Mechanisms and key active ingredients of HeDan capsules in ameliorating MASLD via bile acid metabolism regulation.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: HeDan Capsules(HD), a traditional Chinese medicinal preparation, possesses the effects of resolving phlegm, reducing turbidity, activating blood circulation, and resolving blood stasis. It is clinically indicated for the treatment of hyperlipidemia and metabolic dysfunction-associated steatotic liver disease (MASLD). However, the mechanism by which HD ameliorates MASLD remains unclear. AIM OF THE STUDY: This study aims to systematically elucidate the potential mechanisms and key bioactive ingredients underlying the efficacy of HD in ameliorating MASLD. MATERIAL AND METHODS: Classical rat MASLD model was used to evaluate the therapeutic effects of HD. Non-targeted and targeted quantitative metabolomics approaches were employed to investigate the therapeutic effects of HD from the perspective of bile acid metabolism. Key proteins involved in the bile acid synthesis pathway were identified using RT-qPCR and Western blotting, while differences in gut microbiota composition were analyzed via 16S rDNA sequencing. Network pharmacology combined with Bayesian optimization-based molecular docking was used for screening of active compounds. Molecular dynamics simulations and alanine scanning were subsequently performed to assess binding stability and key residue interactions. Finally, the key active compounds of HD were validated in a zebrafish model, and their mechanism of action was investigated at the cellular level using inhibitors. RESULTS: HD improved liver function in the MASLD rat model by enhancing lipid deposition and inflammatory response, and significantly modulated the bile acid metabolic network in rats with MASLD. Subsequent targeted metabolomics analyses further confirmed that HD markedly alter bile acid profiles in rat serum. Results from RT-qPCR and Western blotting suggested that HD might influence the classical bile acid synthesis pathway by acting on the FXR/CYP7A1/CYP8B1 signaling pathway. Sequencing results of 16S rDNA indicated that HD may also influence bile acid metabolism through affecting the stability of the gut microbiota. Ultimately, nuciferine, ursolic acid, cryptotanshinone, quercetin, salvianolic acid A, and methyl tanshinonate were identified as the key active components, and cellular-level experiments further confirmed that the effects of HD could be blocked by an FXR inhibitor. CONCLUSIONS: HD may effectively reduce hepatic lipid accumulation to ameliorate MASLD. Mechanistically, HD regulates the classical bile acid synthesis pathway through the FXR/CYP7A1/CYP8B1 signaling axis and promotes gut microbiota homeostasis. And this study identified firstly the key bioactive ingredients in HD that underlie its anti-MASLD effect.