| Objective:To Drug line buried on Acupuncture point investigate the effects of decoction formulated on the basis of Principle of composition by traditional Chinese medicine method of discussing blood and eliminating phlegm on the electroencephalogram, seizure behavior epileptic rats induced by kanaic acid, and to explore furtherly the possible molecular pathogenesis mechanism on the intractable epilepsy from the expression and distribution of multiple drug resistance gene1(MDR1) mRNA and related protein. It provides scientific experimental basis For "phlegm blood stagnation" method of traditional Chinese medicine treatment intractable epilepsy, which also provides the theoretical foundation for the development of new traditional Chinese medicine new drug for the treatment of epilepsy.Methods:(1) The establishment of chronic intractable epilepsy rats model: Hippocampal dosing can be located and was injected lμl kainic acid (1.0μg/μl) to induce epilepsy model by microsyringe according to Paxions Watras map and under the guidance of brain stereotaxic apparatus. The rats whose seizure behavior level in Racines standard V level or above were intragastric injection administration with valproate and carbamazepine intervention for14days, Sub-convulsions dose0.5μlKA was injected from micro-catheter again to kindle the seizure, the rats whose seizure behavior level in Racines standard V level or above and electroencephalogram (EEG) test with epileptiform wave discharge were selected to be Successful resistant refractory epilepsy rats model.(2) Methods of EEG detection and behavior observation:Scalp EEG was recorded by Digital video-EEG monitoring system. EEG waveform, frequency, amplitude and epileptiform wave discharge frequency were analyzed.(3) The hippocampus nerve Pathologic morphology observation:Rats brain specimens were fixed and embedded through the hippocampus amygdala complex level5microns thick layer of transverse section, Then respectively observed Under the microscopy on temporal lobe cortex and hippocampus tissue pathological change after HE staining. And the temporal lobe and hippocampus CA1,CA3area is selected five discontinuous vision remaining normal cortex neurons quantitative count.(4) To detect MDRib multi-resistant genes expression by real-time fluorescent quantitative aggregate min chain reaction (RT-PCR) methods. Using immunohistochemical technique.(5)Detection of multi-resistant related protein MRP1and P-gp the expression of different parts in the hippocampus and temporal lobe cortex.Results:(1)It can prolong the Epileptic incubation period, reduce epilepsy seizures, reduce seizure frequency after Drug line buried on Acupuncture point, lamotrigine (LTG)intervention of the kindling induced rats. The dfferences was significantly compared with the model group (P<0.05).(2)Inhibition the discharge of epileptiform wave (sharp/spike wave, sharp/spike complex slow wave and spike wave rhythm) frequency and reduce the proportion of it,reducing the average brain waves amplitude and speed up the frequency of EEG background activities after intervention of YX or LTG, the dfferences was significantly compared with the model group (P<0.05).(3)Nerve pathological change:Temporal lobe cortex neuron degeneration necrosis, loss and gliosis, hippocampus neurons depigmentation, glial hyperplasia (namely, mesial temporal sclerosis (MTS) mossy fiber filaments sprout and brain edema in model group rats. The dfferences of remaining hippocampus neuron count between YX or LTG intervention group and model group was not significantly(P>0.05).(4)Compared with the model group, the expression of MDR1b mRNA,MDRP1,P-gp was significantly reduced in all YX or LTG intervention groups (P<0.05). The expression and distribution of MDR1b mRNA, MDRP1, P-gp was significantly compared between the hippocampus and cerebral cortex of the epileptic rats. There was no significant difference in temporal lobe cortex protein expression Between groups (P>0.05)Conclusion:(1)The expression of multi-resistant genes MDR1b and their drug resistance related proteins MRP1, P-gp were elevated in brain hippocampus in rat models of chronic drug-resistant epilepsy induced by KA.(2)It may reduce the behavior level, reduce epilepsy induced seizures, shorten the onset time, prolong the incubation period, inhibition of electroencephalogram epileptiform wave frequency and reduce the average amplitude, to improve the convulsions threshold, at the same time can protect hippocampal neurons damage of the model rats after intervention of YX;(3) Reversing and reducing multi-resistant multi-resistant genes related protein expression level of induced RE rat hippocampus,which may be one biological mechanism of YX for the treatment of refractory epilepsy.(4) From the perspective of treatment "stagnation phlegm and blood stasis" in the meantime on refractory epilpsy is one of the important traditional Chinese medicine treatment principles. |
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