Antiepileptic Effect Of VPA Combined With The Compound Danshen Dripping Pills On The Epileptic Rats

ObjectiveThe study is based on injected kanic acid（KA）into the hippocampus by Brain stereoscopic positioning technology to induce epileptic rats,then use Compound Danshen Dripping Pills（CDDP）,valproate sodium（VPA）and integrated therapy to assess the effect of integrated therapy on the behavior,learning and memory ability,spatial reference memory ability by water maze and radial-arm maze experiments.To assess the effect of hippocampal neurons injury by Nissl staining and CDDP on the concentration of VPA by UPLC-MS/MS.Determine the content of CRP,IL-6and TNF-αin brain tissue and serum by ELISA and the apoptosis factors Caspase3 and Caspase8in hippocampus by Real-time quantitative fluorescence PCR and Western Blot.In order to verify whether VPA combined with CDDP has a better antiepileptic effect on epileptic rats and provide a certain theoretical basis for clinical medication.Methods1.Injected with KA（saline group was injected with saline）into the hippocampus of SD male rats to establish epileptic model by brain stereoscopic positioning technology and selected the successfully ignited rats according to the Racine level,then rats were randomly divided into model group（Model）,saline group（Saline）,valproate sodium group（VPA）,Compound Danshen Dripping Pills group（CDDP）,valproate combined with Compound Danshen Dripping Pills group（VPA with CDDP）and 8 rats in each group.The model group and the saline group were given a certain amount of saline,and the other groups were given corresponding drugs.To assess the effect of VPA combined with CDDP on the behavior,learning and memory ability,spatial reference memory ability of rats by water maze and radial-arm maze after 30 and 60 days’administration.2.Four rats were randomy selected from each group after 60 days’intragastric administration,then narcosised by 10%chloral hydrate intraperitoneal and cardiac perfusion by 4%polyformaldehyde and saline to stiffness of the limbs.The brain was quickly taken from the head and fixed by polyformaldehyde.Observed the damage of hippocampus neurons and CA3 region by Nissl staining and neuronal counting.3.The epileptic rats were randomly divided into VPA group,VPA with CDDP group and five rats in each group.After the combined group was treated with the CDDP for seven days and given VPA in the eighth day to all the two groups,then collected blood from the eyelids at different timeline,and determined the concentration of VPA by UPLC-MS/MS.Drawing a pharmaceutical time curve by DAS 3.0 software to study the effect of CDDP on the concentration of VPA in epileptic rats.4.Five rats were randomly selected in each group after 60 days’treatment,then collected blood and brain tissue after narcosising by 10%chloral hydrate intraperitoneal and determined the content of CRP,IL-6 and TNF-αby ELISA.5.Six rats were randomly selected in each group after 60 days’treatment,and narcosised by10%chloral hydrate intraperitoneal,taken out the brain and the hippocampus rapidly,determined the expression of the genes and proteins of the apoptosis factors Caspase3 and Caspase8 by real-time fluorescence quantitative PCR and Western Blot.Results1.With the increase of training time in place navigation experiment,the escape latency of each group were decreased,and the remaining groups was significantly decreased compared with the model group in the same period（P<0.01）.The escape latency of the VPA with CDDP group was reduced compared with VPA alone,but there was no significant difference（P>0.05）after 30days’treatment;The VPA with CDDP group was significantly reduced compared with the VPA alone（P<0.05,P<0.01）after 60 days’treatment;In the space probe test,compared with the model group,the swimming frequency of crossing the platform of the other groups was increased with varying degrees and in 60 days experiment the VPA with CDDP group was significantly increased compared with the VPA alone（P<0.01）.In the radial-arm maze experiment,the number of reference memory errors,the number of working memory errors and the total time of eating food in the remaining groups were all lower than those in the model group,and the indicators of the VPA with CDDP group were significantly lower than those of the VPA group in 60days（P<0.05）.2.Compared with the model group,the number of neuronal cells in CA3 region of hippocampus in other groups was increased,and the combined group was significantly increased compared with the VPA group（P<0.05）.3.Compared with the VPA group,the AUC_（0-8h）,AUC_（0-∞）,T_maxax and C_maxax of the VPA with CDDP group were significantly improved（P<0.01,P<0.05）,CL_Z/F/F was significantly reduced（P<0.05）,the T_1/2z,V_z/F/F were also improved in different degrees.4.Compared with the model group,the content of CRP,IL-6,TNF-αin the other groups of brain tissue and serum was significantly reduced（P<0.01）.The CRP in VPA with CDDP group of brain tissues was significantly lower than the VPA group（P<0.01）,the IL-6 and TNF-αlevel of VPA with CDDP group was decreased compared to the VPA group,but there was no significant difference（P>0.05）.5.Compared with the model group,the expression of Caspase3 gene in VPA with CDDP group and saline group was decreased significantly（P<0.01）;Compared with VPA group,Caspase3 gene expression in VPA with CDDP group is reduced significantly（P<0.05）.Compared with the saline group,the gene expression of Caspase8 in the other groups increased significantly（P<0.01,P<0.05）,the VPA with CDDP group was no statistical significance compared with the VPA group（P>0.05）.6.Compared with the model group,the expression of Caspase3 and Caspase 8 protein in the other groups was significantly reduced（P<0.01）and compared with VPA alone,the expression of Caspase3 and Caspase 8 protein in VPA and CDDP group was significantly reduced（P<0.01）.ConclusionVPA combined with CDDP can increase the distribution of VPA in epileptic rats and increase the concentration of VPA and can better improve the learning and memory ability,spatial reference memory ability and reduce neuronal damage.The mechanism may be to regulate inflammatory reactions by reducing inflammatory factors such as CRP in the brain,or to reduce apoptosis by regulating apoptosis factors like Caspase3 and Caspase 8.