| ObjectiveThis paper attempts to study the possible antiepileptic mechanism of Ningxian Particles by the inflammation factors, apoptosis and drug-resistance gene, based on the kainic acid refractory epilepsy rats model by the Ningxian particles, CBZ alone or integrated therapy, observing the kainic acid refractory epilepsy rats behavior and EEG epileptic wave in order to provide a new therapeutic method for combing traditional Chinese and western medicine treatment of refractory epilepsy.MethodPart1:36chronic refractory epilepsy models were made by microinjection of kainic acid hippocampal CA3chemic igniting and PHT and EEG screening, and they were divided randomly into7groups, such as model, CBZ, Ningxian particles, Ningxian particles low, medium and high dose combination CBZ and sham-operated group, and these are6rats each group.Part2:The rats before and after being treated were observed by the weight, the number of seizures, the average duration of seizures, the defense levels, the seizures levels, the spontaneous recurrent seizures total times, and EEG monitoring.Part3:Refractory epilepsy rat model (n=6), each group after treatment with the sham group (n=6), the apical perfusion normal saline before removal of brain, fixed in4%paraformaldehyde, conventional preparation of paraffin sections (containing the cerebral cortex and hippocampus). HE staining was used to observe the changes in the hippocampus and temporal cortex neurons morphology, immunohistochemical observation hippocampus and temporal cortex of c-fos and caspase-3expression.Part4:The experimental group rats serum IL-1β, IL-6, TNF-α content were detected by using the enzyme-linked immunosorbent assay method (Elisa method) after femoral artery blood collected in each experimental group therapy. Part5:The rats were under anaesthesia by10%chloral hydrate to be removed their brains. The MDR1mRNA expression of rats was detected by Real-time quantitative polymerase chain reaction. The P-gp expression was detected by western-blot.ResultPart1:Refractory epilepsy rat model was successfully made by hippocampal local injection of kainic acid and drug screening.Part2:The weight of the rats by combining medium and high-dose Ningxian particles with CBZ group drastically increased, comparing with CBZ group. There was a significant difference (P<0.05). The seizures mean duration of the rats before and after being treated by combining medium and high-dose Ningxian particles with CBZ group were drastically decreased, comparing with CBZ group. There were very significant differences (P<0.01). The mean defense response of the rats before and after being treated by combining medium and high-dose Ningxian particles with CBZ group were drastically decreased, comparing with CBZ group. There were very significant differences (P<0.01). The average seizure changes in the level of rats before and after being treated by combining medium and high-dose Ningxian particles with CBZ group drastically decreased, comparing with CBZ group. There were very significant differences (P<0.01).The spontaneous recurrent seizures total time of the rats being treated by combining medium and high-dose Ningxian particles with CBZ drastically decreased, comparing with CBZ group. There were very significant differences (P<0.01). The EEG showed the Ningxian particles medium and high-dose combination CBZ group epileptic discharge was inhibited.Part3:Minimum c-fos positive cells expression was seen in sham-operated rats’ hippocampus tissue sections. The c-fos positive cells expression in the hippocampus increased significantly in model group. The expression of combining low, medium and high-dose Ningxian particles with CBZ group drastically decreased, comparing with CBZ group. There were very significant differences (P<0.01). The caspase-3 expression of combining low, medium and high-dose Ningxian particles with CBZ group drastically decreased, comparing with CBZ group. There was a significant difference (P<0.05).Part4:Comparing with CBZ group,the Ningxian particles low dose combination CBZ rats serum IL-1β levels were significantly lower (P<0.05).The Ningxian particles medium and high-dose combination CBZ rats serum IL-1β levels were very significantly decreased (P<0.01); Comparing with CBZ group, the Ningxian particles low dose combination CBZ rats serum IL-6levels were significantly lower (P<0.05); The Ningxian particles medium and high dose combination CBZ rats serum IL-6levels very significantly decreased (P<0.01); Comparing with CBZ group, the Ningxian particles low, medium and high-dose combination CBZ rats serum TNF-a levels were very significantly decreased (P<0.01).Part5:Comparing with model group, the remaining groups of rat brain tissue MDR1mRNA was very significantly decreased (P<0.01). Comparison with CBZ group, the Ningxian particles medium and high dose combination CBZ group of rats brain tissue MDR1mRNA content was significantly decreased (P<0.01); Comparing with model group, the Ningxian particles high dose combination CBZ group rats brain P-gp protein expression was very significantly decreased (P<0.01); the Ningxian particles low and medium dose combination CBZ rats brain P-gp protein expression was significantly decreased (P<0.05).Conclusion1. The study successfully establishes refractory epilepsy rat model by microinjecting kainic acid in rats’hippocampal CA3with epilepticus status and by drug screening. The model was easily, economically, relatively simply set up in order to lay a good foundation for the further research.2. The Ningxian particle low, medium and high-dose combined CBZ group can significantly reduce the epileptic rats in each group of seizure average duration, the defense responses score, the spontaneous recurrent seizures total time and the degree of seizures.The Ningxian Particles combined CBZ use better than single effect.The EEG show the Ningxian particles medium and high dose combined CBZ group of rats epileptic discharge was significantly inhibited, only a small amount of spikes or sharp waves, while the Ningxian particles high dose combined CBZ group appeared at least.3. The Ningxian particles low, medium and high-dose combination CBZ group can significantly reduce c-fos and caspase-3gene expression, inhibit apoptosis, to protect nerve cells; Speculated the Ningxian particles may act on multiple targets in the apoptotic pathway synergistic role with CBZ, but apoptotic gene expression did not downward trend in the increase of herb dose.4.IL-1P, IL-6, TNF-a inflamatory cytokines participate in the immune response, the changes in their levels indirectly reflect the degree of the brain tissue damage.The Ningxian particles medium and high doses combination CBZ group of inflammatory cytokines was significantly lower than the CBZ group (P<0.01). The Ningxian particles medium and high-dose combination CBZ have some immunosuppressive effects than with CBZ efficacy significantly, then explain the combination has a synergistic effect on epilepsy treatment, have a certain role in the treatment of epilepsy.5. The Ningxian particles joint CBZ can certainly inhibit MDR1mRNA/P-gp drug transporter function, reduce MDR1mRNA/P-gp expression, and there was a dose-effect relationship. |
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