Study On Neurotoxic Mechanism Of Gelsenicine Based On Phosphoproteomics

Gelsemium elegans Benth.(G.elegans)is a small genus of the Loganiaceae family,which is poisonous.People eat it to cause poisoning,while eating by pigs and sheep can promote fattening.There are many reports of poisoning caused by ingestion of G.elegans in Fujian,Guangdong,Guangxi and Hong Kong,China.However,at present,there is no effective antidote for G.elegans poisoning,and its toxicity mechanism is not clear.In this study,gelsenicine,one of the most toxic alkaloid monomers of G.elegans,was selected as the research object.Based on the phosphoproteomics,the neurotoxicity mechanism of gelsenicine has been fully revealed,which provides theoretical basis for the elucidation of the toxicity mechanism and the development of clinical attenuating and detoxifying drugs.In this study,hippocampal tissues,striatal tissues and brainstem tissues of the control group,the 10 min group and the death group were collected for phosphoproteomics analysis.According to the results of phosphoproteomics,the antidote to prevent gelsenicine poisoning were screened preliminarily,and then the detoxification mechanism was further studied.Phosphoproteomics yielded a total of 17,877 unique identified phosphosites that mapped to 4,170 brain proteins.Through the difference analysis of phosphopeptides in three regions at different time points,it was found that the brain phosphorylation of mice has regional specificity,and gelsenicine has different degrees of influence on the different brain regions of mice,among which it is most influential in the hippocampus.Phosphorylation in the hippocampus increased with the degree of toxicity.The differentially changed phosphopeptides in each comparison group were analyzed by GO annotation and KEGG pathway.The results showed that whether in the early or late stage of poisoning,gelsenicine had significantly affected the synaptic pathways of neurotransmitters in the hippocampus,such as glutamatergic synapses,GABAergic synapses,and downstream molecular signal pathways,such as Rap1,HIF-1,MAPK and calcium signaling pathways.Through the screening of antidotes,it was found that the pretreatment of NMDA,baclofen and sarcosine could significantly prolong the survival time and survival rate of gelsenicine poisoned mice,and the detoxification effect of NMDA was the best.Then,the results of phosphoproteomics were verified by parallel reaction monitoring.31 phosphorylation sites in the hippocampus of four different groups(control,10 min,Death and NMDA experiments)were quantitatively analyzed.The expression trend of 31 phosphorylation sites in mouse hippocampus was basically consistent with the results of phosphoproteomics.It was found that NMDA pretreatment inhibited the phosphorylation of proteins in mouse hippocampus.Because NMDA pretreatment can reduce the release of glutamate,combined with the previous research results,this study found that NMDA receptor-mediated excitotoxicity is one of the key signal pathways for the toxic effects of gelsenicine for the first time.In order to further explore its mechanism,we detected the content of gelsenicine and neurotransmitters in different brain regions of gelsenicine poisoned mice.It was found that the content of gelsenicine in hippocampus was the highest,followed by cortex.The content of aspartate in striatum and cortex increased significantly,and the content of glycine in cerebellum decreased significantly.Further studies showed that gelsenicine poisoning significantly reduced the level of mitochondrial membrane potential and the content of ATP in mouse hippocampus,and the level of mitochondrial membrane potential could be restored by NMDA pretreatment.However,the content of NO in hippocampus did not change.In this paper,it was found that the hippocampus is the key area of neurotoxicity of gelsenicine.After administration of gelsenicine,it led to the over activation of NMDA receptor,and then caused the continuous influx of calcium ions,resulting in mitochondrial damage,and finally caused the death of mice.The results of this study not only provided an important research basis for the study of the toxic mechanism of G.elegans,but also provided an important scientific basis for the development of subsequent clinical attenuating and detoxifying drugs.