柴胡皂苷a对大鼠海马神经元癫痫样放电抑制作用及相关离子通道电流调节作用的研究
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摘要
一、目的：
癫痫是常见的神经系统疾病之一,是一组由大脑神经元反复异常同步化放电所引起的短暂中枢神经系统功能失常为特征的慢性脑部疾病。据1997年WHO统计全球约有5000万癫痫患者,2003年国内癫痫流行病学调查表明：我国癫痫患病率约为7.0‰,以此推算我国约有600万患者正遭受癫痫的困扰。癫痫反复发作、病程长、致残率高严重影响了患者的生活质量。临床常用抗癫痫药物,不能有效控制癫痫发作,仍有约40%的癫痫患者产生耐药性,且多数抗癫痫药有多种急慢性不良反应及毒副作用,部分药物价格昂贵,长期服用给患者家庭和社会带来沉重的心理压力和巨大的经济负担。只有明确癫痫发病机制,才能指导有效性高、安全性好的抗癫痫药物的研发。
癫痫发作是由神经递质及其受体异常、离子通道异常、胶质细胞活性、免疫因素、遗传因素等各种原因引起大脑神经元兴奋性-抑制性动态失衡,神经元兴奋性增高、异常同步化放电引起。离子通道是担负中枢神经系统兴奋性活动(即神经元动作电位的传导)以及形成神经环路(即神经元间突触信号的传递)的核心构件,任何离子通道的基因突变都可能异化通道蛋白的正常功能,造成中枢神经系统兴奋性-抑制性动态失衡,神经元兴奋性增高,最终引起神经元异常同步化放电,导致癫痫发作。离子通道分为电压门控离子通道,如电压依赖性K+通道、电压依赖性Na+通道,和配体依赖性离子通道,如NMDA受体(NMDAR)通道等。近年来,随着分子生物学、膜片钳等技术的发展,作为神经元之间或神经元与效应细胞之间藉以传递各种生理和病理信息的核心媒介,离子通道与癫痫发病机制的研究已愈来愈引起神经科科研工作者的重视。
借助实验性癫痫模型模拟人类癫痫发作,从电生理学角度探讨癫痫的发病机制已成为当前本领域的研究热点之一。无镁外液诱导的原代培养海马神经元自发性反复性癫痫样放电(spontaneous recurrent epileptiform discharges, SREDs)模型是目前国际公认的模拟人类后天获得性癫痫(acquired epilepsy, AE)的细胞模型,而持续性高频癫痫样放电(continuous epileptiform high-frequency bursts,SEs)模型是模拟人类持续性癫痫(status epilepticus, SE)的细胞模型,两种癫痫细胞模型目前被广泛应用于癫痫状态下神经元生物化学、神经电生理学、分子生物学改变的机制研究及抗癫痫药物筛选的研究中；4-氨基毗啶(4-aminopyridine,4AP)诱导的海马脑片神经元癫痫样放电模型与人类颞叶癫痫发作时脑电图记录的放电模式相似,是筛选抗癫痫药物研究的经典体外癫痫模型,能较为客观地反映药物的抗癫痫作用。
癫痫属于中医“痫病”范畴,《黄帝内经素问·卷二十一·六元正纪大论篇第七十一》曰：“木郁之发,太虚埃昏,云物以扰,大风乃至,屋发折木,木有变。故民病胃脘当心而痛,上支两胁,鬲咽不通,食饮不下,甚则耳鸣眩转,目不识人,善暴僵仆”,我国历代医家认为“肝失疏泄、肝气郁结”是癫痫发病的病因病机之一,并提出癫痫“从肝论治”的中医理论,在此理论的指导下,课题组自拟方“柴胡疏肝汤”在临床癫痫的预防和治疗中取得客观疗效,进一步的实验研究发现“柴胡疏肝汤”能够降低戊四氮(PTZ)点燃模型大鼠的痫性发作潜伏期及发作级别,减轻锂-匹罗卡品诱发的难治性癫痫模型大鼠的癫痫发作级别,在前期研究基础上逐步筛选出复方中君药柴胡的有效成分柴胡皂苷a(Saikosaponin a, SSa)并发现SSa能够抑制最大电休克模型(MES)、PTZ急性致痫模型及锂-匹罗卡品模型诱发的痫性发作。
为了进一步评价SSa的抗癫痫作用并探讨SSa的抗癫痫机制,本研究运用全细胞膜片钳技术,从电生理学角度观察了SSa对无镁外液诱导的原代培养大鼠海马神经元癫痫样放电及4AP诱导的大鼠内嗅皮层-海马脑片CA1区锥体神经元癫痫样放电的抑制作用,并进一步观察了SSa对大鼠海马神经元癫痫相关离子通道电流,包括NMDAR电流、Na+通道电流及K+通道电流的调节作用,明确SSa抗癫痫作用的电生理机制,为癫痫“从肝论治”的中医理论提供较为直观的实验依据。
二、方法：
1.原代培养大鼠海马神经元及荧光免疫细胞化学鉴定
出生<24h的新生SD大鼠,无菌条件下断头取脑、分离海马,常规海马组织消化、离散细胞后以2.5×104/ml的密度接种在放有圆形玻片的24孔培养板中,以Neurobasal-A+10%B-27培养基进行培养。选取生长至第10d的海马神经元,用III β-Tubulin/GFAP及DAPI荧光三标免疫细胞化学法进行染色标记,计算绿色荧光标记的阳性细胞占蓝色荧光标记细胞的百分比。
2.无镁诱导原代培养大鼠海马神经元损伤及SSa的干预作用
选取培养至第10d的海马神经元,随机分为空白组、无镁损伤组、苯妥英组和SSa组,空白组以正常细胞外液孵育；无镁损伤组以无镁细胞外液孵育；苯妥英组以含有50μM苯妥英的无镁细胞外液孵育；SSa组以含有1gM SSa的无镁细胞外液孵育,10min后以III β-Tubulin荧光免疫细胞化学法进行染色标记,对比、评估SSa对无镁外液引起的大鼠海马神经元损伤的保护作用。
3.SSa对原代培养大鼠海马神经元静息膜电位(Vm)及兴奋性的影响
选取培养至第10-14d的海马神经元,随机分为空白组(Control)、苯妥英组和SSa组,空白组以正常细胞外液干预；苯妥英组以含有50μM苯妥英的正常细胞外液干预；SSa组以含有1μM SSa的正常细胞外液干预。在全细胞膜片钳电流钳记录模式下,观察并比较不同干预处理后海马神经元Vm的变化率,及以大小为150pA,时间为600ms的去极化方波电流刺激海马神经元诱发的动作电位(action potential, AP)个数的变化率,评估SSa对原代培养大鼠海马神经元兴奋性的影响。
4.无镁外液诱导原代培养大鼠海马神经元SREDs样放电及SSa的干预作用
选取培养至第10-14d的大鼠海马神经元,以无镁外液孵育3h后,置于正常细胞外液中孵育24h,诱发海马神经元SREDs样放电。在全细胞膜片钳电流钳记录模式下,出现典型SREDs样放电的神经元随机分为无镁组、苯妥英组(50μM)及SSa各剂量(分别为0.1μM、0.3μM、0.5μM、μM、2μM和4μM)组,观察并比较不同处理组对海马神经元SREDs样放电的抑制率,评估SSa对无镁外液诱导的原代培养大鼠海马神经元SREDs样放电的抑制作用。
5.无镁外液诱导原代培养大鼠海马神经元SEs样放电及SSa的干预作用
选取培养至第10-14d的大鼠海马神经元,以无镁外液持续灌流孵育>10min,诱发海马神经元SEs样放电。在全细胞膜片钳电流钳记录模式下,出现典型SEs样放电的神经元随机分为无镁组、苯妥英组(50μM)及SSa各剂量(分别为0.μM、0.3μM、0.5μM、1μM、2μM和4gM)组,观察并比较不同处理组对海马神经元SEs样放电的抑制率,评估SSa对无镁外液诱导的原代培养大鼠海马神经元SEs样放电的抑制作用。
6.4AP诱导大鼠内嗅皮层-海马脑片CA1区锥体神经元癫痫样放电及SSa的干预作用
选取出生25-50d的SD大鼠,吸入乙醚麻醉后,于振动切片机上切取厚300-400μm的内嗅皮层-海马脑片,以正常人工脑脊液(ACSF)37℃孵育1h后,以含有100μM4AP的ACSF持续灌流孵育20-40min诱发海马CA1区锥体神经元癫痫样放电。在全细胞膜片钳电流钳记录模式下,出现典型癫痫样放电的海马CA1区锥体神经元随机分为4AP组、丙戊酸钠(VPA)组(1mM)及SSa各剂量(分别为0.1μM、0.3μM、0.5μM、1μM、2μM和4μM)组,观察并比较不同药物干预后,海马神经元癫痫样放电持续时间及放电频率的变化率,评估SSa对4AP诱导大鼠内嗅皮层-海马脑片CA1区锥体神经元癫痫样放电的抑制作用。
7.SSa对原代培养大鼠海马神经元NMDAR电流的调节作用
选取培养至第10-14d的大鼠海马神经元,随机分为NMDA (Control)组、APV组(50μM)、苯妥英组(50μM)及SSa各剂量(分别为0.1μM、0.3μM、0.5μM、1μM、2μM和4μM)组,在全细胞膜片钳电压钳记录模式下,以含有100gMNMDA的无镁细胞外液通过“Y型”给药系统快速给药,诱发海马神经元NMDAR电流,观察并比较不同药物干预后,NMDAR电流的变化率,评估SSa对NMDAR电流的调节作用。
8.SSa对原代培养大鼠海马神经元电压依赖性Na+通道电流的调节作用
选取培养至第10-14d的大鼠海马神经元,随机分为Control组、苯妥英组(50μM)及SSa各剂量(分别为0.1μM、0.3μM、0.5μM、1μM、2μM和4μM)组,在全细胞膜片钳电压钳记录模式下,以大小为-80mV到+20mV,持续时间为2s的缓慢去极化的斜波(Ramp)电压刺激海马神经元,诱发电压依赖性持续性Na+通道电流(INaP);以大小为-70mV到0mV,持续时间为30ms的去极化方波(Step)电压刺激海马神经元,诱发电压依赖性瞬时Na+通道电流(INat),观察并比较不同药物干预后,INaP和INat的变化率,评估SSa对INaP和INat的调节作用。9.SSa对大鼠海马脑片CAl区锥体神经元电压依赖性K+通道电流的调节作用
选取出生25-50d的SD大鼠,吸入乙醚麻醉后,于振动切片机上切取大鼠海马脑片,随机分为Control组及SSa组(1μM),在全细胞膜片钳电压钳记录模式下,以一-120mV,持续时间为300ms的超极化电压脉冲,紧接之后是从-60mV到+60mV,步阶为+10mV,持续时间为400ms的去极化Step脉冲刺激海马CA1区锥体神经元,诱发总电压依赖性K+通道电流(ITotal);给予一-120mV,持续时间为200ms的超极化电压预脉冲,紧接之后是去极化至-40mV,持续时间为100ms的预脉冲,之后是从-60mV到+60mV,步阶为+10mV,持续时间为400ms的去极化Step脉冲刺激神经元,诱发电压依赖性持续性K+通道电流(IK),电压依赖性瞬时K+通道电流(IA)通过Clampfit10.0软件将记录到的ITotal和IK相减得到。并以一持续时间为120ms,从-90mV到+10mV,步阶为+10mV的Step预脉冲,之后是去极化至+50mV,持续时间为80ms的单脉冲刺激神经元,诱发IA失活电流。观察并比较不同处理组对电压门控性ITotal、IK和IA激活及失活特性的影响,评估SSa对电压依赖性外向性K+通道电流的调节作用。
分别以含有20mM四乙铵(tetraethylammonium,TEA)或4mM4AP的ACSF孵育海马脑片2min后,以去极化至+10mV,持续时间为400ms的单脉冲刺激,诱发外向性K+通道电流,观察不同干预组对TEA敏感和4AP敏感的外向性K+通道电流的影响,评估SSa对药物敏感外向性K+通道电流的调节作用。
10.统计学分析
运用SPSS13.0统计软件进行分析,实验数据均采用均数±标准差(x±s)表示,各处理组对神经元Vm、AP的调节作用及对4AP诱导的癫痫样放电持续时间、NMDAR电流、INaP和INat的抑制率均采用单因素方差分析(one-way AVOVA)进行统计分析,组间两两比较,方差齐采用LSD法,方差不齐采用Dunnett s T3法；对SERDs样放电频率抑制率、SE样放电频率抑制率及对4AP诱导的癫痫样放电频率抑制率采用单样本t检验；对电压依赖性K+通道电流特性的调节作用采用配对t检验,P<0.05表示差异具有统计学意义。
三、结果：
1.原代培养大鼠海马神经元及荧光免疫细胞化学鉴定
神经元在培养至第10d分化成熟,可见细胞散在生长,胞体饱满,折光性强,轴突形成密集的神经细胞网络,荧光显微镜下可见大部分细胞呈绿色荧光,Ⅲ β-Tubulin绿色阳性细胞占全部细胞的96.132%±2.164%。
2.SSa对无镁诱导原代培养大鼠海马神经元损伤的保护作用
镜下观,经无镁外液孵育后的海马神经元,神经元胞体皱缩,甚至溶解,轴突断裂,SSa组神经元胞体饱满、突触较完整,提示SSa能抑制无镁外液诱导的原代培养大鼠海马神经元的损伤。
3.SSa对原代培养大鼠海马神经元Vm及兴奋性的影响
各组对原代培养大鼠海马神经元Vm无显著调节作用(F=1.997,P=0.155),与Control组相比较,SSa(1μM)对Vm无显著调节作用(P=0.056)；各组对AP的抑制率有显著性差异(F=108.178,P<0.001),与Control组相比较,1μMSSa作用5min后,能显著抑制去极化电流诱发的平均AP的个数(P<0.001),提示在不影响神经元一般膜特性的前提下,SSa能够抑制原代培养大鼠海马神经元的兴奋性。
4.SSa对无镁外液诱导的原代培养大鼠海马神经元SREDs样放电的抑制作用
在全细胞电流钳记录模式下,Control组海马神经元在灌流正常细胞外液下呈现出单个偶发AP的发放,这是原代培养大鼠海马神经元的基本电生理活性；经无镁外液孵育3h的原代培养海马神经元,经正常培养液孵育24h后,在正常细胞外液下出现多个典型的自发性反复的癫痫样放电即SREDs样放电。
记录图像显示,含有0.1μM、0.3μM、0.5μM、1μM、2μM和4μM浓度SSa的正常细胞外液作用后,海马神经元SREDs发放个数及持续时间均逐渐减少甚至消失,经正常细胞外液进行洗脱后SREDs样放电再次出现,提示SSa对无镁外液诱导的大鼠海马神经元SREDs样放电的抑制作用是可逆的。统计结果显示,不同浓度的SSa作用后,无镁外液诱导的原代培养大鼠海马神经元SREDs样放电的发作有不同程度的减少。与无镁组相比较,除0.1μM作用浓度外(P=0.170),0.3μM、0.5μM、1μM、2μM及4μM浓度的SSa均能够显著抑制海马神经元SREDs样放电,抑制率分别为19.167±4.182%、66.433±4.950%、95.160±3.317%、98.000±1.216%、98.571±1.118%,P值均<0.001,SSa对无镁外液诱导的原代培养大鼠海马神经元SREDs样放电的抑制作用在一定浓度范围内呈现浓度依赖性,半数有效剂量为0.42μM。
5.SSa对无镁外液诱导的原代培养大鼠海马神经元SEs样放电的抑制作用
在全细胞电流钳记录模式下,Control组海马神经元在灌流正常细胞外液下呈现出单个偶发AP的发放,这是原代培养大鼠海马神经元的基本电生理活性,海马神经元经持续无镁外液灌流孵育>10min后,出现典型的持续性高频癫痫样放电即SEs样放电。
记录图像显示,含有0.1μM、0.3μM、0.5μM、1μM、2μM和4μM浓度SSa的无镁细胞外液作用后,原代培养海马神经元SEs样放电频率逐渐减少甚至消失,经无镁外液进行洗脱后,SEs样放电再次出现,提示SSa对无镁诱导的大鼠海马神经元SEs样放电的抑制作用是可逆的。统计结果显示,各浓度的SSa均能抑制无镁外液诱导的原代培养大鼠海马神经元SEs样放电。与无镁组相比较,除0.μMM作用浓度外(P=0.005),0.3gM、0.5μM、1μM、2μM及4μM浓度的SSa均能够显著抑制海马神经元SEs样放电,抑制率分别为12.071±5.794%、43.418±4.441%、73.626±3.384%、93.538±4.474%、97.798±2.023%,P值均<0.001,而苯妥英对原代培养大鼠海马神经元SEs样放电无显著抑制作用(P=0.082)。不同浓度的SSa对无镁诱导的海马神经元SEs样放电的抑制作用不同,呈现一定程度的浓度依赖性,半数有效剂量为0.62μM。
6.SSa对4AP诱导的大鼠内嗅皮层-海马脑片CAl区锥体神经元癫痫样放电的抑制作用
全细胞电流钳记录模式下,Control组大鼠海马CA1区锥体神经元呈现出单个偶发AP的发放,这是大鼠海马脑片CA1区锥体神经元的基本电生理活性；经含有100μM4AP的ACSF持续灌流孵育大鼠海马脑片20-40min后,CA1区锥体神经元逐渐出现稳定的促发性癫痫样放电。
记录图像显示,经0.1μM、0.3μM、0.5gM、1μM、2μM和4μM不同浓度的SSa作用后,锥体神经元促发性癫痫样放电持续时间逐渐减少,癫痫样放电发放间歇期逐渐延长,经含有100μM4AP的ACSF进行洗脱后可见用药前促发性癫痫样放电再次出现,提示SSa对4AP诱导的海马CA1区锥体神经元癫痫样放电的抑制作用是可逆的。统计结果显示,各处理组对CA1区锥神经元癫痫样放电持续时间的抑制作用有显著差异(F=457.286,P<0.001)。与4AP组相比较,除0.1μM作用浓度外(P=0.145),0.3μM、0.5μM、1μM、2μM和4μM作用浓度的SSa均能显著减少CA1区锥体神经元癫痫样放电持续时间,抑制率分别为26.448±6.788%、40.059±3.264%、57.212±3.231%、67.928±1.908、74.876±2.380%,P值均<0.001；与4AP组相比较,各浓度的SSa对4AP诱导的锥体神经元癫痫样放电频率的抑制率均显著升高,抑制率分别为7.750±2.053%、25.875±2.475%、43.250±1.982%、57.250±3.105%、68.625±3.114%、74.750±2.493%,P值均<0.001,而VPA组对4AP诱导的海马CA1区锥体神经元癫痫样放电频率无显著抑制作用(P=0.170)。不同浓度的SSa对4AP诱导的大鼠海马CA1区锥体神经元促发性癫痫样放电的抑制作用不同,呈现一定程度的浓度依赖性,半数有效剂量为0.70μM。
7.SSa对原代培养大鼠海马神经元NMDAR电流的调节作用
在全细胞电压钳记录模式下,通过“Y型”给药系统向培养的海马神经元局部快速滴加含有100μMNMDA的无镁细胞外液后,诱发出一粗壮的快速内向电流,这种内向电流能够被NMDAR阻断剂APV完全阻断,表明所记录到的内向电流为NMDAR电流。
各处理组对NMDAR电流的抑制作用有显著性差异(F=662.526,P=0.000)。与Control组相比较,0.1μM、0.3μM、0.5μM、1μM、2μM和4μM浓度的SSa均能显著减少诱发的NMDAR电流幅度,抑制率分别为1.070±0.136%、6.108±0.245%、15.015±0.219%、29.853±1.679%、37.726±1.956%、39.224±2.433%,P值均<0.001,且SSa对NMDAR电流的抑制作用在一定浓度范围内有剂量依赖性,半数抑制剂量为0.62μM。
8.SSa对原代培养大鼠海马神经元INaP和INat的调节作用
在全细胞电压钳记录模式下,选用-80mV到+20mV的缓慢去极化Ramp脉冲刺激海马神经元,诱发出一种缓慢激活的陡峭的内向电流；选用从-70mV到0mV,持续时间为30ms的去极化Step脉冲刺激海马神经元,诱发出一种在去极化瞬间瞬时激活达到峰值并迅速失活的内向流,这两种内向电流均能被1μM TTX完全阻断,表明所诱发的内向电流分别为INaP和INat。
各处理组对INaP的抑制作用有显著差异(F=4090.738,P<0.001)。与Control组相比较,0.1μM、0.3μM、0.5μM、1μM、2μM和4μM浓度的SSa均能够显著增加INaP抑制率,抑制率分别为3.483±0.097%、6.722±0.186%、21.284±2.012%、58.104±1.546%、70.546±0.991%、72.504±0.991%,P值均<0.001,不同浓度的SSa对INaP的抑制作用不同,在一定浓度范围内SSa对INaP的抑制作用呈现剂量依赖性,半数有效抑制量为0.84μM。各处理组对INat的抑制作用有显著性差异(F=3342.278,P<0.001)。而与Control组相比较,各浓度的SSa对INa,均无显著影响,P值均为1.000。表明SSa对INaP有选择性的抑制作用。
9.SSa对大鼠海马脑片CA1区锥体神经元ITotal、IK和IA大小的调节作用
将细胞膜电位钳制至-50mV,后给予一持续时间为300ms,-120mV的超极化电压脉冲,紧接之后是一从-60mV到+60mV,步阶为+10mV,持续时间为400ms的去极化Step脉冲刺激,诱发出一外向K+通道电流即ITotal'选用一持续时间为200ms,-120mV的超极化电压预脉冲,紧接之后是去极化至-40mV,持续时间为100ms的预脉冲,之后是从-60mV到+60mV,步阶为+10mV,持续时间为400ms的去极化Step脉冲刺激,诱发出一持续性外向K+通道电流成分即IK,运用Clampfit10.0软件将得到的ITotal和IK相减得到瞬时外向K+通道电流成分即IA。
将用药前后ITotal、Ik和IA的电流峰值进行标准化并进行统计分析表明,1μMSSa能够显著升高ITotal和IA电流幅度(t=-23.975,P<0.001；t=-10.281,P<0.001),而用药前后IK电流幅度无显著统计学差异(t=-1.543,P=0.157)。
10.SSa对大鼠海马脑片CA1区锥体神经元ITotal、IK和IA激活特性的调节作用
将ITotal、Ik和IA的刺激电压(V)和诱发出的相应电流值(Ⅰ)运用Clampfit10.0软件绘制I-V曲线,随着刺激电压的去极化,SSa对ITotal和IA电流的增强作用逐渐增大,而SSa对IK的I-V曲线无明显影响。绘制三种电流的激活曲线,用药前后相比较,1μM SSa能够使IA的激活曲线超极化相移位,显著下调V1/2(28.583±0.588vs.22.317±0.662mV,t=36.729,P<0.001),对k值无显著影响(21.933±1.472vs.23.617±1.592,t=-2.139,P=0.085)；对ITotal激活曲线的V1/2和k均无显著性影响(V1/2：32.467±0.432vs.31.700±1.014mV,t=2.43,P=0.059；k:33.483±1.057vs.32.867±1.648,t=2.116,P=0.088)；对IK的激活曲线的V1/2和k也无显著性调节作用(V1/2：28.350±1.058vs.28.450±1.810mV,t=-0.249,P=0.813；k：30.233±0.942vs.31.233±0.977,t=-2.421,P=0.06)。
11.SSa对大鼠海马脑片CA1区锥体神经元IA失活特性的调节作用
进一步观察SSa对IA失活特性的调节作用,将细胞膜电位钳制至-50mV,给予一持续时间为120ms从-90mV到+10mV步阶为+10mV的预脉冲,之后是去极化至+50mV,持续时间为80ms的单脉冲刺激诱发IA失活电流。绘制IA失活曲线,用药前后相比较,1μM SSa能够使IA的失活曲线去极化相移位,能显著上调IA失活曲线的V1/2(t=-82.750,P<0.001),而对IA失活曲线的k值无显著影响(t=-2.436,P=0.059)。
12.SSa对大鼠海马脑片CA1区锥体神经元4AP敏感及TEA敏感K+通道电流的调节作用
选用20mM TEA和4mM4AP,从药理学角度分离TEA敏感的外向性K+通道电流成分和4AP敏感的外向性K+通道电流成分,并观察SSa对这两种药物敏感的K+通道电流的调节作用。20mM TEA作用后总外向性K+通道电流的持续成分减少,仅剩快速激活-失活成分,即4AP敏感K+通道电流,1μM SSa能够显著上调这种外向性快K+通道电流成分(t=-20.457,P<0.001)；4mM4AP作用后总外向性K+通道电流的快速激活-失活成分被阻断,仅剩一持续性外向性K+通道电流成分,即TEA敏感K+通道电流,1μM SSa对这种持续性外向性K+通道电流成分无显著调节作用(t=-2.329,P=0.053)。表明SSa对4AP敏感K+通道电流有选择性增加作用。
四、结论：
1.SSa能够显著抑制无镁外液诱导的原代培养大鼠海马神经元SREDs样放电及SEs样放电,这种抑制作用是可逆的,在一定剂量范围内,呈现浓度依赖性。在SREDs样放电模型中,1μM SSa的抑制作用与常规剂量苯妥英的抑制作用相当；SEs样放电模型对多种临床常用抗癫痫药表现耐药性,而0.1μM浓度的SSa即可显著抑制SEs样放电,常规剂量及高剂量的苯妥英均对SEs无抑制作用,提示SSa可能对难治性癫痫有良好的治疗潜力。
2. SSa能够显著抑制4AP诱导的大鼠内嗅皮层-海马脑片CAl区锥体神经元癫痫样放电,这种抑制作用是可逆的,在一定剂量范围内,呈现浓度依赖性。抑制作用优于VPA,提示SSa有良好的抗癫痫作用。
3.SSa能够抑制NMDA诱发的海马神经元NMDAR电流幅度,抑制N+通道电流幅度,上调K+通道电流幅度,说明SSa可以通过多位点调节发挥抗癫痫作用,不仅对兴奋性配体依赖性离子通道有调节作用,对电压依赖性离子通道也有显著的调节作用。
4.SSa能够选择性的下调INap电流幅度,而对INat无影响；能够选择性的上调IA成分及4AP敏感的K+通道电流成分,使IA激活曲线左移,而失活曲线右移,加快IA的激活而使其失活速度减慢,而对TEA敏感的IA无明显影响,表明SSa可能通过选择性的调节不同的Na+通道、K+通道亚单位发挥抗癫痫作用。SSa对离子通道不同亚单位的调节作用等机制有待于进一步探讨,为明确SSa抗癫痫机制提供实验依据。
Purpose
Epilepsy, one of the most common neurological disorders, is induced by abnormal electrical discharge, manifestes by recurrent seizure. According to the WHO statistics, there were about50million epilepsy patients worldwide. In2003, the national epidemiological survey of epilepsy demonstrated that the morbidity rate of epilepsy was7.0‰. Epilepsy duration long high recurrent disability seriously affects the patient's quality of life. Clinical commonly used antiepileptic drug, cannot effectively control epileptic seizures and there were about40%of intractable epilepsy can not be controlled with drugs. Beside, most antiepileptic drugs have a variety of acute or chronic adverse reactions and side effects, and some drugs are expensive, long-term use to family and society bring heavy psychological pressure and great economic burden. Only clear the pathogenesis of epilepsy, and can guide high efficiency with good security antiepileptic drug research and development.
Seizures are caused by the brain excitatory and inhibitory dynamic imbalance of neurons, and increased neuronal excitability caused abnormal synchronization discharge. Ion channels are responsible for the central nervous system excitability activity (that is, the neurons action potential conduction) and the formation of neural circuits (that is, synapses between neurons signal transmission) at the core of the artifacts. Any ion channel gene mutation may have alienated the normal function of the channel proteins, caused the central nervous system excitability-inhibitory dynamic imbalances, and triggered neurons abnormal synchronization discharge, eventually. Ion channels contain voltage-gated ion channels, such as Voltage-gated K+channel and voltage-gated Na+channels, and ligand-gated ion channel, such as NMDA receptor channel. Recently, With the development of molecular biology patch clamp techniques, ion channels has increasingly aroused people's attention in the pathogenesis of epilepsy research.
From the perspective of electrophysiology taking Experimental models simulating human epilepsy seizures to study the pathogenesis of epilepsy has become the research hotspot in the field. Low-Mg2+solution induced spontaneous recurrent epileptiform discharges (SREDs) in cultured hippocampal neurons model is the internationally recognized imitating humans acquired epilepsy cell model. And Low-Mg2+solution induced continuous epileptiform high-frequency bursts (SEs) model is imitating human status epilepticus cell model. The two cell models have been widely used in biochemical, electrophysiological and the molecular biology mechanism in epilepsy and in the antiepileptic drug screening researchs.4-aminopyridine (4AP) induced epileptiform events in hippocampal neurons sharing electrographic similarities with the seizure discharges seen in patients with temporal lobe epilepsy is a classic in vitro epilepsy model for screening of antiepileptic drug which could objectively reflect the antiepileptic drug effect. Epilepsy is known as "Xian Bing" in Traditional Chinese Medicine (TCM). Ancient doctors take "Stagnation of Gan, Abnormal Dispersion of Gan" as one of the basic pathogenesis of epilepsy and "Treatment from Gan" as a treatment method for epilepsy. Under the guidance of this TCM theory, the prescription of "Chaihu Shugan Tang" displayed objective curative effects in the clinical prevention and treatment of epilepsy. Further studies found that "Chaihu Shugan Tang" could decrease the level and the latency of seizure in PTZ-kindling rats, reduce the level of seizure in Li-pilocarpine incuded rat refractory epilepsy model. Based On these results, we have gradually focused on the effect of saikosaponin a (SSa), a active ingredient from Bupleurum, the monarch drug in the prescription. We found that SSa could inhibit epileptic seizure in the MES model, the acute PTZ model and the Li-pilocarpine model. The results.suggest that SSa has good anticonvulsant effects on experimental epilepsy models.
In order to further investigate the antiepileptic mechanism of SSa, using the patch clamp technique we observed the inhibition effects of SSa on Low-Mg2+induced epileptiform discharges in cultured hippocampal neurons and4AP induced epileptiform discharges in CA1pyramidal neurons in rat entorhinal cortex-hippocampus slices. Furthermore, we observed the moderating effects of SSa on the NMDAR current, the sodium currents and the potassium currents in rat hippocampal neurons. To clarify the antiepileptic mechanism of SSa and provide experimental bases for the TCM theory of "Treatment form Gan".
Methods
1. Primary cultured rat hippocampal neurons and identification of hippocampal neurons with Fluorescent Immunocytochemistry Technique Newborn SD rats (<24h) were sacrificed and the hippocampus was separated in sterile conditions. Cells were plated at a density of2.5×104cells/cm2onto a glial support layer that was previously plated onto poly-L-lysine-coated (0.05mg/ml) glass coverslips and cultured with Neurobasal-A medium supplemented with2%B-27. On the10th day, cultures were utilized for identification with Ⅲ β-Tubulin, GFAP and DAPI by Fluorescent Immunocytochemistry Technique. The percentage of green fluorescence positive cells in blue fluorescent cells was determined.
2. Low-Mg2+induced injury of cultured hippocampal neurons and the protective effects of SSa
Cultures in the10th day were randomly divided into control group, Low-Mg2+group, phenytoin group (phenytoin,50μM), and SSa group (SSa,1μM).The protective effects of SSa on Low-Mg2+induced cultured hippocampal neurons were evaluated with III β-Tubulin and DAPI by Fluorescent Immunocytochemistry Technique.
3. Effects of SSa on resting membrane potential (Vm) and excitability of cultured hippocampal neurons
Cultures in the10th day were randomly divided into control group, phenytoin group (phenytoin,50μM), and SSa group (SSa,1μM). Using whole-cell patch-clamp recording, changes of the Vm before and after different treatments in cultured hippocampal neurons were measured. And the excitability of cultured hippocampal neurons was measured as the number of action potentials (AP) induced by a depolarizing current (150pA,600ms) injection. The change rates of Vm and evoked AP were determined to evaluate the effects of SSa on the excitability of cultured hippocampal neurons.
4. Induction of SREDs by Low-Mg2+treatment of cultured hippocampal neurons and the inhibition effects of SSa on SREDs
Cultures in the10-14th day were utilized for experimentation. A24h maintenance culturing following3h exposure of low-Mg2+solution to cultured hippocampal neurons was employed to induce SREDs. Using whole-cell patch-clamp recording, cultures displayed SREDs were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). The percentage inhibition of SREDs frequency before and after applying SSa was determined to evaluated inhibition effects of SSa on SREDs in cultured hippocampal neurons.
5. Induction of SEs by Low-Mg2+treatment of cultured hippocampal neurons and the inhibition effects of SSa on SEs
Cultures in the10-14th day were utilized for experimentation. SEs was induced by exposing cultured hippocampal neurons to low-Mg2+solution for more than10minutes. Using whole-cell patch-clamp recording, cultures displayed typical SEs were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). The percentage inhibition of SEs frequency before and after applying SSa was determined to evaluated inhibition effects of SSa on SEs in cultured hippocampal neurons.
6. Induction of epileptiform discharges in CA1pyramidal neurons in rat entorhinal cortex-hippocampus slices induced by4AP solution treatment and the inhibition effects of SSa
Sprague-Dawley rats (25-50days old) were anesthetized with isoflurane and decapitated. The brains were rapidly removed and transverse brain slices (300-400 μm in thickness) that included the entorhinal cortex and hippocampus were cut with a Vibratome. The slices were left undisturbed in an incubation chamber for1h for stabilization at37℃in artificial cerebrospinal fluid (ACSF). To induce epileptiform events, slices were superfused with ACSF containing100μM4AP for20-40min. Using whole-cell patch-clamp recording, CA1pyramidal neurons displayed typical epileptiform discharges were randomly divided into normal saline group, valproic acid group (VPA,1mM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). The percentage inhibition of epileptiform discharges frequency and duration before and after applying SSa were quantified to evaluated inhibition effects of SSa on epileptiform discharges induced by4AP treatment entorhinal cortex-hippocampus slices.
7. Effects of SSa on NMDA-evoked current in cultured hippocampal neurons
The10-14d cultured hippocampal neurons were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). Using whole-cell patch-clamp recording, NMDAR current was evoked by rapid local application of100μM NMDA to the cultured hippocampal neurons through a'Y-tube' perfusion system. The percentage inhibition of peak amplitude of NMDAR current was determined to evaluate the moderating effects of SSa.
8. Effects of SSa on Na+current in cultured hippocampal neurons
The10-14d cultured hippocampal neurons were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). Using whole-cell patch-clamp recording, INaP was evoked by slow depolarizing voltage-ramps from-80mV to+20mV, and a30 ms depolarization voltage step from-70mV to0mV was used to evoke INat. The percentage inhibition of peak amplitude of INap and INat were determined to evaluate the moderating effects of SSa.
9. Effects of SSa on K+current in CA1pyramidal neurons in rat hippocampus slices
Sprague-Dawley rats (25-50days old) were anesthetized with isoflurane and decapitated. The brains were rapidly removed and transverse brain slices (300-400μm in thickness) that hippocampus were cut with a Vibratome. The slices were left undisturbed in an incubation chamber for1h for stabilization at37℃in artificial cerebrospinal fluid (ACSF). Using whole-cell patch-clamp recording, to elicit ITotal, a300ms hyperpolarizing prepulse to-120mV was followed by a series of400ms steps from-60to+60mV in10mV increments, delivered every10s, to elicit Ik, a similar protocol was used, but a100ms interval at-40mV was inserted after the prepulse. IA was calculated by point-by-point subtracting Ik from ITotal.he Steady-state deactivation currents of IA were elicited with an80ms test pulse to+50mV proceded by120ms prepulses to potentials between-90mV and+10mV. The effects of SSa on activation and deactivation properties of ITotal, Ik and IA were evaluated.
Outward K+currents were generated by a depolarizing pulse+10mV delivered from a holding potential of-80mV4AP-sensitive and TEA-sensitive K+current were obtained by application of20mM TEA or4mM4AP, respectively. Effects of SSa on4AP-sensitive and TEA-sensitive K+currents were evaluated.
10. Statistical analysis
Statistical analysis was performed using Statistical Product and Service Solutions13.0(SPSS13.0) software. All results were expressed as the mean±SEM. The effects of SSa on Vm, AP, SREDs, SEs,4AP induced epilpetiform discharges, NMDAR current, INaP and Inat were evalulated by one-way ANOVA with LSD or Dunnett's T3, and the effects on K+currents were by paired t-test. Values of P<0.05were considered to be significant.
Results
1. Primary cultured rat hippocampal neurons and identification of hippocampal neurons with Fluorescent Immunocytochemistry Technique
Microscopically, cultures in the10th d scattered in the growth, cell body full, refraction strong, axons form a dense network of nerve cells. Most of the cells were dyed by green fluorescence and III P-Tubulin positive green fluorescence accounted for96.132%±2.164%of the total cells.
2. Protective effects of SSa on Low-Mg2+induced injury of cultured hippocampal neurons
Microscopically, after incubated with Low-Mg2+solution the hippocampal neurons'bodies were shrinkage, even dissolve, axon fracture. After application of SSa the cells'bodies full and synaptics were complete. The results suggested SSa has protective effects on Low-Mg2+induced injury of cultured hippocampal neurons.
3. Effects of SSa on resting membrane potential (Vm) and excitability of cultured hippocampal neurons
There was no significant difference between groups in Vm of cultured hippocampal neurons (F=1.997, P=0.155). Compared with normal saline group, SSa has no significant effects on Vm (P=0.056). There was significant difference between groups in the inhibition rates of AP of cultured hippocampal neurons (F=108.178, P<0.001). Compared with normal saline group, SSa significantly decreased the number of AP in cultured hippocampal neurons (P<0.001). These results suggested that SSa could inhibit the excitability of cultured hippocampal neurons with no effects on the membrane properties of the neurons.
4. Inhibition effects of SSa on SREDs induced by Low-Mg2+treatment of cultured hippocampal neurons
Using whole-cell patch-clamp recording, control neurons displayed intermittent spontaneous action potentials that were consistently observed during basal activity in the hippocampal culture preparations. While,24h following exposure to low-Mg2+solution, whole-cell current-clamp recordings in pBRS showed a permanent plasticity change evidenced by the presence of SREDs.
After application of different does of SSa (0.1μM,0.3μM,0.5μM,1μM,2μM or4μM), the frequency and duration of SREDs reduced gradually or extinguished evenly. The inhibition of SSa on SREDs was reversible after being washed out with pBRS. These results suggested the inhibition effects of SSa on SREDs were reversible. Different dose of SSa had different effects on SREDs. Compared with normal saline group SSa significantly increased the percentage inhibition of SREDs at any does and the percentage inhibitions were19.167±4.182%,66.433±7.95%,95.160±6.317%,98.000±4.216%and98.571±4.518%(P<0.001) except0.1μM (P=0.170). SSa inhibited SREDs in a concentration-dependent manner with an IC50=0.42μM.
5. Inhibition effects of SSa on SEs induced by Low-Mg2+treatment of cultured hippocampal neurons
Using whole-cell patch-clamp recording, control neurons displayed intermittent spontaneous action potentials that were consistently observed during basal activity in the hippocampal culture preparations. Removal of MgCl2(low-Mg2+) from the recording solution for more than10minutes resulted in SEs cultured hippocampal neurons.
After application of different does of SSa (0.1μM,0.3μM,0.5μM,1μM,2μM or4μM), the frequency and duration of SEs reduced gradually or extinguished evenly. The inhibition of SSa on SEs was reversible after being washed out with low-Mg2+solution. These results suggested the inhibition effects of SSa on SEs were reversible. Compared with normal saline group SSa significantly increased the percentage inhibition of SEs at any does (P=0.005and P<0.001), and the percentage inhibitions were0.362±0.314%,12.071±5.794%,43.418±4.441%,73.626±3.384%,93.538±4.474%and97.798±2.323%. SSa inhibited SEs in a concentration-dependent manner and the IC50was0.62μM.
6. Inhibition effects of SSa on epileptiform discharges induced by4AP in CA1pyramidal neurons in entorhinal cortex-hippocampus slices
Using whole-cell patch-clamp recording, control neurons displayed intermittent spontaneous action potentials that were consistently observed during basal activity in CA1pyramidal neurons in entorhinal cortex-hippocampus slices. After superfusion of ACSF containing100μM4AP on entorhinal cortex-hippocampus slices for20-40min a typical epileptiform discharges in CA1pyramidal neurons was caused.
After application of different does of SSa (0.1μM,0.3μM,0.5μM,1μM,2μM or4μM), the frequency and duration of epileptiform discharges reduced gradually or extinguished evenly. The inhibition of SSa on epileptiform discharges was reversible after being washed out with ACSF containing100μM4MAP.These results suggested the inhibition effects of SSa on epileptiform discharges induced by4AP were reversible. There was significant difference between groups in the inhibition rates of the frequency and duration of epileptiform discharges in CA1pyramidal neurons (F=457.286, P<0.001). Compared with normal saline group SSa significantly decreased the duration of epileptiform discharges at any does and the percentage inhibitions were26.448±6.788%,40.059±3.264%,57.212±3.231%,67.928±1.908and74.876±2.380%(P<0.001) except at0.1μM (P=0.145). And SSa significantly decreased the frequency of epileptiform discharges at any does and the percentage inhibitions were7.750±2.053%,25.875±2.475%,43.250±1.982%,57.250±3.105%,68.625±3.114%and74.750±2.493%(P<0.001). there was no significant difference between VPA group and control group in the frequency of epileptiform discharges (P=0.170). SSa inhibited epileptiform activities induced by4AP in CA1pyramidal neurons in entorhinal cortex-hippocampus slices in a concentration-dependent manner and that the IC50was0.70μM.
7. Inhibition effects of SSa on NMDAR current in cultured hippocampal neurons
Using whole-cell patch-clamp recording, after local puff of NMDA (100μM) through the "Y-tube" perfusion system, a robust inward current was evoked which could be completely inhibited by APV, a NMDAR blocker.
There was significant difference between groups in the inhibition rates of NMDAR current of cultured hippocampal neurons (F=662.526, P<0.001). Compared with normal saline group, SSa significantly increased the percentage inhibition of NMDAR current at any does and the percentage inhibitions were1.070±0.136%,6.108±0.245%,15.015±0.219%,29.853±1.679%,37.726±1.956% and39.224±2.433%(P<0.001) and the inhibition effects were in a concentration-dependent manner with an IC50=0.62μM.
8. Inhibition effects of SSa on INaP and INat in cultured hippocampal neurons
Using whole-cell patch-clamp recording, INaP was evoked by slow depolarizing voltage-ramps from-80mV to+20mV and INat was evoked by a30ms depolarization voltage step from-70mV to0mV. The two currents could be abolished by1μM TTX.
There was significant difference between groups in the inhibition rates of INaP of cultured hippocampal neurons (F=4090.738,P<0.001). Compared with normal saline group, SSa significantly increased the percentage inhibition of INap at any does and the percentage inhibitions were3.483±0.097%,6.722±0.186%,21.284±2.012%,58.104±1.546%,70.546±0.991%and72.504±0.991%(P<0.001) and the inhibition effects were in a concentration-dependent manner with an IC50=0.84μM. There was significant difference between groups in the inhibition rates of INat of cultured hippocampal neurons (F=3342.278, P<0.001), while there was no significant difference between normal saline group and any does of SSa group (P<0.001). These results suggested that SSa display a selective inhibition effects on INaP.
9. Effects of SSa on ITotal, Ik and IA in CA1pyramidal neurons in hippocampus slices
Using whole-cell patch-clamp recording, to elicit ITotal, the holding potential was-50mV and a300ms hyperpolarizing prepulse to-120mV was followed by a series of400ms steps from-60to+60mV in10mV increments, delivered every10s and to elicit Ik, a similar protocol was used, but a100ms interval at-40mV was inserted after the prepulse. IA was calculated by point-by-point subtracting Ik from ITotal.After application of1μM SSa, the current amplitudes of ITotal and IA were significant increased (P<0.001), while there was no significant changes in current amplitudes of IK (P=0.157).
10. Regulation effects of SSa on the activation properties of ITotal, Ik and IA in CA1pyramidal neurons in hippocampus slices
The Ⅰ-Ⅴ relationships for ITotal, Ik and IA were determined by Clampfit10.0soft. The increasing effects of SSa on were voltage dependent. As the membrane potential was stepped to more depolarizing values, the amplitudes of ITotal, and IA were significantly increased after application of1μM SSa, while there was no significant change in Ik.1μM SSa caused a significant change in voltage for half-maximal-activation (V1/2) for IA (28.583±0.588vs.22.317±0.662mV, t=36.729, P<0.001) with slope factor (k)(21.933±1.472vs.23.617±1.592, t=-2.139, P=0.085). The steady-state activation curves showed that SSa significantly negative shifted the voltage-dependence of the activation of IA with no change in k. There was no significant change in V1/2of ITotal (32.467±0.432vs.31.700±1.014mV,t=2.43, P=0.059), nor did the k change (33.483±1.057vs.32.867±1.648, t=2.116, P=0.088). Similarly, no significant change was observed in V1/2for Ik (28.350±1.058vs.28.450±1.810mV, t=-0.249, P=0.813) as well as k (30.233±0.942vs.31.233±0.977, t=-2.421,P=0.06).11. Regulation effects of SSa on the steady-state inactivation properties of IA in
CA1pyramidal neurons in hippocampus slices Furthermore the moderate effects of SSa on the steady-state inactivation
properties of IA was observed. The the inactivation current was evoked by the protocol as follow. Neurons were held at-50mV and currents were elicited with an 80ms test pulse to+50mV preceded by120ms prepulses to potentials between-90mV and+10mV. Application of1μM SSa caused a significant depolarizing shift of the voltage-dependent steady-state inactivation of IA, and a significant changes of V1/2(t=-82.750, P<0.001), while there was no significant changes in k (t=-2.436, P=0.059).
12. Regulation effects of SSa on4AP-sensitive and TEA-sensitive K+currents in CA1pyramidal neurons in hippocampus slices
20mM TEA or4mM4AP was applied to separate outward K+currents pharmacologically.1μM SSa significantly enhanced a transient,4AP-sensitive outward K+current in the presence of20mM TEA (t=-20.457, P<0.001), while it did not induce a significant modification in the amplitude of a late, TEA-sensitive K+current after application of4mM4AP (t=-2.329, P=0.053).
Conclusion
1. SSa acted as an anticonvulsant against low-Mg2+or4AP solution induced epileptiform discharges in hippicampal neurons in a concentration-dependent manner, and the inhibition effects were reversible.
2. SSa inhibited the current amplitudes of NMDA-evoked current and Na+current, at the same time increased the current amplitude of K+current in hippocampal neurons, which suggests that SSa acts as an anticonvulsant by moderating on multiple mechanisms sites, not only on excitatory ligand gating ion channels but also on voltage-gated ion channels.
3. SSa could selectively decrease INap while had no effects on INat, at the same SSa selective increase IA that was4AP-sensitive K+current, hyperpolarizing shifted the activation and depolarizing shift the steady-state inactivation of IA, while had no effects on IK. These results suggest that the antiepileptic mechanisms of SSa may be lie in selective regulating different channel subunits of Na+and K+channel.
癫痫是常见的神经系统疾病之一,是一组由大脑神经元反复异常同步化放电所引起的短暂中枢神经系统功能失常为特征的慢性脑部疾病。据1997年WHO统计全球约有5000万癫痫患者,2003年国内癫痫流行病学调查表明：我国癫痫患病率约为7.0‰,以此推算我国约有600万患者正遭受癫痫的困扰。癫痫反复发作、病程长、致残率高严重影响了患者的生活质量。临床常用抗癫痫药物,不能有效控制癫痫发作,仍有约40%的癫痫患者产生耐药性,且多数抗癫痫药有多种急慢性不良反应及毒副作用,部分药物价格昂贵,长期服用给患者家庭和社会带来沉重的心理压力和巨大的经济负担。只有明确癫痫发病机制,才能指导有效性高、安全性好的抗癫痫药物的研发。
癫痫发作是由神经递质及其受体异常、离子通道异常、胶质细胞活性、免疫因素、遗传因素等各种原因引起大脑神经元兴奋性-抑制性动态失衡,神经元兴奋性增高、异常同步化放电引起。离子通道是担负中枢神经系统兴奋性活动(即神经元动作电位的传导)以及形成神经环路(即神经元间突触信号的传递)的核心构件,任何离子通道的基因突变都可能异化通道蛋白的正常功能,造成中枢神经系统兴奋性-抑制性动态失衡,神经元兴奋性增高,最终引起神经元异常同步化放电,导致癫痫发作。离子通道分为电压门控离子通道,如电压依赖性K+通道、电压依赖性Na+通道,和配体依赖性离子通道,如NMDA受体(NMDAR)通道等。近年来,随着分子生物学、膜片钳等技术的发展,作为神经元之间或神经元与效应细胞之间藉以传递各种生理和病理信息的核心媒介,离子通道与癫痫发病机制的研究已愈来愈引起神经科科研工作者的重视。
借助实验性癫痫模型模拟人类癫痫发作,从电生理学角度探讨癫痫的发病机制已成为当前本领域的研究热点之一。无镁外液诱导的原代培养海马神经元自发性反复性癫痫样放电(spontaneous recurrent epileptiform discharges, SREDs)模型是目前国际公认的模拟人类后天获得性癫痫(acquired epilepsy, AE)的细胞模型,而持续性高频癫痫样放电(continuous epileptiform high-frequency bursts,SEs)模型是模拟人类持续性癫痫(status epilepticus, SE)的细胞模型,两种癫痫细胞模型目前被广泛应用于癫痫状态下神经元生物化学、神经电生理学、分子生物学改变的机制研究及抗癫痫药物筛选的研究中；4-氨基毗啶(4-aminopyridine,4AP)诱导的海马脑片神经元癫痫样放电模型与人类颞叶癫痫发作时脑电图记录的放电模式相似,是筛选抗癫痫药物研究的经典体外癫痫模型,能较为客观地反映药物的抗癫痫作用。
癫痫属于中医“痫病”范畴,《黄帝内经素问·卷二十一·六元正纪大论篇第七十一》曰：“木郁之发,太虚埃昏,云物以扰,大风乃至,屋发折木,木有变。故民病胃脘当心而痛,上支两胁,鬲咽不通,食饮不下,甚则耳鸣眩转,目不识人,善暴僵仆”,我国历代医家认为“肝失疏泄、肝气郁结”是癫痫发病的病因病机之一,并提出癫痫“从肝论治”的中医理论,在此理论的指导下,课题组自拟方“柴胡疏肝汤”在临床癫痫的预防和治疗中取得客观疗效,进一步的实验研究发现“柴胡疏肝汤”能够降低戊四氮(PTZ)点燃模型大鼠的痫性发作潜伏期及发作级别,减轻锂-匹罗卡品诱发的难治性癫痫模型大鼠的癫痫发作级别,在前期研究基础上逐步筛选出复方中君药柴胡的有效成分柴胡皂苷a(Saikosaponin a, SSa)并发现SSa能够抑制最大电休克模型(MES)、PTZ急性致痫模型及锂-匹罗卡品模型诱发的痫性发作。
为了进一步评价SSa的抗癫痫作用并探讨SSa的抗癫痫机制,本研究运用全细胞膜片钳技术,从电生理学角度观察了SSa对无镁外液诱导的原代培养大鼠海马神经元癫痫样放电及4AP诱导的大鼠内嗅皮层-海马脑片CA1区锥体神经元癫痫样放电的抑制作用,并进一步观察了SSa对大鼠海马神经元癫痫相关离子通道电流,包括NMDAR电流、Na+通道电流及K+通道电流的调节作用,明确SSa抗癫痫作用的电生理机制,为癫痫“从肝论治”的中医理论提供较为直观的实验依据。
二、方法：
1.原代培养大鼠海马神经元及荧光免疫细胞化学鉴定
出生<24h的新生SD大鼠,无菌条件下断头取脑、分离海马,常规海马组织消化、离散细胞后以2.5×104/ml的密度接种在放有圆形玻片的24孔培养板中,以Neurobasal-A+10%B-27培养基进行培养。选取生长至第10d的海马神经元,用III β-Tubulin/GFAP及DAPI荧光三标免疫细胞化学法进行染色标记,计算绿色荧光标记的阳性细胞占蓝色荧光标记细胞的百分比。
2.无镁诱导原代培养大鼠海马神经元损伤及SSa的干预作用
选取培养至第10d的海马神经元,随机分为空白组、无镁损伤组、苯妥英组和SSa组,空白组以正常细胞外液孵育；无镁损伤组以无镁细胞外液孵育；苯妥英组以含有50μM苯妥英的无镁细胞外液孵育；SSa组以含有1gM SSa的无镁细胞外液孵育,10min后以III β-Tubulin荧光免疫细胞化学法进行染色标记,对比、评估SSa对无镁外液引起的大鼠海马神经元损伤的保护作用。
3.SSa对原代培养大鼠海马神经元静息膜电位(Vm)及兴奋性的影响
选取培养至第10-14d的海马神经元,随机分为空白组(Control)、苯妥英组和SSa组,空白组以正常细胞外液干预；苯妥英组以含有50μM苯妥英的正常细胞外液干预；SSa组以含有1μM SSa的正常细胞外液干预。在全细胞膜片钳电流钳记录模式下,观察并比较不同干预处理后海马神经元Vm的变化率,及以大小为150pA,时间为600ms的去极化方波电流刺激海马神经元诱发的动作电位(action potential, AP)个数的变化率,评估SSa对原代培养大鼠海马神经元兴奋性的影响。
4.无镁外液诱导原代培养大鼠海马神经元SREDs样放电及SSa的干预作用
选取培养至第10-14d的大鼠海马神经元,以无镁外液孵育3h后,置于正常细胞外液中孵育24h,诱发海马神经元SREDs样放电。在全细胞膜片钳电流钳记录模式下,出现典型SREDs样放电的神经元随机分为无镁组、苯妥英组(50μM)及SSa各剂量(分别为0.1μM、0.3μM、0.5μM、μM、2μM和4μM)组,观察并比较不同处理组对海马神经元SREDs样放电的抑制率,评估SSa对无镁外液诱导的原代培养大鼠海马神经元SREDs样放电的抑制作用。
5.无镁外液诱导原代培养大鼠海马神经元SEs样放电及SSa的干预作用
选取培养至第10-14d的大鼠海马神经元,以无镁外液持续灌流孵育>10min,诱发海马神经元SEs样放电。在全细胞膜片钳电流钳记录模式下,出现典型SEs样放电的神经元随机分为无镁组、苯妥英组(50μM)及SSa各剂量(分别为0.μM、0.3μM、0.5μM、1μM、2μM和4gM)组,观察并比较不同处理组对海马神经元SEs样放电的抑制率,评估SSa对无镁外液诱导的原代培养大鼠海马神经元SEs样放电的抑制作用。
6.4AP诱导大鼠内嗅皮层-海马脑片CA1区锥体神经元癫痫样放电及SSa的干预作用
选取出生25-50d的SD大鼠,吸入乙醚麻醉后,于振动切片机上切取厚300-400μm的内嗅皮层-海马脑片,以正常人工脑脊液(ACSF)37℃孵育1h后,以含有100μM4AP的ACSF持续灌流孵育20-40min诱发海马CA1区锥体神经元癫痫样放电。在全细胞膜片钳电流钳记录模式下,出现典型癫痫样放电的海马CA1区锥体神经元随机分为4AP组、丙戊酸钠(VPA)组(1mM)及SSa各剂量(分别为0.1μM、0.3μM、0.5μM、1μM、2μM和4μM)组,观察并比较不同药物干预后,海马神经元癫痫样放电持续时间及放电频率的变化率,评估SSa对4AP诱导大鼠内嗅皮层-海马脑片CA1区锥体神经元癫痫样放电的抑制作用。
7.SSa对原代培养大鼠海马神经元NMDAR电流的调节作用
选取培养至第10-14d的大鼠海马神经元,随机分为NMDA (Control)组、APV组(50μM)、苯妥英组(50μM)及SSa各剂量(分别为0.1μM、0.3μM、0.5μM、1μM、2μM和4μM)组,在全细胞膜片钳电压钳记录模式下,以含有100gMNMDA的无镁细胞外液通过“Y型”给药系统快速给药,诱发海马神经元NMDAR电流,观察并比较不同药物干预后,NMDAR电流的变化率,评估SSa对NMDAR电流的调节作用。
8.SSa对原代培养大鼠海马神经元电压依赖性Na+通道电流的调节作用
选取培养至第10-14d的大鼠海马神经元,随机分为Control组、苯妥英组(50μM)及SSa各剂量(分别为0.1μM、0.3μM、0.5μM、1μM、2μM和4μM)组,在全细胞膜片钳电压钳记录模式下,以大小为-80mV到+20mV,持续时间为2s的缓慢去极化的斜波(Ramp)电压刺激海马神经元,诱发电压依赖性持续性Na+通道电流(INaP);以大小为-70mV到0mV,持续时间为30ms的去极化方波(Step)电压刺激海马神经元,诱发电压依赖性瞬时Na+通道电流(INat),观察并比较不同药物干预后,INaP和INat的变化率,评估SSa对INaP和INat的调节作用。9.SSa对大鼠海马脑片CAl区锥体神经元电压依赖性K+通道电流的调节作用
选取出生25-50d的SD大鼠,吸入乙醚麻醉后,于振动切片机上切取大鼠海马脑片,随机分为Control组及SSa组(1μM),在全细胞膜片钳电压钳记录模式下,以一-120mV,持续时间为300ms的超极化电压脉冲,紧接之后是从-60mV到+60mV,步阶为+10mV,持续时间为400ms的去极化Step脉冲刺激海马CA1区锥体神经元,诱发总电压依赖性K+通道电流(ITotal);给予一-120mV,持续时间为200ms的超极化电压预脉冲,紧接之后是去极化至-40mV,持续时间为100ms的预脉冲,之后是从-60mV到+60mV,步阶为+10mV,持续时间为400ms的去极化Step脉冲刺激神经元,诱发电压依赖性持续性K+通道电流(IK),电压依赖性瞬时K+通道电流(IA)通过Clampfit10.0软件将记录到的ITotal和IK相减得到。并以一持续时间为120ms,从-90mV到+10mV,步阶为+10mV的Step预脉冲,之后是去极化至+50mV,持续时间为80ms的单脉冲刺激神经元,诱发IA失活电流。观察并比较不同处理组对电压门控性ITotal、IK和IA激活及失活特性的影响,评估SSa对电压依赖性外向性K+通道电流的调节作用。
分别以含有20mM四乙铵(tetraethylammonium,TEA)或4mM4AP的ACSF孵育海马脑片2min后,以去极化至+10mV,持续时间为400ms的单脉冲刺激,诱发外向性K+通道电流,观察不同干预组对TEA敏感和4AP敏感的外向性K+通道电流的影响,评估SSa对药物敏感外向性K+通道电流的调节作用。
10.统计学分析
运用SPSS13.0统计软件进行分析,实验数据均采用均数±标准差(x±s)表示,各处理组对神经元Vm、AP的调节作用及对4AP诱导的癫痫样放电持续时间、NMDAR电流、INaP和INat的抑制率均采用单因素方差分析(one-way AVOVA)进行统计分析,组间两两比较,方差齐采用LSD法,方差不齐采用Dunnett s T3法；对SERDs样放电频率抑制率、SE样放电频率抑制率及对4AP诱导的癫痫样放电频率抑制率采用单样本t检验；对电压依赖性K+通道电流特性的调节作用采用配对t检验,P<0.05表示差异具有统计学意义。
三、结果：
1.原代培养大鼠海马神经元及荧光免疫细胞化学鉴定
神经元在培养至第10d分化成熟,可见细胞散在生长,胞体饱满,折光性强,轴突形成密集的神经细胞网络,荧光显微镜下可见大部分细胞呈绿色荧光,Ⅲ β-Tubulin绿色阳性细胞占全部细胞的96.132%±2.164%。
2.SSa对无镁诱导原代培养大鼠海马神经元损伤的保护作用
镜下观,经无镁外液孵育后的海马神经元,神经元胞体皱缩,甚至溶解,轴突断裂,SSa组神经元胞体饱满、突触较完整,提示SSa能抑制无镁外液诱导的原代培养大鼠海马神经元的损伤。
3.SSa对原代培养大鼠海马神经元Vm及兴奋性的影响
各组对原代培养大鼠海马神经元Vm无显著调节作用(F=1.997,P=0.155),与Control组相比较,SSa(1μM)对Vm无显著调节作用(P=0.056)；各组对AP的抑制率有显著性差异(F=108.178,P<0.001),与Control组相比较,1μMSSa作用5min后,能显著抑制去极化电流诱发的平均AP的个数(P<0.001),提示在不影响神经元一般膜特性的前提下,SSa能够抑制原代培养大鼠海马神经元的兴奋性。
4.SSa对无镁外液诱导的原代培养大鼠海马神经元SREDs样放电的抑制作用
在全细胞电流钳记录模式下,Control组海马神经元在灌流正常细胞外液下呈现出单个偶发AP的发放,这是原代培养大鼠海马神经元的基本电生理活性；经无镁外液孵育3h的原代培养海马神经元,经正常培养液孵育24h后,在正常细胞外液下出现多个典型的自发性反复的癫痫样放电即SREDs样放电。
记录图像显示,含有0.1μM、0.3μM、0.5μM、1μM、2μM和4μM浓度SSa的正常细胞外液作用后,海马神经元SREDs发放个数及持续时间均逐渐减少甚至消失,经正常细胞外液进行洗脱后SREDs样放电再次出现,提示SSa对无镁外液诱导的大鼠海马神经元SREDs样放电的抑制作用是可逆的。统计结果显示,不同浓度的SSa作用后,无镁外液诱导的原代培养大鼠海马神经元SREDs样放电的发作有不同程度的减少。与无镁组相比较,除0.1μM作用浓度外(P=0.170),0.3μM、0.5μM、1μM、2μM及4μM浓度的SSa均能够显著抑制海马神经元SREDs样放电,抑制率分别为19.167±4.182%、66.433±4.950%、95.160±3.317%、98.000±1.216%、98.571±1.118%,P值均<0.001,SSa对无镁外液诱导的原代培养大鼠海马神经元SREDs样放电的抑制作用在一定浓度范围内呈现浓度依赖性,半数有效剂量为0.42μM。
5.SSa对无镁外液诱导的原代培养大鼠海马神经元SEs样放电的抑制作用
在全细胞电流钳记录模式下,Control组海马神经元在灌流正常细胞外液下呈现出单个偶发AP的发放,这是原代培养大鼠海马神经元的基本电生理活性,海马神经元经持续无镁外液灌流孵育>10min后,出现典型的持续性高频癫痫样放电即SEs样放电。
记录图像显示,含有0.1μM、0.3μM、0.5μM、1μM、2μM和4μM浓度SSa的无镁细胞外液作用后,原代培养海马神经元SEs样放电频率逐渐减少甚至消失,经无镁外液进行洗脱后,SEs样放电再次出现,提示SSa对无镁诱导的大鼠海马神经元SEs样放电的抑制作用是可逆的。统计结果显示,各浓度的SSa均能抑制无镁外液诱导的原代培养大鼠海马神经元SEs样放电。与无镁组相比较,除0.μMM作用浓度外(P=0.005),0.3gM、0.5μM、1μM、2μM及4μM浓度的SSa均能够显著抑制海马神经元SEs样放电,抑制率分别为12.071±5.794%、43.418±4.441%、73.626±3.384%、93.538±4.474%、97.798±2.023%,P值均<0.001,而苯妥英对原代培养大鼠海马神经元SEs样放电无显著抑制作用(P=0.082)。不同浓度的SSa对无镁诱导的海马神经元SEs样放电的抑制作用不同,呈现一定程度的浓度依赖性,半数有效剂量为0.62μM。
6.SSa对4AP诱导的大鼠内嗅皮层-海马脑片CAl区锥体神经元癫痫样放电的抑制作用
全细胞电流钳记录模式下,Control组大鼠海马CA1区锥体神经元呈现出单个偶发AP的发放,这是大鼠海马脑片CA1区锥体神经元的基本电生理活性；经含有100μM4AP的ACSF持续灌流孵育大鼠海马脑片20-40min后,CA1区锥体神经元逐渐出现稳定的促发性癫痫样放电。
记录图像显示,经0.1μM、0.3μM、0.5gM、1μM、2μM和4μM不同浓度的SSa作用后,锥体神经元促发性癫痫样放电持续时间逐渐减少,癫痫样放电发放间歇期逐渐延长,经含有100μM4AP的ACSF进行洗脱后可见用药前促发性癫痫样放电再次出现,提示SSa对4AP诱导的海马CA1区锥体神经元癫痫样放电的抑制作用是可逆的。统计结果显示,各处理组对CA1区锥神经元癫痫样放电持续时间的抑制作用有显著差异(F=457.286,P<0.001)。与4AP组相比较,除0.1μM作用浓度外(P=0.145),0.3μM、0.5μM、1μM、2μM和4μM作用浓度的SSa均能显著减少CA1区锥体神经元癫痫样放电持续时间,抑制率分别为26.448±6.788%、40.059±3.264%、57.212±3.231%、67.928±1.908、74.876±2.380%,P值均<0.001；与4AP组相比较,各浓度的SSa对4AP诱导的锥体神经元癫痫样放电频率的抑制率均显著升高,抑制率分别为7.750±2.053%、25.875±2.475%、43.250±1.982%、57.250±3.105%、68.625±3.114%、74.750±2.493%,P值均<0.001,而VPA组对4AP诱导的海马CA1区锥体神经元癫痫样放电频率无显著抑制作用(P=0.170)。不同浓度的SSa对4AP诱导的大鼠海马CA1区锥体神经元促发性癫痫样放电的抑制作用不同,呈现一定程度的浓度依赖性,半数有效剂量为0.70μM。
7.SSa对原代培养大鼠海马神经元NMDAR电流的调节作用
在全细胞电压钳记录模式下,通过“Y型”给药系统向培养的海马神经元局部快速滴加含有100μMNMDA的无镁细胞外液后,诱发出一粗壮的快速内向电流,这种内向电流能够被NMDAR阻断剂APV完全阻断,表明所记录到的内向电流为NMDAR电流。
各处理组对NMDAR电流的抑制作用有显著性差异(F=662.526,P=0.000)。与Control组相比较,0.1μM、0.3μM、0.5μM、1μM、2μM和4μM浓度的SSa均能显著减少诱发的NMDAR电流幅度,抑制率分别为1.070±0.136%、6.108±0.245%、15.015±0.219%、29.853±1.679%、37.726±1.956%、39.224±2.433%,P值均<0.001,且SSa对NMDAR电流的抑制作用在一定浓度范围内有剂量依赖性,半数抑制剂量为0.62μM。
8.SSa对原代培养大鼠海马神经元INaP和INat的调节作用
在全细胞电压钳记录模式下,选用-80mV到+20mV的缓慢去极化Ramp脉冲刺激海马神经元,诱发出一种缓慢激活的陡峭的内向电流；选用从-70mV到0mV,持续时间为30ms的去极化Step脉冲刺激海马神经元,诱发出一种在去极化瞬间瞬时激活达到峰值并迅速失活的内向流,这两种内向电流均能被1μM TTX完全阻断,表明所诱发的内向电流分别为INaP和INat。
各处理组对INaP的抑制作用有显著差异(F=4090.738,P<0.001)。与Control组相比较,0.1μM、0.3μM、0.5μM、1μM、2μM和4μM浓度的SSa均能够显著增加INaP抑制率,抑制率分别为3.483±0.097%、6.722±0.186%、21.284±2.012%、58.104±1.546%、70.546±0.991%、72.504±0.991%,P值均<0.001,不同浓度的SSa对INaP的抑制作用不同,在一定浓度范围内SSa对INaP的抑制作用呈现剂量依赖性,半数有效抑制量为0.84μM。各处理组对INat的抑制作用有显著性差异(F=3342.278,P<0.001)。而与Control组相比较,各浓度的SSa对INa,均无显著影响,P值均为1.000。表明SSa对INaP有选择性的抑制作用。
9.SSa对大鼠海马脑片CA1区锥体神经元ITotal、IK和IA大小的调节作用
将细胞膜电位钳制至-50mV,后给予一持续时间为300ms,-120mV的超极化电压脉冲,紧接之后是一从-60mV到+60mV,步阶为+10mV,持续时间为400ms的去极化Step脉冲刺激,诱发出一外向K+通道电流即ITotal'选用一持续时间为200ms,-120mV的超极化电压预脉冲,紧接之后是去极化至-40mV,持续时间为100ms的预脉冲,之后是从-60mV到+60mV,步阶为+10mV,持续时间为400ms的去极化Step脉冲刺激,诱发出一持续性外向K+通道电流成分即IK,运用Clampfit10.0软件将得到的ITotal和IK相减得到瞬时外向K+通道电流成分即IA。
将用药前后ITotal、Ik和IA的电流峰值进行标准化并进行统计分析表明,1μMSSa能够显著升高ITotal和IA电流幅度(t=-23.975,P<0.001；t=-10.281,P<0.001),而用药前后IK电流幅度无显著统计学差异(t=-1.543,P=0.157)。
10.SSa对大鼠海马脑片CA1区锥体神经元ITotal、IK和IA激活特性的调节作用
将ITotal、Ik和IA的刺激电压(V)和诱发出的相应电流值(Ⅰ)运用Clampfit10.0软件绘制I-V曲线,随着刺激电压的去极化,SSa对ITotal和IA电流的增强作用逐渐增大,而SSa对IK的I-V曲线无明显影响。绘制三种电流的激活曲线,用药前后相比较,1μM SSa能够使IA的激活曲线超极化相移位,显著下调V1/2(28.583±0.588vs.22.317±0.662mV,t=36.729,P<0.001),对k值无显著影响(21.933±1.472vs.23.617±1.592,t=-2.139,P=0.085)；对ITotal激活曲线的V1/2和k均无显著性影响(V1/2：32.467±0.432vs.31.700±1.014mV,t=2.43,P=0.059；k:33.483±1.057vs.32.867±1.648,t=2.116,P=0.088)；对IK的激活曲线的V1/2和k也无显著性调节作用(V1/2：28.350±1.058vs.28.450±1.810mV,t=-0.249,P=0.813；k：30.233±0.942vs.31.233±0.977,t=-2.421,P=0.06)。
11.SSa对大鼠海马脑片CA1区锥体神经元IA失活特性的调节作用
进一步观察SSa对IA失活特性的调节作用,将细胞膜电位钳制至-50mV,给予一持续时间为120ms从-90mV到+10mV步阶为+10mV的预脉冲,之后是去极化至+50mV,持续时间为80ms的单脉冲刺激诱发IA失活电流。绘制IA失活曲线,用药前后相比较,1μM SSa能够使IA的失活曲线去极化相移位,能显著上调IA失活曲线的V1/2(t=-82.750,P<0.001),而对IA失活曲线的k值无显著影响(t=-2.436,P=0.059)。
12.SSa对大鼠海马脑片CA1区锥体神经元4AP敏感及TEA敏感K+通道电流的调节作用
选用20mM TEA和4mM4AP,从药理学角度分离TEA敏感的外向性K+通道电流成分和4AP敏感的外向性K+通道电流成分,并观察SSa对这两种药物敏感的K+通道电流的调节作用。20mM TEA作用后总外向性K+通道电流的持续成分减少,仅剩快速激活-失活成分,即4AP敏感K+通道电流,1μM SSa能够显著上调这种外向性快K+通道电流成分(t=-20.457,P<0.001)；4mM4AP作用后总外向性K+通道电流的快速激活-失活成分被阻断,仅剩一持续性外向性K+通道电流成分,即TEA敏感K+通道电流,1μM SSa对这种持续性外向性K+通道电流成分无显著调节作用(t=-2.329,P=0.053)。表明SSa对4AP敏感K+通道电流有选择性增加作用。
四、结论：
1.SSa能够显著抑制无镁外液诱导的原代培养大鼠海马神经元SREDs样放电及SEs样放电,这种抑制作用是可逆的,在一定剂量范围内,呈现浓度依赖性。在SREDs样放电模型中,1μM SSa的抑制作用与常规剂量苯妥英的抑制作用相当；SEs样放电模型对多种临床常用抗癫痫药表现耐药性,而0.1μM浓度的SSa即可显著抑制SEs样放电,常规剂量及高剂量的苯妥英均对SEs无抑制作用,提示SSa可能对难治性癫痫有良好的治疗潜力。
2. SSa能够显著抑制4AP诱导的大鼠内嗅皮层-海马脑片CAl区锥体神经元癫痫样放电,这种抑制作用是可逆的,在一定剂量范围内,呈现浓度依赖性。抑制作用优于VPA,提示SSa有良好的抗癫痫作用。
3.SSa能够抑制NMDA诱发的海马神经元NMDAR电流幅度,抑制N+通道电流幅度,上调K+通道电流幅度,说明SSa可以通过多位点调节发挥抗癫痫作用,不仅对兴奋性配体依赖性离子通道有调节作用,对电压依赖性离子通道也有显著的调节作用。
4.SSa能够选择性的下调INap电流幅度,而对INat无影响；能够选择性的上调IA成分及4AP敏感的K+通道电流成分,使IA激活曲线左移,而失活曲线右移,加快IA的激活而使其失活速度减慢,而对TEA敏感的IA无明显影响,表明SSa可能通过选择性的调节不同的Na+通道、K+通道亚单位发挥抗癫痫作用。SSa对离子通道不同亚单位的调节作用等机制有待于进一步探讨,为明确SSa抗癫痫机制提供实验依据。
Purpose
Epilepsy, one of the most common neurological disorders, is induced by abnormal electrical discharge, manifestes by recurrent seizure. According to the WHO statistics, there were about50million epilepsy patients worldwide. In2003, the national epidemiological survey of epilepsy demonstrated that the morbidity rate of epilepsy was7.0‰. Epilepsy duration long high recurrent disability seriously affects the patient's quality of life. Clinical commonly used antiepileptic drug, cannot effectively control epileptic seizures and there were about40%of intractable epilepsy can not be controlled with drugs. Beside, most antiepileptic drugs have a variety of acute or chronic adverse reactions and side effects, and some drugs are expensive, long-term use to family and society bring heavy psychological pressure and great economic burden. Only clear the pathogenesis of epilepsy, and can guide high efficiency with good security antiepileptic drug research and development.
Seizures are caused by the brain excitatory and inhibitory dynamic imbalance of neurons, and increased neuronal excitability caused abnormal synchronization discharge. Ion channels are responsible for the central nervous system excitability activity (that is, the neurons action potential conduction) and the formation of neural circuits (that is, synapses between neurons signal transmission) at the core of the artifacts. Any ion channel gene mutation may have alienated the normal function of the channel proteins, caused the central nervous system excitability-inhibitory dynamic imbalances, and triggered neurons abnormal synchronization discharge, eventually. Ion channels contain voltage-gated ion channels, such as Voltage-gated K+channel and voltage-gated Na+channels, and ligand-gated ion channel, such as NMDA receptor channel. Recently, With the development of molecular biology patch clamp techniques, ion channels has increasingly aroused people's attention in the pathogenesis of epilepsy research.
From the perspective of electrophysiology taking Experimental models simulating human epilepsy seizures to study the pathogenesis of epilepsy has become the research hotspot in the field. Low-Mg2+solution induced spontaneous recurrent epileptiform discharges (SREDs) in cultured hippocampal neurons model is the internationally recognized imitating humans acquired epilepsy cell model. And Low-Mg2+solution induced continuous epileptiform high-frequency bursts (SEs) model is imitating human status epilepticus cell model. The two cell models have been widely used in biochemical, electrophysiological and the molecular biology mechanism in epilepsy and in the antiepileptic drug screening researchs.4-aminopyridine (4AP) induced epileptiform events in hippocampal neurons sharing electrographic similarities with the seizure discharges seen in patients with temporal lobe epilepsy is a classic in vitro epilepsy model for screening of antiepileptic drug which could objectively reflect the antiepileptic drug effect. Epilepsy is known as "Xian Bing" in Traditional Chinese Medicine (TCM). Ancient doctors take "Stagnation of Gan, Abnormal Dispersion of Gan" as one of the basic pathogenesis of epilepsy and "Treatment from Gan" as a treatment method for epilepsy. Under the guidance of this TCM theory, the prescription of "Chaihu Shugan Tang" displayed objective curative effects in the clinical prevention and treatment of epilepsy. Further studies found that "Chaihu Shugan Tang" could decrease the level and the latency of seizure in PTZ-kindling rats, reduce the level of seizure in Li-pilocarpine incuded rat refractory epilepsy model. Based On these results, we have gradually focused on the effect of saikosaponin a (SSa), a active ingredient from Bupleurum, the monarch drug in the prescription. We found that SSa could inhibit epileptic seizure in the MES model, the acute PTZ model and the Li-pilocarpine model. The results.suggest that SSa has good anticonvulsant effects on experimental epilepsy models.
In order to further investigate the antiepileptic mechanism of SSa, using the patch clamp technique we observed the inhibition effects of SSa on Low-Mg2+induced epileptiform discharges in cultured hippocampal neurons and4AP induced epileptiform discharges in CA1pyramidal neurons in rat entorhinal cortex-hippocampus slices. Furthermore, we observed the moderating effects of SSa on the NMDAR current, the sodium currents and the potassium currents in rat hippocampal neurons. To clarify the antiepileptic mechanism of SSa and provide experimental bases for the TCM theory of "Treatment form Gan".
Methods
1. Primary cultured rat hippocampal neurons and identification of hippocampal neurons with Fluorescent Immunocytochemistry Technique Newborn SD rats (<24h) were sacrificed and the hippocampus was separated in sterile conditions. Cells were plated at a density of2.5×104cells/cm2onto a glial support layer that was previously plated onto poly-L-lysine-coated (0.05mg/ml) glass coverslips and cultured with Neurobasal-A medium supplemented with2%B-27. On the10th day, cultures were utilized for identification with Ⅲ β-Tubulin, GFAP and DAPI by Fluorescent Immunocytochemistry Technique. The percentage of green fluorescence positive cells in blue fluorescent cells was determined.
2. Low-Mg2+induced injury of cultured hippocampal neurons and the protective effects of SSa
Cultures in the10th day were randomly divided into control group, Low-Mg2+group, phenytoin group (phenytoin,50μM), and SSa group (SSa,1μM).The protective effects of SSa on Low-Mg2+induced cultured hippocampal neurons were evaluated with III β-Tubulin and DAPI by Fluorescent Immunocytochemistry Technique.
3. Effects of SSa on resting membrane potential (Vm) and excitability of cultured hippocampal neurons
Cultures in the10th day were randomly divided into control group, phenytoin group (phenytoin,50μM), and SSa group (SSa,1μM). Using whole-cell patch-clamp recording, changes of the Vm before and after different treatments in cultured hippocampal neurons were measured. And the excitability of cultured hippocampal neurons was measured as the number of action potentials (AP) induced by a depolarizing current (150pA,600ms) injection. The change rates of Vm and evoked AP were determined to evaluate the effects of SSa on the excitability of cultured hippocampal neurons.
4. Induction of SREDs by Low-Mg2+treatment of cultured hippocampal neurons and the inhibition effects of SSa on SREDs
Cultures in the10-14th day were utilized for experimentation. A24h maintenance culturing following3h exposure of low-Mg2+solution to cultured hippocampal neurons was employed to induce SREDs. Using whole-cell patch-clamp recording, cultures displayed SREDs were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). The percentage inhibition of SREDs frequency before and after applying SSa was determined to evaluated inhibition effects of SSa on SREDs in cultured hippocampal neurons.
5. Induction of SEs by Low-Mg2+treatment of cultured hippocampal neurons and the inhibition effects of SSa on SEs
Cultures in the10-14th day were utilized for experimentation. SEs was induced by exposing cultured hippocampal neurons to low-Mg2+solution for more than10minutes. Using whole-cell patch-clamp recording, cultures displayed typical SEs were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). The percentage inhibition of SEs frequency before and after applying SSa was determined to evaluated inhibition effects of SSa on SEs in cultured hippocampal neurons.
6. Induction of epileptiform discharges in CA1pyramidal neurons in rat entorhinal cortex-hippocampus slices induced by4AP solution treatment and the inhibition effects of SSa
Sprague-Dawley rats (25-50days old) were anesthetized with isoflurane and decapitated. The brains were rapidly removed and transverse brain slices (300-400 μm in thickness) that included the entorhinal cortex and hippocampus were cut with a Vibratome. The slices were left undisturbed in an incubation chamber for1h for stabilization at37℃in artificial cerebrospinal fluid (ACSF). To induce epileptiform events, slices were superfused with ACSF containing100μM4AP for20-40min. Using whole-cell patch-clamp recording, CA1pyramidal neurons displayed typical epileptiform discharges were randomly divided into normal saline group, valproic acid group (VPA,1mM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). The percentage inhibition of epileptiform discharges frequency and duration before and after applying SSa were quantified to evaluated inhibition effects of SSa on epileptiform discharges induced by4AP treatment entorhinal cortex-hippocampus slices.
7. Effects of SSa on NMDA-evoked current in cultured hippocampal neurons
The10-14d cultured hippocampal neurons were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). Using whole-cell patch-clamp recording, NMDAR current was evoked by rapid local application of100μM NMDA to the cultured hippocampal neurons through a'Y-tube' perfusion system. The percentage inhibition of peak amplitude of NMDAR current was determined to evaluate the moderating effects of SSa.
8. Effects of SSa on Na+current in cultured hippocampal neurons
The10-14d cultured hippocampal neurons were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). Using whole-cell patch-clamp recording, INaP was evoked by slow depolarizing voltage-ramps from-80mV to+20mV, and a30 ms depolarization voltage step from-70mV to0mV was used to evoke INat. The percentage inhibition of peak amplitude of INap and INat were determined to evaluate the moderating effects of SSa.
9. Effects of SSa on K+current in CA1pyramidal neurons in rat hippocampus slices
Sprague-Dawley rats (25-50days old) were anesthetized with isoflurane and decapitated. The brains were rapidly removed and transverse brain slices (300-400μm in thickness) that hippocampus were cut with a Vibratome. The slices were left undisturbed in an incubation chamber for1h for stabilization at37℃in artificial cerebrospinal fluid (ACSF). Using whole-cell patch-clamp recording, to elicit ITotal, a300ms hyperpolarizing prepulse to-120mV was followed by a series of400ms steps from-60to+60mV in10mV increments, delivered every10s, to elicit Ik, a similar protocol was used, but a100ms interval at-40mV was inserted after the prepulse. IA was calculated by point-by-point subtracting Ik from ITotal.he Steady-state deactivation currents of IA were elicited with an80ms test pulse to+50mV proceded by120ms prepulses to potentials between-90mV and+10mV. The effects of SSa on activation and deactivation properties of ITotal, Ik and IA were evaluated.
Outward K+currents were generated by a depolarizing pulse+10mV delivered from a holding potential of-80mV4AP-sensitive and TEA-sensitive K+current were obtained by application of20mM TEA or4mM4AP, respectively. Effects of SSa on4AP-sensitive and TEA-sensitive K+currents were evaluated.
10. Statistical analysis
Statistical analysis was performed using Statistical Product and Service Solutions13.0(SPSS13.0) software. All results were expressed as the mean±SEM. The effects of SSa on Vm, AP, SREDs, SEs,4AP induced epilpetiform discharges, NMDAR current, INaP and Inat were evalulated by one-way ANOVA with LSD or Dunnett's T3, and the effects on K+currents were by paired t-test. Values of P<0.05were considered to be significant.
Results
1. Primary cultured rat hippocampal neurons and identification of hippocampal neurons with Fluorescent Immunocytochemistry Technique
Microscopically, cultures in the10th d scattered in the growth, cell body full, refraction strong, axons form a dense network of nerve cells. Most of the cells were dyed by green fluorescence and III P-Tubulin positive green fluorescence accounted for96.132%±2.164%of the total cells.
2. Protective effects of SSa on Low-Mg2+induced injury of cultured hippocampal neurons
Microscopically, after incubated with Low-Mg2+solution the hippocampal neurons'bodies were shrinkage, even dissolve, axon fracture. After application of SSa the cells'bodies full and synaptics were complete. The results suggested SSa has protective effects on Low-Mg2+induced injury of cultured hippocampal neurons.
3. Effects of SSa on resting membrane potential (Vm) and excitability of cultured hippocampal neurons
There was no significant difference between groups in Vm of cultured hippocampal neurons (F=1.997, P=0.155). Compared with normal saline group, SSa has no significant effects on Vm (P=0.056). There was significant difference between groups in the inhibition rates of AP of cultured hippocampal neurons (F=108.178, P<0.001). Compared with normal saline group, SSa significantly decreased the number of AP in cultured hippocampal neurons (P<0.001). These results suggested that SSa could inhibit the excitability of cultured hippocampal neurons with no effects on the membrane properties of the neurons.
4. Inhibition effects of SSa on SREDs induced by Low-Mg2+treatment of cultured hippocampal neurons
Using whole-cell patch-clamp recording, control neurons displayed intermittent spontaneous action potentials that were consistently observed during basal activity in the hippocampal culture preparations. While,24h following exposure to low-Mg2+solution, whole-cell current-clamp recordings in pBRS showed a permanent plasticity change evidenced by the presence of SREDs.
After application of different does of SSa (0.1μM,0.3μM,0.5μM,1μM,2μM or4μM), the frequency and duration of SREDs reduced gradually or extinguished evenly. The inhibition of SSa on SREDs was reversible after being washed out with pBRS. These results suggested the inhibition effects of SSa on SREDs were reversible. Different dose of SSa had different effects on SREDs. Compared with normal saline group SSa significantly increased the percentage inhibition of SREDs at any does and the percentage inhibitions were19.167±4.182%,66.433±7.95%,95.160±6.317%,98.000±4.216%and98.571±4.518%(P<0.001) except0.1μM (P=0.170). SSa inhibited SREDs in a concentration-dependent manner with an IC50=0.42μM.
5. Inhibition effects of SSa on SEs induced by Low-Mg2+treatment of cultured hippocampal neurons
Using whole-cell patch-clamp recording, control neurons displayed intermittent spontaneous action potentials that were consistently observed during basal activity in the hippocampal culture preparations. Removal of MgCl2(low-Mg2+) from the recording solution for more than10minutes resulted in SEs cultured hippocampal neurons.
After application of different does of SSa (0.1μM,0.3μM,0.5μM,1μM,2μM or4μM), the frequency and duration of SEs reduced gradually or extinguished evenly. The inhibition of SSa on SEs was reversible after being washed out with low-Mg2+solution. These results suggested the inhibition effects of SSa on SEs were reversible. Compared with normal saline group SSa significantly increased the percentage inhibition of SEs at any does (P=0.005and P<0.001), and the percentage inhibitions were0.362±0.314%,12.071±5.794%,43.418±4.441%,73.626±3.384%,93.538±4.474%and97.798±2.323%. SSa inhibited SEs in a concentration-dependent manner and the IC50was0.62μM.
6. Inhibition effects of SSa on epileptiform discharges induced by4AP in CA1pyramidal neurons in entorhinal cortex-hippocampus slices
Using whole-cell patch-clamp recording, control neurons displayed intermittent spontaneous action potentials that were consistently observed during basal activity in CA1pyramidal neurons in entorhinal cortex-hippocampus slices. After superfusion of ACSF containing100μM4AP on entorhinal cortex-hippocampus slices for20-40min a typical epileptiform discharges in CA1pyramidal neurons was caused.
After application of different does of SSa (0.1μM,0.3μM,0.5μM,1μM,2μM or4μM), the frequency and duration of epileptiform discharges reduced gradually or extinguished evenly. The inhibition of SSa on epileptiform discharges was reversible after being washed out with ACSF containing100μM4MAP.These results suggested the inhibition effects of SSa on epileptiform discharges induced by4AP were reversible. There was significant difference between groups in the inhibition rates of the frequency and duration of epileptiform discharges in CA1pyramidal neurons (F=457.286, P<0.001). Compared with normal saline group SSa significantly decreased the duration of epileptiform discharges at any does and the percentage inhibitions were26.448±6.788%,40.059±3.264%,57.212±3.231%,67.928±1.908and74.876±2.380%(P<0.001) except at0.1μM (P=0.145). And SSa significantly decreased the frequency of epileptiform discharges at any does and the percentage inhibitions were7.750±2.053%,25.875±2.475%,43.250±1.982%,57.250±3.105%,68.625±3.114%and74.750±2.493%(P<0.001). there was no significant difference between VPA group and control group in the frequency of epileptiform discharges (P=0.170). SSa inhibited epileptiform activities induced by4AP in CA1pyramidal neurons in entorhinal cortex-hippocampus slices in a concentration-dependent manner and that the IC50was0.70μM.
7. Inhibition effects of SSa on NMDAR current in cultured hippocampal neurons
Using whole-cell patch-clamp recording, after local puff of NMDA (100μM) through the "Y-tube" perfusion system, a robust inward current was evoked which could be completely inhibited by APV, a NMDAR blocker.
There was significant difference between groups in the inhibition rates of NMDAR current of cultured hippocampal neurons (F=662.526, P<0.001). Compared with normal saline group, SSa significantly increased the percentage inhibition of NMDAR current at any does and the percentage inhibitions were1.070±0.136%,6.108±0.245%,15.015±0.219%,29.853±1.679%,37.726±1.956% and39.224±2.433%(P<0.001) and the inhibition effects were in a concentration-dependent manner with an IC50=0.62μM.
8. Inhibition effects of SSa on INaP and INat in cultured hippocampal neurons
Using whole-cell patch-clamp recording, INaP was evoked by slow depolarizing voltage-ramps from-80mV to+20mV and INat was evoked by a30ms depolarization voltage step from-70mV to0mV. The two currents could be abolished by1μM TTX.
There was significant difference between groups in the inhibition rates of INaP of cultured hippocampal neurons (F=4090.738,P<0.001). Compared with normal saline group, SSa significantly increased the percentage inhibition of INap at any does and the percentage inhibitions were3.483±0.097%,6.722±0.186%,21.284±2.012%,58.104±1.546%,70.546±0.991%and72.504±0.991%(P<0.001) and the inhibition effects were in a concentration-dependent manner with an IC50=0.84μM. There was significant difference between groups in the inhibition rates of INat of cultured hippocampal neurons (F=3342.278, P<0.001), while there was no significant difference between normal saline group and any does of SSa group (P<0.001). These results suggested that SSa display a selective inhibition effects on INaP.
9. Effects of SSa on ITotal, Ik and IA in CA1pyramidal neurons in hippocampus slices
Using whole-cell patch-clamp recording, to elicit ITotal, the holding potential was-50mV and a300ms hyperpolarizing prepulse to-120mV was followed by a series of400ms steps from-60to+60mV in10mV increments, delivered every10s and to elicit Ik, a similar protocol was used, but a100ms interval at-40mV was inserted after the prepulse. IA was calculated by point-by-point subtracting Ik from ITotal.After application of1μM SSa, the current amplitudes of ITotal and IA were significant increased (P<0.001), while there was no significant changes in current amplitudes of IK (P=0.157).
10. Regulation effects of SSa on the activation properties of ITotal, Ik and IA in CA1pyramidal neurons in hippocampus slices
The Ⅰ-Ⅴ relationships for ITotal, Ik and IA were determined by Clampfit10.0soft. The increasing effects of SSa on were voltage dependent. As the membrane potential was stepped to more depolarizing values, the amplitudes of ITotal, and IA were significantly increased after application of1μM SSa, while there was no significant change in Ik.1μM SSa caused a significant change in voltage for half-maximal-activation (V1/2) for IA (28.583±0.588vs.22.317±0.662mV, t=36.729, P<0.001) with slope factor (k)(21.933±1.472vs.23.617±1.592, t=-2.139, P=0.085). The steady-state activation curves showed that SSa significantly negative shifted the voltage-dependence of the activation of IA with no change in k. There was no significant change in V1/2of ITotal (32.467±0.432vs.31.700±1.014mV,t=2.43, P=0.059), nor did the k change (33.483±1.057vs.32.867±1.648, t=2.116, P=0.088). Similarly, no significant change was observed in V1/2for Ik (28.350±1.058vs.28.450±1.810mV, t=-0.249, P=0.813) as well as k (30.233±0.942vs.31.233±0.977, t=-2.421,P=0.06).11. Regulation effects of SSa on the steady-state inactivation properties of IA in
CA1pyramidal neurons in hippocampus slices Furthermore the moderate effects of SSa on the steady-state inactivation
properties of IA was observed. The the inactivation current was evoked by the protocol as follow. Neurons were held at-50mV and currents were elicited with an 80ms test pulse to+50mV preceded by120ms prepulses to potentials between-90mV and+10mV. Application of1μM SSa caused a significant depolarizing shift of the voltage-dependent steady-state inactivation of IA, and a significant changes of V1/2(t=-82.750, P<0.001), while there was no significant changes in k (t=-2.436, P=0.059).
12. Regulation effects of SSa on4AP-sensitive and TEA-sensitive K+currents in CA1pyramidal neurons in hippocampus slices
20mM TEA or4mM4AP was applied to separate outward K+currents pharmacologically.1μM SSa significantly enhanced a transient,4AP-sensitive outward K+current in the presence of20mM TEA (t=-20.457, P<0.001), while it did not induce a significant modification in the amplitude of a late, TEA-sensitive K+current after application of4mM4AP (t=-2.329, P=0.053).
Conclusion
1. SSa acted as an anticonvulsant against low-Mg2+or4AP solution induced epileptiform discharges in hippicampal neurons in a concentration-dependent manner, and the inhibition effects were reversible.
2. SSa inhibited the current amplitudes of NMDA-evoked current and Na+current, at the same time increased the current amplitude of K+current in hippocampal neurons, which suggests that SSa acts as an anticonvulsant by moderating on multiple mechanisms sites, not only on excitatory ligand gating ion channels but also on voltage-gated ion channels.
3. SSa could selectively decrease INap while had no effects on INat, at the same SSa selective increase IA that was4AP-sensitive K+current, hyperpolarizing shifted the activation and depolarizing shift the steady-state inactivation of IA, while had no effects on IK. These results suggest that the antiepileptic mechanisms of SSa may be lie in selective regulating different channel subunits of Na+and K+channel.
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