Bruceine D ameliorates cholestatic liver injury by selectively modulating bile acid synthesis and activating FXR-SHP signaling.

Abstract

BACKGROUND AND AIMS: Cholestatic liver diseases (CLDs) are characterized by impaired bile acid (BA) homeostasis, chronic inflammation, and progressive fibrosis, for which effective pharmacological options remain limited. Ursodeoxycholic acid (UDCA) offers modest benefits and obeticholic acid (OCA) is constrained by tolerability issues, underscoring the need for novel therapeutics. This study evaluated the hepatoprotective effects and underlying mechanisms of Bruceine D (BD), a natural quassinoid compound, in murine models of cholestasis. METHODS: Two complementary models were employed: Multidrug resistance protein 2 knockout (Mdr2) mice and α-naphthylisothiocyanate (ANIT)-induced cholestasis. Serum biochemistry, histopathology, bile acid profiling, and ultrastructural analyses were performed to assess hepatocellular injury and fibrosis. Mechanistic studies included gene and protein expression analyses, functional FXR luciferase reporter assays with pharmacological antagonism, and in vitro hepatocyte assays to interrogate BA metabolism, inflammatory responses, and FXR-dependent signaling. RESULTS: BD significantly reduced alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL) in both models, whereas direct bilirubin (DBIL) remained unaffected. Histological analyses demonstrated marked attenuation of hepatocellular injury, bile duct proliferation, fibrosis, and macrophage infiltration. Notably, BD selectively suppressed classical BA synthesis enzymes (CYP7A1, CYP8B1, CYP27A1), while sparing CYP7B1 and major BA transporters. Quantitative BA profiling revealed a shift from hydrophobic, hepatotoxic species (CDCA, DCA, CA) toward hydrophilic and conjugated bile acids (TUDCA, TDCA, β-TMCA), accompanied by restoration of canalicular ultrastructure. BD attenuated inflammatory cytokines and chemokines and reduced fibrogenic responses. Mechanistically, BD functionally restored FXR signaling and reactivated the FXR-SHP-FGF15/19 feedback axis suppressed under cholestatic conditions. Short-term toxicological evaluation revealed no significant adverse effects in major organs. CONCLUSIONS: BD ameliorates cholestatic liver injury by selectively inhibiting classical BA synthesis, quantitatively remodeling the BA pool toward a less hepatotoxic profile, and suppressing inflammation and fibrosis through functional restoration of FXR-dependent feedback signaling. Its synthesis-centered mechanism and favorable short-term safety profile support BD as a promising therapeutic candidate for cholestatic liver diseases.