Protopine-induced neurotoxicity in female mice is associated with blood-brain barrier disruption, gut barrier impairment, cholinergic signaling alterations and gut microbiota dysbiosis.

Protopine is a major isoquinoline alkaloid of Macleaya cordata (Willd.) R. Br. and a constituent of botanical preparations used in veterinary and human medicine. Its occurrence in phytogenic feed additives makes characterization of its potential neurotoxicity important. Female ICR mice received protopine orally at 31.3 or 62.6mg/kg/day for seven days. These regimens, corresponding to one-tenth and one-fifth of the mouse acute oral LD50, were selected for exploratory hazard identification and do not represent documented human environmental, dietary or occupational exposure. Protopine reduced body weight, increased gait scores and caused histopathological injury in the cerebral cortex and hippocampus. Evans blue leakage and reduced Cldn1 expression indicated impaired blood-brain barrier integrity. Brain transcriptomics identified differentially expressed genes enriched in cholinergic synapse and tight junction pathways, supported by RT-qPCR and Western blotting. Protopine also damaged colonic structure, reduced tight junction-related markers, altered cholinergic synapse-related genes and proteins in the colon, changed acetylcholinesterase activity in a tissue-dependent manner and induced oxidative stress-related biochemical changes. 16S rRNA sequencing showed increased Bacteroidetes and decreased Firmicutes. Collectively, short-term high-dose protopine exposure induced neurobehavioral impairment and brain injury in female mice, accompanied by blood-brain barrier and colonic barrier impairment, cholinergic alterations, oxidative stress and gut microbiota dysbiosis. These findings identify candidate neurotoxicity endpoints for component-specific safety re-evaluation but do not establish risk at documented human exposure levels.