柴胡皂苷a对谷氨酸激活体外培养大鼠海马星形胶质细胞的干预作用
摘要
一目的与意义
癫痫是一种严重危害人类健康的神经系统疾病,在我国癫痫的患病率为4.6‰。由于社会传统的偏见,癫痫病人及家属常隐瞒或否认自己的病情,并承受很大的心理压力。由癫痫引起的医疗、家庭和社会问题极为显著。
目前常用一线抗癫痫药(AEDs)可使大部分癫痫患者发作得以控制,但长期应用安全性较差,对于一些难治性癫痫行外科手术治疗,也受到适应症的限制,风险大,费用昂贵,还存在复发的可能。因此难治性癫痫仍然以药物治疗为主要手段,这就需要使用新的抗癫痫药,但其研究前提是对于癫痫发病机制的深入认识。
近年来癫痫的发病机制研究有了长足进步。神经元兴奋性增高和高度同步化发放是癫痫发病的两个重要特征已得到公认,国内外很多学者试图从多个方面来探索引起神经元兴奋性增加和高度同步化发放的形成机制,提出了不少有益的见解。其中对星形胶质细胞与神经信号传递的认识逐步深入,位于突触联合处的星形胶质细胞被认为是突触三联体的重要组成部分。谷氨酸(Glu)作为中枢神经系统的主要的兴奋性神经递质,是重要的致痫原因,既可导致癫痫发作,又可能在癫痫的继发性脑损害中起重要作用。正常情况下,神经元将Glu释放到突触间隙,然后由星形胶质细胞摄取后再转化为谷氨酰胺,这种神经元与星形胶质细胞间的谷氨酸-谷氨酰胺循环在正常神经系统功能中起着重要作用,这种循环一旦遭破坏即可导致Glu在突触间隙大量堆积,使神经元处于高度兴奋的状态,从而致使惊厥形成。神经系统损伤时,往往伴随星形胶质细胞的活化,又称反应性胶质增生(Reactive gliosis),首要特征是其重要的骨架蛋白胶质纤维酸性蛋白(GFAP)表达增加,激活的星形胶质细胞对神经突触传递施加影响,可以通过多种途径来作用于神经元,增加神经元兴奋性,进而引起癫痫的发作。
柴胡皂苷a(SSa)为我国传统中药柴胡主要药理成分柴胡皂苷的一种单体成分,柴胡皂甙具有公认的抗炎、免疫调节作用,并且在我们前期的研究基础上发现柴胡总皂苷和SSa可以抗实验性癫痫、抑制慢性点燃大鼠GFAP的表达,调节海马Glu的表达,故我们推测SSa可以通过抑制Glu对神经系统作用和抑制其激活的星形胶质细胞来发挥抗实验性癫痫的作用。
而目前体外SSa抗癫痫机制研究尚不明确,本实验由广东省中管局课题基金(NO.1060133)资助研究了SSa对Glu激活体外培养大鼠海马星形胶质细胞的干预作用,探讨SSa可能的抗癫痫机制。
二方法与内容
1大鼠海马星形胶质细胞的培养与鉴定
星形胶质细胞的原代培养参考McCarthy法。取新生SD大鼠海马,剪碎,消化,离心,差速贴壁40 min后,按10~6个/cm~2接种。37.0℃,5%CO_2孵箱中培养7-9 d,待培养的细胞70%-80%融合时,将培养瓶置于恒温旋转摇床上摇18 h(37.0℃,240 r/min)后,进行传代培养即得纯化的星形胶质细胞,纯化的细胞进一步运用免疫细胞化学SABC法进行鉴定。
2 SSa对Glu激活体外培养的大鼠海马星形胶质细胞活性的影响
运用MTT比色法检测各组星形胶质细胞活细胞数量。将传代细胞按8×10~4个/cm~2的密度接种于96孔培养板中,每孔200μl。完全培养基培养48 h后,用含5%胎牛血清的培养基配制药物干预组共6组:a组为正常组(control组),b组为L-Glu(1.5 mmol/L)的激活组,c组为L-Glu+SSa剂量组(分别为1.5mmol/L,10 mg/L),d组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,5 mg/L),e组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,2.5 mg/L),f组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,1.25 mg/L)。每组6孔,每孔加入200μl,继续培养72 h后运用MTT法测OD值,以OD值反映各组星形胶质细胞活细胞数量。
3 SSa对Glu激活体外培养大鼠海马星形胶质细胞分裂周期的影响
运用流式细胞技术检测各组星形胶质细胞的细胞周期。将传代细胞以5×10~5个/cm~2密度种植于直径10 mm的培养皿中。完全培养基培养48 h后,用DMEM/F_(12)培养基配制药物干预组共5组:a组为正常组(control组),b组为L-Glu(1 mmol/L)的激活组,c组为L-Glu+SSa剂量组(分别为1 mmol/L,5mg/L),d组为L-Glu+SSa剂量组(分别为1 mmol/L,2.5 mg/L),e组为含L-Glu+SSa剂量组(分别为1 mmol/L,1.25 mg/L)。在加药24 h后取材,PI染色并调整细胞密度为10~6个/cm~2,而后用流式细胞技术检测各组星形胶质细胞细胞周期,每组样本检测10~4个细胞,以细胞指数表示分布于细胞周期各时相的细胞百分比数,以增殖指数反映细胞增殖状态。
4 SSa对Glu激活体外培养大鼠海马星形胶质细胞GFAP表达的影响
运用Western-blot法检测各组星形胶质细胞GFAP表达量。将传代细胞以5×10~5个/ml的密度种植于培养皿中,完全培养基培养48 h后,用DMEM/F_(12)培养基配制药物干预组共5组:a组为正常组(control组),b组为L-Glu(1.5 mmol/L)的激活组,c组为L-Glu+SSa剂量组(分别为1.5 mmol/L,5 mg/L),d组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,2.5 mg/L),e组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,1.25 mg/L)。作用24 h后,提取蛋白,运用western-blot技术检测GFAP表达的变化,对结果进行灰度分析,计算GFAP与β-actin的比值。每组蛋白检测6次。
三统计方法
应用SPSS 11.5统计软件进行处理。各组MTT比色法结果和western-blot检测结果比较采用单因素方差分析(One-way ANOVA),进一步组间比较用LSD法;流式细胞技术检测结果比较采用R×C表资料的卡方检验,P＜0.05表示差异具有统计学意义。
四结果
1星形胶质细胞的培养和鉴定
原代培养的大鼠海马星形胶质细胞,镜下观察可见星形胶质细胞生长较好,所有细胞均贴壁。原代培养的星形胶质细胞运用免疫组化染色法鉴定,细胞的GFAP免疫反应阳性率为98%以上。
2 Glu对体外培养大鼠海马星形胶质细胞活性的影响及SSa的干预作用
体外培养大鼠海马星形胶质细胞在不同处理因素处理下各组OD值,与a组比较,b组、c组、d组、f组OD值均高于a组,差异有显著性意义(P＜0.05);与b组比较,a组、c组、d组、e组OD值均低于b组,而f组则高于b组,差异有显著性意义(P＜0.05)。结果提示:Glu可以显著激活体外培养星形胶质细胞,促进细胞增殖,而SSa对Glu激活的细胞增殖具有显著抑制作用。
3 Glu对体外培养大鼠海马星形胶质细胞分裂周期的影响及SSa的干预作用
各组流式细胞技术检测的细胞周期结果:各组细胞增殖指数差异有显著性意义(P＜0.001)。各组细胞增殖指数均高于a组,各组细胞增殖指数均低于b组。结果提示:Glu(1 mmol/L)可以显著激活大鼠海马星形胶质细胞,加速星形胶质细胞的分裂周期。SSa对激活细胞的分裂周期均存在一定的阻滞作用。
4 Glu对体外培养大鼠海马星形胶质细胞GFAP表达的影响及SSa的干预作用
各组western-blot检测结果:GFAP表达量b组、c组、e组与a组比较差异均有显著性意义(P＜0.05);a组、c组、d组GFAP表达量低于b组,差异有显著性意义(P＜0.05),而e组高于b组,差异有显著性意义(P＜0.05)。提示:Glu可以显著激活大鼠海马星形胶质细胞,促使星形胶质细胞GFAP过度表达,而SSa对其激活存在抑制作用。
五结论
1 Glu可促进体外培养大鼠海马星形胶质细胞的活性,SSa可抑制激活星形胶质细胞的活性。
2 Glu可促进体外培养大鼠海马星形胶质细胞的分裂周期,SSa可阻滞激活星形胶质细胞的分裂周期。
3 Glu可促进体外培养大鼠海马星形胶质细胞GFAP的表达,SSa可抑制过度表达的GFAP。
Objective and significance
Epilepsy is a kind of neurological disease that is harmful to the health of human being. There is about 4.6‰of the population in our country affected by this neurological disease. Epileptic and their family have possibly to hide or to deny the heath condition because of orthodox prejudice of society,and epileptic beared mental stress from the world. It cant't be ignored that the problems of medical treament,family and social problem induced by epilepsy.
In now, epileptic treatment still dependent on chemicals. General anti-epileptic drug(AEDs) can control epileptic attack,but security is a problem in treatment of long-term, and the method to treat refractoriness epilepsy by surgery may be confined by indication, high risk, expensive, and possible to recur. So epileptic treatment still dependent on chemicals, but it must know clearly that is pathogenesy of epilepsy before develop new chemicals.
For the past few years, research of epileptic pathogenesy has been advanced. Increase in neuronal excitability and synchronize firing are two important characteristic of epileptic, which had be generally accepted. Many scholars try them best to study mechanism of form, and introduced many view. Recognition of relation of astrocyte with synaptic signal transmission have been gradually clear. Synaptically associated astrocytes should be viewed as integral modulatory elements of tripartite synapses. Glu, one of main excitatory neurotransmitters in central nervous system, is a etiological factor of epilepsy. It can not cause epilepsy but also may play a important part in the brain damage caused by epilepsy. In normal, glu which is released by neuron into synaptic cleft is intaked by astrocyte and then turned into glutamine. This cycle of glutamate- glutamine between neuron and astrocyte plays a important role in normal nervous system. Once the cycle is blocked, glu will cumulate in synaptic cleft that would excite neuron and then lead to convulsion. Astrocytes can become reactive gliosis when nervous system have been damaged. Histologic characteristics of reactive gliosis is greaten and hyperplasia of astrocyte, biochemical marker is increase in content of glial fibrillary acidic protein(GFAP)of astrocyte. The reactive gliosis have effect on synaptic transmission that excits neuron by many kinds of pathway, and is epileptogenous.
SSa is a effective monomer in saikosaponin which is a main pharmaco -ingredient of traditional Chinese drug-- Bupleurum chinense. In the past research, we have found that saikosaponins and saikosaponin a can inhibit experimental epilepsy; saikosaponins can inhibit expression of glial fibrillary acidic protein and modulate expression of Glu in hippocampus. So. We suppose that saikosaponin a may inhibit experimental epilepsy by block the effect of Glu on nervous system and reactive gliosis.
Up to now,the mechanism why SSa can inhibit experimental epilepsy is not clear.So our experiment invest the effects of SSa on glu actived-astrocyte in vitro to discuss the mechanism that the role of SSa in epilepsy. This expriment is supported by the State Administration of Traditional Chinese Medicine Of GuangDong Province (NO.1060133) .
Method and content
1 Cultivation and identification of rat' hippocampal astrocyte in vitro
The primary culture method of astrocytes was improved on the basis of McCarthy's way. Hippocamp part was obtained from neonatal Spague-Dawley rats(l-3day),which was snipped, digested and centrifuged. After adherencing 40min, the cells were implanted into culture flask by the density of 10~6 /cm~2. Cells were cultured at 37℃in a humidified 5% (v/v) CO_2 atmosphere for 7-9 days. When the cells coalesced in the percentage of 70-80,put the cultures on the rocking bed which kept shaking 18 h (37.0℃, 240 r/min) .And then transferred of culture,so we could obtain pure cells which were identified by way of immunohistochemistry.
2 The effect of glu on activity of rats' hippocampal astrocyte in vitro culture and the role of SSa.
The quantity of alive cells were detected by technology of MTT assay. Cells were transferred of culture into 96-well by the density of 8×10~4 /cm~2 200μl per well. After cells were cultivated in the complete medium 48h,the different groups treated with drugs which were disposed by complete medium containing 5% FBS:a control,b L-Glu(1.5 mmol/L),c L-Glu + SSa(1.5 mmol/L,10 mg/L), d L-Glu + SSa(1.5mmol/L,5mg/L), e L-Glu+SSa( 1.5 mmol/L ,2.5 mg/L), f L-Glu+SSa(1.5 mmol/L,1.25 mg/L). Six wells per group,200μl per well. Being incubated for 72 hours, the cells were detected the optical density which reacts the quantity of living cells by technology of MTT.
3 The effect of glu on cell division of rats' hippocampal astrocyte in vitro culture and the role of SSa.
The cell division of rats' hippocampal astrocyte was detected by flow cytometer. Cells were transferred of culture by the density of 10~5/cm~2 .After cells were cultured in the complete medium 48h, the different groups treated with drugs were disposed by DMEM/F_(12) medium: a(control);b L-Glu(1 mmol/L);c L-Glu+SSa(1 mmol/L,5 mg/L);d L-Glu+SSa(1 mmol/L.2.5 mg/L);e L-Glu+SSa(1 mmol/L,1.25 mg/L). 24 hours later, the cells were stained by PI, and transferred the cells density to 10~6/cm~2 .Detected the cells division by flow cytometer 10~4 cells per group, and we evaluated the condition of proliferation by proliferation index.
4 The effect of glu on express of GFAP in rats' hippocampal astrocyte in vitro culture and the role of SSa.
The express of GFAP was detected by way of western bolt. Cells were transferred of culture by the density of 10~5/cm~2 .After cells were cultured in the complete medium 48h, the different groups treated with drugs were disposed by DMEM/F12 medium: a(control);b L-Glu(1.5 mmol/L);c L-Glu+SSa(1.5 mmol/L,5 mg/L);d L-Glu+SSa(1.5 mmol/L,2.5 mg/L);e L-Glu+SSa(1.5 mmol/L,1.25 mg/L). 24h later, proteins in the cells were extracted from the cells. The express of GFAP was detected by way of western bolt. The result of western-blot was analyzed, demonstrated in value of gray scale, and calculated the ratio of the value of gray scale of GFAP with the value of gray scale, ofβ-actin.
Result
1 Cultivation and identification of rats' hippocampal astrocyte in vitro
We observe the rats" hippocampal astrocyte in vitro primary culture through the microscope. From the glass we could see that cells were growing and adherencing well. The cells were identified by way of imrnunocytochemistry, showed over 98% positive staining.
2 The effect of glu on activity of rats' hippocampal astrocyte in vitro culture and the role of SSa.
Rats' hippocampal astrocyte in vitro culture were dealt with different factors. Compared with group a,the optical density of group b.c,d,f were obviously higher than that of group a(P<0.05 );Compared with group b, optical density of group a,c,d,e were obviously lower (P<0.05) ,and group f was obviously higher than group b (P<0.05) .The results showed that glu can obviously active astrocyte in vitro culture,and SSa can inhibit the cell proliferation.
3 The effect of glu on cell division of rats' hippocampal astrocyte in vitro culture and the role of SSa.
The results in different groups of flow cytometer: the proliferation index of each groups were obviously different (P<0.001) .The proliferation index of other groups were obviously higher than that of group a;compared with group b,the other groups' index were obviously lower. It showed that glu can promote the cell division of rats' hippocampal astrocyte invitro culture,different doses of SSa can block the cell division of glu-actived cells in some way.
4 The effect of glu on express of GFAP in rats' hippocampal astrocyte in vitro culture and the role of SSa.
The results of western blot between different groups: compared with a,the quatity of GFAP expressed in cells were obviously different in b,c,e groups (P<0.05) ;the quatity of group a,c,d was obviously lower than group b (P<0.05) ;the quatity of group e was obviously higher than group b(P<0.05) .It showed that glu can obviously enhance the express of GFAP in cells and make them active, SSa can inhibit the excess express.
Conclusions
1 The activity of rats' hippocampal astrocyte in vitro culture was encouraged by glu,and this proliferation of glu-actived cell was inhabited by SSa. It also could maintain the cell quantity in normal.
2 The cell division of rats' hippocampal astrocyte in vitro culture cycle was promoted by glu,and the cell division of glu-actived cell was blocked by SSa.It also could maintain the cell division in normal.
3 Glu could enhance the express of GFAP in rats' hippocampal astrocyte invirto culture,and the excess express of GFAP could be controlled by SSa. It also could maintain the quantity of GFAP in normal.
癫痫是一种严重危害人类健康的神经系统疾病,在我国癫痫的患病率为4.6‰。由于社会传统的偏见,癫痫病人及家属常隐瞒或否认自己的病情,并承受很大的心理压力。由癫痫引起的医疗、家庭和社会问题极为显著。
目前常用一线抗癫痫药(AEDs)可使大部分癫痫患者发作得以控制,但长期应用安全性较差,对于一些难治性癫痫行外科手术治疗,也受到适应症的限制,风险大,费用昂贵,还存在复发的可能。因此难治性癫痫仍然以药物治疗为主要手段,这就需要使用新的抗癫痫药,但其研究前提是对于癫痫发病机制的深入认识。
近年来癫痫的发病机制研究有了长足进步。神经元兴奋性增高和高度同步化发放是癫痫发病的两个重要特征已得到公认,国内外很多学者试图从多个方面来探索引起神经元兴奋性增加和高度同步化发放的形成机制,提出了不少有益的见解。其中对星形胶质细胞与神经信号传递的认识逐步深入,位于突触联合处的星形胶质细胞被认为是突触三联体的重要组成部分。谷氨酸(Glu)作为中枢神经系统的主要的兴奋性神经递质,是重要的致痫原因,既可导致癫痫发作,又可能在癫痫的继发性脑损害中起重要作用。正常情况下,神经元将Glu释放到突触间隙,然后由星形胶质细胞摄取后再转化为谷氨酰胺,这种神经元与星形胶质细胞间的谷氨酸-谷氨酰胺循环在正常神经系统功能中起着重要作用,这种循环一旦遭破坏即可导致Glu在突触间隙大量堆积,使神经元处于高度兴奋的状态,从而致使惊厥形成。神经系统损伤时,往往伴随星形胶质细胞的活化,又称反应性胶质增生(Reactive gliosis),首要特征是其重要的骨架蛋白胶质纤维酸性蛋白(GFAP)表达增加,激活的星形胶质细胞对神经突触传递施加影响,可以通过多种途径来作用于神经元,增加神经元兴奋性,进而引起癫痫的发作。
柴胡皂苷a(SSa)为我国传统中药柴胡主要药理成分柴胡皂苷的一种单体成分,柴胡皂甙具有公认的抗炎、免疫调节作用,并且在我们前期的研究基础上发现柴胡总皂苷和SSa可以抗实验性癫痫、抑制慢性点燃大鼠GFAP的表达,调节海马Glu的表达,故我们推测SSa可以通过抑制Glu对神经系统作用和抑制其激活的星形胶质细胞来发挥抗实验性癫痫的作用。
而目前体外SSa抗癫痫机制研究尚不明确,本实验由广东省中管局课题基金(NO.1060133)资助研究了SSa对Glu激活体外培养大鼠海马星形胶质细胞的干预作用,探讨SSa可能的抗癫痫机制。
二方法与内容
1大鼠海马星形胶质细胞的培养与鉴定
星形胶质细胞的原代培养参考McCarthy法。取新生SD大鼠海马,剪碎,消化,离心,差速贴壁40 min后,按10~6个/cm~2接种。37.0℃,5%CO_2孵箱中培养7-9 d,待培养的细胞70%-80%融合时,将培养瓶置于恒温旋转摇床上摇18 h(37.0℃,240 r/min)后,进行传代培养即得纯化的星形胶质细胞,纯化的细胞进一步运用免疫细胞化学SABC法进行鉴定。
2 SSa对Glu激活体外培养的大鼠海马星形胶质细胞活性的影响
运用MTT比色法检测各组星形胶质细胞活细胞数量。将传代细胞按8×10~4个/cm~2的密度接种于96孔培养板中,每孔200μl。完全培养基培养48 h后,用含5%胎牛血清的培养基配制药物干预组共6组:a组为正常组(control组),b组为L-Glu(1.5 mmol/L)的激活组,c组为L-Glu+SSa剂量组(分别为1.5mmol/L,10 mg/L),d组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,5 mg/L),e组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,2.5 mg/L),f组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,1.25 mg/L)。每组6孔,每孔加入200μl,继续培养72 h后运用MTT法测OD值,以OD值反映各组星形胶质细胞活细胞数量。
3 SSa对Glu激活体外培养大鼠海马星形胶质细胞分裂周期的影响
运用流式细胞技术检测各组星形胶质细胞的细胞周期。将传代细胞以5×10~5个/cm~2密度种植于直径10 mm的培养皿中。完全培养基培养48 h后,用DMEM/F_(12)培养基配制药物干预组共5组:a组为正常组(control组),b组为L-Glu(1 mmol/L)的激活组,c组为L-Glu+SSa剂量组(分别为1 mmol/L,5mg/L),d组为L-Glu+SSa剂量组(分别为1 mmol/L,2.5 mg/L),e组为含L-Glu+SSa剂量组(分别为1 mmol/L,1.25 mg/L)。在加药24 h后取材,PI染色并调整细胞密度为10~6个/cm~2,而后用流式细胞技术检测各组星形胶质细胞细胞周期,每组样本检测10~4个细胞,以细胞指数表示分布于细胞周期各时相的细胞百分比数,以增殖指数反映细胞增殖状态。
4 SSa对Glu激活体外培养大鼠海马星形胶质细胞GFAP表达的影响
运用Western-blot法检测各组星形胶质细胞GFAP表达量。将传代细胞以5×10~5个/ml的密度种植于培养皿中,完全培养基培养48 h后,用DMEM/F_(12)培养基配制药物干预组共5组:a组为正常组(control组),b组为L-Glu(1.5 mmol/L)的激活组,c组为L-Glu+SSa剂量组(分别为1.5 mmol/L,5 mg/L),d组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,2.5 mg/L),e组为含L-Glu+SSa剂量组(分别为1.5 mmol/L,1.25 mg/L)。作用24 h后,提取蛋白,运用western-blot技术检测GFAP表达的变化,对结果进行灰度分析,计算GFAP与β-actin的比值。每组蛋白检测6次。
三统计方法
应用SPSS 11.5统计软件进行处理。各组MTT比色法结果和western-blot检测结果比较采用单因素方差分析(One-way ANOVA),进一步组间比较用LSD法;流式细胞技术检测结果比较采用R×C表资料的卡方检验,P＜0.05表示差异具有统计学意义。
四结果
1星形胶质细胞的培养和鉴定
原代培养的大鼠海马星形胶质细胞,镜下观察可见星形胶质细胞生长较好,所有细胞均贴壁。原代培养的星形胶质细胞运用免疫组化染色法鉴定,细胞的GFAP免疫反应阳性率为98%以上。
2 Glu对体外培养大鼠海马星形胶质细胞活性的影响及SSa的干预作用
体外培养大鼠海马星形胶质细胞在不同处理因素处理下各组OD值,与a组比较,b组、c组、d组、f组OD值均高于a组,差异有显著性意义(P＜0.05);与b组比较,a组、c组、d组、e组OD值均低于b组,而f组则高于b组,差异有显著性意义(P＜0.05)。结果提示:Glu可以显著激活体外培养星形胶质细胞,促进细胞增殖,而SSa对Glu激活的细胞增殖具有显著抑制作用。
3 Glu对体外培养大鼠海马星形胶质细胞分裂周期的影响及SSa的干预作用
各组流式细胞技术检测的细胞周期结果:各组细胞增殖指数差异有显著性意义(P＜0.001)。各组细胞增殖指数均高于a组,各组细胞增殖指数均低于b组。结果提示:Glu(1 mmol/L)可以显著激活大鼠海马星形胶质细胞,加速星形胶质细胞的分裂周期。SSa对激活细胞的分裂周期均存在一定的阻滞作用。
4 Glu对体外培养大鼠海马星形胶质细胞GFAP表达的影响及SSa的干预作用
各组western-blot检测结果:GFAP表达量b组、c组、e组与a组比较差异均有显著性意义(P＜0.05);a组、c组、d组GFAP表达量低于b组,差异有显著性意义(P＜0.05),而e组高于b组,差异有显著性意义(P＜0.05)。提示:Glu可以显著激活大鼠海马星形胶质细胞,促使星形胶质细胞GFAP过度表达,而SSa对其激活存在抑制作用。
五结论
1 Glu可促进体外培养大鼠海马星形胶质细胞的活性,SSa可抑制激活星形胶质细胞的活性。
2 Glu可促进体外培养大鼠海马星形胶质细胞的分裂周期,SSa可阻滞激活星形胶质细胞的分裂周期。
3 Glu可促进体外培养大鼠海马星形胶质细胞GFAP的表达,SSa可抑制过度表达的GFAP。
Objective and significance
Epilepsy is a kind of neurological disease that is harmful to the health of human being. There is about 4.6‰of the population in our country affected by this neurological disease. Epileptic and their family have possibly to hide or to deny the heath condition because of orthodox prejudice of society,and epileptic beared mental stress from the world. It cant't be ignored that the problems of medical treament,family and social problem induced by epilepsy.
In now, epileptic treatment still dependent on chemicals. General anti-epileptic drug(AEDs) can control epileptic attack,but security is a problem in treatment of long-term, and the method to treat refractoriness epilepsy by surgery may be confined by indication, high risk, expensive, and possible to recur. So epileptic treatment still dependent on chemicals, but it must know clearly that is pathogenesy of epilepsy before develop new chemicals.
For the past few years, research of epileptic pathogenesy has been advanced. Increase in neuronal excitability and synchronize firing are two important characteristic of epileptic, which had be generally accepted. Many scholars try them best to study mechanism of form, and introduced many view. Recognition of relation of astrocyte with synaptic signal transmission have been gradually clear. Synaptically associated astrocytes should be viewed as integral modulatory elements of tripartite synapses. Glu, one of main excitatory neurotransmitters in central nervous system, is a etiological factor of epilepsy. It can not cause epilepsy but also may play a important part in the brain damage caused by epilepsy. In normal, glu which is released by neuron into synaptic cleft is intaked by astrocyte and then turned into glutamine. This cycle of glutamate- glutamine between neuron and astrocyte plays a important role in normal nervous system. Once the cycle is blocked, glu will cumulate in synaptic cleft that would excite neuron and then lead to convulsion. Astrocytes can become reactive gliosis when nervous system have been damaged. Histologic characteristics of reactive gliosis is greaten and hyperplasia of astrocyte, biochemical marker is increase in content of glial fibrillary acidic protein(GFAP)of astrocyte. The reactive gliosis have effect on synaptic transmission that excits neuron by many kinds of pathway, and is epileptogenous.
SSa is a effective monomer in saikosaponin which is a main pharmaco -ingredient of traditional Chinese drug-- Bupleurum chinense. In the past research, we have found that saikosaponins and saikosaponin a can inhibit experimental epilepsy; saikosaponins can inhibit expression of glial fibrillary acidic protein and modulate expression of Glu in hippocampus. So. We suppose that saikosaponin a may inhibit experimental epilepsy by block the effect of Glu on nervous system and reactive gliosis.
Up to now,the mechanism why SSa can inhibit experimental epilepsy is not clear.So our experiment invest the effects of SSa on glu actived-astrocyte in vitro to discuss the mechanism that the role of SSa in epilepsy. This expriment is supported by the State Administration of Traditional Chinese Medicine Of GuangDong Province (NO.1060133) .
Method and content
1 Cultivation and identification of rat' hippocampal astrocyte in vitro
The primary culture method of astrocytes was improved on the basis of McCarthy's way. Hippocamp part was obtained from neonatal Spague-Dawley rats(l-3day),which was snipped, digested and centrifuged. After adherencing 40min, the cells were implanted into culture flask by the density of 10~6 /cm~2. Cells were cultured at 37℃in a humidified 5% (v/v) CO_2 atmosphere for 7-9 days. When the cells coalesced in the percentage of 70-80,put the cultures on the rocking bed which kept shaking 18 h (37.0℃, 240 r/min) .And then transferred of culture,so we could obtain pure cells which were identified by way of immunohistochemistry.
2 The effect of glu on activity of rats' hippocampal astrocyte in vitro culture and the role of SSa.
The quantity of alive cells were detected by technology of MTT assay. Cells were transferred of culture into 96-well by the density of 8×10~4 /cm~2 200μl per well. After cells were cultivated in the complete medium 48h,the different groups treated with drugs which were disposed by complete medium containing 5% FBS:a control,b L-Glu(1.5 mmol/L),c L-Glu + SSa(1.5 mmol/L,10 mg/L), d L-Glu + SSa(1.5mmol/L,5mg/L), e L-Glu+SSa( 1.5 mmol/L ,2.5 mg/L), f L-Glu+SSa(1.5 mmol/L,1.25 mg/L). Six wells per group,200μl per well. Being incubated for 72 hours, the cells were detected the optical density which reacts the quantity of living cells by technology of MTT.
3 The effect of glu on cell division of rats' hippocampal astrocyte in vitro culture and the role of SSa.
The cell division of rats' hippocampal astrocyte was detected by flow cytometer. Cells were transferred of culture by the density of 10~5/cm~2 .After cells were cultured in the complete medium 48h, the different groups treated with drugs were disposed by DMEM/F_(12) medium: a(control);b L-Glu(1 mmol/L);c L-Glu+SSa(1 mmol/L,5 mg/L);d L-Glu+SSa(1 mmol/L.2.5 mg/L);e L-Glu+SSa(1 mmol/L,1.25 mg/L). 24 hours later, the cells were stained by PI, and transferred the cells density to 10~6/cm~2 .Detected the cells division by flow cytometer 10~4 cells per group, and we evaluated the condition of proliferation by proliferation index.
4 The effect of glu on express of GFAP in rats' hippocampal astrocyte in vitro culture and the role of SSa.
The express of GFAP was detected by way of western bolt. Cells were transferred of culture by the density of 10~5/cm~2 .After cells were cultured in the complete medium 48h, the different groups treated with drugs were disposed by DMEM/F12 medium: a(control);b L-Glu(1.5 mmol/L);c L-Glu+SSa(1.5 mmol/L,5 mg/L);d L-Glu+SSa(1.5 mmol/L,2.5 mg/L);e L-Glu+SSa(1.5 mmol/L,1.25 mg/L). 24h later, proteins in the cells were extracted from the cells. The express of GFAP was detected by way of western bolt. The result of western-blot was analyzed, demonstrated in value of gray scale, and calculated the ratio of the value of gray scale of GFAP with the value of gray scale, ofβ-actin.
Result
1 Cultivation and identification of rats' hippocampal astrocyte in vitro
We observe the rats" hippocampal astrocyte in vitro primary culture through the microscope. From the glass we could see that cells were growing and adherencing well. The cells were identified by way of imrnunocytochemistry, showed over 98% positive staining.
2 The effect of glu on activity of rats' hippocampal astrocyte in vitro culture and the role of SSa.
Rats' hippocampal astrocyte in vitro culture were dealt with different factors. Compared with group a,the optical density of group b.c,d,f were obviously higher than that of group a(P<0.05 );Compared with group b, optical density of group a,c,d,e were obviously lower (P<0.05) ,and group f was obviously higher than group b (P<0.05) .The results showed that glu can obviously active astrocyte in vitro culture,and SSa can inhibit the cell proliferation.
3 The effect of glu on cell division of rats' hippocampal astrocyte in vitro culture and the role of SSa.
The results in different groups of flow cytometer: the proliferation index of each groups were obviously different (P<0.001) .The proliferation index of other groups were obviously higher than that of group a;compared with group b,the other groups' index were obviously lower. It showed that glu can promote the cell division of rats' hippocampal astrocyte invitro culture,different doses of SSa can block the cell division of glu-actived cells in some way.
4 The effect of glu on express of GFAP in rats' hippocampal astrocyte in vitro culture and the role of SSa.
The results of western blot between different groups: compared with a,the quatity of GFAP expressed in cells were obviously different in b,c,e groups (P<0.05) ;the quatity of group a,c,d was obviously lower than group b (P<0.05) ;the quatity of group e was obviously higher than group b(P<0.05) .It showed that glu can obviously enhance the express of GFAP in cells and make them active, SSa can inhibit the excess express.
Conclusions
1 The activity of rats' hippocampal astrocyte in vitro culture was encouraged by glu,and this proliferation of glu-actived cell was inhabited by SSa. It also could maintain the cell quantity in normal.
2 The cell division of rats' hippocampal astrocyte in vitro culture cycle was promoted by glu,and the cell division of glu-actived cell was blocked by SSa.It also could maintain the cell division in normal.
3 Glu could enhance the express of GFAP in rats' hippocampal astrocyte invirto culture,and the excess express of GFAP could be controlled by SSa. It also could maintain the quantity of GFAP in normal.
引文
[1]周永,刘民,梁万年.癫痫流行病学研究进展[J].中华流行病学杂志.2007,28(1):92-94.
[2]Salpekar JA,Dunn DW.Psychiatric and psychosocial consequences of pediatric epilepsy[J].Semin Pediatr Neurol.2007,14(4):181-8.
[3]Kimiskidis VK,Triantafyllou NI,Kararizou E,et al.Depression and anxiety in epilepsy:the association with demographic and seizure-related variables[J].Ann Gen Psychiatry.2007,30(6):28.
[4]Laporte A,Rouvel-Tallec A,Grosdidier E,et al.Epilepsy among the homeless:prevalence and characteristics[J].Eur J Public Health.2006,16(5):484-486.
[5]Perucca E.The treatment of the first seizure:the risks[J].Epilepsia.2008,49(1):29-34.
[6]Wilson SJ,Bladin PF,Saling MM.The burden of normality:a framework for rehabilitation after epilepsy surgery[J].Epilepsia.2007,48(9):13-6.
[7]Sasa M.A new frontier in epilepsy:novel antiepileptogenic drugs[J].J Pharmacol Sci,2006,100:487-494.
[8]Sierra-Paredes G,Sierra-Marcuno G.Extrasynaptic GABA and glutamate receptors in epilepsy[J].CNS Neurol Disord Drug Targets.2007,6(4):288-300.
[9]Volterra,A.and Meldolesi J.Astrocytes,from brain glue to communication elements:the revolution continues[J].Nat.Rev.Neurosci.2005,(6):626-640.
[10]Bjφrnsen LP,Eid T,Holmseth S,et al.Changes in glial glutamate transporters in human epileptogenic hippocampus:inadequate explanation for high extracellular glutamate during seizures[J].Neurobiol Dis.2007,25(2):319-330.
[11]Angeka CE,Robert KS,Shih-Chieh C,et al.Role of synaptic metabotropic glutamate receptors in epileptiorm discharges in hippocampal slices[J].J Neurophysiol.2002,88(4):1625-1633.
[12]付欣鸽.谷氨酸转运体与癫痫[J].国外医学·生理、病理与临床分册.2003,23(2):162-164.
[13]朱长庚.胶质细胞与神经元间的信号交流及其与癫痫发病机制的关系[J].中国组织化学与细胞化学杂志.2003.12(3):323-326.
[14]CamachoA,MassieuL.Role of glutamate transporters in the clearance and release of glutamate during ischemia and its relation to neuronal death[J].ArchMed Res.2006,37(1):11-18.
[15]王静敏,姜玉武,姜茜,等.发育中星形胶质细胞兴奋在惊厥中的作用[J].生理科学进展.2007,38(1):75-77.
[16]Eduardo E,Benarroch.Neuron-astrocyte interactions:partnership for normalfunction and disease in the central nervous system[J].Mayo Clin Proc.2005,80(10):1326-1338.
[17]Pascual Juan M.Fever,molecular excitability and epilepsy[J].Neurosci Lett.2004,368(1):121.
[18]Araque A,Parpura V,Sanzgiri RP,et al.Tripartite synapses:glia,the unacknowledged partner[J].Trends Neurosci.1999,22(5):208-215.
[19]Araque A,Martin ED,Perea G,et al.Synaptically Released Acetylcholine Evokes Ca~(2+) Elevations in Astrocytes in Hippocampal Slices[J].J Neurosci,2002,22(7):2443-2450.
[20]Perea G,Araque A.Glial calcium signaling and neuron-glia communication [J].Cell Calcium.2005,38(3-4):375-382.
[21]Araque A,Perea G.Glial modulation of synaptic transmission in culture[J].Glia,2004,47(3):241-248.
[22]Binder DK,Steinh(a|¨)user C.Functional changes in astroglial cells in epilepsy[J].-Glia.2006,54(5):358-68.
[23]Yoshida Y,Tsuchiya R,Matsumoto N,et al.Ca~(2+)-Dependent induction of intracellular Ca~(2+) oscillation in hippocampal astrocytes during metabotropic glutamate receptor activation.[J]J Pharmacol Sci 2005;97(2):212-218.
[24]Tian GF,Azmi H,Takano T,et al.An astrocytic basis of epilepsy.NatMed 2005,11(9):973-981.
[25]Vessal M,Dugani CB,Solomon DA,et al.Might astrocytes play a role in maintaining the seizure-prone state?[J]Brain Res.2005,1044(2):190-196.
[26]Matute C,DomercqM,Sanchez-gomezMV.Glutamate mediated glia injury:mechanisms and clinical importance[J].Glia 2006,53(2):212-224.
[27]Ding S,Fellin T,Zhu Y,Lee SY,et al.Enhanced astrocytic Ca~(2+) signals contribute to neuronal excitotoxicity after status epilepticus[J].J Neurosci.2007,27(40):10674-10684.
[28]Van Damme P,Bogaert E,Dewil M,et al.Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity[J].Proc Natl Acad Sci U S A.2007,104(37):14825-30.
[29]Somera-Molina KC,Robin B,Somera CA,et al.Glial activation links early-life seizures and long-term neurologic dysfunction:evidence using a small molecule inhibitor of proinflammatory cytokine upregulation[J].Epilepsia.2007,48(9):1785-1800.
[30]谢炜,鲍勇,于礼建.柴胡总皂甙及柴胡皂甙a、c、d对MES惊厥小鼠的影响[J].中药药理与临床[J].2006,22(1):39-40.
[31]McCarthy KD,De Vellis J.Preparation of separate astroglial and oligodendroglial astroglial cell cultures from rat cerebral tissue[J].J Cell Biol.1980.85(3):890-902.
[32]黄仁栋,张横柳,李巨奇.仲景学说与癫痫证治的探讨[J].湖北中医杂.2003, 23(4):5-6.
[33]黄运生,陈宝田,谢炜.柴胡疏肝汤添加治疗原发性癫痫108例疗效观察[J].中药材.2002.25(5):374-376.
[34]谢炜,鲍勇,于礼建,等.柴胡总皂甙对戊四氮慢性点燃大鼠海马GFAP表达的影响[J].南方医科大学学报.2006,26(4):452-455.
[35]谢炜,鲍勇,于礼建,等.柴胡总皂甙对戊四氮慢性点燃大鼠海马谷氨酸细胞的影响[J].中华神经医学杂志.2006,5(8):761-763,774.
[36]李长征,谢炜,鲍勇,等.柴胡皂苷a对实验性癫痫大鼠模型的干预作用[J].南方医科大学学报.2007,27(6):839-840.
[37]Tsai YJ,Chen IL,Homg LY,et al.Induction of differention in rat C6 glioma cells with Saikosaponins[J].Phytother Res.2002,16(2):117-121.
[38]Bernardinelli Y,Magistretti PJ,Chatton JY.Astrocytes generate Na~+-mediated metabolic waves[J].Proc Natl Acad Sci.2004,101(41):14937-14942.
[39]Mothet JP,Pollegioni L,Ouanounou G,et al.Glutamate receptor activation triggers a calciumdependent and SNARE protein-dependentrelease of the gliotransmitter D-serine[J].Proc Natl Acad Sci.2005,02(15):5606-5611.
[40]Colombo JA.Glutamate uptake by rat brain astroglia incubated in human cerebrospinal fluid[J].Med Sci Monit.2005,11(1):BR13-17.
[41]Bowser DN,Khakh BS.ATP Excites Interneurons and Astrocytes to Increase Synaptic Inhibition in Neuronal Networks[J].J Neuro.2004,24(39):8606-8620.6]
[42]Beart PM,O'Shea RD.Transporters for L-glutamate:an update on their molecular pharmacology and pathological involvement[J].Br J Pharmacol.2007,150(1):5-17.
[43]Hausmann R,Riess R,Fieguth A,et al.Immunohistochemical investigations on the course of astroglia GFAP expression following human brain injury[J].Int J Legal Med.2000,113(2):70-75.
[44]王国卿,杜景卫,夏作理.星形胶质细胞生物学功能和活化[J].泰山医学院学报.2005,26(2):184-186.
[2]Salpekar JA,Dunn DW.Psychiatric and psychosocial consequences of pediatric epilepsy[J].Semin Pediatr Neurol.2007,14(4):181-8.
[3]Kimiskidis VK,Triantafyllou NI,Kararizou E,et al.Depression and anxiety in epilepsy:the association with demographic and seizure-related variables[J].Ann Gen Psychiatry.2007,30(6):28.
[4]Laporte A,Rouvel-Tallec A,Grosdidier E,et al.Epilepsy among the homeless:prevalence and characteristics[J].Eur J Public Health.2006,16(5):484-486.
[5]Perucca E.The treatment of the first seizure:the risks[J].Epilepsia.2008,49(1):29-34.
[6]Wilson SJ,Bladin PF,Saling MM.The burden of normality:a framework for rehabilitation after epilepsy surgery[J].Epilepsia.2007,48(9):13-6.
[7]Sasa M.A new frontier in epilepsy:novel antiepileptogenic drugs[J].J Pharmacol Sci,2006,100:487-494.
[8]Sierra-Paredes G,Sierra-Marcuno G.Extrasynaptic GABA and glutamate receptors in epilepsy[J].CNS Neurol Disord Drug Targets.2007,6(4):288-300.
[9]Volterra,A.and Meldolesi J.Astrocytes,from brain glue to communication elements:the revolution continues[J].Nat.Rev.Neurosci.2005,(6):626-640.
[10]Bjφrnsen LP,Eid T,Holmseth S,et al.Changes in glial glutamate transporters in human epileptogenic hippocampus:inadequate explanation for high extracellular glutamate during seizures[J].Neurobiol Dis.2007,25(2):319-330.
[11]Angeka CE,Robert KS,Shih-Chieh C,et al.Role of synaptic metabotropic glutamate receptors in epileptiorm discharges in hippocampal slices[J].J Neurophysiol.2002,88(4):1625-1633.
[12]付欣鸽.谷氨酸转运体与癫痫[J].国外医学·生理、病理与临床分册.2003,23(2):162-164.
[13]朱长庚.胶质细胞与神经元间的信号交流及其与癫痫发病机制的关系[J].中国组织化学与细胞化学杂志.2003.12(3):323-326.
[14]CamachoA,MassieuL.Role of glutamate transporters in the clearance and release of glutamate during ischemia and its relation to neuronal death[J].ArchMed Res.2006,37(1):11-18.
[15]王静敏,姜玉武,姜茜,等.发育中星形胶质细胞兴奋在惊厥中的作用[J].生理科学进展.2007,38(1):75-77.
[16]Eduardo E,Benarroch.Neuron-astrocyte interactions:partnership for normalfunction and disease in the central nervous system[J].Mayo Clin Proc.2005,80(10):1326-1338.
[17]Pascual Juan M.Fever,molecular excitability and epilepsy[J].Neurosci Lett.2004,368(1):121.
[18]Araque A,Parpura V,Sanzgiri RP,et al.Tripartite synapses:glia,the unacknowledged partner[J].Trends Neurosci.1999,22(5):208-215.
[19]Araque A,Martin ED,Perea G,et al.Synaptically Released Acetylcholine Evokes Ca~(2+) Elevations in Astrocytes in Hippocampal Slices[J].J Neurosci,2002,22(7):2443-2450.
[20]Perea G,Araque A.Glial calcium signaling and neuron-glia communication [J].Cell Calcium.2005,38(3-4):375-382.
[21]Araque A,Perea G.Glial modulation of synaptic transmission in culture[J].Glia,2004,47(3):241-248.
[22]Binder DK,Steinh(a|¨)user C.Functional changes in astroglial cells in epilepsy[J].-Glia.2006,54(5):358-68.
[23]Yoshida Y,Tsuchiya R,Matsumoto N,et al.Ca~(2+)-Dependent induction of intracellular Ca~(2+) oscillation in hippocampal astrocytes during metabotropic glutamate receptor activation.[J]J Pharmacol Sci 2005;97(2):212-218.
[24]Tian GF,Azmi H,Takano T,et al.An astrocytic basis of epilepsy.NatMed 2005,11(9):973-981.
[25]Vessal M,Dugani CB,Solomon DA,et al.Might astrocytes play a role in maintaining the seizure-prone state?[J]Brain Res.2005,1044(2):190-196.
[26]Matute C,DomercqM,Sanchez-gomezMV.Glutamate mediated glia injury:mechanisms and clinical importance[J].Glia 2006,53(2):212-224.
[27]Ding S,Fellin T,Zhu Y,Lee SY,et al.Enhanced astrocytic Ca~(2+) signals contribute to neuronal excitotoxicity after status epilepticus[J].J Neurosci.2007,27(40):10674-10684.
[28]Van Damme P,Bogaert E,Dewil M,et al.Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity[J].Proc Natl Acad Sci U S A.2007,104(37):14825-30.
[29]Somera-Molina KC,Robin B,Somera CA,et al.Glial activation links early-life seizures and long-term neurologic dysfunction:evidence using a small molecule inhibitor of proinflammatory cytokine upregulation[J].Epilepsia.2007,48(9):1785-1800.
[30]谢炜,鲍勇,于礼建.柴胡总皂甙及柴胡皂甙a、c、d对MES惊厥小鼠的影响[J].中药药理与临床[J].2006,22(1):39-40.
[31]McCarthy KD,De Vellis J.Preparation of separate astroglial and oligodendroglial astroglial cell cultures from rat cerebral tissue[J].J Cell Biol.1980.85(3):890-902.
[32]黄仁栋,张横柳,李巨奇.仲景学说与癫痫证治的探讨[J].湖北中医杂.2003, 23(4):5-6.
[33]黄运生,陈宝田,谢炜.柴胡疏肝汤添加治疗原发性癫痫108例疗效观察[J].中药材.2002.25(5):374-376.
[34]谢炜,鲍勇,于礼建,等.柴胡总皂甙对戊四氮慢性点燃大鼠海马GFAP表达的影响[J].南方医科大学学报.2006,26(4):452-455.
[35]谢炜,鲍勇,于礼建,等.柴胡总皂甙对戊四氮慢性点燃大鼠海马谷氨酸细胞的影响[J].中华神经医学杂志.2006,5(8):761-763,774.
[36]李长征,谢炜,鲍勇,等.柴胡皂苷a对实验性癫痫大鼠模型的干预作用[J].南方医科大学学报.2007,27(6):839-840.
[37]Tsai YJ,Chen IL,Homg LY,et al.Induction of differention in rat C6 glioma cells with Saikosaponins[J].Phytother Res.2002,16(2):117-121.
[38]Bernardinelli Y,Magistretti PJ,Chatton JY.Astrocytes generate Na~+-mediated metabolic waves[J].Proc Natl Acad Sci.2004,101(41):14937-14942.
[39]Mothet JP,Pollegioni L,Ouanounou G,et al.Glutamate receptor activation triggers a calciumdependent and SNARE protein-dependentrelease of the gliotransmitter D-serine[J].Proc Natl Acad Sci.2005,02(15):5606-5611.
[40]Colombo JA.Glutamate uptake by rat brain astroglia incubated in human cerebrospinal fluid[J].Med Sci Monit.2005,11(1):BR13-17.
[41]Bowser DN,Khakh BS.ATP Excites Interneurons and Astrocytes to Increase Synaptic Inhibition in Neuronal Networks[J].J Neuro.2004,24(39):8606-8620.6]
[42]Beart PM,O'Shea RD.Transporters for L-glutamate:an update on their molecular pharmacology and pathological involvement[J].Br J Pharmacol.2007,150(1):5-17.
[43]Hausmann R,Riess R,Fieguth A,et al.Immunohistochemical investigations on the course of astroglia GFAP expression following human brain injury[J].Int J Legal Med.2000,113(2):70-75.
[44]王国卿,杜景卫,夏作理.星形胶质细胞生物学功能和活化[J].泰山医学院学报.2005,26(2):184-186.