| Objective:This study observed the cell morphology change in hippocampal CA3neurons of the epilepsic rats and detected voltage-gated potassium channel kv1.1of this area expression level, to examine the activity of triptolide (TL) on kv1.1influence.To further research the effect and mechanism of triptolide (TL) on neuronal cell death in epilepsy.Methods:Thirty SD rats were randomly divided into three group:control, model and TL group(n=10). The TL group were injected TL15μg/(kg·d) by intraperitoneally injection for7days,and at the same time the rats of control group were injected physiological saline with the equivalence of TL in the same way. Then the model group and TL group were made epilepsy by the internal carotid subcutaneously injecting Kainic Acid (KA), the concentration is5mg/ml,but the rats of control group were injected physiological saline in the same way.72h later, the rats by intraperitoneal injection of chloral hydrate anesthesia, after the left ventricular perfusion were decapitated, getting the brain tissue. The brains were separated into two hemispheres. The left hemisphere fixed and done paraffin sections, part of which made crystal violet staining to observe neuronal morphology of rat hippocampal CA3region, another part by immunohistochemistry detected voltage-gated potassium channels kv1.1protein expression in rat hippocampal CA3region. The right hemisphere were cryopreserved and detected kv1.1protein expression levels rat hippocampal CA3region using Western Blot.Results:1-The result of epilepsy model:After subcutaneous injection of KA,the TL and model group rats appeared spastic limbs, erection,falls,flips,reaching the Racine six evaluation criteriaⅣ-Ⅴ, and EEG appeared the high-tip wave,the complexed sharp-slow wave and other abnormal wave.The incubation period of seizures in TL rats were longer than in model group; the seizures of epilepsy were slight compared with model group; the onset time and duration is also shorter than in model group. Control rats without any reaction.2. The change of crystal violet staining of hippocampal neurons:There were no neuronal morphology changes and reducation of the neuron number of no handling with KA control group using low power lens(LP) observation. The hippocampal overall shape of model group and TL group were bigger than the injected by KA group, and the hippocampus of TL group was larger obviously. Part of pyramidal cells in epilepsy rat hippocampal CA3region, which were pretreated by TL was saved, and the hippocampal overall shape of TL was similar to normal neurons. After injecting KA72h> hippocampal neurons shrink and got small, also pyramidal cells degenerated or lost. Neurons of the CA3region lost more obviously.3. The result of immunohistochemical staining kv1.1protein expression in rat hippocampus:The control, model and TL group voltage-gated potassium channel kv1.1protein in rat hippocampus, are widely distributed, and the pyramidal cell layer is main. Immunohistochemical staining at high power lens(HP) showed positive cells and the cytoplasm with brown particles on the membrane to the CA3region obviously. The number of positive cells in model group was less than the control and TL group, lighter colored positive cells, few protrusions, the average optical density values of model group were compared with the control and TL group, significantly reduced (P<0.05); control and TL group had no significant difference (P>0.05).4. The result of Western blot detection of rat hippocampus kvl.1protein concentration:voltage-gated potassium channel kv1.1protein in hippocampal neurons is expressed. In model group kv1.1protein expression decreased in rat hippocampal neurons, and the rats of TL group express more than the model group. In model group kv1.1protein of hippocampal CA3region relative concentration were significantly different compared with TL group and control group (P<0.05), control group and TL group kv1.1protein relative concentrations were non-significant different (P>0.05).Conclusion:TL can protect neuron in the hippocampus of epileptic rats which maybe increase the expression of kv1.1in hippocampus CA3neuron. |
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