Chemistry of Bairui granules and its mechanisms in the protective effect against methotrexate-induced liver injury

Background: Bairui granules (BRKL), a traditional Chinese medicine preparation, possess a range of pharmacological effects. However, its impact on methotrexate (MTX)-induced liver damage remains unexplored. Purpose: The present work focused on investigating the potential protection of BRKL on MTX-induced liver damage, along with its potential active ingredients and underlying mechanisms. Methods: We evaluated the hepatoprotective activities of BRKL in liver-damaged Wistar rats induced by intraperitoneal MTX injection, observing the liver's morphological and pathological features. Additionally, we measured serum ALT, AST, and LDH levels using kits. Ultra High-Performance Liquid Chromatography-Q-Exactive Orbitrap Mass Spectrometry (UHPLC-Q-Exactive Orbitrap MS) analyzed BRKL composition, and network pharmacology strategy predicted and analyzed BRKL's targets and pathways. Thereafter, we conducted molecular docking for analyzing affinity of bioactive ingredients for targets with Autodock. At last, results were verified through in vitro experiments. Results: The animal experiments revealed the significant protection of BRKL against MTX-mediated rat liver damage. A total of 64 major chemical constituents were identified in BRKL by UHPLC-Q-Exactive Orbitrap MS. We then applied the network-based pharmacological strategy to clarify BRKL's molecular mechanism on liver damage based on the identified components. The targets EGFR, SRC, PIK3R1, AKT1, and ESR1, as well as compounds isorhamnetin 3,7-O-diglucoside, beta-ecdysone, chrysoeriol, apigenin, and diosmetin, may play pivotal roles in treating MTX-mediated liver damage. According to our in vitro experiments, isorhamnetin 3,7-O-diglucoside may exert its liver-protective effect via AKT/NF-kappa B pathway. Conclusion: BRKL protected against MTX-mediated liver injury, and the bioactive ingredients, key pathways, and liver injury-related molecular targets have been identified. These findings provide new insights into using BRKL in treating liver damage and propose a feasible approach to exploring phytomedicine's chemical and pharmacological foundation.