| I .The foundation of theoryEpilepsy is a kind of common diseases in the nervous system. Epilepsy is a kind of chronic disorders in pars enciphalica caused by various etiological factors, which is characterized by sudden and repeated and temporal malfunction of central nervous system led by excess discharges of neurons. According to encroached neuron's location and discharges' suffused range, this malfunction can be divided into the disorder of one or more aspects such as movement, sense, consciousness, behavior and autonomic nerves, and so on. Epilepsy, as one of the ten big world-wide medical problems, has not still a unite recognition in pathogenesis and development process up to now. therefore, there are rather a few patients existing treatment difficulty or unsatisfactory curative effect, which not only does seriously harm to the patient's physical and mental health but also brings heavy burden to families and societies.To go in for fundamental research in epilepsy, it is essential to establish good animal model.PTZ-induced epileptic rat model is a kind of ideal one for generalized tonic-clonic seizures, whie PTZ has no special nervous poison itself not accompanying by damage and necrosis of nerones. Therefore PTZ has been generally used in epileptic etiopathogenesis and selection of AEDs.Epileptic discharges traced by electroencephalogram(EEG) are unique objective evidences to diagnose epilepsy, which play a important assistant role in the diagnosisof epilepsy. With the application and improvement of all kinds of epileptic EEG's provocation test,the epileptic diagnosis rate has risen from 50% to 80%. In recent years, the application of active Video EEG recording system and Video EEG monitoring system had made epileptic diagnosis more accurate. Intracal electrodes EEG, which directly traces electrical activities of brain by the electrodes in the surface or deep part of cerebral cortex, has better space resolution and anti-interference and anti-artifact, and can detect electrophysiogy activities in the deep part of the cerebra, which contributes to the research of epileptic electrophysiogy activities.At present it still is unclear of the epileptic pathogenesis. In recent years, wih the rapid development of molecular biology technique and its constant pervasion into neurology, large amounts of researches indicated that epileptic occurrence is closely related with many controlling genes in the encephalon. Epileptic seizure is the result of excessive synchronization led by the increase of neuronal excitability. While immediate early gene such as c-fos and c-jun, which are expressed actively in the during of neuronal activity, has been generally used as the marker of neuronal activity and been used in epileptic research. The changes of fos protein during epileptogenesis represent neuronal emotional state and produce a marked effect on neuronal apoptosis and the changes of synaptic plasticity.hippocampus sclerosis is the most attractive patho-change in temporal epilepsy since this change is closely related with neuronal apoptosis during epileptic seizure. Both bcl-2 and bax ,as a pair of positive and negative apoptosis genes, can been expressed in central and peripheral nervous system. Bcl-2 gene can repress the apoptosis caused by multisystem and multi-damage and multi-stimulationprlong corpuscular duration, while the expression of bcl-2 can remarkably repress the apoptosis induced by the increase of peroxide and free radical. Bax protein can be combined with bcl-2 and be changed into homodimier, thus bax can make bcl-2 inactive and indirectly promote the apoptosis. Bax also canbe changed into homodimier through itself and directly promote the apoptosis. The ratio of bcl-2/bax not only determine the cell's susceptibility to apoptosis stimulation but also determine the cell's survival. The descent of the ratio means to promote the apoptosis and the ascent of the one means to repress the apoptosis. Therefore, it is very significant for revealing the pathogenesis of epilepsy from gene level and treatment to find out the function and regulation mechanism of related genes which influence the occurrence and development of epilepsy.The majorest therapy of epilepsy is to control seizure. The latter-day AEDs can control about 80% patients' seizure, but all kinds of the chemical drugs in common uses have noxious and side effects. The medicine of epilepsy Chinese Herbal Medicine is a great treasury. The Chinese Herbal Medicine of treating epilepsy is worth being studied because of their predominance of the undoubted curative effect and the lack of noxious and side effects.It has a centuries-old history using Chinese Herbal Medicine to treat epilepsy. For many years doctors treated epilepsy from Feng and Tan, but the effects were not ideal. It is the innovation in theory to use Chaihushugantang(CHSGT) treating epilepsy. In recent years Professor Chen Baotian got some new cognitions about the reason of epilepsy, he considered that the reason was the angry and gloomy of liver and that we should treat the epilepsy from liver. Professor Chen founded the CHSGT, which made up of Chaihu, Guizhi, Shenglonggu, Shengmuli, Chuanxiong, Danggui, Shengdi, Baishao, Banxia, Huangqin,Dangshen,Gouteng, Shengjiang, Dazao and Gancao, and he also got good results in useing the CHSGT to treat the refractory epilepsy. So we took some animal experiments to research about how the CHSGT treat epilepsy in order to get more scienti& proof to extend the usage^of th^CHSGT in clinic and to benefit the epilepsy patients.II. Research contentsl.To establish the mode of epileptic rats with PTZ-induced2.Effect of CHSGT on the EEG of the PTZ-induced epileptic rats3.Effect of CHSGT on the expression of c-fos in hippocampaus and frontal lobe cortex of the PTZ-induced epileptic rats4.Effect of CHSGT on the expression of bax and bcl-2 in hippocampus and frontal lobe cortex of the PTZ-induced epileptic ratsIII. Methods and Resultsl.To establish the model of epileptic rats induced by PTZ A dose of second- eclampsia PTZ(37.5mg/kg) was injected intraperitoneally into the rats every morning until the kindling criterion was reached. Once some rats occurred the lasting V-grade epileptic seizure that caused by injecting overdose of PTZ carelessly or the rat's higher drug sensitivity to PTZ in the course of making the model, rescued them by injection of 10% hydration chlorine aldehyde.80 rats were induced epilepsy by intraperitoneal inject PTZ continuously for four weeks, 60 rats were induced epilepsy successfully, 16 rats died. The achievement ratio of making the model is about 91%. Epileptic seizures and epileptic discharge of EEG can be observed in the epileptic rats. Rats would be in status epilepticus to die if the dose of PTZ injection were higher than eclampsia dosage (60mg/kg). 2. Effect of CHSGT on the EEG of the PTZ-induced epileptic rats Methods to dividing groups and taking medicine: There were seven groups in the experiment. The 48 PTZ-induced epileptic rats were randomly divided into six groups as following, epileptic model group(II), Valproate(VPA) group(III), Dingxianwan(DXW) group(IV), low-dose CHSGT group(V), moderate-dose CHSGT group(VI) and high-dose CHSGT group(VII). There was a normal group(I) in addition. Eeach group were given intragastric administration with drugs continuously for 5 weeks. The standard dosage of drugs showed as following: VPA's dosage is175mg/kg, DXW's dosage is 1.2g/kg, low-dosage, moderate-dosage, and high-dosage of CHSGT is lg/kg, 2g/kg, 4g/kg respectively. The software SPSS 12.0 was adopted to do statistical analysis. Methods to the EEG seizure detection: Imbedding the frontal lobe cortical electrode and the hippocampal electrode into the brain of rats, then detecting the EEG after one day. The EEG detection system using the electroencephalograph (type 4218) of NIHON KOHDE Company, set nose as the reference electrode. Calibrating: lOuv/mm, paper speed: 3cm/s, wave smoothing: 70Hz.The cortex of frontal lobe and hippocampal EEG of the normal group were mainly composed of the 9-12Hz a wave and the 17-19Hz 6 wave, which were called the basic wave. The voltage of wave ranges from 20uv to 80uv. Epileptic waves such as spike and sharp wave were not observed. There observed the paroxysmal epileptic discharge in the cortex of frontal lobe and hippocampal EEG of rats in model group, shown as high-potential spike waves and sharp waves which voltage ranges from 120uv to 250uv. While the epileptic discharge couldn't be observed in the EEG of rats in middle-dose group and high-dose group of CHSGT, VPA group and DXW group, in which only the a wave and B wave can be observed. The difference was significant compared with model group (P<0.001). But the low-dose CHSGT group had epileptic discharge, shown as the spike wave and sharp wave, and the epileptic discharge was more obviously in hippocampus than in cortex. The difference is significant compared with normal group (P<0.001), and there is no obvious difference compared with model group.3.Effect of CHSGT on the expression of c-fos in hippocampaus and frontal lobecortex of the PTZ-induced epileptic ratsPrepared paraffin sections of hippocampus and frontal lobe cortex, then stained paraffin sections by the immunohistochemical ABC method. The quantitative analysis of experiment result was carried out according to the OD value of c-fos stained cells. The expression of c-fos in hippocampus and frontal lobe cortex of the PTZ-induced epileptic rats were significantly increased. Treating with middle-dose and high-doseof CHSGT and VPA and DXW could decrease the expression of c-fos in the hippocampus and the frontal lobe cortex of epileptic rats. The difference was significant compared with model group(P<0.001). The expression of c-fos in the hippocampus and the frontal lobe cortex of epileptic rats in the low-lose group of CHSGT was not decreased obviously. It showed that CHSGT was able to decrease the expression of c-fos in epileptic rats and had good antiepileptic effects4. Effect of CHSGT on the expression of bax and bcl-2 in hippocampaus and frontal lobe cortex of the PTZ-induced epileptic ratsNerve cells in hippocampus and frontal lobe cortex of both normal rats and epileptic rats existed apoptosis, but the latter was more obviously. The expression of bax in hippocampus and frontal lobe cortex of epileptic rats increased evidently. Treating with middle-dose and high-dose of CHSGT and VPA and DXW could decrease the expression of bax, while increased the expression of bcl-2 and the ratio of bcl-2/bax. The difference was significant compared with model group(P<0.001). The percentage of apoptosis cells in lower-dose of CHSGT treated epileptic rats were not decreased obviously, the ratio of bcl-2/bax was not increased obviously too. There was no significant difference compared with model group. Low-dose group of CHSGT had no good therapeutic efficacy on epileptic rats, that may be related with the underdosage of drugs.IV. Conclusionl.PTZ of second convulsion dose(37.5mg/kg) can induce successfully epileptic rats and achievement ratio of epileptic animal models reach to 91%, for all of who epileptic seizure can be observed and stable epileptic discharge also can be traced.2.A11 groups including middle-dose and high-dose group of CHSGT and VPA and DXW have good therapeutic effect on epileptic rats and there is no significant difference between each of them, while each of them can repress successfullyepileptic seizure of epileptic rats and improve epileptic discharge. However, low-dose group of CHSGT can't do it, and hasn't good therapeutic effect on epileptic rats, that may be related with the underdosage of drugs.3.A11 groups including middle-dose group and high-dose group of CHSGT and VPA group and DXW group can lower the expression c-fos in the hippocampus and the frontal lobe cortex of the epileptic rats, but the action of low-lose group of CHSGT isn't significant. The antiepileptic mechanism of action of CHSGT can be related with lowering the expression of c-fos in brain.4.Both normal rats and epileptic rats exist the phenomena of neuronal apoptosis, but the latter is more manifest. The expression of bax in the hippocampus and the frontal lobe cortex of the epileptic rats increase evidently, while middle-dose group and high-dose group of CHSGT and VPA group and DXW group can lower the expression of bax and raise the ratio of bcl-2/bax and lower evidently the percentage of apoptosis on epileptic rats. Low-dose group of CHSGT have no clear influence on this. That CHSGT and VPA and DXW have good antiepileptic effect can be through lowering the percentage of neuronal apoptosis in epileptic rats and raising the ratio of bcl-2/bax.5.Both middle-doze group and high-doze group of CHSGT have good therapeutic effect on epileptic rats and there is no significant difference between them, but low-dose group have worse one on epileptic rats compared with the forward two groups and there is significant difference between them. All groups such as middle-dose and high-dose group of CHSGT and VAP group and DXW group have good therapeutic effect on epileptic rats and there is no significant difference among them.6.CHSGT have its own superiority to DXW in treatment of epilepsy.the application of CHSGT in treatment of epilepsy is a kind of innovation andbreakthrough in therapeutic theory on epilepsy. That CHSGT treats epilepsy is based on the epileptic fundamental pathogenesis and is to aim directly at "ben". However, that DXW treats epilepsy is based on pathological output such as sputum muddy and stagnant blood etc and is to aim at "biao". |
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