The Interfering Effect And Mechanism Of Heterotherapy For Homopathy On Differernt Animal Models

Objective and significanceEpilepsy, a functional disturbance of the CNS and induced by abnormal electrical discharge, manifestes by recurrent seizure. The morbidity rate in developed countries is relatively lower than in developing countries.The morbidity rate in China is 3.5‰～4.8‰,and the disease incidence is 23/100,000 every year in China. According to the epidemiology statistics, at present the incidence of epilepsy patients erery year in China is approximately 300,000 to 400,000. In a word,the number of patients suffer from epilepsy in our country is enormous.Compare to these,50% or more patients in the country can not be treated normly.Further more,its prominent clinical characteristic has the unpredictability which repeatedly paroxysmic and each time manifests suddenly,which affected epilepsy sickness patient's life, the work and social interaction ability enormously, reduced patient's quality of life. It is thus clear that treating epilepsy patient is a difficult and important mission.At present we controlled epilepsy still by chemistry medicine primarily, commonly used antiepileptic like benzene proper UK sodium, the third propyl-acetic acid sodium, the card Mussey equality which could control 70%-80% epilepsy patient to manifest suddenly epileptic seizure.But the pharmacokinetics effects and the side effect for example fetal malformationas,cognitive effects and bone density effecta limited their application.Compare with traditional chemical medicine,some new antiepileptic drugs,such as Gabapentin,Lamotrigine and Oxcarbazepine,which is degraded the drug tolerance,appeared in the end of last century.But because of the expensive price,the application is limited.Regarding some difficult epilepsy, although may choose the surgical operation to treat,because its indication is limited, which has great risk, while the expense is expensive and recrudescence possibility, therefore the difficult epilepsy still by the pharmacological treatment primarily,so it needs to research and develop the new ant-iepileptic drugs. The traditional Chinese medicine has the good anti-epilepsy function, but its mechanism is not often explicit, obtaining from the single taste traditional Chinese medicine active constituent to study not can only be clear about the anti-epilepsy mechanism, but is also advantageous to promote modernization of traditional Chinese medicine.The pentetrazole(PTZ) convulsion model,the MES model and the vauqueline convulsion model are epilepsia major model used mostly and study transparently.It is clear that the drug which can prevent the epilepsia major made from the above animal model,can treat the epilepsia major patients in clinic.Pathologic change of chronicity PTZ kindling model,such as loss of neuron in hippocampus,the proliferation of glial cell, regeneration of neuron,and mossy fiber shaped proliferation,is approximate to the human temporal epilepsy.To some animal, repeatly epileptic seizure can lead to the change of synaptic plasticity,the cognition functional lesion and the tolerance of antiepilepsy drug. At present,PTZ kindling model is widely used in bolting antiepileptic drugs and research of epileptic mechanism.GABA,which is generated from glutamic acid decarboxylase,is the most important inhibitive neurotransmitter in CNS.GABA is released into synaptic cleft after combining in pro-synapse nerve terminal between GABA-energic interneuron,activing GABA receptor in post-synapse, opening Cl- channnels,thereby causing a rapid hyperpolarization,then producing depressive effect.It Is thus clear that much stage of GABA synapse function is correlated with epilepsy,such as GABA combining, GABA releasing, GABA transmitting,GABAR(A and B) activing.The drugs which can affect above conduction iteral have antipileptic function.Heterotherapy for homopathy is one of the most important rationale of traditional Chinese medicine.There are two main antiepileptic method:treating from sputum and treating from liver.The represent complex,Cha hu shu gan tang and Ding xian wan have fine therapeutic effect in clinic.While in the animal experiment,the therapeutic effect of the two complex need to be identified. So the experiment obserbes and compares the therapeutic effect of the two complex in three different acute epilepsy animal models,and obeserve the effect of inhibitive neurotransmitte conduction iteral in chronic PTZ kinding model.Method and content1. Making the cute PTZ convulsion rat model60 of wistar rats were divided into 6 groups,A,B,C,D E,and F group,and there were 10 rats in each group. All of rats in ach group were administered by intragastric administration 7 days at 8:00am and 20:00pm.The group of A was given equal volume of physiological saline,while the B was given VPA by 200mg/(kg·d),and the C,D,E are given Chai hu shu gan tang by different concentration, such 2.5g/(kg·d) and 5g/(kg·d) and 10g/(kg·d). The group of F was given Ding xian wan by 12g/(kg·d). After 1.5 hours of the last administration,all groups of rats were injected PTZ by peritoneal,according to 50mg/kg.And then observed the ethology change of the rats immediately in 30 min,recording the incubative stage of clonus and the number of rats in each group in different stroke grade.2.Embedding electrode and recording the EKG 21 of wistar rats,was divided into 7 groups evenly. All of rats in ach group were administered by intragastric administration 7 days at 8:00am and 20:00pm except G group,and the G group was given normal saline.The group of A was given equal volume of physiological saline,while the B was given VPA suspl by 200mg/(kg·d),and the C,D,E were given Chai hu shu gan tang by different concentration, such 2.5g/(kg·d) and 5g/(kg·d) and 10g/(kg·d). The group of F was given Ding xian wan by 12g/(kg·d).After 3 days every group of rats were anesthetized by 10% chloral hydrate,according to 350mg/kg,then fixed stainless steel electrode in hippocampus and cortex frontal lobe,thereby fixed by dental base acrylic resin powder and dentistry portland cement,then sutured scalp.One week later,recorded the EKG until 5 min.3.Making the MES model.Bolting sixty fit mouse according pharmacological multi-functional implement,and then dividing into 6 groups, The group of A was given equal volume of physiological saline,while the B was given VPA suspl by 400mg/(kg·d),and the C,D,E are given Chai hu shu gan tang by different concentration, such 3.75g/(kg·d) and 7.5g/(kg·d) and 15g/(kg·d). The group of F was given Ding xian wan by 20g/(kg·d). After 1.5 hours of the last administration,giving electric stimulation,4.Making the convulsive mouse model by vauquelineBolting sixty SPF grade KM mouse. The groups and the administration are the same as MES mouse model. After 1.5 hours of the last administration,all groups of rats were injected vauqueline by peritoneal, recording the number of convulsion and death,then calculating the rate of convulsion and death.5. Making of chronic PTZ kindling rat modelInjected PTZ according intraperitoneal until 3 weeks, then observed the incubation period of seizure and recorded seizure grade.The rats which convulsed at least 3 consecutive 4 or 5 times are kindling models. Selecting kindled rats convulsed between 15 and 21 days, stimulated again after 7 days, and the rats convulsed again could be thought to successed model. The group of A was normal,breeding at the same time with the other groups.The kindling rats were divided randomly into 4 groups,such as B,C,D and E.Every group was intragastrical 2 times a day for 4 weeks. Group of A and B was given equal volume saline,and group of C was given VPA solution by 200mg/(kg·d),while group of D was given Dingxianwan by 12g/(kg d),and group of E was gived Chaihushugantang.6.Detecting GAD65 content by Western blottingAfter 4 weeks, six rats in each group were randomly taken, separating the cortex and hippocampus. The expression of GAD65 andβ-actin was detected by way of Western blotting. We evaluated the expression of protein by analyzing the gray scale, and calculated the ratio of the value of gray scale of the protein with the value of gray scale ofβ-actin.7.Measuring the content of GABA by HPLCAfter 4 weeks, six rats in each group were randomly taken, separating the cortex and hippocampus. The content of GABA was detected by way of HPLC.8. The immunohistochemical staining of GABAARα1After 4 weeks, six rats in each group were randomly taken.Then seprated the brain and fixed the brain by PFA phosphate buffer.And prepared paraffin section following routine method(content cortex and hippocampus).We made the immunohistochemical staining of GABAARal by instruction.We counted the masculine cell in CA1 area and calculated the OD value.9. Statistical methodsThe data analysis and treatment was used the SPSS13.0 statistics software. The measurement data indicated by the mean value and standard deviation, and the result comparison of each group by three grade incubation period in PTZ acute convulsion model and wester blotting test uses the single factor variance analysis (One-way ANOVA), the multiple comparisons with the LSD law,P<0.05 expressed that the difference has statistics significance.The comparision of different seizure grade in each group uses the independent sampler non-parametric test.The comparison of rate uses chi square test.Result1.the effect of different groups on seizure incubation period and grade in PTZ acute convulsion model.The incubation period of PTZ acute convulsion could increase in the group of B, C, D, E, F (F=5.145, P=0.001), but the group of C, D, E and has no statistical differences. The three groups of Chaihushugantang had dose-effect relationship.2.the EEG of different groups in PTZ acute convulsion model.The EEG in group of G (normal group) was base wave,such asα,βwave, and scattered 0 wave, and had no obvious rhythm. The EEG in group of A had a large number of high amplitude sharp waves, spikes, spike-slow wave. The epileptic discharges in group of B and E was inhibited, and there was only a very small number of spikes or sharp waves. The EEG in group of C had more sharp waves, spikes, spike-slow wave, but the amplitude was lower than the model group. The EEG in group of D, F had scattered spikes or sharp waves and low amplitude.3.the effect of different groups in MES mouse model.The convulsion rate in differernt groups was statistically significant (X2=24.000, P=0.000), The convulsion rate in group of B, C, D, E and F was lower than in the group of A,.The convulsion rate in group of B was the lowest,and convulsion rate in the group of D, E was lower than in the group of F. And C, D, E group had close dose-effect relationship. 4. the effect of different groups on acute convulsion mouse model by strychnineThe convulsion and death rate in differernt groups was statistically significant (X2=15.173 and 15.250, P=0.001 and 0.009), The convulsion and death rate in group of E was the lowest,and the convulsion and death rate in the group of B, D, E,F was lower than in the group of A. And C, D, E group had close dose-effect relationship.5. the expression of GAD65 in the hippocampus measured by Western blotting.The expression of GAD65 in differernt groups was statistically significant (F=6.409, P=0.001).The expression of GAD65 in group of B was significant lower than in group of A,and there was statistically significant between the two groups (P< 0.01).The expression of GAD65 in group of C,D,E was higher than in the group of B(P<0.05).But there was no statistically significance between three groups.6. the content of GABA in hippocampus measured by HPLC.The content of GABA in hippocampus in differernt groups was statistically significant (F=5.736, P=0.002).The content of GABA in group of B was significant lower than in group of A,and there was statistically significant between the two groups (P<0.01).The content of GABA in group of C,D,E was higher than in the group of B(P<0.05).But there was no statistically significance between three groups.7.the results of immunohistochemistry of GABAARα1.The immunoreactive cells of GABAARal in the CA1 area of hippocampal was mainly expressed on the cell membrane, and some expressed in the cytoplasm.The positive cell body was round, oval, triangular and so on,but some cells had one or more processes.There was statistically significant between the different groups (F/Welch=4.563 and 2.961, P=0.017 and 0.039).Compared with group of B,the number of immunoreactive cells and OD value in groups of A,C,E was statistically significant(P<0.05),But there was no statistically significance between three groups (P>0.05). Conclusion1.Both Chai hu shu gan tang and Ding xian wan have the effect of antiepilepsy on PTZ seizures in rats, MES seizures in mice, strychnine convulsions in mice.2.The antiepilepsy mechanism of Chai hu shu gan tang:(1)increasing the protein expression of GAD65 and GABA synthesis in hippocampus of kindling rat;(2) increasing the expression of GABAARα1 in hippocampus of kindling rat and enhancing its activity.3.The antiepilepsy mechanism of Ding xian wan is increasing the protein expression of GAD65 and GABA synthesis in hippocampus of kindling rat.