| BackgroundEpilepsy is a general term for many etiologies with high precision synchronization of cerebral neuron and self-limiting abnormal discharge. Epilepsy syndromes are paroxysmal, transient, repeatability and abnormal of central nervous system. Repeated epilepsy causes injuries not only to body but also to mind. The ratio of suicide by patients with epilepsy is3-5times higher than normal people. Incidence of epilepsy may occurred anywhere, anytime, therefore, it causes really heavy burden to patients’family. Epilepsy is not only medical problem but also a social event.The prevalence rates of epilepsy in developed countries, economic transition countries, developing countries and undeveloped countries are5.0‰、6.1‰、7.2‰、11.2%o finding in a1997report of WHO. There are50million patiences with epilepsy approximately all around the world. In China, the morbidity of epilepsy is290per million each year which means there are additional0.35million epilepsy patients each year. The prevalence is between3.6‰-7‰. There are more than9millions patiences in China. Mortality caused by epilepsy is24.2-78.2per million.20%die of continuous epilepsy,40.21%die of accidents,5.51%die of suicide,4.13%die of unknown cause.The control of epilepsy is still dominated by chemical drugs, about70%of patients could obtain complete remission by drug therapy, with poor safety of long-term use of anti-epileptic drugs,however,about33.5%of patients taking antiepileptic drugs appear all sorts of side effects,about25.3%of adult patients and13.4%of children patients which appear drug resistance become intractable epilepsy. Surgical operations are often applied to some of the treatment of intractable epilepsy, but which due to the high risk, high cost and existing postoperative recurrence,their use are limited, therefore the treatment of epilepsy is still dominated by drugs, which need to explore a new safety antiepileptic drugs. Clinical practice of traditional Chinese medicine for thousands of years have proved:treatment of epilepsy has a low toxicity and side effects the characteristics of traditional Chinese medicine, which bring new opportunities to the development of new antiepileptic.Epilepsy belongs to Chinese areas of the epilepsy disease, the pathogenesis of epilepsy is related to the liver disfunction. Clinical studies have shown that treatmeant of epilepsy from the liver and the use of Chaihu Shugan Tang in treatment of refractory epilepsy achieved good effects, and could decrease the epileptic seizure of intractable epilepsy rats and inhibit the expression of multi-drug resistance protein(P-GP).Bupleurum,the usage amount of which is the biggest in Chaihu Shugan Tang, tastes pungent and bitter,slight cold, belongs to liver and guts channel.It can relieve superficies,soothe the liver and disperse the depression, invigorate splenic yang and prevent malaria. Our early research showed that bupleurum and saikosaponins had sedative and antiepileptic effect, while saikosaponin a is a main constituents of saikosaponins.It had been confirmed that saikosaponin a could reduce blood lipid, anti-seepage, anti-granuloma, protection liver cell, immune regulation and antibacterial.our early research showed that SSa could effectively inhibit the hippocampal astrocytes from decreasing Ca2+and secreting IL-6activated by glutamate,inhibit the high release of TNF-α and expression of TNFR1in cultured hippocampal astrocytes induced by pentylenetetrazol,and found that SSa could ruduce the number of seizure,the average time of seizure,the level of seizure,impacting the EEG,and down-regulate the expression of P-GP in hippocampus area in intractable epilepsy rats by building the pilocarpine-induced chronic models of intractable seizure models,however,whether SSa has good anticonvulsant effect is unknown. It’s a main way for the study of neurology to screen the antiepileptic drugs with the help of using animal models.In this study, four kinds of epilepsy mouse models were used to evaluate the anticonvulsant effect of SSa:①The Maximal electroshock model is currently the most effective model for screening the drugs against tonic-clonic drugs;②Pentylenetrazol induced models:pentylenetetrazol is the GABA receptor antagonist, it is generally believed that one can significantly against pentylenetetrazol induced convulsions can often against clinical petit mal;③Strychnine induced models:strychnine is the inhibitory Neurotransmitter glycine antagonist in the central nervous system, the model is a good model of refractory epilepsy;④Pilocarpine induced models:pilocarpine is the muscarinic cholinergic receptor agonist, administration systematically can cause status epilepticus, now used to build refractory epilepsy model. we use the locomotor activity test and the threshold dose of pentobarbital sodium induced sleep experiment to evaluate the effect of sedative and hypnotic.PurposeThere had been established four models in mice,including maximal electroshock seizures (MES),pentylenetetrazol,strychnine and pilocarpine induced seizure models, in order to evaluate anticonvulsant effect of SSa multi-angle and scientifically and sedative and hypnotic effect of SSa through locomotor activity experiment and pentobarbital-induced sleep test in the threshold range,and attempt to infer the mechanism of anticonvulsant potentially, providing experimental foundation for the clinical therapy of epilepsy.Methods1.Effect of SSa on Maximal electroshock seizures (MES) models in miceThe day before experiment, fifty qualified mice that were selected by screening were randomly divided into five groups:the control group,the diazepam(4mg/kg) group,saikosaponin a high-dose(4.36mg/kg),middle-dose(2.18mg/kg),low-dose(1.09mg/kg)group.5%Tween-80solution of equal volume was injected into intraperitoneal(i.p.) in control group dealt with electrical stimulation after sixty minutes. In the light of stated method in the literature, MES experiment is advanced by using experimental spectrometer (YSD-4G) of pharmacological and physiological in a single stimulation way, stimulation parameter for100V,2Hz, tonic hind limb extension was considered as convulsions target, the number of convulsions,the duration of tonic hind limb extension and convulsions rate would be recorded and calculated.2.Effect of SSa on pentylenetetrzol-induced convulsion models in miceGrouping and the way of administration were the same as MES model, mice were i.p. injected pentylenetetrzol (85mg/kg) after administration of5%tween-80,diazepam,SSa of different doses for sixty minutes, and the latency of convulsions including the duration from administration to seizures of grade Ⅰ-Ⅲ (the latency of clonic convulsions) and the duration from administration to tonic convulsions(the latency of tonic convulsions) was recorded through continuous observation for thirty minutes, meanwhile the mortality rate of mouse is recorded and calculated within thirty minutes.3.Effect of SSa on strychnine-induced convulsion models in miceGrouping and the way of administration are the same as MES model, mice were i.p. injected strychnine (2.5mg/kg) after administration of5%tween-80,diazepam,SSa of different doses for sixty minutes, the latency of convulsions,i.g. the duration from administration to seizures of grade Ⅰ-Ⅲ(the latency of clonic convulsions) and the mortality rate were separately recorded and calculated through continuous observation for fifteen minutes.4.Effect of SSa on pilocarpine-induced convulsion models in miceGrouping and the way of administration were the same as MES model, mice were i.p. injected pilocarpine(400mg/kg) after administration of5%tween-80,diazepam,SSa of different doses for sixty minutes, the latency of convulsions, i.g. the duration from adminstration to seizures of grade Ⅰ-Ⅲ (the latency of clonic convulsions) and the mortality rate were separately recorded and calculated through continuous observation for fifteen minutes. 5.Effect of SSa on locomotor activity test in miceGrouping and the way of administration were the same as MES model, mice were put into the active box to adapt for five minutes before administration, recording time while administrating, walking time and the number of lifting of upper limbs in mice were needed to record within two minutes.6.Effect of SSa on pentobarbital sodium-induced sleep in the threshold range in miceGrouping and the way of administration were the same as MES model. After administration for sixty minutes,the mice of each group were treated with pentobarbital sodium(45mg/kg).Animals were observed after administration whether less activity,quiet,sleepiness and sleeping will appear.Sleeping were defined as the disappearance of righting reflex as indicator, the latency(the disappearance of righting reflex) and the duration(from disappearance of righting reflex to restoration) of sleep should be observed and recorded.7.Statistical analysisThe data were statistically analyzed by using SPSS13.0software, One-Way ANOVA was applied to the compare of mean among several samples, Tukey was used if multiple comparision among groups was in keeping with homogeneity of variance,if not, Dunnett’ T3would be applied,chi-square test was utilized in the comparision of enumeration data and rate. The value of P<0.05was considered statistically significant.Result1.Effect of SSa on Maximal electroshock seizures (MES) models in miceCompared with the control group,different dose of SSa could inhibit MES in different levels as a result of the occurrence of hind limbs extension of mice; compared with the control group,SSa(4.36mg/kg) exists statistically significant difference (P<0.05),the duration among groups of tonic hind limb extension had no significant difference.2.Effect of SSa on pentylenetetrzol-induced epilepsy models in miceCompared with the control group, the high and middle dose groups of SSa could significantly prolong the latency of clonic and tonic of pentylenetetrzol-induced convulsion as well as the diazepam group(P<0.01), the high group of SSa could reduce the mortality of mice (P<0.05).3.Effect of SSa on strychnine-induced epilepsy models in miceCompared with the control group, each dose group of SSa could not prolong the latency of clonic and death and also reduce the mortality of mice except diazepam on strychnine-induced seizures.4.Effect of SSa on pilocarpine-induced epilepsy models in miceCompared with the control group, the high-dose group of SSa and diazepam group prolonged the latency of clonic and death of pilocarpine-induced seizures(P<0.01), each group of SSa could not reduce the mortality of mice except diazepam group.5.Effect of SSa on locomotor activity test in miceCompared with the blank control group, the high and middle dose groups of SSa and diazepam group could significantly reduce walking time of mice(P<0.01).However, compared with the control group, the number of lifting of forelimb of mouse could obviously be reduced in the high and middle dose groups of SSa and diazepam group(P<0.05or P<0.01). Compared with the blank control group, the low-dose group can reduced walking time and the number of lifting of forelimbs,but the difference had no statistically significant(.P>0.05).6.Effect of SSa on pentobarbital sodium-induced sleep in the threshold range in miceCompared with the blank control group, the high and middle dose groups of SSa and diazepam group could significantly reduce the latency of sleep of mice(P<0.01). However, compared with the blank control group, the duration of sleep of mice was obviously increased in high and middle dose groups of SSa and diazepam group(P<0.01). Compared with the control group, the low-dose group of SSa could reduce the latency of sleep and increase the duration of sleep in mice, but the difference had no statistically significant(P>0.05).Conclusion1.Acute epilepsy models which were induced by maximal electroshock convulsions, pentylenetetrazol,strychnine and pilocarpine were successfully established, the technology of model of establishment is mature, the operation is relatively simple, it has a higher success rate and a certain representation.2.SSa could decrease the occurrence of tonic hind limb extension in different levels,and the high dose group had the best effect;SSa could prolong the latency of convulsions and tonic-clonic convulsions induced by pentylenetetrazol with a slightly dose-dependent manner,and the middle and high dose groups had better effect;high dose group of SSa could prolong the latency of convulsions and death induced by pilocarpine.3.SSa had significant sedative and hypnotic effects.It could decrease the walking time, the number of lifting of upper limbs,and the latency of sleep,increase the duration of sleep with a dose-dependent manner,middle and high dose group had a better effect.4.SSa could inhibit the maximal electric shock,pentylenetetrazol and pilocarpine-induced seizure models,indicating that it may be effective to against the general tonic-clonic seizures,absence seizures and part of the intractable epilepsy. |
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