阴阳分治发作期癫痫的临床与实验研究
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摘要
“阴阳分治法”是根据发作期癫痫不同病机属性,分为阴痫和阳痫两型进行治疗的一种中医疗法。这种辨治方法最早由元代巢元方提出。后来,元代危亦林《世医得效方》提出用五生饮治疗阴痫,清代程国彭《医学心悟》提出用定痫丸治疗阳痫,至此阴阳分治法基本确立。时至今日,已历经700多年的临床验证。目前《中医内科学》教材已将五生饮合二陈汤列为治疗阴痫的代表方,黄连解毒汤合定痫丸列为治疗阳痫的代表方。由于这些方剂中含有生南星、生半夏、生白附子、生川乌、朱砂等多味毒性药物,当今临床应用较少。在癫痫的中医药基础研究中,目前国内使用的动物实验模型均是单纯的西医病理模型,尚未建立符合中医证候特点的癫痫动物模型。本研究分为临床研究和实验研究两部分。其中,实验研究又分为动物实验研究和细胞培养实验研究两节。目的是探讨阴阳分治法的抗痫疗效、安全性及作用机理,同时建立阴痫和阳痫两个中医证候癫痫动物模型。
第一部分阴阳分治发作期癫痫的临床研究
目的：
观察阴阳分治法治疗发作期癫痫的临床疗效及安全性。
方法：
将入院当天癫痫发作≥3次的癫痫患者随机分为治疗组与对照组,对照组仅给予常规抗痫西药治疗,治疗组给予常规抗痫西药的同时,按阴阳分治法辨证口服中药(阴痫予减味五生饮合二陈汤,阳痫予黄连解毒汤合定痫丸)及醒脑静注射液静滴。通过治疗前后癫痫发作次数减少率、中医证候评价、抗癫痫西药平均总用量及NHS3、MRS、MBI等多种量表进行评价。
结果：
共纳入合格受试者12例,有11例完成治疗方案,其中对照组5例,治疗组6例,脱落1例。治疗组与对照组治疗前后癫痫发作次数减少率、抗癫痫西药平均总用量及NHS3、MRS、MBI等量表得分比较均无统计学意义。中医证候评价结果显示,对照组改善闭证疗效较好(P<0.05),治疗组改善风、闭、肾阴虚、痰、脾气虚等证疗效较好(P<0.05),其中以痰证、脾气虚证最显著(P<0.01)。治疗组所有病例未见药物不良反应。
结论：
阴阳分治法可能能够较好地改善发作期癫痫患者中医证候,其中以痰证和脾气虚证效果最好,且未见不良反应。
第二部分阴阳分治发作期癫痫的实验研究
第一节动物实验研究
一、实验一：减味五生饮合二陈汤治疗阴痫型癫痫大鼠的实验研究
目的：
建立阴痫型癫痫大鼠模型,探讨减味五生饮合二陈汤的抗痫疗效和机理。
方法：
长期予大鼠喂饲高脂饲料及寒性中药稀释煎液,并以匹罗卡品致痫,以制作阴痫型癫痫大鼠模型。以治疗阴痫的代表方减味五生饮合二陈汤治疗阴痫型癫痫大鼠,同时以治疗阳痫的代表方黄连解毒汤合定痫丸(汤剂)为对照,记录癫痫发作潜伏期和到达癫痫持续状态(SE)时间；高效液相色谱(HPLC)法检测脑内氨基酸神经递质；HE染色切片观察病理变化；免疫组化技术半定量分析caspase-3表达情况,以评价建模质量及探讨减味五生饮合二陈汤的抗痫疗效和机制。
结果：
与正常对照组比较,各致痫组大鼠脑内谷氨酸(Glu)、天门冬氨酸(Asp)、γ-氨基丁酸(GABA)、甘氨酸(G1y)、丙氨酸(A1a)等氨基酸类神经递质浓度均下降,caspase-3表达量均上升,其中以阴痫中药组变化幅度最小,阴痫中药对照组次之,但均无统计学意义。各致痫组大鼠癫痫发作潜伏期和到达SE时间比较均无统计学意义。各组癫痫大鼠大脑皮质、海马、肺、肝、肾等组织均出现较严重损伤,但组间比较无明显差异。
结论：
本实验初步建立了阴痫型癫痫大鼠模型。减味五生饮合二陈汤及黄连解毒汤合定痫丸(汤剂)均可抑制阴痫型癫痫大鼠脑内Glu、Asp、GABA、Gly、Ala等氨基酸类神经递质的下降及caspase-3的表达,其中以减味五生饮合二陈汤作用较强,提示这些方剂均可能对阴痫型癫痫大鼠具有一定的治疗作用,其中可能以减味五生饮合二陈汤疗效较佳。
二、实验二：黄连解毒汤合定痫丸(汤剂)治疗阳痫型癫痫大鼠的实验研究
目的：
建立阳痫型癫痫大鼠模型,探讨黄连解毒汤合定痫丸(汤剂)的抗痫疗效和机理。
方法：
长期予大鼠喂饲高脂饲料及热性中药稀释煎液,并以匹罗卡品致痫,以制作阳痫型癫痫大鼠模型。以治疗阳痫的代表方黄连解毒汤合定痫丸(汤剂)治疗阳痫型癫痫大鼠,同时以治疗阴痫的代表方减味五生饮合二陈汤为对照,记录癫痫发作潜伏期和到达SE时间；HPLC法检测脑内氨基酸类神经递质；HE染色切片观察病理变化；免疫组化技术半定量分析caspase-3表达情况,以评价建模质量及黄连解毒汤合定痫丸(汤剂)的抗痫疗效和机制。
结果：
与正常对照组比较,各组致痫大鼠脑内Glu、Asp、GABA、Gly、Ala等氨基酸类神经递质浓度均下降,caspase-3表达量均上升,其中阳痫中药组Asp、Ala下降幅度及caspase-3上升幅度在各组中最小,阴痫中药对照组各种氨基酸类神经递质浓度下降及caspase-3上升幅度最大,但均无统计学意义。各致痫组大鼠癫痫发作潜伏期和到达SE时间比较均无统计学意义。各组致痫大鼠大脑皮质、海马、肺、肝、肾等组织均出现较严重损伤,但组间比较无明显差异。
结论：
本实验初步建立了阳痫型癫痫大鼠模型。黄连解毒汤合定痫丸(汤剂)可抑制阳痫型癫痫大鼠脑内Glu、Asp、GABA、Gly、Ala等氨基酸类神经递质的下降及caspase-3的表达,提示其可能对阳痫型癫痫大鼠有一定的治疗作用。减味五生饮合二陈汤可促进阳痫型癫痫大鼠脑内Glu、Asp、GABA、Gly、Ala等氨基酸类神经递质的下降及caspase-3的表达,提示其可能会加重阳痫型癫痫大鼠的病情。
第二节细胞培养实验研究
一、实验一：谷氨酸损伤PC12细胞模型的建立实验研究
目的：
探讨Glu损伤PC12细胞模型的建立方法。
方法：
以不同浓度的Glu作用PC12细胞,分别于24小时、48小时和72小时三个时间点观察细胞形态学；MTT法检测细胞活力,以确立Glu对PC12细胞活力影响的量效及量时关系,探讨合适的造模时间及浓度。
结果：
细胞形态的观察结果显示,在同一时间点中,自5mMGlu组开始,随Glu浓度的升高,细胞折光性及立体感逐渐下降,轮廓逐渐模糊,生长逐渐稀疏,但不同时间点同一Glu浓度组的细胞形态未发现有明显差别。
细胞活力检测结果显示,与正常对照组比较,24小时、48小时和72小时三个时间点均自Glu浓度为5mM开始,随着浓度的逐渐升高,细胞活力逐渐下降。除40mM组外,正常对照组和各不同浓度的Glu模型组细胞随着培养时间的延长,细胞活力逐渐增加,即Glu对PC12细胞的活力存在剂量、时间依赖作用：细胞活力随Glu浓度的递增而下降,随培养时间的延长而上升。浓度为10mmol/L以上的Glu处理PC12细胞,在24小时、48小时和72小时三个时间点,其细胞活力与正常对照组比较均显著降低(P<0.01)。
结论：
Glu对PC12细胞的作用呈剂量和时间依赖关系：细胞活力随Glu浓度增高而降低,随培养时间延长而增强。10mmol/L的Glu作用PC12细胞24小时可能是建立Glu损伤PC12细胞模型较理想的实验条件。
二、实验二：减味五生饮合二陈汤含药血清对谷氨酸损伤PC12细胞治疗作用的实验研究
目的：
在细胞水平上进一步探讨减味五生饮合二陈汤的抗痫疗效和机理。
方法：
将实验细胞随机分为6组,分别是正常对照组、Glu损伤模型组、正常大鼠血清组、寒痰型大鼠血清组、阴痫中药对照血清组、阴痫中药血清组。以长期喂饲高脂饲料及寒性中药稀释煎液的方法建立“寒痰”型体质大鼠,并以这些大鼠制作的减味五生饮合二陈汤含药血清处理Glu损伤PC12细胞模型,同时以黄连解毒汤合定痫丸(汤剂)含药血清为对照。MTT法检测细胞活力；流式细胞技术检测细胞内游离Ca2+浓度。
结果：
与正常对照组比较,各组细胞活力均下降,其中以阴痫中药血清组下降幅度最小,阴痫中药对照血清组次之,除阴痫中药血清组外,均有显著差异(P<0.01)。阴痫中药血清组与阴痫中药对照血清组比较亦有统计学意义(P<0.05)。
与正常对照组比较,除G1u损伤模型组细胞内游离Ca2+浓度升高外,各组均下降,以阴痫中药血清组最低,阴痫中药对照血清组次之。其中,Glu损伤模型组、阴痫中药对照血清组、阴痫中药血清组与正常对照组比较均有显著差异(P<0.01)。阴痫中药血清组与中阴痫中药对照血清组比较亦有显著差异(P<0.01)。
结论：
以“寒痰”型体质大鼠制作的减味五生饮合二陈汤含药血清及黄连解毒汤合定痫丸含药血清均能提高Glu损伤PC12细胞活力及减少Ca2+内流,提示上述方剂的含药血清可能均有治疗Glu损伤PC12细胞的作用,其中可能以减味五生饮合二陈汤含药血清疗效较佳。
三、实验三：黄连解毒汤合定痛丸(汤剂)含药血清对谷氨酸损伤PC12细胞治疗作用的实验研究
目的：
在细胞水平上进一步探讨黄连解毒汤合定痫丸(汤剂)的抗痫疗效和机理。
方法：
将实验细胞随机分为6组,分别是正常对照组、Glu损伤模型组、正常大鼠血清组、痰热型大鼠血清组、阳痫中药对照血清组、阳痫中药血清组。以长期喂饲高脂饲料及热性中药稀释煎液的方法建立“痰热”型体质大鼠,并以之制作的黄连解毒汤合定痫丸(汤剂)含药血清处理Glu损伤PC12细胞模型,同时以减味五生饮合二陈汤含药血清为对照。MTT法检测细胞活力；流式细胞技术检测细胞内游离Ca2+浓度。
结果：
与正常对照组比较,各组细胞活力均下降,其中以阳痫中药对照血清组下降幅度最小,阳痫中药血清组次之。各组与正常对照组比较,均有显著差异(P<0.01)。阳痫中药对照血清组与阳痫中药血清组比较亦有显著差异(P<0.01)。
与正常对照组比较,除G1u损伤模型组外,各组细胞内游离Ca2+浓度均下降,痰热型大鼠血清组最低,阳痫中药血清组次之,阳痫中药对照血清组再次之。除阳痫中药对照血清组外,痰热型大鼠血清组、阳痫中药血清组与正常对照组比较均有统计学意义(P<0.05)。阳痫中药对照血清组与阳痫中药血清组比较无统计学意义。
结论：
以“痰热”型体质大鼠制作的黄连解毒汤合定痫丸含药血清和减味五生饮合二陈汤含药血清均能提高Glu损伤PC12细胞活力,其中黄连解毒汤合定痫丸含药血清尚能减少Ca2+内流,提示上述方剂的含药血清均可能对Glu损伤PC12细胞有一定治疗,但黄连解毒汤合定痫丸含药血清与减味五生饮合二陈汤含药血清疗效高低尚需进一步研究。
The therapy based on yin and yang differentiation is a kind of traditional therapy in Chinese medicine (CM) which treats epilepsy attacking frequently under two conditions that are yinxian and yangxian, one of approaches of classification of epilepsy from the respect of CM on the basis of different pathogenesis between cold and heat property in terms of CM. This therapy originated from the suggestion of Chao Yuanfang in the Yuan era. Then Wushengying was put forward to treating yinxian in Shi Yi De Xiao Fang writed by Wei Yilin in the Yuan period. And Dingxianwan was recommended to treat yangxian in Yi Xue Xin Wu authored by Cheng Guopeng in the qing dynasty. It is advisable to treat yinxian in wushengyin (WSY) and Erchentang (ECT), while yangxian in huanglianjiedutang (HLJDT) and dingxianwan (DXW) in the teaching material of Internal of Medicine of CM. However, clinical appliance for these decoctions is little, the reason for which may result from their inclusion of certain toxic Chinese medicines like Rhizoma Arisaematis Erubescentis, Rhizoma Pinelliae, Rhizoma Typhonii Gigantei, all of which have not been processed, and Cinnabaris. Besides, domestic researches on anti-epileptic effects and mechanisms of Chinese medicine are carried on by pathological animal models of western medicine (WM) nowadays, and animal models characterized by CM differentiation of symptoms and signs, which is named zheng hou in traditional Chinese medicine, have not been established. From what had been discussed above, we designed and undertook this program which was consisted of two parts that were clinical trial and empirical study which included the animal experiment and the cell culture experiment. And the main aim was to explore the effects, safety, and mechanisms of the therapy based on yin and yang differentiation, and set up epileptic animal models which were yinxian and yangxian characterized by zheng hou of CM.
Part1Clinic Trial of The Therapy Based on Yin and Yang Differentiation
Objective
To observe clinical effects and safety of the therapy based on yin and yang differentiation in epilepsy attacking frequently.
Methods
Patients who suffered from epileptic seizure more than three times were randomly divided into the treatment group and the control group. The control group only received conventional AEDs, while the treatment group received conventional AEDs, Chinese herbal drugs that were JWWSY and ECT, or HLJDT and DXW on the basis of the therapy based on yin and yang differentiation, and intravenous drip of xingnaojing parenteral solution as well. Therapeutic effects were measured by the decrease in seizure rate between prior and post-treatment, zheng hou evaluation, average total dosages of different AEDs, NHS3, MBI, and MRS respectively.
Results
There were12cases who participated in this trial, and11of them completed the program, of which5were in the control group, and6were in the treatment group, while1lost due to sudden death. Although most of parameters like the decrease in seizure rate between prior and post-treatment, average total dosages of different AEDs, NHS3, MBI, MRS, and etc we measured were no statistical difference between the two groups. In terms of evaluation of zheng hou, the control group adepted in improving asthenia(P<0.05), while the treatment group was effective at ameliorating wind, asthenia, renal yin deficiency, phlegm, and spleen energy deficiency(P<0.05), especially at phlegm and spleen energy deficiency (P<0.01). There was no side effect in all the cases in the treatment group.
Conclusion
It is superior for the therapy based on yin and yang differentiation in terms of improvement in relative symptoms and signs of zheng hou of patients suffering from epilepsy attacking frequently, especially in zhenghou of phlegm and spleen energy deficiency, and no side effect was observed.
Part2Experimental Research of The Therapy Based on Yin and Yang Differentiation
Section1Animal Research
Experiment1Research on Effects of JWSY and ECT on Yinxian Epileptic Rats
Objective
To explore anti-epileptic effects and mechanisms of JWWSY and ECT, and establish yinxian epileptic rat model.
Methods
The yinxian epileptic rat model was made by the method of feeding on high fat diet and CM in cold property for a long time, and then the being induced seizure by PILO. Yinxian epileptic rats induced by PILO were treated with JWWSY and ECT, the representative recipe for treatment of yinxian, while HLJDT and DXW, the representative recipe for treatment of yangxian, was used as the control formula. Time spots of seizure latency period and seizure status were record, and concentrations of amino acid neurotransmitters were measured by HPLC method, and the expression of caspase-3was determined by immunohistochemisty technology, and pathological changes were observed by hematoxylin and eosin stain. All of indexes mentioned above were adopted to evaluate the anti-epileptic effects and mechanisms of JWWSY and ECT, and assess the quality of epileptic animal model characterized by zheng hou.
Results
All kinds of concentrations of amino acid neurotransmitters measured in this experiment were lower, and the expression of caspase-3was higher in all the epileptic rats, in all of which the variation of yinxian CM group was the least, and the yinxian CM control group was the second, when compared with normal control group. However, there was no statistical difference in concentrations of amino acid neurotransmitters of Glu, Asp, GABA, Gly, and Ala, and in the expression of caspase-3between epileptic rats and normal control ones. Also, There was no recognizable difference in time spots of seizure latency period and seizure status among epileptic groups. And the tissue of the cerebral cortex and hippocampus, lung, liver, and kidney of all epileptic rats was injured severely, but there was no significant difference in all epileptic groups.
Conclusion
JWWSY and ECT, as well as HLJDT and DXW can inhibit the declination of different sorts of amino acid neurotransmitters, including Glu, Asp, GABA, Gly, and Ala, and apoptosis in the central nervous system, and effects of JWWSY and ECT on which are better, which indicates that such decoctions may have certain effects in treating the yinxian epileptic rats induced by PILO, and JWWSY and ECT seems to be superior. Yinxian epileptic rat model was set up in this experiment initially.
Experiment2Research on Effects of HLJDT and DXW on Yangxian Epileptic Rats Objective
To explore anti-epileptic effects and mechanisms of HLJDT and DXW, and establish yangxian epileptic rat model.
Methods
The yangxian epileptic rat model was made by the method of feeding on high fat diet and CM in heat property for a long time, and then being induced seizure by PILO. Yangxian epileptic rats induced by PILO were treated with HLJDT and DXW, the representative recipe for treatment of yangxian, while JWWSY and ECT, the representative recipe for treatment of yinxian, was used as the control formula. Time spots of seizure latency period and seizure status were record, and concentrations of amino acid neurotransmitters were measured by HPLC method, and the expression of caspase-3was determined by immunohistochemisty technology, and pathological changes were observed by hematoxylin and eosin stain. All of indexes mentioned above were adopted to evaluate the anti-epileptic effects and mechanisms of HLJDT and DXW, and assess the quality of epileptic animal model characterized by zheng hou.
Results
All kinds of concentrations of amino acid neurotransmitters measured in this experiment were lower, and the expression of caspase-3was higher in all the epileptic rats, when compared with normal control group. In all of epileptic groups, the variation of amino acid neurotransmitters including Asp and Ala and the expression of caspase-3of yangxian CM group was the least. In contrast, the variation of the indexes concerned of yangxian CM control group was the biggest. However, there was no statistical difference in concentrations of amino acid neurotransmitters of Glu, Asp, GABA, Gly, and Ala, and in the expression of caspase-3between epileptic rats and normal control ones. And there was no meaningful difference in time spots of seizure latency period and seizure status among epileptic groups. The tissue of the cerebral cortex and hippocampus, lung, liver, and kidney of all epileptic rats was injured severely, but there was also no significant difference in all of the epileptic groups.
Conclusion
HLJDT and DXW can inhibit the declination of different sorts of amino acid neurotransmitters, including Glu, Asp, GABA, Gly, and Ala, and apoptosis in the central nervous system, which shows that such decoctions may have certain effects in treating the yangxian epileptic rats induced by PILO. The yangxian epileptic rats model was established initially in this experiment.
Section2Cell Culture Research
Experiment1Research on Establishment of PC12Cell Trauma Model Induced by Glutamate Objective
To explore a proper methods to establish PC12cell trauma model for the following experiments.
Methods
PC12cells were treated with various concentrations of Glu, and cell morphology and viability were measured by MTT assay at24h,48h, and72h respectively so that the concentration-effect and concentration-time relationship between the effects of Glu neurotoxicity and the viability of PC12cells was investigated.
Results
The results of the observation of cell morphology indicated that the cell reflection, dimensionality, sketch, and the growth condition become bad to worse step by step as the increasing concentrations of Glu at the same time spot from5mM Glu group, while there was on obvious difference in cell morphology at different time spots comparison. The outcomes of the vitality of cells manifested that the cell vitality declined gradually as the increase of the concentration of Glu from5mM at24h,48h, and72h time spots. However, the vitality of cells in normal control group and trauma model groups induced by Glu in different concentrations. The relationship between effects of Glu neurotoxicity and viability of PC12cells was in manner of concentration and time-dependence:the higher the concentration of Glu is, the lower the viability of PC12cells becomes;the longer the incubation time is, the higher the viability of PC12cells becomes. There was significant difference in the cell vitality between PC12cells administered by Glu in the concentration of10mmol/L and in the normal control group.
Conclusion
The relationship between effects of Glu neurotoxicity and viability of PC12cells was in manner of concentration and time-dependence:the higher the concentration of Glu is, the lower the viability of PC12cells becomes;the longer the incubation time is, the higher the viability of PC12cells becomes. PC12cells administered by Glu neurotoxicity in the concentration of10mmol/L for24h maybe the ideal conditions for the following experiment.
Experiment2Therapeutic effects of JWSY and ECT drug-containing serum on PC12cell trauma model induced by Glu neurotoxicity
Objective
To move forward a single step to explore the therapeutic action and mechanisms of anti-epileptic effects of JWWSY and ECT at the cellular level.
Methods
The experimental cells were randomly devided into six groups, which were the normal control group, the Glu trauma model group, the normal rat serum group, the cold phlegm rat group, the yinxian CM control group, and the yinxian CM group. The PC12cell trauma models induced by Glu neurotoxicity were treated in JWWSY and ECT drug-containing serum made by rats with body constitution in cold phlegm, which were made by the method of feeding on high fat diet and CM in cold property for a long time, while HLJDT and DXW drug-containing serum was used as the control formula. The MTT assay was utilized to detect the viability of cells, and flow cytometry was adopted to assess the concentration of intra-cellular calcium.
Results
The cell viability of all groups administrated by Glu declined, in all of which the yinxian CM serum group was the least, and the yinxian CM control serum group was the second, when compared with the normal control group. There was a significant difference between groups administrated by Glu and the normal control group (P<0.01), except the yinxian CM serum group. And there was a meaningful difference between the yinxian CM serum group and the yinxian CM control serum group as well (P<0.01).
Except the Glu trauma model group, the concentrations of intra-cellular calcium of all groups administrated by Glu decreased, in all of which the yinxian CM serum group was the most, and the yinxian CM control serum group was the second, when compared with the正常对照group. Both the yinxian CM control serum group and the yinxian CM serum group had a recognizable difference in contrast to the正常对照group(P<0.01). And there also was a noticeable difference between the yinxian CM control serum group and the yinxian CM serum group(P<0.01).
Conclusion
JWWSY and ECT, as well as HLJDT and DXW drug-containing serum made by rats with body constitution in cold phlegm can increase the vitality and inhabit internal flow of calcium of the PC12cell trauma models induced by Glu neurotoxicity, which indicates that the drug-containing serum of such decoctions, especially JWWSY and ECT drug-containing serum, may have certain therapeutic effects in treating the PC12cell trauma models.
Experiment3Therapeutic effects of HLJDT and DXW drug-containing serum on PC12cell trauma model induced by Glu neurotoxicity Objective
To further explore the therapeutic action and mechanisms of anti-epileptic effects of HLJDT and DXW at the cellular level.
Methods
The experimental cells were randomly devided into six groups, which were the normal control group, the Glu trauma model group, the normal rat serum group, the heat phlegm rat serum group, the yangxian CM control serum group, and the yangxian CM serum group. The PC12cell trauma models induced by Glu neurotoxicity were treated in HLJDT and DXW drug-containing serum made by rats with body constitution in heat phlegm, which were made by the method of feeding on high fat diet and CM in heat property for a long time while JWWSY and ECT drug-containing serum was used as the control formula. The MTT assay was utilized to detect the viability of cells, and flow cytometry was adopted to assess the concentration of intra-cellular calcium.
Results
The cell viability of all groups administrated by Glu declined, in all of which the range in declination of the yangxian CM control serum group was the least, and the yangxian CM serum group was the second, when compared with the normal control group. There was a significant difference between groups administrated by Glu and the normal control group(P<0.01). And there was a meaningful difference between yangxian CM serum group and the yangxian CM control serum group as well (P<0.01).
Except the Glu trauma model group, the concentrations of intra-cellular calcium of all groups administrated by Glu decreased, in all of which, the range of the heat phlegm rat serum group was the most, the yangxian CM serum group was the second, and the yangxian CM control serum group was the last, when compared with the normal control group. And except the yangxian CM control serum group, both the heat phlegm rat serum group and the yangxian CM serum group had a statistical difference in contrast to the normal control group(P <0.05), while there was no noticeable difference between the yangxian CM control serum group and the yangxian CM serum group.
Conclusion
HLJDT and DXW, as well as JWWSY and ECT drug-containing serum made by rats with body constitution in heat phlegm can increase the vitality of the PC12cell trauma models induced by Glu neurotoxicity, and the HLJDT and DXW drug-containing serum can also inhabit the internal flow of calcium, which manifests that there seems to be certain the therapeutic effects for the drug-containing serum of such decoctions in treating the PC12cell trauma models, but which decoction, the HLJDT and DXW, or JWWSY and ECT drug-containing serum of yangxian pre-seizure rats,is better needs further exploration.
第一部分阴阳分治发作期癫痫的临床研究
目的：
观察阴阳分治法治疗发作期癫痫的临床疗效及安全性。
方法：
将入院当天癫痫发作≥3次的癫痫患者随机分为治疗组与对照组,对照组仅给予常规抗痫西药治疗,治疗组给予常规抗痫西药的同时,按阴阳分治法辨证口服中药(阴痫予减味五生饮合二陈汤,阳痫予黄连解毒汤合定痫丸)及醒脑静注射液静滴。通过治疗前后癫痫发作次数减少率、中医证候评价、抗癫痫西药平均总用量及NHS3、MRS、MBI等多种量表进行评价。
结果：
共纳入合格受试者12例,有11例完成治疗方案,其中对照组5例,治疗组6例,脱落1例。治疗组与对照组治疗前后癫痫发作次数减少率、抗癫痫西药平均总用量及NHS3、MRS、MBI等量表得分比较均无统计学意义。中医证候评价结果显示,对照组改善闭证疗效较好(P<0.05),治疗组改善风、闭、肾阴虚、痰、脾气虚等证疗效较好(P<0.05),其中以痰证、脾气虚证最显著(P<0.01)。治疗组所有病例未见药物不良反应。
结论：
阴阳分治法可能能够较好地改善发作期癫痫患者中医证候,其中以痰证和脾气虚证效果最好,且未见不良反应。
第二部分阴阳分治发作期癫痫的实验研究
第一节动物实验研究
一、实验一：减味五生饮合二陈汤治疗阴痫型癫痫大鼠的实验研究
目的：
建立阴痫型癫痫大鼠模型,探讨减味五生饮合二陈汤的抗痫疗效和机理。
方法：
长期予大鼠喂饲高脂饲料及寒性中药稀释煎液,并以匹罗卡品致痫,以制作阴痫型癫痫大鼠模型。以治疗阴痫的代表方减味五生饮合二陈汤治疗阴痫型癫痫大鼠,同时以治疗阳痫的代表方黄连解毒汤合定痫丸(汤剂)为对照,记录癫痫发作潜伏期和到达癫痫持续状态(SE)时间；高效液相色谱(HPLC)法检测脑内氨基酸神经递质；HE染色切片观察病理变化；免疫组化技术半定量分析caspase-3表达情况,以评价建模质量及探讨减味五生饮合二陈汤的抗痫疗效和机制。
结果：
与正常对照组比较,各致痫组大鼠脑内谷氨酸(Glu)、天门冬氨酸(Asp)、γ-氨基丁酸(GABA)、甘氨酸(G1y)、丙氨酸(A1a)等氨基酸类神经递质浓度均下降,caspase-3表达量均上升,其中以阴痫中药组变化幅度最小,阴痫中药对照组次之,但均无统计学意义。各致痫组大鼠癫痫发作潜伏期和到达SE时间比较均无统计学意义。各组癫痫大鼠大脑皮质、海马、肺、肝、肾等组织均出现较严重损伤,但组间比较无明显差异。
结论：
本实验初步建立了阴痫型癫痫大鼠模型。减味五生饮合二陈汤及黄连解毒汤合定痫丸(汤剂)均可抑制阴痫型癫痫大鼠脑内Glu、Asp、GABA、Gly、Ala等氨基酸类神经递质的下降及caspase-3的表达,其中以减味五生饮合二陈汤作用较强,提示这些方剂均可能对阴痫型癫痫大鼠具有一定的治疗作用,其中可能以减味五生饮合二陈汤疗效较佳。
二、实验二：黄连解毒汤合定痫丸(汤剂)治疗阳痫型癫痫大鼠的实验研究
目的：
建立阳痫型癫痫大鼠模型,探讨黄连解毒汤合定痫丸(汤剂)的抗痫疗效和机理。
方法：
长期予大鼠喂饲高脂饲料及热性中药稀释煎液,并以匹罗卡品致痫,以制作阳痫型癫痫大鼠模型。以治疗阳痫的代表方黄连解毒汤合定痫丸(汤剂)治疗阳痫型癫痫大鼠,同时以治疗阴痫的代表方减味五生饮合二陈汤为对照,记录癫痫发作潜伏期和到达SE时间；HPLC法检测脑内氨基酸类神经递质；HE染色切片观察病理变化；免疫组化技术半定量分析caspase-3表达情况,以评价建模质量及黄连解毒汤合定痫丸(汤剂)的抗痫疗效和机制。
结果：
与正常对照组比较,各组致痫大鼠脑内Glu、Asp、GABA、Gly、Ala等氨基酸类神经递质浓度均下降,caspase-3表达量均上升,其中阳痫中药组Asp、Ala下降幅度及caspase-3上升幅度在各组中最小,阴痫中药对照组各种氨基酸类神经递质浓度下降及caspase-3上升幅度最大,但均无统计学意义。各致痫组大鼠癫痫发作潜伏期和到达SE时间比较均无统计学意义。各组致痫大鼠大脑皮质、海马、肺、肝、肾等组织均出现较严重损伤,但组间比较无明显差异。
结论：
本实验初步建立了阳痫型癫痫大鼠模型。黄连解毒汤合定痫丸(汤剂)可抑制阳痫型癫痫大鼠脑内Glu、Asp、GABA、Gly、Ala等氨基酸类神经递质的下降及caspase-3的表达,提示其可能对阳痫型癫痫大鼠有一定的治疗作用。减味五生饮合二陈汤可促进阳痫型癫痫大鼠脑内Glu、Asp、GABA、Gly、Ala等氨基酸类神经递质的下降及caspase-3的表达,提示其可能会加重阳痫型癫痫大鼠的病情。
第二节细胞培养实验研究
一、实验一：谷氨酸损伤PC12细胞模型的建立实验研究
目的：
探讨Glu损伤PC12细胞模型的建立方法。
方法：
以不同浓度的Glu作用PC12细胞,分别于24小时、48小时和72小时三个时间点观察细胞形态学；MTT法检测细胞活力,以确立Glu对PC12细胞活力影响的量效及量时关系,探讨合适的造模时间及浓度。
结果：
细胞形态的观察结果显示,在同一时间点中,自5mMGlu组开始,随Glu浓度的升高,细胞折光性及立体感逐渐下降,轮廓逐渐模糊,生长逐渐稀疏,但不同时间点同一Glu浓度组的细胞形态未发现有明显差别。
细胞活力检测结果显示,与正常对照组比较,24小时、48小时和72小时三个时间点均自Glu浓度为5mM开始,随着浓度的逐渐升高,细胞活力逐渐下降。除40mM组外,正常对照组和各不同浓度的Glu模型组细胞随着培养时间的延长,细胞活力逐渐增加,即Glu对PC12细胞的活力存在剂量、时间依赖作用：细胞活力随Glu浓度的递增而下降,随培养时间的延长而上升。浓度为10mmol/L以上的Glu处理PC12细胞,在24小时、48小时和72小时三个时间点,其细胞活力与正常对照组比较均显著降低(P<0.01)。
结论：
Glu对PC12细胞的作用呈剂量和时间依赖关系：细胞活力随Glu浓度增高而降低,随培养时间延长而增强。10mmol/L的Glu作用PC12细胞24小时可能是建立Glu损伤PC12细胞模型较理想的实验条件。
二、实验二：减味五生饮合二陈汤含药血清对谷氨酸损伤PC12细胞治疗作用的实验研究
目的：
在细胞水平上进一步探讨减味五生饮合二陈汤的抗痫疗效和机理。
方法：
将实验细胞随机分为6组,分别是正常对照组、Glu损伤模型组、正常大鼠血清组、寒痰型大鼠血清组、阴痫中药对照血清组、阴痫中药血清组。以长期喂饲高脂饲料及寒性中药稀释煎液的方法建立“寒痰”型体质大鼠,并以这些大鼠制作的减味五生饮合二陈汤含药血清处理Glu损伤PC12细胞模型,同时以黄连解毒汤合定痫丸(汤剂)含药血清为对照。MTT法检测细胞活力；流式细胞技术检测细胞内游离Ca2+浓度。
结果：
与正常对照组比较,各组细胞活力均下降,其中以阴痫中药血清组下降幅度最小,阴痫中药对照血清组次之,除阴痫中药血清组外,均有显著差异(P<0.01)。阴痫中药血清组与阴痫中药对照血清组比较亦有统计学意义(P<0.05)。
与正常对照组比较,除G1u损伤模型组细胞内游离Ca2+浓度升高外,各组均下降,以阴痫中药血清组最低,阴痫中药对照血清组次之。其中,Glu损伤模型组、阴痫中药对照血清组、阴痫中药血清组与正常对照组比较均有显著差异(P<0.01)。阴痫中药血清组与中阴痫中药对照血清组比较亦有显著差异(P<0.01)。
结论：
以“寒痰”型体质大鼠制作的减味五生饮合二陈汤含药血清及黄连解毒汤合定痫丸含药血清均能提高Glu损伤PC12细胞活力及减少Ca2+内流,提示上述方剂的含药血清可能均有治疗Glu损伤PC12细胞的作用,其中可能以减味五生饮合二陈汤含药血清疗效较佳。
三、实验三：黄连解毒汤合定痛丸(汤剂)含药血清对谷氨酸损伤PC12细胞治疗作用的实验研究
目的：
在细胞水平上进一步探讨黄连解毒汤合定痫丸(汤剂)的抗痫疗效和机理。
方法：
将实验细胞随机分为6组,分别是正常对照组、Glu损伤模型组、正常大鼠血清组、痰热型大鼠血清组、阳痫中药对照血清组、阳痫中药血清组。以长期喂饲高脂饲料及热性中药稀释煎液的方法建立“痰热”型体质大鼠,并以之制作的黄连解毒汤合定痫丸(汤剂)含药血清处理Glu损伤PC12细胞模型,同时以减味五生饮合二陈汤含药血清为对照。MTT法检测细胞活力；流式细胞技术检测细胞内游离Ca2+浓度。
结果：
与正常对照组比较,各组细胞活力均下降,其中以阳痫中药对照血清组下降幅度最小,阳痫中药血清组次之。各组与正常对照组比较,均有显著差异(P<0.01)。阳痫中药对照血清组与阳痫中药血清组比较亦有显著差异(P<0.01)。
与正常对照组比较,除G1u损伤模型组外,各组细胞内游离Ca2+浓度均下降,痰热型大鼠血清组最低,阳痫中药血清组次之,阳痫中药对照血清组再次之。除阳痫中药对照血清组外,痰热型大鼠血清组、阳痫中药血清组与正常对照组比较均有统计学意义(P<0.05)。阳痫中药对照血清组与阳痫中药血清组比较无统计学意义。
结论：
以“痰热”型体质大鼠制作的黄连解毒汤合定痫丸含药血清和减味五生饮合二陈汤含药血清均能提高Glu损伤PC12细胞活力,其中黄连解毒汤合定痫丸含药血清尚能减少Ca2+内流,提示上述方剂的含药血清均可能对Glu损伤PC12细胞有一定治疗,但黄连解毒汤合定痫丸含药血清与减味五生饮合二陈汤含药血清疗效高低尚需进一步研究。
The therapy based on yin and yang differentiation is a kind of traditional therapy in Chinese medicine (CM) which treats epilepsy attacking frequently under two conditions that are yinxian and yangxian, one of approaches of classification of epilepsy from the respect of CM on the basis of different pathogenesis between cold and heat property in terms of CM. This therapy originated from the suggestion of Chao Yuanfang in the Yuan era. Then Wushengying was put forward to treating yinxian in Shi Yi De Xiao Fang writed by Wei Yilin in the Yuan period. And Dingxianwan was recommended to treat yangxian in Yi Xue Xin Wu authored by Cheng Guopeng in the qing dynasty. It is advisable to treat yinxian in wushengyin (WSY) and Erchentang (ECT), while yangxian in huanglianjiedutang (HLJDT) and dingxianwan (DXW) in the teaching material of Internal of Medicine of CM. However, clinical appliance for these decoctions is little, the reason for which may result from their inclusion of certain toxic Chinese medicines like Rhizoma Arisaematis Erubescentis, Rhizoma Pinelliae, Rhizoma Typhonii Gigantei, all of which have not been processed, and Cinnabaris. Besides, domestic researches on anti-epileptic effects and mechanisms of Chinese medicine are carried on by pathological animal models of western medicine (WM) nowadays, and animal models characterized by CM differentiation of symptoms and signs, which is named zheng hou in traditional Chinese medicine, have not been established. From what had been discussed above, we designed and undertook this program which was consisted of two parts that were clinical trial and empirical study which included the animal experiment and the cell culture experiment. And the main aim was to explore the effects, safety, and mechanisms of the therapy based on yin and yang differentiation, and set up epileptic animal models which were yinxian and yangxian characterized by zheng hou of CM.
Part1Clinic Trial of The Therapy Based on Yin and Yang Differentiation
Objective
To observe clinical effects and safety of the therapy based on yin and yang differentiation in epilepsy attacking frequently.
Methods
Patients who suffered from epileptic seizure more than three times were randomly divided into the treatment group and the control group. The control group only received conventional AEDs, while the treatment group received conventional AEDs, Chinese herbal drugs that were JWWSY and ECT, or HLJDT and DXW on the basis of the therapy based on yin and yang differentiation, and intravenous drip of xingnaojing parenteral solution as well. Therapeutic effects were measured by the decrease in seizure rate between prior and post-treatment, zheng hou evaluation, average total dosages of different AEDs, NHS3, MBI, and MRS respectively.
Results
There were12cases who participated in this trial, and11of them completed the program, of which5were in the control group, and6were in the treatment group, while1lost due to sudden death. Although most of parameters like the decrease in seizure rate between prior and post-treatment, average total dosages of different AEDs, NHS3, MBI, MRS, and etc we measured were no statistical difference between the two groups. In terms of evaluation of zheng hou, the control group adepted in improving asthenia(P<0.05), while the treatment group was effective at ameliorating wind, asthenia, renal yin deficiency, phlegm, and spleen energy deficiency(P<0.05), especially at phlegm and spleen energy deficiency (P<0.01). There was no side effect in all the cases in the treatment group.
Conclusion
It is superior for the therapy based on yin and yang differentiation in terms of improvement in relative symptoms and signs of zheng hou of patients suffering from epilepsy attacking frequently, especially in zhenghou of phlegm and spleen energy deficiency, and no side effect was observed.
Part2Experimental Research of The Therapy Based on Yin and Yang Differentiation
Section1Animal Research
Experiment1Research on Effects of JWSY and ECT on Yinxian Epileptic Rats
Objective
To explore anti-epileptic effects and mechanisms of JWWSY and ECT, and establish yinxian epileptic rat model.
Methods
The yinxian epileptic rat model was made by the method of feeding on high fat diet and CM in cold property for a long time, and then the being induced seizure by PILO. Yinxian epileptic rats induced by PILO were treated with JWWSY and ECT, the representative recipe for treatment of yinxian, while HLJDT and DXW, the representative recipe for treatment of yangxian, was used as the control formula. Time spots of seizure latency period and seizure status were record, and concentrations of amino acid neurotransmitters were measured by HPLC method, and the expression of caspase-3was determined by immunohistochemisty technology, and pathological changes were observed by hematoxylin and eosin stain. All of indexes mentioned above were adopted to evaluate the anti-epileptic effects and mechanisms of JWWSY and ECT, and assess the quality of epileptic animal model characterized by zheng hou.
Results
All kinds of concentrations of amino acid neurotransmitters measured in this experiment were lower, and the expression of caspase-3was higher in all the epileptic rats, in all of which the variation of yinxian CM group was the least, and the yinxian CM control group was the second, when compared with normal control group. However, there was no statistical difference in concentrations of amino acid neurotransmitters of Glu, Asp, GABA, Gly, and Ala, and in the expression of caspase-3between epileptic rats and normal control ones. Also, There was no recognizable difference in time spots of seizure latency period and seizure status among epileptic groups. And the tissue of the cerebral cortex and hippocampus, lung, liver, and kidney of all epileptic rats was injured severely, but there was no significant difference in all epileptic groups.
Conclusion
JWWSY and ECT, as well as HLJDT and DXW can inhibit the declination of different sorts of amino acid neurotransmitters, including Glu, Asp, GABA, Gly, and Ala, and apoptosis in the central nervous system, and effects of JWWSY and ECT on which are better, which indicates that such decoctions may have certain effects in treating the yinxian epileptic rats induced by PILO, and JWWSY and ECT seems to be superior. Yinxian epileptic rat model was set up in this experiment initially.
Experiment2Research on Effects of HLJDT and DXW on Yangxian Epileptic Rats Objective
To explore anti-epileptic effects and mechanisms of HLJDT and DXW, and establish yangxian epileptic rat model.
Methods
The yangxian epileptic rat model was made by the method of feeding on high fat diet and CM in heat property for a long time, and then being induced seizure by PILO. Yangxian epileptic rats induced by PILO were treated with HLJDT and DXW, the representative recipe for treatment of yangxian, while JWWSY and ECT, the representative recipe for treatment of yinxian, was used as the control formula. Time spots of seizure latency period and seizure status were record, and concentrations of amino acid neurotransmitters were measured by HPLC method, and the expression of caspase-3was determined by immunohistochemisty technology, and pathological changes were observed by hematoxylin and eosin stain. All of indexes mentioned above were adopted to evaluate the anti-epileptic effects and mechanisms of HLJDT and DXW, and assess the quality of epileptic animal model characterized by zheng hou.
Results
All kinds of concentrations of amino acid neurotransmitters measured in this experiment were lower, and the expression of caspase-3was higher in all the epileptic rats, when compared with normal control group. In all of epileptic groups, the variation of amino acid neurotransmitters including Asp and Ala and the expression of caspase-3of yangxian CM group was the least. In contrast, the variation of the indexes concerned of yangxian CM control group was the biggest. However, there was no statistical difference in concentrations of amino acid neurotransmitters of Glu, Asp, GABA, Gly, and Ala, and in the expression of caspase-3between epileptic rats and normal control ones. And there was no meaningful difference in time spots of seizure latency period and seizure status among epileptic groups. The tissue of the cerebral cortex and hippocampus, lung, liver, and kidney of all epileptic rats was injured severely, but there was also no significant difference in all of the epileptic groups.
Conclusion
HLJDT and DXW can inhibit the declination of different sorts of amino acid neurotransmitters, including Glu, Asp, GABA, Gly, and Ala, and apoptosis in the central nervous system, which shows that such decoctions may have certain effects in treating the yangxian epileptic rats induced by PILO. The yangxian epileptic rats model was established initially in this experiment.
Section2Cell Culture Research
Experiment1Research on Establishment of PC12Cell Trauma Model Induced by Glutamate Objective
To explore a proper methods to establish PC12cell trauma model for the following experiments.
Methods
PC12cells were treated with various concentrations of Glu, and cell morphology and viability were measured by MTT assay at24h,48h, and72h respectively so that the concentration-effect and concentration-time relationship between the effects of Glu neurotoxicity and the viability of PC12cells was investigated.
Results
The results of the observation of cell morphology indicated that the cell reflection, dimensionality, sketch, and the growth condition become bad to worse step by step as the increasing concentrations of Glu at the same time spot from5mM Glu group, while there was on obvious difference in cell morphology at different time spots comparison. The outcomes of the vitality of cells manifested that the cell vitality declined gradually as the increase of the concentration of Glu from5mM at24h,48h, and72h time spots. However, the vitality of cells in normal control group and trauma model groups induced by Glu in different concentrations. The relationship between effects of Glu neurotoxicity and viability of PC12cells was in manner of concentration and time-dependence:the higher the concentration of Glu is, the lower the viability of PC12cells becomes;the longer the incubation time is, the higher the viability of PC12cells becomes. There was significant difference in the cell vitality between PC12cells administered by Glu in the concentration of10mmol/L and in the normal control group.
Conclusion
The relationship between effects of Glu neurotoxicity and viability of PC12cells was in manner of concentration and time-dependence:the higher the concentration of Glu is, the lower the viability of PC12cells becomes;the longer the incubation time is, the higher the viability of PC12cells becomes. PC12cells administered by Glu neurotoxicity in the concentration of10mmol/L for24h maybe the ideal conditions for the following experiment.
Experiment2Therapeutic effects of JWSY and ECT drug-containing serum on PC12cell trauma model induced by Glu neurotoxicity
Objective
To move forward a single step to explore the therapeutic action and mechanisms of anti-epileptic effects of JWWSY and ECT at the cellular level.
Methods
The experimental cells were randomly devided into six groups, which were the normal control group, the Glu trauma model group, the normal rat serum group, the cold phlegm rat group, the yinxian CM control group, and the yinxian CM group. The PC12cell trauma models induced by Glu neurotoxicity were treated in JWWSY and ECT drug-containing serum made by rats with body constitution in cold phlegm, which were made by the method of feeding on high fat diet and CM in cold property for a long time, while HLJDT and DXW drug-containing serum was used as the control formula. The MTT assay was utilized to detect the viability of cells, and flow cytometry was adopted to assess the concentration of intra-cellular calcium.
Results
The cell viability of all groups administrated by Glu declined, in all of which the yinxian CM serum group was the least, and the yinxian CM control serum group was the second, when compared with the normal control group. There was a significant difference between groups administrated by Glu and the normal control group (P<0.01), except the yinxian CM serum group. And there was a meaningful difference between the yinxian CM serum group and the yinxian CM control serum group as well (P<0.01).
Except the Glu trauma model group, the concentrations of intra-cellular calcium of all groups administrated by Glu decreased, in all of which the yinxian CM serum group was the most, and the yinxian CM control serum group was the second, when compared with the正常对照group. Both the yinxian CM control serum group and the yinxian CM serum group had a recognizable difference in contrast to the正常对照group(P<0.01). And there also was a noticeable difference between the yinxian CM control serum group and the yinxian CM serum group(P<0.01).
Conclusion
JWWSY and ECT, as well as HLJDT and DXW drug-containing serum made by rats with body constitution in cold phlegm can increase the vitality and inhabit internal flow of calcium of the PC12cell trauma models induced by Glu neurotoxicity, which indicates that the drug-containing serum of such decoctions, especially JWWSY and ECT drug-containing serum, may have certain therapeutic effects in treating the PC12cell trauma models.
Experiment3Therapeutic effects of HLJDT and DXW drug-containing serum on PC12cell trauma model induced by Glu neurotoxicity Objective
To further explore the therapeutic action and mechanisms of anti-epileptic effects of HLJDT and DXW at the cellular level.
Methods
The experimental cells were randomly devided into six groups, which were the normal control group, the Glu trauma model group, the normal rat serum group, the heat phlegm rat serum group, the yangxian CM control serum group, and the yangxian CM serum group. The PC12cell trauma models induced by Glu neurotoxicity were treated in HLJDT and DXW drug-containing serum made by rats with body constitution in heat phlegm, which were made by the method of feeding on high fat diet and CM in heat property for a long time while JWWSY and ECT drug-containing serum was used as the control formula. The MTT assay was utilized to detect the viability of cells, and flow cytometry was adopted to assess the concentration of intra-cellular calcium.
Results
The cell viability of all groups administrated by Glu declined, in all of which the range in declination of the yangxian CM control serum group was the least, and the yangxian CM serum group was the second, when compared with the normal control group. There was a significant difference between groups administrated by Glu and the normal control group(P<0.01). And there was a meaningful difference between yangxian CM serum group and the yangxian CM control serum group as well (P<0.01).
Except the Glu trauma model group, the concentrations of intra-cellular calcium of all groups administrated by Glu decreased, in all of which, the range of the heat phlegm rat serum group was the most, the yangxian CM serum group was the second, and the yangxian CM control serum group was the last, when compared with the normal control group. And except the yangxian CM control serum group, both the heat phlegm rat serum group and the yangxian CM serum group had a statistical difference in contrast to the normal control group(P <0.05), while there was no noticeable difference between the yangxian CM control serum group and the yangxian CM serum group.
Conclusion
HLJDT and DXW, as well as JWWSY and ECT drug-containing serum made by rats with body constitution in heat phlegm can increase the vitality of the PC12cell trauma models induced by Glu neurotoxicity, and the HLJDT and DXW drug-containing serum can also inhabit the internal flow of calcium, which manifests that there seems to be certain the therapeutic effects for the drug-containing serum of such decoctions in treating the PC12cell trauma models, but which decoction, the HLJDT and DXW, or JWWSY and ECT drug-containing serum of yangxian pre-seizure rats,is better needs further exploration.
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