Analgesic Of Three Kinds Of Gelsemium Alkaloids On Prostaglandin E2-induced Hyperalgesia In Mice

G.elegans is the whole grass of Gelsemium elegans Benth,a plant of Loganiacease.It is recorded in the medical book Compendium of Materia Medica,which said that"people who eat its leaves by mistake all die,while goat eat its seedlings will fat".In traditional Chinese applications,G.legans has been used to treat a variety of intractable pain disorders,and in western homeopathy,as an effective treatment for anxiety disorders.At the same time,modern pharmacological studies have also confirmed the analgesic effects of gelsemine and koumine in a variety of animal pain models.Gelsenicine is one of the most toxic alkaloids in G.elegans.Whether it can produce good analgesic effect has not been fully certified.Therefore,this study aims to compare and analyze the analgesic effects of gelsemine,koumine and gelsenicine on prostaglandin E2（PGE₂）induced hyperalgesia model,observe whether there are differences in analgesic effects through behavioral methods,and explore the molecular mechanism through relevant molecular research methods.In this study,intrathecal injection of PGE₂was used to construct hyperalgesia model in mice,and pain score was used to evaluate the analgesic potential of intraperitoneal injection of gelsemine,koumine and gelsenicine in mice with pain threshold in behavior.Median effective dose(ED₅₀)of the three G.legans alkaloids were determined simultaneously.The results showed that gelsemine,koumine and gelsenicine had good analgesic effects on mechanical pain score and heat pain threshold test measured by hot plate method,with ED₅₀of 0.82 mg/kg,0.60 mg/kg and 8.43μg/kg,respectively.In order to explore whether the three alkaloids and PGE₂administration would cause damage to the spinal cord tissue,each experimental group was observed and found that the experiment did not cause histopathological damage to the mice.Then,in order to further explore whether the three alkaloids’anti-hyperalgesia induced by PGE₂is related to Gly Rα3 receptor.Strychnine,a glycine receptor antagonist,was used to interfere,and it was found that the analgesic effect of the three alkaloids on hyperalgesia mice was blocked by strychnine.These results suggest that the anti-hyperalgesia effect of alkaloids is related to Gly Rα3 receptor.On this basis,the contents of the neurotransmitter Glycine（Gly）and protein kinase C（PKC）as well as the expression levels of Gly Rα3 and Gephyrin were detected by High Performance Liquid Chromatography（HPLC）,ELISA and Western Blot.The results showed that there was no significant change in the content of Gly in the spinal cord of mice.Compared with the normal control group,the content of PKC and the expression levels of Gly Rα3 and Gephyrin were significantly decreased in the model group,while the content of PKC,the expression levels of Gly Rα3 and Gephyrin were significantly increased in the G.legans alkaloid group.N-methyl-D-aspartic acid（NMDA）receptor is also one of the important targets of pain pathway.In order to explore whether NMDA receptors involved in the regulation of G.legans alkaloid in anti-PGE₂-induced hyperalgesia,this study used animal pain threshold as the measurement index in the PGE₂-induced hyperalgesia model in mice.And to observe the possible interference of intraperitoneal injection of MK801 on the analgesic effect of three alkaloids.The results showed that MK801 could block the analgesic effect of three alkaloids on pain model mice and increase hyperalgesia,suggesting that the anti-PGE₂-induced hyperalgesia of the three alkaloids may be related to NMDA receptor.Sarcosine is an inhibitor of glycine transpoter-1（Gly T-1）,which can increase the concentration of Gly on NMDA receptors.To further understand the role of NMDA receptors,the interference of Sarcosine in the analgesic experiment of G.elegans alkaloids was observed.The result shows that sarcosine has analgesic effect in PGE₂induced hyperalgesia.In addition,it was found that after the administration of sarcosine and the combination of three alkaloids,the analgesic effect of gelsemine was still significant in the mouse pain model,while koumine and gelsenicine had no significant effect on pain sensitivity in mice,and the analgesic effect of sarcosine itself was eliminated.In conclusion,the results of this study suggest that gelsemine,koumine and gelsenicine have good analgesic effects on PGE₂-induced hyperalgesia,and gelsemine ED₅₀>koumine ED₅₀>gelsenicine ED₅₀.On the one hand,the expression of Gly Rα3 and Gephyrin in the spinal cord is increased by increasing the content of PKC.On the other hand,it may be that the alkaloids indirectly increase the concentration of Gly around NMDA receptors,and then activate NMDA receptors and enhance the activity of NMDA receptors.Finally,the glycine neurotransmitter system and NMDA neurotransmitter system of spinal cord were involved in the mechanism of PGE₂-mediated pain sensitization.It provides a certain basis and direction for the pharmacological research of G.elegans.It also provides reference for pain research and clinical development of G.elegans alkaloids.