β-Sitosterol protects against lithocholic acid-induced hepatotoxicity and cholestasis via farnesoid X receptor-mediated regulation of transporters and enzymes in vitro and in vivo.

Cholestasis arises as a clinical syndrome triggered by the accumulation and aggregation of bile acids. Currently, there are only a few treatment options available for cholestasis. Therefore, it is necessary to explore novel therapeutic strategies. β-sitosterol (SIT), the phytosterol most abundantly found in plants, exhibits diverse pharmacological activities. This study examined SIT's protective role against hepatotoxicity and cholestasis induced by lithocholic acid (LCA). LCA was administered twice a day to male C57BL/6 mice for four days to cause hepatotoxicity and cholestasis. Assessment of the improvement in cholestasis following SIT treatment used H&E staining and serum biomarkers. Mice hepatocyte culture, real-time PCR, immunofluorescence staining, and Western blot were utilized to clarify the mechanisms of SIT hepatoprotection. Furthermore, molecular docking and dual-luciferase reporter gene analysis were utilized to show that SIT would activate the farnesoid X receptor (FXR). In vivo, SIT reduced bile acid accumulation by inducing the bile salt export pump (Bsep), multidrug resistance-related protein 2 (Mrp2), and reduced hepatic uptake of bile acids by inhibiting Na+/taurocholate co-transporting polypeptide (Ntcp), and cholesterol 7α-hydroxylase (Cyp7a1) and oxysterol 12α-hydroxylase (Cyp8b1) while in vitro, it restored FXR expression and transcriptional activity. Besides, SIT decreased hepatic inflammation by suppressing the inflammatory genes NF-κB p65 and p-NF-κB p65, TNF-α, IL-6, and IL-1β. However, the hepatoprotective effects of SIT were abolished by the FXR antagonist guggulsterone in vivo and FXR siRNA in vitro, confirming FXR-dependent mechanisms. In conclusion, SIT protects against LCA-induced hepatotoxicity and cholestasis via FXR activation. These findings highlight SIT as a promising therapeutic candidate for cholestasis.