Integrative Toxicology Profiling of <i>Thuja sutchuenensis</i> Essential Oil: From <scp>LD50</scp> Safety Assessment to Hepatic Apoptosis and Gut Microbiota Modulation

In traditional Chinese medicine, the leaves and essential oil of <i>Thuja sutchuenensis</i> Franch. are documented in the <i>Supplement to the Compendium of Materia Medica</i> for their properties of "cooling blood, detoxifying, and healing wounds". However, documentation regarding its potential toxicity is lacking. This study aims to evaluate the toxicity of <i>Thuja sutchuenensis</i> essential oil (TEO) in mice. The half-lethal dose of TEO was determined through acute toxicity assessment in mice. Changes in blood and serum parameters were evaluated, and histopathological examination using hematoxylin-eosin staining (HE) was performed to observe the effects of TEO on the liver and kidneys (<i>n</i> = 6 per group). A network toxicology workflow identified hepatotoxicity-related targets and pathways, which were validated by untargeted liver metabolomics and differential analysis with FDR control. 16S rRNA sequencing profiled gut microbiota (alpha diversity, beta diversity). TEO did not elevate systemic inflammatory cytokines (all <i>p</i> > 0.05), but decreased leukocytes and increased red cells. HE revealed focal necrosis and mild inflammatory infiltration in the liver of mice, with no notable effects on the kidneys. Network toxicology highlighted PPARG/CYP3A4/TNF axes; enrichment included. Metabolomics detected 611 differential metabolites (log₂FC ≥ 2, <i>p</i> < 0.05); representative changes were Picric acid and Gemfibrozil 1-O-beta-Glucuronide, consistent with oxidative stress. Notably, TEO did not alter the overall composition of the intestinal microbiota in mice but increased the abundance of lactobacilli-associated taxa, including <i>Ligilactobacillus</i>, <i>Lactobacillus</i>, and <i>Limosilactobacillus</i> (<i>p</i> < 0.05). The median lethal dose (LD₅₀) of TEO in mice was found to be approximately 3800 mg/kg. These findings provide a foundation for further studies on the protective effects and potential applications of TEO.