Rehmannioside D directly targets PTP1B to ameliorate metabolic dysfunction-associated fatty liver disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Rehmanniae Radix Praeparata, the processed root of Rehmannia glutinosa Steud. (Orobanchaceae), a traditional Chinese medicine, has traditionally been used for metabolic disorders related deficiency patterns in traditional Chinese medicine. As a pharmacopoeial quality control marker of Rehmanniae Radix Praeparata, rehmannioside D (RD) possesses hepatoprotective and metabolic regulatory potential; however, its therapeutic role in MAFLD remains unclear. AIM OF THE STUDY: This study aimed to evaluate the therapeutic effects of RD against MAFLD and to elucidate its underlying molecular mechanisms. MATERIALS AND METHODS: db/db mice and free fatty acid (FFA)-induced HepG2 cells were used to assess the effects of RD in vivo and in vitro. Lipid accumulation, liver injury, insulin signaling, and endoplasmic reticulum (ER) stress were evaluated. Limited proteolysis-mass spectrometry (LiP-MS) was used to identify potential targets of RD. Molecular docking, cellular thermal shift assay (CETSA), and molecular dynamics (MD) simulations were performed to validate the interaction between RD and the target protein. RESULTS: Marked amelioration of lipid metabolic disorders was observed in both db/db mice and FFA-induced HepG2 cells following RD treatment. This intervention effectively attenuated ALT and AST elevations while mitigating histopathological hepatic injury. LiP-MS identified PTP1B as a potential target of RD; molecular docking, CETSA, and MD simulation further corroborated stable binding between RD and PTP1B, identifying Asp181 as an important residue contributing to the RD-PTP1B interaction. Mechanistically, RD inhibited PTP1B activity, suppressed SREBP-1c transcription and expression, and downregulated lipogenesis-related genes. RD also inhibited the PERK/eIF2α pathway, alleviated ER stress, and restored the PI3K/AKT insulin signaling. CONCLUSION: RD ameliorates MAFLD by inhibiting PTP1B, orchestrating a concerted improvement in lipid metabolism, ER stress resolution, and insulin signaling, supporting its potential as a therapeutic agent for MAFLD management.