Exploring the Molecular Mechanism of Ganoderic Acid B for Insomnia Based on Metabolomics, Network Pharmacology, Molecular Docking, and Experimental Validation

Abstract Objective: Ganoderma lucidum, a fungus of the genus Ganoderma in the family Polyporaceae, is often used in traditional medicine for the treatment of insomnia and amnesia, etc. Ganoderic acid B, a characteristic lanostane-type triterpenoid isolated from Ganoderma lucidum, has been preliminarily verified to possess mild sedative activity in existing studies. This study aims to explore the potential regulatory mechanism of Ganoderic acid B against PCPA-induced insomnia-like damage via multiple preclinical research strategies, and provide preliminary molecular and experimental clues for subsequent basic mechanistic research of this compound. Methods: Taking Ganoderic acid B as the research object, we established a PCPA-induced mouse insomnia model and evaluated its protective effects on insomnia-associated pathological lesions through behavioral assays, biochemical detection, and cerebral histopathological observation. Subsequently, UHPLC-MS/MS serum metabolomics analysis was performed to screen characteristic biomarkers and pivotal metabolic pathways linked to insomnia pathology. Next, compound and disease target databases were combined to obtain overlapping targets correlated with the alleviation of insomnia lesions by Ganoderic acid B; network pharmacology analyses, including GO and KEGG enrichment, were implemented to screen core hub targets, followed by molecular docking and molecular dynamics simulation to preliminarily verify the binding interaction between Ganoderic acid B and these targets. Finally, an in vitro CORT-triggered HT22 neuronal injury model was adopted for experimental validation, and Western blot assays were conducted on mouse brain tissue to detect the expression of core regulatory proteins. Results: In vivo experiments showed Ganoderic acid B alleviated weight loss and abnormal behaviors, raised serum 5-HT and GABA, and lowered DA and NE in PCPA insomnia model mice. Metabolomics screened six key insomnia-related serum markers, with phenylalanine, tyrosine, and tryptophan biosynthesis as the core pathway. CCK-8 assays showed that 4 and 8 μM Ganoderic acid B had no cytotoxicity and relieved CORT-induced neuronal injury, while 16 μM inhibited HT22 cell viability. Network pharmacology identified six core regulatory proteins (EP300, HDAC1, ESR1, IL-6, TNF, NCoR1) mediating Ganoderic acid B’s protective effects against insomnia lesions, with Pathways in cancer enriched. Molecular docking verified stable binding between Ganoderic acid B and these targets. Western blot results indicated Ganoderic acid B downregulated NCoR1, EP300, HDAC1, IL-6, and TNF and upregulated ESR1 expression Conclusion: Ganoderic acid B alleviates PCPA-triggered behavioral abnormalities, neurotransmitter disturbance, and hippocampal neuronal injury in mice via multitarget, multipathway synergistic regulation of NCoR1, EP300, ESR1, HDAC1, IL-6, and TNF, and restores the disrupted phenylalanine, tyrosine, and tryptophan biosynthetic network. This work offers preliminary preclinical and molecular evidence to clarify how Ganoderic acid B relieves insomnia-related pathological injuries