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

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. <b>Results:</b> 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 <b>Conclusion:</b> 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