Effect Of Chaihushugantang On Epileptic Seizure Of Refractory Epilepsy And Expression Of Multidrug Resistance Protein P-GP And MRP1

BackgroundEpilepsy, a functional disturbance of the CNS and induced by abnormal electr-ical discharge,manifestes by recurrent seizure. According to the WHO statisties, the prevalences of epilepsy in developed countries, economies intransition countries, developing countries and least developed countries were 5.0‰,6.1‰,7.2%o and 11.2‰.It was estimated that there were about 50 million epilepsy patients worldwide. In 2001, the national epidemiological survey of epilepsy demonstrated that the mothidity rate of epilepsy was 7.0‰.and the number of epileptic patients has risen to as many as 5-6million in our country.Most patients with epilepsy were treated standardized for a long time, attack can be controlled. But a part of patients are not controlled by drugs, the type of patients is called refractory epilepsy(RE).RE is called intractable epilepsy,response to antiepileptic drugs unsensitively, and epilepsy is still attacking by first-line drugs of best dose for two years. The incidence of RE is 10% to 20%, long time, repeatedly, high morbidity, and affect the quality of life of patients. Learning the mechanism of epilepsy and seeking the treatment methods are necessary.The mechanism of epilepsy is complex. Multidrug transporter in brain of epilepsy patients is attentioned by the tip of tumor multidrug resistance, the most thoroughly are P-GP, MRP1. Transmembrane structure of the proteins has the function of medication pumps that can transport drugs and hydrophobic compounds out cell.Most antiepileptic drugs are Fat-soluble highly, and through the blood brain barrier, natural substrate P-GP and MRP1.If the expression of P-GP, MRP1of luminal side of the capillary endothelial cellsis high, they can pump AEDs to blood,and limit Fat-soluble drugs into parenchymal.Because the high expression of P-GP, MRP1,these proteins can transport most antiepileptic drugs,and reduce the plasma concentration in brain tissue, leding to the emergence of resistant epilepsy.The reversal agents of P-GP and MRP1 are payed attention in the treatment of RE. The reversal agents of P-GP include calcium channel blockers, calmodulin inhibitors, cyclosporine, lipophilic compounds and hormones.The reversal agents of MRP1 include probenecid, isoflavones.The application of reversal agents is a good method to solve MDR,but these are limit to use for toxic and side effect. Pathogenesis of epilepsy is related to liver catharsis function,and the Wind, phlegm, blood stasis is the Pathological changes of liver catharsis function out of tune.So epilepsy can select to treat "from the liver ".Professor Chen Baotian pays attention to treat epilepsy "from the liver",and use Chaihushugantang to treat RE, and achieved good effects.Traditional Chinese medicine has a exact effect for RE, may be a multi-channel integrated targets. In this study, rat model of RE in the medical treatment of epilepsy as a breakthrough in clinical research, multi-drug resistance protein P-GP, MRP1 as the starting point, to discus the possible mechanism of herb intervention.ObjectivesThis study is based on li-Pilocarpine-induced refractory epilepsy model rats, to observe Chaihushugantang epileptic rats in the improvement of behavior and EEG epileptic wave,to discus the effect of Chaihushugantang protecting neurons, Inhibiting the expression of multidrug resistance protein P-GP, MRP1 and the upstream gene mRNA.And evaluating the valuation of Chaihushugantang treating RE "from the liver ", to provide theoretical basis for the treatment of epilepsy. Methods:1. The establishion of intractable epilepsy modelTo make models using lithium-pilocarpine hydrochloride method. Concrete steps: (1). firstly, the intraperitoneal injection of lithium chloride 127mg/kg;(2).The intraperi-toneal injection of atropine lmg/kg after 18-24h;(3)The intraperitoneal injection of 10% pilocarpine 10mg/kg after 30min,if no epilepsy IV or above attacks, intraperito-neal injection of 10% pilocarpine 10mg/kg every 30min, and stop injecting until status epilepticus of the rats;(4).Status epilepticus for 60 min, intraperitoneal injection 10% chloral hydrate 300mg/kg to terminate attack.(5).After the acute phase after 24h, get into the incubation period of 10-15 days;(6)get into the chronic phase after 15 days, monitor the rats continuously with the video surveillance system to screen laboratory animals. The time is 4 weeks of getting into the chronic phase from 8am to 8pm.If rats seizure spontaneously during this time, these are successful models of chronic refractory epilepsy. And if rats no seizures spontaneously, these are abandoned.2. Effect of Chaihushugantang on epileptic seizure of refractory epilepsy and EEGThe 60 rats with refractory epilepsy were random divided into 5 groups, model, VPA, Chaihushugantang low,medium,high dose, and these are 12 rats each group,still 12 blank control rats.All rats in each group were administered by intragastric administration for 56 days at 9am and 6pm after chronic phase.Blank control and model group were administered equal volume of physiological saline, while the VPA group was given by 200mg/(kg·d), Chaihushugantang low,medium,high dose group was given by different concentration, such 4.5g/(kg·d),9g/(kg·d),18g/(kg·d). Video record Seizure behavior,including The number of epileptic seizures, The average duration, Attack level and weight change.After 8 weeks treatment, two epileptic rats were randomly selected from each group for recording the EEG..3. Effect of Chaihushugantang on hippocampus and temporal lobe neurons. The 30 rats:with refractory epilepsy after administration, including model,VPA, Chaihushugantang low,medium,high dose, and these are 6 rats each group,still 6 blank control rats. Fixed rat brain by aortic perfusion of 4% paraformaldehyde,Prepa-ring paraffin sections including hippocampus and temporal cortex. HE staining observe the morphological change of hippocampus and temporal cortex neurons,nissl staining observe the neuronal survival and loss of nissl bodies of hippocampus and temporal cortex.4. Effect of Chaihushugantang on multidrug resistance protein P-GP,MRP1 and upstream gene in the brain of epileptic rats.The 30 rats with refractory epilepsy after administration, including model,VPA, Chaihushugantang low,medium,high dose, and these are 6 rats each group,still 6 blank control rats.To detect multidrug resistance protein P-GP,MRP1 and upstream gene mRNA in the brain of epileptic rats by immunohistochemistry,Western blotting, quantitative PCR.5. Statistical methodThe data analysis was used the SPSS 13.0 statistics software. The measurement data indicated by the mean value gentleman standard deviation, and the result comparison of each group about attack frequency, time, weight was by the repeater measure variance analysis and the multiple comparisons with the LSD, the the seizure serious degree by Nonparametric Test Kruskal-Wallis.Nissl bodies,Immunohi-stochemistry, the western blotting, and PCR were by the single factor variance analysis (One-way ANOVA). P<0.05 expressed the difference has statistics significance.Results1. The establishion of intractable epilepsy models88 rats were intraperitoneally injected li-pilocarpine hydrochloride to make intractable epilepsy models. The 6 rats can not induced status epilepticus by injecting pilocarpine repeatedly, and were given up.The other 82 rats began seizures for the first time or additional pilocarpine, gradually to reach status epilepticus. In the acute period (24h),9 rats died for epileptic seizures frequently. In the incubation period,13 rats died for frequent convulsions, and can not self-feeding in the one week after status epilepticus, others get into chronic phase. In the chronic phase, there were not rats died, and all 60 rats showed spontaneous seizures, attack level from the gradeⅠto gradeⅤ.2. Effect of Chaihushugantang on seizures of refractory epilepsy model2.1 Changes in seizure frequency of each group rats before and after treatmentThree time points was significantly (F=207.535,P=0.000) using single factor repeated measures analysis of variance, there was significant between the groups (F=2.755, P=0.037), there was interaction between each time point and each group (F=48.098,P=0.000).Before treatment, there was no significance in seizure frequency of each group (P>0.05), there was significance after treatment of 4 and 8 weeks(P<0.05 or P<0.01).Multiple comparisons between groups, after treatment of 4 weeks, compared with model group, VPA and Chaihushugantang high dose group had reduced the frequency of seizures in rats (P< 0.01 or P< 0.05),Chaihushugantang low and middle dose group had no significance (P> 0.05),but had significance(P<0.05) compared with Chaihushugantang high dose group. After treatment of 8weeks, compared with model group, all groups had reduced the frequency of seizures in rats(P< 0.01).VPA,Chaihushugantang low and middle dose group had significance (P< 0.05 or P<0.01) compared with Chaihushugantang high dose group. Compared with Before treatment, all groups had significance except for model group (P<0.01)2.2 Changes in average attack time of each group rats before and after treatment Three time points was significantly (F=182.683, P=0.000) using single factor repeated measures analysis of variance, there was significant between the groups (F=4.706, P=0.002), there was interaction between each time point and each group (F=26.995, P=0.000).Before treatment, there was no significance in seizure time of each group (P>0.05), there was significance after treatment of 4 and 8 weeks (P <0.01).Multiple comparisons between groups, after treatment of 4 weeks, compared with model group, VPA and Chaihushugantang high dose group had reduced the time of seizures in rats (P<0.01),Chaihushugantang low and middle dose group had significance(P<0.05 or P<0.01) compared with Chaihushugantang high dose group. After treatment of 8weeks, compared with model group, all groups had reduced the time of seizures in rats(P<0.05 or P<0.01).VPA,Chaihushugantang low and middle dose group had significance (P<0.05 or P<0.01) compared with Chaihushugantang high dose group. Compared with before treatment, all groups had significance except for model and Chaihushugantang low dose group (P<0.01)2.3 Changes in attack levels of each group rats before and after treatmentBefore and after treatment, epilepsy models all showⅣorⅤlevel attack by Kruskal-Wallis, the attack mean rank of model,VPA,Chaihushugantang low,middle and high is 31.00,28.50,33.50,31.00,28.50, five groups had no significance (χ2=0.919, P=0.922); after treatment of 4 weeks, the attack mean rank of five groups 42.25,22.75,35.08,29.75,22.67,there were significance (χ2=12.945, P=0.012), attack level of Chaihushugantang high dose group was low, secondly, VPA group, attack level of model group was highest. after treatment of 8 weeks, the attack mean rank of five groups 44.63,26.38,34.13,28.13,19.25,there were significance(χ2=15.776, P=0.003), attack level of Chaihushugantang high dose group was low, secondly, VPA group, attack level of model group was highest.3. Changes in weight of rats before and after treatment Three time points was significantly (F=672.873,P=0.000) using single factor repeated measures analysis of variance, there was significant between the groups (F=203.671, P=0.000),there was interaction between each time point and each group (F=51.172, P=0.000). There were significance before treatment,after treatment of 4 and 8 weeks (P<0.01).Multiple comparisons, before treatment, there were significance compared with blank control in all groups(P<0.01), there were no significance in other groups (P>0.05).After treatment of 4 weeks, there were significance in other groups compared with blank control(P<0.01),compared with model group, VPA and Chaihushugantang middle,high dose group had gained weight (P<0.01), Chaihushugantang low,middle dose had significance compared with high dose(P<0.01).After treatment of 8weeks, there were significance in other groups compared with blank control(P<0.01),compared with model group, all groups had gained weight(P< 0.01). Chaihushugantang low and middle dose group had significance (P<0.01) compared with Chaihushugantang high dose group. Compared with before treatment,all groups had significance except for model group (P<0.01).4. Effect of Chaihushugantang on EEG of refractory epilepsy modelThe EEG of blank control group rats was the basis ofα,βwave, model groups had a large number of high amplitude wave, mostly spikes, sharp wave, spike slow wave complex, there were epileptic discharge. Chaihushugantang low,middle dose had more spikes, sharp wave, spike slow wave complex, and had lower volatility compared with the model group. The epileptic discharge was inhibited in VPA and Chaihushugantang high dose group, and had small spikes or sharp waves, and Chaihushugantang high dose group showed the least.5. Effect of Chaihushugantang on neurons in the hippocampus and temporal lobe of refractory epilepsy model.5.1 HE staining shows, Hippocampal CA1 and DG area of blank control group rats had the large number of Granule cells and pyramidal cells, lightly stained nuclei, prominent nucleoli, glial cells can be seen occasionally. Temporal cortex nucleus were round or oval, nucleolus clear, normal morphology. Hippocampal and temporal cortex slight edema of the stromal cells, nuclear fragmentation, dissolution, a small amount of neuronal degeneration and necrosis in the model,VPA, each dose group of Chaihushugantang. Degree of neuronal damage of VPA, each dose group of Chaihushugantang is lower than model group.5.2 Nissl staining shows, Hippocampal CA1 and DG area of blank control group rats had the large number of Granule cells and pyramidal cells, normal distribution of the nissl bodies within the cytoplasm. Neurons in temporal cortex morphology rules,nissl bodies rich in cytoplasm. Hippocampal and temporal cortex neurons Irregular morphology, cell swelling, rupture, cell outline vague, ill-defined, nissl bodies decreased in cytoplasm,in the model,VPA, each dose group of Chaihushugantang. Degree of neuronal damage of VPA, each dose group of Chaihushugantang is lower than model group.5.3 The number of nissl bodies in hippocampal CA1, DG and temporal lobe cortexNissl staining showed nissl bodies had significance in hippocampal CA1, DG ar-ea and temporal lobe cortex of rats (P< 0.01). Multiple comparisons between groups, nissl bodies of blank control,VPA, Chaihushugantang high dose group were higher than model group (P<0.05 or P<0.01), Chaihushugantang low and middle dose group had no significance compared with model group (P>0.05), Chaihushugan-tang high dose group was higher than low and middle dose group (P<0.05 or P< 0.01). In hippocampal DG area, Chaihushugantang high dose group had no signifi-cance compared with middle dose group (P>0.05)6. Effect of Chaihushugantang on multidrug resistance protein P-GP and upstream gene MDR1mRNA IOD values by immunohistochemistry:expression of P-GP had significance (P <0.01) in hippocampal CA1, DG area and temporal lobe cortex of rats. Multiple comparisons between groups, in hippocampal CA1, DG area, blank control and Chaihushugantang high dose group were lower model group (P<0.01).VPA group was higher model group (P<0.05 or P<0.01), Chaihushugantang high dose group was lower low and middle dose group (P<0.05 or P<0.01), was higher than blank control (P<0.05). In temporal lobe cortex, blank control and Chaihushugantang high dose group were lower model group (P<0.01), VPA, Chaihushugantang low and middle dose group had no significance compared with model group (P>0.05) Chaihushugantang high dose group was lower than VPA,low and middle dose group (P<0.05 or P<0.01), had no significance compared with blank control (P>0.05).Positive cells by immunohistochemistry:expression of P-GP had significance (P <0.01) in hippocampal CA1, DG area and temporal lobe cortex of rats. Multiple comparisons between groups, blank control and Chaihushugantang high dose group were lower model group (P<0.05 or P<0.01), VPA group was higher than model group (P<0.05 or P<0.01), Chaihushugantang high dose group was lower than low dose group (P<0.05), and had no significance compared with blank control group and middle dose group (P>0.05)Expression of Protein by Western Blotting:expression of P-GP had significance in hippocampal area and temporal lobe cortex of rats (P<0.01). Multiple comparisons between groups, In hippocampal area, VPA group was higher than model group (P<0.05), blank control and Chaihushugantang low, middle, high dose group were lower model group (P<0.05 or P<0.01),and Chaihushugantang high dose group was lower low, middle dose group (P<0.05 or P<0.01),but higher than blank control (P<0.01). In temporal lobe cortex, VPA group had no significance compared with model group (P>0.05), blank control and Chaihushugantang middle, high dose group were lower model group (P<0.01). Chaihushugantang high dose group was lower than low, middle dose group (P<0.05 or P<0.01), had no significance compared with blank control (P>0.05)Expression of MDR1 mRNA by quantitative PCR:expression of MDR1-mRNA had significance in hippocampal area and temporal lobe cortex of rats (P<0.01) Multiple comparisons between groups, VPA group was higher than model group (P< 0.01), blank control and Chaihushugantang high dose group were lower model group (P<0.05 or P<0.01), Chaihushugantang low,middle dose group had no significance compared with model group (P>0.05), Chaihushugantang high dose group was higher blank control (P<0.05). In hippocampal area, Chaihushugantang high dose group was lower than low,middle dose group (P<0.05 or P<0.01) 7. Effect of Chaihushugantang on multidrug resistance-associated protein MRP1 and upstream gene mRNAIOD values by immunohistochemistry:expression of MRP1 had significance (P <0.01) in hippocampal CA1, DG area and temporal lobe cortex of rats. Multiple comparisons between groups, in hippocampal CA1 area, VPA group was higher model group (P<0.05), blank control and Chaihushugantang high dose group were lower model group (P<0.01).Chaihushugantang high dose group was lower low dose group (P<0.05),there were no significance compared with blank control and Chaihushugantang middle dose group (P>0.05). In hippocampal DG area, VPA group was higher model group (P<0.01), blank control and Chaihushugantang high dose group were lower than model group (P<0.01). Chaihushugantang high dose group was lower low and middle dose group (P<0.01), was higher than blank control (P<0.01). In temporal lobe cortex, blank control and Chaihushugantang high dose group were lower model group (P<0.01), VPA, Chaihushugantang low and middle dose group had no significance compared with model group (P>0.05) Chaihushugantang high dose group was lower low and middle dose group (P<0.05), higher blank control (P<0.05)Positive cells by immunohistochemistry:expression of MRP 1 had significance (P<0.01) in hippocampal CA1, DG area and temporal lobe cortex of rats. Multiple comparisons between groups, blank control and Chaihushugantang high dose group were lower model group (P<0.05 or P<0.01), VPA group was higher than model group (P<0.05), Chaihushugantang high dose group was lower than low dose group (P<0.05 or P<0.01), and had no significance compared with blank control group and middle dose group (P>0.05). In hippocampal DG area, VPA group had no significance compared with model group (P>0.05)Expression of Protein by Western Blotting:expression of MRP1 had significance in hippocampal area and temporal lobe cortex of rats (P<0.01). Multiple comparisons between groups, blank control and Chaihushugantang middle, high dose group were lower model group (P<0.05 or P<0.01), VPA group and Chaihushugan-tang low dose group had no significance compared with model group (P> 0.05),Chaihushugantang high dose group was lower than low, middle dose group (P <0.05 or P<0.01).In hippocampal area, Chaihushugantang high dose group was higher than blank control (P<0.01)Expression of MRP 1 mRNA by quantitative PCR:expression of MRP1-mRNA had significance in hippocampal area and temporal lobe cortex of rats (P<0.01) Multiple comparisons between groups, in hippocampal area, VPA group was higher than model group (P<0.05), blank control and Chaihushugantang high dose group were lower model group (P<0.05 or P<0.01), Chaihushugantang high dose group was lower than low dose (P<0.05), had no significance compared with blank control and Chaihushugantang middle group (P>0.05). In temporal lobe cortex, VPA group was higher than model group (P<0.01), blank control and Chaihushugantang middle,high dose group were lower model group (P<0.05 or P<0.01), Chaihushu-gantang low dose group had no significance compared with model group (P> 0.05),Chaihushugantang high dose group had no significance compared with low, middle dose group (P>0.05),but was higher than blank control (P<0.05)Conclusions1. The refractory epilepsy model after status epilepticus was stablished by Li-PILO was simple and its achievement ratio and stability was fine, laying the good foundation for follow-up experiments.2. Chaihushugantang had good effect of antiepilepsy, can reduce seizure frequency of refractory epilepsy model, the average attack time, attack level,and Improve the overall state of epileptic rats, Increase body weight in rats,and it tends to present a dose-effect and time-effect relationship between them.3. Chaihushugantang can reduce neuronal damage in hippocampal CA1, DG area and temporal lobe cortex by HE staining and nissl staining, reduce the loss of nissl bodies.4. Chaihushugantang can inhibit drug transporter function of P-GP and MRP1, reduce the expression of P-GP and MRP1 protein. By immunohistochemistry, Western Blotting, quantitative PCR, chaihushugantang can inhibit the expression of P-GP and MRP I protein in hippocampal CA1, DG area and temporal lobe cortex of rats, inhibit the expression of MDR1mRNA and MRP1mRNA, and it tends to present a dose-effect relationship between them. VPA can strengthen the expression of P-GP and MRP1 protein and upstream gene mRNA in corresponding positions.