabolism is regulated by various factors, including receptors, transporters, genes, and proteins involved in liver circulation. Farnesoid X receptor (FXR) is a key regulator, with chenodeoxycholic acid (CDCA) as a potent ligand. BAs are pleiotropic signal metabolites, influencing metabolism and inflammation via microflora and host receptor interactions. Intestinal microflora modifies BAs through deconjugation and dehydroxylation, converting cholic acid (CA) and CDCA into dicarboxylic acid (DCA) and tricarboxylic acid (TCA). Conjugated BAs are deconjugated in the ileum by bile salt hydrolase, increasing BA activity. The intestinal barrier, particularly the mucus layer produced by goblet cells, maintains intestinal stability and microflora balance, influencing lipid metabolism. High-fat diets (HFDs) disrupt intestinal barrier function, affecting the gut-liver axis. Phytosterols, similar in structure and function to cholesterol, reduce intestinal cholesterol absorption and lower LDL-C. Stigmasterol (ST), a phytosterol, shows therapeutic effects in hyperlipidemia and obesity, potentially by regulating intestinal barrier function and cholesterol metabolism. This study hypothesizes that ST treatment regulates intestinal barrier function, affecting cholesterol and BA metabolism.

Existing research demonstrates that stigmasterol (ST) offers therapeutic benefits in hyperlipidemia and obesity. However, the underlying mechanisms remain unclear. Studies have shown ST's positive impact on fatty liver and metabolic abnormalities caused by high-fat diets. Another study suggests ST's role in attenuating inflammatory bowel disease by activating the butyrate-PPARγ axis, restoring immune balance in Treg/Th17 cells and improving gut microbiota disequilibrium. Previous work has explored the role of bile acids in cholesterol metabolism and the gut-liver axis. Research indicates that gut microbiota significantly impacts bile acid metabolism, influencing the BA pool through modifications like deconjugation and dehydroxylation. The importance of intestinal barrier function in maintaining the gut environment and its influence on lipid metabolism is well-documented. Phytosterols, including ST, have been shown to reduce cholesterol absorption and improve lipid profiles.

This study used Sprague-Dawley (SD) rats fed a high-fat diet (HFD) for 11 weeks to investigate the effects of stigmasterol (ST). Rats were divided into groups: control (normal diet), HFD, HFD + low-dose ST, HFD + high-dose ST, and HFD + simvastatin. Body weight, visceral fat weight, liver weight, serum lipid levels (TC, TG, LDL-C, HDL-C), and liver function markers (AST, ALT) were measured. Histological analysis (H&E, Oil Red O staining) assessed hepatic steatosis. Intestinal barrier function was evaluated using H&E and AB-PAS staining to assess goblet cell numbers, and immunohistochemistry (IHC) for occludin and ZO-1 protein expression. 16s rDNA gene sequencing analyzed gut microbiota composition. Liquid chromatography-mass spectrometry (LC-MS) analyzed serum and fecal bile acid (BA) profiles. A fecal microbiota transplantation (FMT) study used stool samples from ST-treated and HFD-fed rats to determine the role of the gut microbiota. Gene expression (cyp7a1, nr1h4, cyp27a1, cyp7b, cyp8b) was analyzed by RT-qPCR and protein expression by Western blotting and immunofluorescence. A second animal study investigated the combined effect of CDCA and ST on HFD-fed rats, assessing similar parameters. Statistical analysis employed one-way ANOVA and Tukey's test.

ST significantly reduced weight gain, visceral fat, and liver weight in HFD-fed rats. Serum TC, TG, and LDL-C levels were significantly reduced by ST treatment. ST improved hepatic steatosis, as evidenced by H&E and Oil Red O staining. ST increased the number of goblet cells and the expression of occludin and ZO-1 in the ileum, indicating improved intestinal barrier function. ST treatment altered the composition of the gut microbiota, reducing the abundance of Erysipelotrichaceae and Peptostreptococcaceae. ST treatment modified serum and fecal BA profiles, increasing CDCA levels and decreasing TCA levels. FMT from ST-treated rats improved hepatic steatosis and intestinal barrier function in recipient rats. ST upregulated hepatic cyp7a1 mRNA expression and increased CDCA levels. Combined CDCA/ST treatment further improved lipid profiles and increased the expression of CYP7A1, suggesting synergistic effects.

This study demonstrates that ST effectively alleviates HFD-induced hyperlipidemia and hepatic steatosis. The mechanism involves improving intestinal barrier function, altering gut microbiota composition, and modulating BA metabolism, particularly through the CYP7A1 pathway. The increase in CDCA, an FXR agonist, and the decrease in TCA, associated with liver cirrhosis, are significant findings. The upregulation of ABCG1 suggests ST's involvement in cholesterol efflux. Combined CDCA/ST treatment shows enhanced therapeutic effects. The FMT results indicate a crucial role for the gut microbiota in mediating ST's effects. The observed changes in gut microbiota composition, particularly the reduction of Erysipelotrichaceae and Allobaculum, are consistent with improved metabolic health.

Stigmasterol (ST) effectively mitigates HFD-induced hyperlipidemia and hepatic steatosis through a multi-faceted mechanism involving improved intestinal barrier function, modulation of gut microbiota, and enhanced bile acid metabolism via the CYP7A1 pathway. Combination therapy with CDCA amplifies these benefits. Future research should explore the precise mechanisms governing ST's effects on the gut-liver axis and investigate the potential of ST as a therapeutic agent for obesity-associated disorders. Further studies could focus on identifying specific microbial species involved and exploring the clinical applications of ST supplementation.

The study used a rat model, which may not fully translate to human physiology. The mechanisms underlying ST's effects on the gut microbiota remain partially unclear. Further research is needed to determine the optimal dosage and long-term effects of ST in humans.