Digeda-8 formula attenuates cholestatic liver injury in rats by restoring bile acid transport and suppressing inflammation: An integrated metabolomics and proteomics approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Digeda-8 (DGD8) is a traditional polyherbal formulation with a long history of clinical use in Mongolian medicine for the treatment of hepatobiliary disorders and cholestatic conditions. Despite its traditional reputation, the pharmacological basis and molecular mechanisms underlying its effects against cholestatic liver injury (CLI) remain scientifically uncharacterized.

AIM OF THE STUDY: This study aimed to evaluate the hepatoprotective efficacy of DGD8 against alpha-naphthylisothiocyanate (ANIT)-induced CLI in rats and to systematically elucidate its multi-target mechanisms of action using an integrated systems pharmacology approach.

MATERIALS AND

METHODS

A rat model of ANIT-induced CLI was established to assess the efficacy of DGD8 via biochemical markers (ALT, AST, TBA, TBIL), histopathological analysis, and immunological assessments. The chemical profile of DGD8 was characterized using HPLC-QTOF-MS/MS combined with GNPS molecular networking. To explore the underlying mechanisms, an integrated framework was employed, including metabolomics, TMT-labeled proteomics, Western blot, and immunofluorescence microscopy. Furthermore, network pharmacology integrated with human cholestasis transcriptomics and molecular docking was used to identify and validate disease-relevant molecular targets.

RESULTS

DGD8 treatment, particularly at a high dose (700 mg/kg), significantly reduced ANIT-induced elevations in biochemical markers, demonstrating a protective effect in the ANIT-induced acute CLI model, with UDCA included as a reference treatment. Histopathology confirmed a marked reduction in hepatocyte necrosis and early fibrotic changes. Mechanistically, DGD8 restored dysregulated arachidonic acid and lipid metabolism, suppressed pro-inflammatory cytokines (IL-1β and TNF-α) via COX-2 inhibition, and enhanced antioxidant capacity. Proteomic analysis revealed that DGD8 activated FXR, subsequently upregulating hepatic bile acid transporters (BSEP, MRP2, OATP1A1) and the conjugation enzyme UGT1A1, thereby restoring bile acid homeostasis. Importantly, SAMHD1 and S100A16 were identified as novel therapeutic targets in CLI; their suppression by DGD8 correlated with improved hepatoprotection. Molecular docking indicated that 31 drug-like constituents, primarily alkaloids and phenolics, exhibit strong binding affinities to these key targets.

CONCLUSION

DGD8 ameliorates CLI through a coordinated multi-targeted mechanism involving the suppression of COX-2-mediated inflammation, restoration of bile acid transport and detoxification via FXR activation, and modulation of novel pathogenic mediators (SAMHD1 and S100A16). This study provides a scientific validation for the traditional use of DGD8 and offers a systems-level paradigm for understanding the polypharmacological architecture of multi-herb Mongolian medicines.