Network Pharmacology and Experimental Verification to Unveil the Mechanism of N-Methyl-D-Aspartic Acid Rescue Humantenirine-Induced Excitotoxicity

Gelsemium is a medicinal plant that has been used to treat various diseases, but it is also well-known for its high toxicity. Complex alkaloids are considered the main poisonous components in Gelsemium. However, the toxic mechanism of Gelsemium remains ambiguous. In this work, network pharmacology and experimental verification were combined to systematically explore the specific mechanism of Gelsemium toxicity. The alkaloid compounds and candidate targets of Gelsemium, as well as related targets of excitotoxicity, were collected from public databases. The crucial targets were determined by constructing a protein–protein interaction (PPI) network. Subsequently, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to explore the bioprocesses and signaling pathways involved in the excitotoxicity corresponding to alkaloids in Gelsemium. Then, the binding affinity between the main poisonous alkaloids and key targets was verified by molecular docking. Finally, animal experiments were conducted to further evaluate the potential mechanisms of Gelsemium toxicity. A total of 85 alkaloids in Gelsemium associated with 214 excitotoxicity-related targets were predicted by network pharmacology. Functional analysis showed that the toxicity of Gelsemium was mainly related to the protein phosphorylation reaction and plasma membrane function. There were also 164 pathways involved in the toxic mechanism, such as the calcium signaling pathway and MAPK signaling pathway. Molecular docking showed that alkaloids have high affinity with core targets, including MAPK3, SRC, MAPK1, NMDAR2B and NMDAR2A. In addition, the difference of binding affinity may be the basis of toxicity differences among different alkaloids. Humantenirine showed significant sex differences, and the LD50 values of female and male mice were 0.071 mg·kg−1 and 0.149 mg·kg−1, respectively. Furthermore, we found that N-methyl-D-aspartic acid (NMDA), a specific NMDA receptor agonist, could significantly increase the survival rate of acute humantenirine-poisoned mice. The results also show that humantenirine could upregulate the phosphorylation level of MAPK3/1 and decrease ATP content and mitochondrial membrane potential in hippocampal tissue, while NMDA could rescue humantenirine-induced excitotoxicity by restoring the function of mitochondria. This study revealed the toxic components and potential toxic mechanism of Gelsemium. These findings provide a theoretical basis for further study of the toxic mechanism of Gelsemium and potential therapeutic strategies for Gelsemium poisoning