Tian Wang Bu Xin Dan Attenuates Neuronal Injury in LPS-Treated HT22 Cells and PCPA-Induced Insomnia-Model Rats

Tian Wang Bu Xin Dan (TWBXD) is traditionally used for sleep disorders, but its neuroprotective mechanisms remain unclear. This study evaluated a cinnabar-free modified TWBXD preparation in cellular and animal models, focusing on phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) expression, autophagy-related markers, inflammation, and apoptosis. An aqueous TWBXD extract was profiled by untargeted liquid chromatography-mass spectrometry (LC-MS). Candidate compounds annotated by LC-MS and checked against PubChem records were used for target prediction, followed by intersection with insomnia-related genes, protein-protein interaction analysis, and functional enrichment. TWBXD-containing serum was evaluated in lipopolysaccharide (LPS)-treated HT22 hippocampal neuronal cells. Rapamycin and 3-methyladenine were used to probe autophagy-related processes. In p-chlorophenylalanine (PCPA)-treated rats, pentobarbital-induced sleep responses, locomotor activity, anxiety-like behavior, spatial learning and memory, and hippocampal CA1 pathology were assessed. Twenty high-confidence candidate compounds yielded 553 predicted human targets, of which 144 overlapped with insomnia-related genes. Network analysis identified 10 candidate hub genes, including AKT1, MTOR, BCL2, TNF, and IL6, and highlighted PI3K-Akt, autophagy, apoptosis, and inflammatory pathways. In LPS-treated cells, rapamycin shifted several autophagy-related and inflammatory markers toward control levels. TWBXD-containing serum increased Beclin 1 expression and the microtubule-associated protein 1 light chain 3-II to light chain 3-I (LC3-II/LC3-I) ratio, decreased p62, interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), Bax, and caspase 3, and increased interleukin-10 (IL-10) and Bcl-2. PI3K and AKT protein and messenger RNA (mRNA) levels also shifted toward control levels. Several TWBXD-associated molecular changes were attenuated by 3-methyladenine. In PCPA-treated rats, TWBXD improved pentobarbital-induced sleep responses, anxiety-like behavior, and spatial learning and memory and alleviated hippocampal CA1 neuronal injury. These findings support the neuroprotective effects of cinnabar-free modified TWBXD in the models examined. Behavioral and histological improvements were accompanied by changes in PI3K/AKT expression and autophagy-, inflammation-, and apoptosis-related markers.