| Objective:The model of traumatic brain injury was established to observe the effect of Rhynchophylline on early epilepsy in mice with brain injury,and to explore the mechanism of the action.Method:The closed brain injury model was prepared with reference to the feeney free-fall method,and the successful mice were randomly divided into the model group(MOD group),the low-dose group(RPD group),the middle-dose group(RPZ group),the high-dose group(RPG group)and the sodium valproate group(VPA group).In addition,a blank group(CON group)was set,and only after the scalp was cut,no blow was made.10 in each group.The blank group was treated with normal saline,and the treated group was treated with the corresponding different dose of the drug,and 7 days(in which the water content of the brain was detected,and the model was made after the first dose).After the last dose,the intraperitoneal injection of a sub-alarming dose of pentylenetetrazole(PTZ)was ignited.Behavioral observation and recording of tetanus,clonic seizure latency in mice.The electroencephalogram(EEG)was analyzed by frontal cortex discharge activity.Brain dry-wet weight method was detected the changes of brain tissue water content.The expression of level of inflammatory factor(TNF-α,IL-1β)、apoptotic protein(Caspase-3,Bax,Bcl-2)、glial fibrillary acidic protein(GFAP)and brain-derived neurotrophic factor(BDNF)in hippocampus detected by Western blotting.The fluorescence density of GFAP in the dentate gyrus(DG)and CA1 region and the positive cells of BDNF in the CA3 region were measured by immunofluorescence.Result:1.Behavioral observationMost of the mice in the blank group had normal behavior and a few had convulsive behavior,while the mice in the model group had more large seizures of degree 5 after TBI,and the other groups presented 2-4 grade recurrent seizures and a few grade-5 seizures.Compared with the model group,the administration group could prolong the latent period of clonic and tonic seizure in epileptic mice(P<0.05 or P<0.01).Among them,the improvement effect of high-dose group was better than that of low and medium dose group,and there was a certain dose-dependence in them.2.EEG recording and brain water content changeIn the blank group,EEG showed the basic wave mainly,no obvious epileptiform wave appeared and the amplitude of the wave was stable,while the model group showed obvious paroxysmal epileptiform discharge wave,which showed single spike wave,multiple spike wave,spike wave and paroxysmal rhythm wave with high potential in the model group.Compared with the model group,EEG in each group showed spikes in different degrees,the number of discharges was significantly reduced,and the latency of epileptiform waves was significantly prolonged.the epileptiform discharge occurred occasionally in VPA group with a smaller amplitude.Compared with the blank group,the water content of brain tissue in the model group was significantly higher than that in the model group(P<0.01).Compared with the model group,the water content of the brain tissue in the high-dose group decreased significantly(P<0.01).The low-dose group could decrease the brain water content,but had no statistical significance(P>0.05).3.Western blot assayChanges of inflammatory factors expression:compared with the blank group,the expression of TNF-α and IL-1β protein in the model group was significantly up-regulated(P<0.01).The expression of TNF-a and IL-1β was decreased in each group after different doses of drug treatment,especially in the high dose group(P<0.01),and the therapeutic effect was slightly lower than that of sodium valproate group(P<0.01).Changes of apoptosis factors expression:compared with the blank group,the expression of pro-apoptotic protein Caspase-3 and Bax protein in the model group increased significantly(P<0.01 or P<0.05),while the bcl-2 content of the antiapoptotic protein decreased significantly(P<0.01).The expression of caspase-3 and bax protein was down-regulated and the expression of bcl-2 protein was up-regulated in the high dose group.The improvement effect was the best in the high dose group(P<0.01).Changes of GFAP and BDNF expression:compared with the blank group,the protein content of GFAP and BDNF the model group increased significantly compared with the model group,the low dose group was was no significant difference(P>0.05).There was significant difference in the high dose group(P<0.01 or P<0.05).4.Immunofluorescence detectionChanges of GFAP fluorescence density:Under microscope,In the DG region and CA1 region,the fluorescence density of blank group was the smallest and that of model group was the largest.There was significant difference between them(P<0.01).In the treatment group,the high dose of RP inhibited the positive reaction of GFAP best(P<0.05),and had a certain dose-dependent effec.Changes in the number of bdnf positive cells:the expression of BDNF positive cells was the highest in the model group.After RP intervention,the number of BDNF positive cells in the high dose group was significantly lower than that in the model group(P<0.05),but there was no significant difference between the low dose group and the model group(P>0.05).Conclusion:1.Rhynchophylline can prolong the latency of ankylosing and paroxysmal seizures in brain-injured mice.2.Rhynchophylline can reduce the amplitude and frequency of epileptic discharge in the early stage of brain injury mice to inhibit the abnormal discharge of the brain,reduce the degree of brain edema,and promote the repair of blood-brain barrier.3.Rhynchophylline can down-regulate the expression of epileptic inflammation and apoptosis-related factors in the early stage of brain injury in mice,and participate in the regulation of multiple signal pathways to play an anti-epileptic role.4.Rhynchophylline can inhibit the activation and proliferation of astrocytes by decreasing the fluorescence density of GFAP in DG and CA1 regions of hippocampus and the number of BDNF positive cells in CA3 region of epileptic hippocampus in early stage of brain injury in mice,thus inhibiting the activation and proliferation of astrocytes. |
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