| Epilepsy is a chronic disease of brain dysfunction caused by sudden abnormal discharge of brain neurons.At present,the treatments of epilepsy include drug therapy,surgical treatment,neuromodulation therapy,etc.,but there are still 30% of patients who are not suitable for surgery or no effective drugs available.Therefore,there is an urgent need for new and effective antiepileptic drugs and new treatments.Our previous study has shown that the natural terpenoid maslinic acid exhibits anti-epileptic activity on mouse acute seizure models induced by three classic drugs.And on the zebrafish acute seizure model induced by pentylenetetrazol(PTZ),and its may be that by increasing the M current to reduce the excitability of cortical pyramidal neurons to show anti-epileptic activity.The above results indicate that the natural product maslinic acid may be a new lead compound for the treatment of epilepsy,but its protein target in the body is unknown.In response to this problem,this research paper uses chemical proteomics methods,biophysical methods and genomics methods to find the antiepileptic target of maslinic acid and explore its mechanism of action.First,we established a pentetrazole-induced(PTZ)model of zebrafish with acute epileptic seizures,and tested the antiepileptic activity of maslinic acid on this model.Similar to previous studies,maslinic acid showed significant antiepileptic activity.Subsequently,Thirteen probe molecules,including biotin labeled probes,photoaffinity labeled probes and click-chemical labeled probes,were synthesized from the active fragment of maslinic acid.It is planned to apply the method of chemical proteomics to study the target of maslinic acid.The results showed that the anti-epileptic activity of maslinic acid was lost after being labeled with biotin;the maslinic acid probes modified with photoaffinity groups and click chemistry groups had poor water solubility,which made it impossible to verify the activity of the probes.Therefore,we use transcriptomics methods that do not require chemical modification of maslinic acid,combined with gene expression profile database(CMap)to predict the possible targets of maslinic acid.The model cell line MCF-7 was treated with maslinic acid and compared with the gene expression profile treated with DMSO,and the differential genes were input into the CMap library for prediction.As a result,the gene expression profiles of lycorine and maslinic acid were the most similar.A literature survey of lycorine found that lycorine can reduce the spontaneous activities of mice and present a significant sedative effect,and its target of action is sodium channel protein.Epilepsy is thought to be related to an increase in sodium ion-dependent action potentials,which continue to occur during slow depolarization.Therefore,we speculate that the anti-epileptic target protein of maslinic acid may also be sodium channel protein.The results of the electrophysiology experiments showed that the amplitude of sodium current could be significantly inhibited by maslinic acid.It is speculated that maslinic acid may change the excitability of neurons by affecting the sodium ion current,resulting in the antiepileptic activity.There are 9 highly homologous Nav channel subtypes(Nav1.1～ Nav1.9)in human body,of which the ones associated with epilepsy are Nav1.1,Nav1.2,Nav1.3,Nav1.5,Nav1.6 and Nav1.7.By constructing CHO cells expressing different subtypes of sodium channel proteins,we used the whole-cell patch clamp method to detect the effect of maslinic acid on the functional characteristics of these six subtypes of Nav channel.The results showed that maslinic acid has selective activity on Nav1.2 and Nav1.7,and it is known from the activation curve and inactivation curve that maslinic acid may affect the basic characteristics of Nav1.2 and Nav1.7,thereby inhibiting the sodium current. |
PDF Full Text Request