Stigmasterol mitigates cholesterol gallstone formation by modulating bile acid metabolism and gut microbiota in mice fed a lithogenic diet.

Stigmasterol (STG) has demonstrated cholesterol-lowering properties; however, its influence on the bile acid (BA) metabolic profile and its potential in preventing cholesterol gallstone formation remain unclear. This study explores the inhibitory effects of STG on cholesterol gallstone development under a lithogenic diet (LD). Female C57BL/6 mice were fed either an LD or an LD supplemented with STG (L-STG) for 16 weeks. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed to quantify BAs in colon contents, serum, and gallbladder bile, while 16S rRNA gene sequencing analyzed gut microbiota composition. The results demonstrated that STG supplementation significantly reduced the cholesterol saturation index in mouse bile compared to the LD group, thereby inhibiting cholesterol crystal formation and mitigating gallbladder and liver damage. In the L-STG group, BA hydrophobicity was decreased, and secondary BA levels were markedly reduced, particularly the hepatotoxic deoxycholic acid. Additionally, STG enhanced gut microbiota α-diversity, reversed LD-induced alterations in microbial relative abundance, and especially curbed the abnormal expansion of BA-metabolizing bacteria. These findings suggest that STG can attenuate the overproduction of secondary BAs, thereby alleviating liver injury and facilitating BA excretion. Ultimately, this leads to reduced biliary cholesterol saturation and inhibition of gallstone formation.