| BackgroundEpilepsy is a group by different causes brain neurons highly synchronized, and often with self-limiting abnormal discharge, to attack, transient, repetitive, and usually for the rigidity of the central nervous system dysfunction syndrome characterized. The attack refers to the sudden onset of epilepsy, after a period of rapid recovery of normal intermittent; brief refers to the duration of the acute attack are very short, a few seconds, minutes or tens of minutes at the status epilepticus, rarely more than half hours; repeatability refers to the characteristics of epilepsy have recurrent seizures, only can not be diagnosed as epilepsy; stereotype refers to the clinical manifestations of each type of attack, almost unanimously. Due to its clinical onset of seizures, transient, repetitive characteristics of long-term repeated episodes can cause mental retardation, mental retardation, and even then dementia.Epilepsy, the prevalence of various countries and regions vary. The1997WHO report shows that the prevalence of epilepsy in developed countries, countries with economies in transition, developing countries and least developed countries is5.0‰,6.1‰,7.2‰,11.2‰, estimated about50million people with epilepsy. Asian countries, the prevalence is generally between5‰to10%of our epilepsy prevalence rate of7%, over900million existing patients with epilepsy, active epilepsy (ie, within one year the number of seizures in more than2times) prevalence rate of4.6‰over600million active epilepsy patients, at the same time, the annual incidence of25/100000, the additional number of patients,35million. Reported in China the majority of patients with epilepsy has not been a formal treatment, the treatment gap in epilepsy34.8%-75%range, the mortality was2.42/100000-7.82/100000, but the real death (died because of epilepsy status epilepticus) accounted for only20%of all causes of death,40.21%died due to accident, died of suicide by5.51%, unexplained death of4.13%.About70%to80%of patients under the guidance of the physician by regular antiepileptic drugs (AEDs) and scientific and standardized treatment can be engaged in normal work, study or life, of which about30%of epilepsy patients, the clinically used antiepileptic drugs difficult to control the seizures, known as refractory epilepsy (Refractory epilepsy, RE). At present, the treatment of refractory epilepsy is still the lack of a unified and accurate definition of the clinical will be through the rational use of multiple antiepileptic drugs, plasma concentration has reached the maximum tolerated dose, but the seizures still can not get effective control known as the refractory epilepsy. Domestic scholars have proposed a more comprehensive definition:frequent seizures, at least4times a month; formal treatment of the first-line anti-epileptic drugs and the drug plasma concentration to achieve an effective range observed for at least2years and still can not control the seizures, affecting the daily life; the central nervous system disease, or space-occupying lesions. Refractory seizures frequently, seriously affect the patient’s learning, working and living, even serious disability, so study the pathogenesis of refractory epilepsy and find effective treatment is very important.Ordinary epilepsy, refractory epilepsy pathogenesis remains unclear, in recent years to study the brain in patients with refractory epilepsy multidrug transporter expression may be involved in the resistance of refractory epilepsy, the most in-depth studyis the P-glycoprotein (P-GP). This protein is a membrane-bound protein, belongs to one of the transporter protein ATP-binding material superfamily of MDR1(multidrug resistance) gene encoding exists in normal human and mammalian secretory function of the organization and barrier structure, has a broad substrate specificity with a variety of structural passive diffusion into the intracellular drugs and toxic substances in combination, to rely on the conversion of ATP energy will arrive to pump out the drug within the cell and cytotoxic substances, so that the P-GP at the same time protects normal tissue from toxin damage, acting on the target concentration of the drug decreased, resulting in drug resistance.P-GP inhibitors can significantly increase the concentration of antiepileptic drugs in the brain, have been found so far hundreds of MDR reversal agents, reversal agents can be divided into seven categories:calcium channel blockers, cyclosporine, hormoneand anti-hormonal compound, protein kinase inhibitor, calmodulin antagonist, isoquinoline alkaloids and other. Down of these drugs can be different degrees of P-GP expression. but due to significant neurotoxic effects in humans so far have not yet approved the use of these drugs. Therefore, how the drug of choice is the focus of the development of resistance protein inhibitors as anti-epileptic drugs. Epilepsy belong to Chinese areas of the epilepsy disease, epilepsy, in theory, from the liver, the Neijing long records. Pathogenesis of epilepsy and the catharsis of the liver disorders, wind phlegm silt pathological changes in liver failure catharsis. Clinical studies have shown that epilepsy from the liver and the use of Shugan Tang treatment of refractory epilepsy, and achieved good effect. On this basis, gradually filter out the herbs Bupleurum the active ingredient saikosaponin a (of SSa), preliminary studies of our group found SSa inhibit pentylenetetrazol seizures of epileptic rats.Purpose The clinical practice of Chinese medicine treatment of intractable epilepsy have certain advantages, and found that Chinese herbs can enhance the sensitivity of refractory epilepsy on AEDs, reducing the amount of AEDs. So whether SSa P-GP/MDR1the expression of a certain degree of inhibition?Can be used as multi-drug resistance gene and its protein blocker? This study as a breakthrough in clinical research of Chinese medicine treatment of epilepsy, refractory epilepsy rat model to the SSa on P-GP/MDR1intervention role as a starting point to explore the possible mechanism of the the SSa intervention role.Method1. A lithium chloride-hydrochloric acid pilocarpine refractory epilepsy modelChemical lithium chloride-hydrochloric horses pilocarpine lit production model. As follows:the first day of the SD rats by intraperitoneal injection of lithium chloride (127mg/kg),18-24h after injection of atropine1mg/kg,30minutes after intraperitoneal injection of pilocarpine (10mg/kg). Without abdominal status epilepticus, is every30min to continue intraperitoneal injection of pilocarpine (10mg/kg), until the rats stop injecting after status epilepticus. Be status epilepticus for60min, intraperitoneal injection of10%chloral hydrate (300mg/kg) to terminate the attack. Rats after status epilepticus in the acute phase after24h into the incubation period of about10-15d, given daily intraperitoneal injection of5ml of5%sugar, salt,2times/d.15d after application into the chronic period of four weeks to start screening the animals, and real-time video monitoring, five rats per cage put a different tag for each rat, picric acid, continuous monitoring, the monitoring time8:00-20:00, and timely for video playback, recording IV level and above issued epilepsy as a successful spontaneous seizures model. Does not meet the onset of the grade IV abandoned.2. Effect of SSa on refractory epilepsy rats seizures and electroencephalographic The successful preparation of48SD refractory epilepsy model rats were randomly divided into4group, model group (B), VPA group (C), SSa (D) in low dose group, high dose of SSa group (E), with12rats in each group, and blank control group (A)12. Group A and group B daily received an equal volume of normal saline lavage, group C direct daily intragastric administration of VPA200mg/kg, group D daily intraperitoneal injection of SSa1.09mg/kg, group E daily intraperitoneal injection of SSa2.18mg/kg, the video recorded in rats with chronic period of4weeks, drug treatment for four weeks, eight weeks after the epileptic seizure frequency, the average attack time and attack level changes. At8weeks after treatment, each group randomly from each of2rats underwent EEG.3. Effect of SSa on P-GP and MDR1geneRats in each group8weeks after administration, the rat model of refractory epilepsy24were randomly divided into four group, model group (B), sodium valproate group (C), of SSa low-dose group (D), of SSa high dose group (E), n=6, otherwise blank control group (A)6, a total of five groups. By immunohistochemical method, Western blotting, quantitative PCR method to detect each rat hippocampus and temporal lobe P-GP and MDR1gene expression.4. Statistical methodsThe data were statistically analyzed using SPSS13.0software or SPSS17.0software measurement data with a mean±standard deviation (x±s) said.Each group before and after treatment, the number of attacks, attack the average time, using repeated measures analysis of variance; immunohistochemistry, immunoblotting, fluorescent PCR, different treatment group were compared using single factor analysis of variance (One-Ways ANOVA), homogeneity of variance multiplecomparison of selection of LSD-t test; heterogeneity of variance, the Welch method with approximate variance analysis, multiple comparisons and Dunnett T3 Examine. Ordered ranked data using generalized estimating equations, P<0.05was statistically significant.Result1. With intractable epilepsy rat model establishedA total of70rats during the experiment, except the blank control group (A)12only ignite the preparation of refractory epilepsy model. Five rats repeatedly given pilocarpine, always can not be induced status epilepticus, and therefore abandoned, and the remaining65rats in the initial or additional pilocarpine seizures, and gradually reached epilepticus state (status epilepticus, SE). In the acute period (24h), nine rats were due to frequent convulsions and death. The incubation period (two weeks), failure due to frequent convulsions, and can not be independent eating a total of eight deaths, more than a week after SE, death, into the chronic phase. The remaining48survived rats, according to the random number table, divided into model group (B), VPA group (C) of SSa low-dose group (D) of SSa high dose group (E), each group of12. Four weeks in the chronic phase, survival of rat spontaneous attack, attack level gradually developed from grade I to V grade, non-epileptic rats died.2. effect of SSa on refractory epilepsy in rats epileptic seizures and EEG2.1effect of SSa on the number of rats seizures before and after treatment of refractory epilepsy in ratsUsing a repeated measures analysis of variance showed that the three different time points in rat epileptic attack times the difference was statistically significant (P<0.05). Differences between the groups was statistically significant (P<0.05) at all time points is statistically significant (P<0.05) difference between the grouping; each time point analysis of variance shows:each group before treatment, caused by epilepsy group rats The seizures were no significant differences (P>0.05). Before treatment, the rats with induced epilepsy groups epileptic number of attacks was no significant difference (P>0.05). After four weeks, compared with group B, group C and group E can reduce the number of seizures in rats, there was significant difference (P<0.05), Group D and Group E difference was statistically significant (P>0.05); after8weeks, compared with group B, each group can reduce the number of seizures in rats, the difference was statistically significant (P<0.05), C, D group and E group differences were statistically significance (P<0.05). Each group compared with before treatment, with the exception of group B (P>0.05) differences were statistically significant (P<0.05).2.2Effect of SSa on average time of rats seizures before and after treatment of refractory epilepsy in ratsUsing the single-factor repeated measurements analysis of variance showed that the three time points in rats seizures average time difference was statistically significant (P<0.05), the difference was not statistically significant (P<0.05) between groups. At each time point and group differences were statistically significant (P<0.05). Before injection, the rats with induced epilepsy groups seizures the average time difference was not statistically significant (P>0.05). After four weeks, compared with group B, group C and group D can reduce epileptic seizures the average time difference was statistically significant (P<0.05), group D and group E difference was statistically significant (P<0.05); after8weeks, compared with group B, group C, D, E can reduce the rat epileptic seizures the average time difference was significant (P<0.05) in group C, D group and E-group differences statistically significant (P<0.05). Each group compared with before treatment, with the exception of group B (P>0.05) differences were statistically significant (P<0.05).2.3Effect of SSa on level of rat epileptic seizures after treatment for refractory epilepsy in ratsThe generalized estimating equation with ordered data analysis:three different points in time rat seizures rating difference was statistically significant (P<0.05). The difference between the groups was statistically significant (P<0.05). Between group comparisons revealed, C, D group compared with B, the difference was not statistically significant (P>0.05), Group E and group B difference was statistically significant (P<0.05). Compared with4weeks before treatment, after4weeks of treatment and treatment after8weeks the difference was statistically significant (P<0.05).2.4Effect of SSa on EEG of refractory epilepsy model ratThe rats in group A cortical and hippocampal EEG based waves namely alpha, beta, no obvious rhythmicity; group B rats showed a diffuse epileptiform waves, much as spike, spike, spike and slow wave complex, the emergence of a large number of epileptic discharge; C, D, E groups can be seen scattered in spike waves, sharp waves appear, while in group E at least. Show that valproate, and the SSa high, low-dose group could inhibit the epileptic discharge in rats, with a high dose of SSa group was most obvious.2.5Effect of SSa on P-GP and MDR1mRNA effectsImmunohistochemical detection of IOD value:each rat hippocampal area CA1, DG and temporal lobe cortex, immunohistochemical detection of P-GP protein expression of IOD values had a significant difference (P<0.05). Group multiple comparison, in CA1, DG area, C group in P-GP IOD value is higher than that of B group (P<0.05), A, E group in P-GP IOD value lower than that of group B (P<0.05). E group in P-GP IOD value lower than that of group D (P<0.05). In the temporal cortex area, C, D, E group in P-GP IOD and B groups have no significant difference (P>0.05), E group in P-GP IOD value is lower than that of group D (P<0.05).Immunohistochemical detection of the number of positive cells:each group rat hippocampal area CA1, DG area and temporal lobe cortex, Immunohistochemical detection of P-GP protein-positive cell count difference was statistically significant (P<0.05). Multiple comparisons between groups, in CA1and DG district, C group in P-GP number of positive cells was higher than that of B group (P<0.05), A, E group in P-GP positive cell number lower than group B (P<0.05), E group in P-GP positive cell number lower than group D (P<0.05), compared with the A group had no significant difference (P>0.05). In the temporal cortex area, C group in P-GP number of positive cells was higher than that of B group (P<0.05), E group in P-GP positive cell number and A, B, D group showed no significant difference (P>0.05).Western blotting method for the detection of P-GP expression in the hippocampus and temporal cortex:expression of P-GP, each gray level ratio difference is statistically significant (P<0.05). Multiple comparison group, in the hippocampus,C group in P-GP gray value ratio was higher than that of group B (P<0.05), A, D, E group in P-GP gray value ratio lower than group B (P<0.05), E group in P-GP gray value ratio lower than group D (P<0.05), but higher than that of A group (P<0.05).In the rat temporal cortex, the gray level ratio of the P-GP expression difference is statistically significant (P<0.05). In C group P-GP gray value ratio and B group were not statistically significant (P>0.05), A, E group in P-GP gray value ratio lower than group B (P<0.05), in P-GP gray value ratio lower than group D (P<0.05), E group and A group were not statistically significant (P>0.05).Fluorescent quantitative PCR of MDR1-mRNA expression:each group rat hippocampus, analysis of variance showed that real-time PCR detection of MDR1-mRNA expression difference statistically significant (P<0.05). in group C t he expression of MDR1-mRNA is higher than that of B group (P<0.05), MDR1-mRNA expression in group A was lower than that in B group (P<0.05), expression of MDR1-mRNA in group D, E and group B were not statistically significant (P>0.05), MDR1-mRNA expression in D group and E group were not statistical significance (P>0.05). Groups in the rat brain temporal lobe cortex, analysis of variance, real-time PCR detection of MDR1-mRNA expression was statistically significant (P<0.05). Between group multiple comparison showed that in the C group, the expression of MDR1-mRNA is higher than that of B group (P<0.05), MDR1-mRNA expression in group A was lower than that in B group (P<0.05),MDR1-mRNA in group D, E and group B were not statistically significant (P>0.05),MDR1-mRNA expression in D group and E group were not statistical significance (P>0.05).Conclusion:1.This study has successfully established chloride lithium-pilocarpine status epilepticus chronic refractory epilepsy model, a relatively simple operation, high success rate, good stability and lay the foundation for subsequent experiments.2.SSa had significant antiepilepitic effect, can reduce the refractory epilepsy rat model of seizure frequency, seizure onset time and average level.3.SSa can down-regulate P-GP expression of hippocampus, without significant downregulation of temporal lobe cortical P-GP expression, without significant downregulation of hippocampus and temporal lobe MDR1mRNA expression. |
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