| With 65 million people affected worldwide,epilepsy is among the most common,chronic,serious neurological disorders characterized by an enduring predisposition to generate epileptic seizures,with neurobiologic,cognitive,psychological,and social consequences.It has to be acknowledged that more than 90%of people with epilepsy live in developing countries,where even higher rates have been reported in rural areas.These regional differences probably result from differences in risk factors for epilepsy,including infections and inadequate antenatal and perinatal care.Chinese latest epidemiological data showed that the overall prevalence of epilepsy was 7.0 ‰ and the annual incidence was 28.8 per 100,000 populations,which was one of the most common disease in neurology only after the headache.Epilepsy is one of the most prevalent neurological disorders which affect people of all ages,especially for the children and adolescent.The impact of epilepsy rests on not only the individual patient,but also the family and indirectly the community.Etiology of epilepsy is very complex,and many different conditions are known to be risk factors,which vary with age and geographic location.In the 2010 ILAE proposal,the causes of the epilepsy,previously classified as idiopathic,symptomatic,or cryptogenic,are replaced by the following three categories:i.Genetic:The genetic epilepsy is the direct result of a known or presumed genetic defect(s)in which seizures are the core symptom of the disorder;ⅱ.Structural/metabolic:Conceptually,there is a distinct other structural or metabolic condition or disease that has been demonstrated to be associated with a substantially increased risk of developing epilepsy in appropriately designed studies;ⅲ.Unknown cause:Unknown is meant to be viewed neutrally and to designate that the nature of the underlying cause is as yet unknown;it may have a fundamental genetic defect at its core or it may be the consequence of a separate as yet unrecognized disorder.Little is known about the pathogenesis of epilepsy,however,recent studies show that it is closely related with the abnormality of neurotransmitter,ion channel,glial cells,synapse,genetic and immune factors.Although approximately 65-70%of patients with new-onset epilepsy eventually enter remission with current anti-epileptic drugs(AEDs),these medications fail to control seizures in 25-30%of patients.Understanding the causes of the pathology underlying the occurrence of seizures is necessary and urgent for finding effective and safe treatments for drug-resistant epilepsy.Osthole,7-methoxy-8-(3-methyl-2-butenyl)-2H-1-benzopyran-2-one,is a natural coumarin first derived from Cnidium plant,which is commonly applied in clinical practice of Traditional Chinese Medicine(TCM)to treat skin diseases and sexually transmitted diseases.Modern researches have suggested that osthole exhibits multiple pharmacological actions including cardiovascular protective,immunomodulatory,antimicrobial,anticancer,osteogenic,hepatoprotective,and neuroprotective activities.Accumulating evidence indicates that imperatorin[9-(3.methylbut-2-enyloxy)-7H-furo[3,2-g]chromen-7-one],a natural furanocoumarin derivative possesses the anticonvulsant activity in preclinical studies by elevating the threshold for electroconvulsions and enhancing the antielectroshock action of carbamazepine,phenobarbital,phenytoin and lamotrigine in mice.Since osthole and imperatorin(two naturally occurring coumarin derivatives)have similar chemical structures,we expected that osthole would also exert the anticonvulsant action in mice.Therefore,we firstly examined the dose-response relationship of action of osthole on the maximal electroshock seizure(MES)and pentylenetetrazole(PTZ)seizure tests,which have been used as gatekeepers in novel antiepileptic drugs(AED)discovery for over 6 decades.Results indicate that osthole at a dose of 40 mg/kg and 80mg/kg,i.p.,60 min before the test had significant effect on the anticonvulsant action in the MES(p<0.05):control,1.800±1.033(n=10);vehicle,2.300±0.9487(n=10);20mg/kg,2.000±1.054(n=10);40mg/kg,0.7778±0.6667(n=9);80mg/kg,0.9000±0.8756(n=10)and PTZ seizure test(p<0.01):control,3.556±1.333(n=9);vehicle,3.222±0.9718(n=9);20mg/kg,3.444±1.424(n=9);40mg/kg,1.778±1.787(n=9);80mg/kg,0.222±0.6667(n=9).However,there was no significant difference in both vehicle and 20mg/kg groups when compared wiht control in both above test..Temporal lobe epilepsy(TLE)is the most common refractory epilepsy syndrome characterized by progressive development of recurrent seizures and hippocampal sclerosis.Over one third of patients with TLE will not have adequate seizure control with medication or even surgical excision.Given the significant anticonvulsant effect of osthole in acute epilepsy models,we further explore it’s antiepileptic in TLE model.4-aminopyridine(4-AP)induced epileptiform events in hippocampal neurons share electrographic similarities with the seizure discharges seen in patients with TLE,so 4AP epilepsy model is a classic in vitro epilepsy model for screening AED objectively.Whole-cell voltage-clamp recording was obtained from CA1 pyramidal neurons in artificial cerebrospinal fluid(aCSF)for 10 min followed by a 10-20 min perfusion of 4-AP(100μM).Afterward,osthole(10μM,20μM,50μM)was coapplied with 4-AP(100μM)for 10-20 min.Finally,a 10-20 min perfusion of 4-AP(100μM)alone was performed as a washout for observations of recovery.Data showed that seizure-like events(SLEs)were induced by 4-AP(100μM)for a minimum of 5 min,after being coapplied with 10μM,20μM and 50μM osthole,the normalized SLEs decreased significantly to 39.25±10.92%(n=3,P<0.05),37.59±16.00%(n=3,P<0.05)and 20.92±5,80%(n=4,p<0.01).However,the inhibition of osthole on SLEs was reversible after washout with ACSF containing 100μM 4-AP.How about the effect of osthole on chronic intractable TLE animal model?TLE animal model was induced with pilocarpine hydrochloride(PILO).One week later.Mice with status epilepticus(SE)were randomly divided into vehicle and osthole group and video-monitored for spontaneous generalized seizures(GS)for 12h/day for three weeks.During the second week,0.2ml sunflower oil with 10%alcohol(vehicle)or osthole(80mg/kg)were given through intraperitoneal injection once a day at 9:00;Videotapes were reviewed by observers blinded to the model and the total number of GS in control or osthole group in these three weeks was calculated.Surprisingly,osthole(80mg/kg)inhibited the occurrence of recurrent spontaneous GS during the week after appliance of osthole(0.22±0.2)compared with vehicle(4.5±2.0)(p<0.05).To further analyze when osthole exerted its action,we investigated its effects at different times.Our results showed that osthole inhibited GS at 15-21 days(p<0.05),in which the number of GS decreased significantly from 4.3±1.2 before treatment to 0.2±0.2 after treatment with osthole(p<0.05).Thus,these results demonstrated that osthole inhibited pilocarpine-induced spontaneous seizures,and no rebound after drug withdrawal,however,there was no significant difference in these three stages in the vehicle group.As we known,the majority of idiopathic epilepsies were demonstrated to be channelopathies associated with mutations of some ion channels,where mutations disrupt normal electrical transmission between neurons.For example,decreased availability of A-type potassium ion channels may contribute to the initiation and/or propagation of seizures in TLE.Therefore,we examined the effects of osthole on different ion channels and found that osthole(50μM and 100μM)had no inhibitory effect on calcium channel and sodium channel,but interestingly,osthole(10μM)could accelerate inactivation of potassium channels in the hippocampal neurons,cortex nexurons,dorsal root ganglion(DRG),and NG108 cells.Next,we tested the effect of osthole on different subtypes of delayed rectifier potassium channels,and found that osthole could accelerate inactivation of delayed rectifier potassium channel(Kv1.1,Kv1.2,Kv1.3,Kv2.1,etc.)transfected on the Hek293 cells.At a high concentration of 100μM,osthole produced a rapid inhibition of Kv2.1 channel which is readily reversible with the time constant of 8.7 s upon removal of the drug.A-type potassium channels are also called transient outward potassium channels,which undergo rapid activation and inactivation.A-type channels are usually silent at resting membrane potentials.However,they transiently activate during the decay of the after-hyperpolarization phase of the action potential(AP),consequently delaying depolarization and thus prolonging the period between APs and control the neuronal firing frequency.We then detected the effect of osthole on neuronal excitability.Data showed that osthole at low concentrations reduced the firing frequency and extend the interval of action potentials in hippocampal neurons.Thus,the above findings indicated that osthole could decrease the neuronal excitability by increasing functional A-type channels.We further investigated the mechanism of osthole action on potassium channels.The impact of osthole on deactivation kinetics of potassium channels was detected by substituting K+with low permeability potassium ion(Rb+,Cs+)in intracellular solution.It was found that the impact of osthole on the inactivation of potassium channel currents mediated by these three different ions was significantly different.The fact that the effect of osthole on the inactivation was associated with the permeability of ions indicated that binding sites for osthole were on the channel pore,which directly resulted in a eonformational change of the channels.When ions with different permeability flow through the channel,the deactivation kinetics of currents show discrepancies because of the conformational change of the channel.In addition,our results also found that osthole at high concentrations(100μM,200μM)only inhibited the inward rectifier potassium channel currents and large conductance calcium-activated potassium channel currents,but not changing the current inactivation kinetics.To analysis the sequence alignment of the pore region of the above three types of potassium channels,we found that the main difference in the pore structure between them is a hydrophobic ring structure localized to the S6 domain that line the channel vestibule.Suspicious residues located in the S6 domain were mutated to alanine through alanine-scanning mutagenesis.Osthole showed no effect on the inactivation of Kv2.1 mutants with the mutations of V505A and I508A,indicating that the sites of V505 and 1508 are crucial for inactication.In summary,the main findings of this study are as follows.First,in vivo,osthole decreases seizure classes in the MES or PTZ seizure test and seizure frequency in the TLE model induced by PILO.Secondly,in vitro,osthole suppresses 4AP induced epileptiform events in hippocampal CA1 pyramidal neurons.Finally,osthole modulates the functional properties of Kv channels,converting channels with slowly inactivating current into apparent fast inactivation.Our study will reveal a new mechanism for the treatment of intractable epilepsy:osthole converts delayed rectifier channels into A-type channels within the nervous system,which will compensate the decreased availability of A-type channels in TLE.The current study provides new therapeutie targets for refractory epilepsy by the functional eonversion of intrinsic currents to compensate the decreased availability of A-type currents. |
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