Mechanisms of Longdan Xiegan decoction-aggravated D-Galactosamine-induced liver injury: Gut microbiota-mediated geniposide biotransformation and TLR4/NF-κB pathway activation.

This study aimed to evaluate the aggravating effect of overdosed Longdan Xiegan Decoction (LXD) on D-galactosamine-induced liver injury and to investigate the underlying mechanisms. Histopathological examination, serum biochemical analysis (ALT, AST), serum LPS levels, inflammatory cytokine expression, 16S rRNA gene sequencing of gut microbiota, and untargeted fecal metabolomics were performed to assess the effects of LXD at doses of 150, 300, and 600 mg/kg. LC-MS was employed to analyze gut bacterial biotransformation products of LXD. Network pharmacology and molecular docking were used to predict potential genipin-interacting proteins. An LPS-induced LX-2 cell model was established to evaluate the effects of excessive genipin on cell viability and ROS production. Flow cytometry, Western blot, and immunofluorescence were applied to investigate the activation of the TLR4/NF-κB pathway. 600 mg/kg LXD significantly aggravated liver injury and enhanced the gut biotransformation of geniposide to genipin by altering gut microbiota composition. TLR4 was identified as a potential mediator of genipin effects. Moreover, excessive genipin reduced cell viability, increased ROS levels, and elevated expression of TLR4, MyD88, and NF-κB p65, along with promoting nuclear translocation of NF-κB p65 in LX-2 cells. Overdosed LXD aggravates D-galactosamine-induced liver injury through gut microbiota-mediated biotransformation of geniposide to genipin, which subsequently activates the TLR4/NF-κB inflammatory pathway.