Effects of coriaria lactone-activated,astrocyte-conditioned medium on estrogen receptor and progesterone receptor expression in rat cortical and hippocampal neurons

BACKGROUND： Coriaria lactone-activated astrocytes released bioactive substances that eventually caused epilepsy. OBJECTIVE： It has been suggested that activated astrocytes alter the expression of the estrogen receptor and progesterone receptor by releasing bioactive substances during epilepsy, thereby affecting neuronal activity in the brain. This study was designed to observe the expression of the estrogen receptor and the progesterone receptor in rat brain following lateral ventricle injection of coriaria lactone-activated, astrocyte-conditioned medium. DESIGN AND SETTING： This immunohistochemical, randomized, controlled, animal study was conducted at the Department of Pathology, Hospital Affiliated to Binzhou Medical College, China. MATERIAL： Coriaria lactone was provided by Huaxi Pharmaceutical Factory, China. METHODS： Forty adult, healthy, male, Sprague Dawley rats were randomly assigned into two groups. Astrocyte-conditioned medium （10 μ L） was injected into rat lateral ventricle in the control group （n = 8）. Coriaria lactone-activated, astrocyte-conditioned medium （10 μL） was infused into the rat lateral ventricle in the coriaria lactone group （n = 32）. At 2, 4, 8 and 12 hours following injection, rats were sacrificed and subjected to immunohistochemistry. Eight rats were studied at each time point. MAIN OUTCOME MEASURES： Behavioral changes were observed in rats of both groups. Expression of the estrogen receptor and the progesterone receptor in rat cortical and hippocampal neurons was measured using immunohistochemistry. RESULTS： Four hours after injection, estrogen receptor levels in rat cortical and hippocampal neurons were significantly higher in the coriaria lactone group than in the control group （P 〈 0.05）. Progesterone receptor levels were significantly lower in the coriaria lactone group than in the control group （P 〈 0.05）. Seizures were not observed in the control group. In the coriaria lactone group, convulsions appeared 30 minutes after injection; seizures reached grade Ⅲ at 45 minutes rat behavior was nearly normal at 2 hours. CONCLUSION： Activated astrocytes can induce seizures in the rat by enhancing estrogen receptor expression and decreasing progesterone receptor expression in cerebral cortical and hippocampal neurons.

Effect of Coriaria Lactone on Membrane Potential of Hippocampal Neurons in Rats

Intracellular recording method of electrophysiology was used to study the effect of microinjection of coriaria lactone (CL) into hippocampus on membrane potential of hippocampal neurons in rats. The membrane potential in 34 neurons of 12 rats were recorded. The results showed that CL induced depolarization of all of 34 neurons examined. Among the 34 neurons, 15 had paroxysmal depolarization accompanied by spike discharge in cluster and spike discharge in cluster ceased when membrane was hyperpolarized, 7 showed continuous depolarization accompanied by continuous spike discharge and 12 had only depolarizing response. The results suggest that injection of CL into hippocampus may lead to an increase of excitability of local neurons accompanied by different patterns of epileptioform discharge.

The Effect of Coriaria Lactone on NMDA Receptor Mediated Currents in Rat Hippocampal CA1 Neurons

Summary: To investigate the exact mechanism of epileptogenesis induced by coriaria lactone (CL), the effect of CL on NMDA receptor mediated current (IAsp) in rat hippocampal CA1 neu- rons was investigated by using nystatin perforated whole-cell patch clamp. 10-6-10-4 mol/L Asp acted on NMDA receptors and elicited an inward current (IAsp) at a holding potential (VH) of -40 mV in presence of 10-6 mol/L glycine and absence of Mg2+ extracellularly. CL enhanced NMDA receptor mediated current induced by Asp, but had no effect on threshold concentration, EC50, Hill coefficient as well as maximal-effect concentration and reversal potential of IAsp. The effect had no relationship with holding potential. These results showed that CL could enhance NMDA receptor mediated current to increase [Ca2+]i of neurons by acting on Gly site, thereby inducing epilepsy.

Synaptic and blood-brain barrier structural changes in a rat epilepsy model induced by coriaria lacton Replication experiment with animals

BACKGROUND： Structural and functional synaptic changes, as well as blood-brain barrier （BBB） changes, affect the micro-environment of nervous tissue and excitation, both of which play an important role in epilepsy. OBJECTIVE： To observe synaptic and BBB ultrastructural changes in the motor cortex of a rat epilepsy model induced by coriaria lacton, and to investigate the synaptic and BBB effects on the mechanism of epilepsy. DESIGN： A randomized controlled animal experiment. SETTING： Department of Histology and Embryology, Luzhou Medical College; and Electron Microscopy Laboratory, Luzhou Medical College. MATERIALS： Twenty healthy male Sprague Dawley rats, aged 8 weeks, were chosen for this study. The rats weighed （280 ± 50） g and were supplied by the Experimental Animal Center of Luzhou Medical College. Experimentation was performed in accordance with the ethical guidelines for the use and care of animals. The animals were randomly divided into a control group and an epilepsy group, with 10 rats in each group. METHODS： This study was performed at the Department of Histology and Embryology, and Electron Microscopy Laboratory, Luzhou Medical College between February and December 2006. According to the protocol, the epilepsy group was injected with 10 μ L/100 g coriaria lacton into the lateral ventricles to establish an epileptic model. The control group rats were not administered anything. Eight days after the model was established, all rats were anesthetized with ether. The motor cortex was removed and sectioned into ultrathin sections. Synaptic and BBB ultrastructural changes were observed by electron microscopy. MAIN OUTCOME MEASURES： （1）Structural changes of three different parts of the synapses, synaptic cleft width, postsynaptic density thickness, proportion of perforation synapses, curvature of synaptic interface, and length of active zones. （2）Capillary and BBB changes （endothelium, basement membrane, pericyte, and the astrocyte endfeet）. RESULTS： （1）Curvature of synaptic interface, length of active zones, thickness of postsynaptic density, and percentage of perforation synapses increased significantly. （2）There was significant edema in the endothelium, basement membrane, and the pericyte of the epilepsy group; the electron density of the basement membrane was reduced. CONCLUSION： （1） The coriaria lacton treatment altered synaptic ultrastructure, as well as BBB characteristics, in the epileptic rat model, and also improved synaptic transmission efficiency, as well as BBB permeability; （2）Synaptic and BBB ultrastructural changes might play an important role in the mechanism of epilepsy.

FMOC-Cl柱前衍生高效液相色谱法测定L-高丝氨酸内酯盐酸盐

建立了一种简捷、快速测定L-高丝氨酸内酯盐酸盐的分析方法,L-高丝氨酸内酯盐酸盐经氯甲酸笏甲酯(FMOC-Cl)衍生化后用HPLC定量分析。采用Wonda Sil C18色谱柱,流动相为乙腈-水(体积比为50∶50),流量为1.0m L/min,柱温为25℃,检测波长为210nm进行检测。该方法的线性相关系数为0.9991,标准偏差为0.0016,平均回收率为99.39%。

Effect of Coriaria lactone on cytosolic free calcium of cultured neurons from rat cerebral cortex

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癫痫大鼠海马和大脑皮质糖皮质激素受体变化的观察

为了探讨糖皮质激素受体（ＧＲ）与癫痫发病机制间的关系，本研究应用免疫细胞化学ＰＡＰ法显示马桑内酯致痫大鼠海马和大脑皮质内ＧＲ的变化，并用显微图像分析仪进行检测分析。结果显示：ＧＲ主要表达在海马回的锥体细胞层、齿状回颗粒细胞层及大脑皮质各层，特别是Ⅳ、Ⅴ层。ＧＲ免疫反应主要定位于细胞核内，胞浆内有较弱的免疫反应。图像分析及统计学处理表明：癫痫大鼠海马回、齿状回、大脑皮质含ＧＲ免疫反应阳性细胞数目、平均总面积、平均光密度均明显低于对照鼠，有极显著性差异。

马桑内酯致痫大鼠海马内c－fos原癌基因表达及谷氨酸免疫反应的变化

用免疫细胞化学双重染色法对马桑内酯致痫大鼠齿状回及海马回ＣＡ３区内原癌基因表达、谷氨酸免疫反应的变化及其相互关系进行了研究。一侧侧脑室内注射马桑内酯诱发癫痫后，在双重免疫细胞化学染色的切片上，齿状回及海马回ＣＡ３区内均有３种不同类型的细胞：谷氨酸（Ｇｌｕ）单标细胞、Ｆｏｓ单标细胞和Ｆｏｓ／Ｇｌｕ双标细胞。癫痫发作后１ｈ，注射侧齿状回有大量Ｆｏｓ／Ｇｌｕ双标细胞，而海马回ＣＡ３区仅有散在的双标细胞；癫痫发作后１．５ｈ，海马回ＣＡ３区双标细胞数明显增多。Ｆｏｓ单标细胞数及谷氨酸免疫反应性与双标细胞数是平行的。根据以上结果，本文对马桑内酯致痫的机制进行了讨论。

马桑毒素致毒后试虫酯酶同工酶变化的时序分析

采用 Pearson相似性比较及欧氏聚类法 ,对马桑毒素处理后试虫血液、中肠及组织酯酶同工酶谱及酶带数量随时间的变化进行了分析和比较。结果表明 :处理组侧柏毒蛾幼虫的中肠、组织、血液的酯酶同工酶在处理后 1～ 4h之间酶带数减少 ,8～ 1 2 h内酶带增多 ,2 4h以后这种激应性变化相对稳定。试虫受马桑毒素干扰后可使虫肠组织的分子量相对较小、组织及血液中分子量为中等的酯酶的酶谱变化较大 ,但是该毒素侵入虫体后的后期致毒性与前期基本接近 。

体内和体外实验中马桑内酯对大鼠大脑皮质神经元白细胞介素-2受体表达的影响

为了探讨免疫调质与癫痫发病机制的关系，本文应用免疫细胞化学PAP法对体内和体外实验中马桑内酯（致痫剂）对大鼠大脑皮质种经元白细胞介素-2受体表达的影响进行了研究．在体内实验中，对照经大鼠大脑皮质仅见少量弱阳性白细胞介素-2受体免疫反应神经元，其免疫反应性定位于种经元膜上．大鼠一侧侧脑室注射马桑内酯诱发癫痫后，大脑皮质白细胞介素-2受体阳性神经元明显增多，免疫反应增强．在体外实验中，用马桑内酯温育培养大双皮质神经元24h，其白细胞介素2受体阳性神经元也比对照组增加了1．5倍，免疫染色加深．本实验结果提示，大脑皮质神经元上的白细胞介素-2受体可能参与癫痫的病理机制．

小胶质细胞的分离培养及马桑内酯对其增殖的影响

目的 :探讨马桑内酯 (CL)对分离培养的小胶质细胞的生存和增殖的影响。方法 :采用振摇结合贴壁分离法获得纯化的小胶质细胞 ,MTT比色法观察了CL对培养的小胶质细胞增殖影响的量效 曲线、细胞的存活能力以及小胶质细胞刺激后的形态学变化。结果 :经凝集素细胞化学鉴定 ,分离培养的小胶质细胞纯度达 95 %以上 ;以CL( 1 0 - 5mol/L)可显著诱导小胶质细胞的增殖 ,小胶质细胞的存活率达 99% ;刺激后 ,以反应性小胶质细胞为主。当CL >1 0 - 5mol/L时 ,细胞存活率 <85 %。结论 :CL在一定剂量范围内具有促进小胶质细胞增殖作用 ,可能伴随着细胞功能的活跃 ;

马桑内酯所致慢性癫痫大鼠海马星形胶质细胞胶质纤维酸性蛋白的表达

用免疫组织化学方法研究了系统应用马桑内酯所致的慢性癫痫大鼠海马中星形胶质细胞的胶质纤维酸性蛋白的表达。结果证明，整个海马的胶质纤维酸性蛋白免疫反应明显增强，并可见阳性细胞增生、胞体肥大，尤以齿状回门区和海马分子腔隙层及始层为甚。此外，本实验还发现海马胶质纤维酸性蛋白的阳性反应强度随发作后不同时间间隔（２ ｈ～９ ｄ）而不同，且直至发作后９ ｄ，胶质纤维酸性蛋白阳性反应程度仍高于对照组。此结果表明，马桑内酯所致癫痫反复发作可使海马星形胶质细胞内胶质纤维酸性蛋白的表达长期保持在较高水平。

人参皂甙Rb1对马桑内酯致痫大鼠海马组织蛋白质组的影响

目的:运用双向电泳技术获得可能与马桑内酯(CL)癫痫发作及人参皂甙Rb1(GRb1)神经保护作用有关的蛋白质成分,探讨海马组织蛋白质在癫痫发生发展过程中的作用和地位以及GRb1对其的影响。方法:分别提取对照组、CL致痫组和GRb1+CL致痫组动物海马组织蛋白,双向电泳分离,ImageMaster 2D Platinum 5.0软件进行差异表达蛋白质组分析,基质辅助激光解吸附离子化飞行时间质谱(MALDI-TOF-MS)鉴定蛋白质。结果:成功鉴定出6个蛋白质分别是:脑肌酸激酶(brain creatine kinase)、内吞蛋白A1(endophilin-A1)、UPF0628蛋白C10orf96同源物(UPF0628 protein C10orf96 homolog)、细胞色素P-450(cytochrome P-450)、光导素样蛋白(phosducin-like protein)及桥整合蛋白3(bridging integrator 3)。其中前3个蛋白质在GRb1+CL致痫组表达低于CL致痫组,后3个蛋白质在GRb1+CL致痫组表达低于对照组。结论:CL致痫组、GRb1+CL致痫组和对照组表达的蛋白质存在明显差异。鉴定出的6个差异表达的蛋白质可能与GRb1的神经保护作用有关,其中brain crea-tine kinase、endophilin-A1和UPF0628 protein C10orf96 homolog可能与CL所致癫痫发作有关。

马桑内酯在大鼠离体海马脑片上引起的癫痫样放电活动

采用大鼠离体海马脑片技术,建立了马桑内酯(Coriaria lactone,CL)引起的海马CA 1区锥体细胞癫痫样放电活动模型,并初步探讨了CL的致痫作用机理,可能与直接影响锥体细胞固有爆发放电能力有关。

马桑内酯对大鼠大脑皮层突触体和脑片谷氨酸摄取、释放功能的影响

目的：研究致痫剂马桑内酯对谷氨酸摄取和释放功能的影响，以探讨马桑内酯致痫的可能机制。方法：放射性同位素分析技术。结果：在０．３５～３５０μｍｏｌ·Ｌ－１浓度范围内，马桑内酯使高钾（５０ｍｍｏｌ·Ｌ－１）去极化引起的脑片谷氨酸释放增加，增加率为１１．２％～３３．２％，剂量效应关系明显，３５μｍｏｌ·Ｌ－１和３５０μｍｏｌ·Ｌ－１组与对照组相比谷氨酸释放显著增加（Ｐ＜０．０１），各浓度马桑内酯对静息态谷氨酸基础释放均无明显作用。在３５０μｍｏｌ·Ｌ－１马桑内酯作用下，突触体谷氨酸摄取降低，降低率为７．９４％，与对照组差异显著（Ｐ＜０．０５），其它浓度马桑内酯作用不明显。结论：马桑内酯使谷氨酸释放增加，摄取减少，突触间隙谷氨酸聚积。

糖皮质激素对癫痫大鼠海马c-fos表达的影响

用免疫组织化学方法观察了糖皮质激素对马桑内酯致痫大鼠海马 c- fos的影响。侧脑室注射马桑内酯 2 h后 ,导致动物急性癫痫样发作。海马内出现 c- fos的高度表达。单纯向侧脑室注射糖皮质激素 10 0μg以后 ,动物不出现癫痫行为。海马内也几乎无 c- fos表达。但先向侧脑室注射糖皮质激素 10 0μg 1h,再向侧脑室内注射马桑内酯 2 h后 ,海马内 c- fos表达明显减少。摘除双侧肾上腺一周 ,再向侧脑室注射马桑内酯 2 h后 ,动物癫痫发作更明显 ,海马内 c- fos表达较单纯用马桑内酯致痫动物更强。结果提示 :糖皮质激素对马桑内酯致痫有明显的抑制作用 ,为临床应用糖皮质激素治疗癫痫提供了形态学参考。

马桑内酯对培养的大鼠海马星形胶质细胞的激活

实验研究了马桑内酯对纯化培养的大鼠海马星形胶质细胞的影响。发现 :1免疫细胞化学染色证实马桑内酯作用2 h可引起胶质细胞核内 NF- κBp6 5的表达 ,8h达高峰 ,至 2 4h NF- κBp6 5阳性胞核的百分率和平均光密度仍维持在高水平 ;预先用 PDTC作用 1h可完全抑制 NF- κBp6 5的核表达 ,而 TNFα单抗则可延迟 NF- κBp6 5免疫反应阳性胞核出现的时间 ,阳性胞核百分率及其平均光密度也有所下降 ;2酶联免疫吸附实验发现马桑内酯可促进星形胶质细胞培养基内 TNFα含量升高 ,PDTC可部分对抗此作用。本实验表明马桑内酯激活星形胶质细胞的作用部分是由

超高效液相色谱-高分辨质谱法测定蜂蜜中的3种马桑内酯

建立了采用超高效液相色谱-高分辨质谱测定蜂蜜中3种马桑内酯残留的方法。样品采用0.2 mol／L磷酸盐缓冲溶液（ pH＝7.5）提取，经Waters HLB小柱净化，以Phenomenex C18色谱柱进行色谱分离，通过高分辨质谱t-MS2负离子扫描模式进行定性和定量分析。结果表明3种目标化合物的检出限（ LOD）均为0.05 mg／kg，定量限（ LOQ）均为0.1 mg／kg。空白蜂蜜样品在0.1～0.5 mg／kg 范围内的3个加标水平的平均回收率为86.3％～95.6％，相对标准偏差为3.0％～8.4％。应用该方法对从新西兰进口的麦卢卡蜂蜜进行检测，检出一份样品含羟基马桑毒素0.3 mg／kg。该方法适用于蜂蜜中马桑内酯残留的检测。

马桑内酯对大鼠海马锥体神经细胞内钙稳态的影响

目的 了解钙离子及钙通道在癫痫发病中的作用 ,探讨马桑内酯 (coriaria lactone,CL)调节神经细胞内钙稳态的机理。方法 利用激光扫描共聚焦显微镜和细胞培养技术 ,观察致痫剂 CL 对 Wistar乳鼠海马单个锥体神经细胞内游离钙离子 (Ca2 +)浓度的影晌。结果 经致痫剂 CL 作用后 ,锥体神经细胞内 Ca2 +浓度高于正常对照组 (P<0 .0 1) ,但其升高不呈剂量依赖性 (P>0 .0 5 ) ;经钙通道阻滞剂尼莫地平处理后 ,CL 虽然仍能引起神经元胞内 Ca2 +浓度升高 (P<0 .0 1) ,但与单纯使用 CL 组相比仍有明显差异 (P<0 .0 0 1)。结论 L-型钙通道开放在痫性放电的发生、发展上起了重要作用 ,促进钙通道开放可能是马桑内酯致痫的重要途径之一。

马桑内酯致痫大鼠皮层及海马神经元的放电模式

在腹腔注射马桑内酯所致的急性癫痫和慢性点燃癫痫大鼠模型上，观察了大脑皮层和海马神经元膜电位的变化及放电模式。结果显示：皮层脑电图出现明显的阵发性痫样放电，胞内微电极记录皮层及海马神经元放电的模式为：①单纯去极化；②去极化并伴有阵发性或持续性放电；③去极化与超极化交替出现等。表明马桑内酯可以引起大脑皮层及海马神经元生物电活动的改变，其改变形式不一，但主要引起去极化。