The Biochemical Mechanisms Of Audiogenic Epilepsy In Fmr1 And The Intervention Effect Of Bupleuri And Ramuli Cinnamomi Decoction

ObjectiveEpilepsy is a common chronic brain disease of the nervous system, which is caused by a variety of reasons. It is affecting the health of a large numbers of human beings,which is characterized by suddenly, repeated and temporal malfunction of central nervous system led by excess discharges of neurons. The abnormal discharge is closed related to neurotransmitter imbalances and the abnormal of ion channel, glial cells, immunity and heredity. The current experimental study is mostly used epileptic animal model to research epilepsy, which is kindled by meidicine.The traditional medicine include Pentamethazol, Pilocarpine, Penicillin ,Malysol and so on.The treatment to epilepsy in clinic is mostly controled epileptic seizure by western medicine.But common western medicine are almost have obvious side effects.The traditional medicine are superior to treat epilepsy in effect of treatment and less side effect. Bupleuri and Ramuli Cinnamomi Decoction is obviously react on antiepileptic in clinic ,but its mechanism is unknown. Clearing the mechanism of epilepsy is propitious to the diagnosis, preventability and treatment of epilepsy.The experiment used FMR1 gene kockout mouse to reaserch the mechanism of epilepsy and intervention effect of Bupleuri and Ramuli Cinnamomi Decoction in some fields, such as the function of spatial cognition , synaptic rebuiltion, the animal behavior , production and elimination of free radical and the level of amino acid.The research helps to build the substantial basics of opening up new antiepileptic medicine.Methods1. The preliminary study of FMR1 gene knockout and epileptic mouse model FVB strain four-week mice, six-week mice, eight-week mice,including FMR1 knockout (KO) and wild type (WT) counterparts were used and genotyped by PCR before experiments. They were trained in a five-day Morris water maze learning and memory tests, icluding the first four days of hiddern-platform acquisition training and the fifth probe trial testing.FVB strain 0-week mice, two-week mice, four-week mice,including FMR1 knockout (KO) and wild type (WT) counterparts were used and genotyped by PCR.Then we used immunohistochemical method to observe the expression of EphA2 in brain of two different mice groups.2. Reaserch about biochemical mechanism in audiogenic epilepsy of FMR1 konckout mice and antiepileptic effect of Bupleuri and Ramuli Cinnamomi DecoctionFVB strain four-week mice,including FMR1 knockout (KO) and wild type (WT) counterparts were used. They were intraperitoneal injected by different concentrations Bupleuri and Ramuli Cinnamomi Decoction to observe animal behavior indicators and establish an effective drug concentration curve.Experimented anmimals were divided into three groups ,including control group, blank group and treatment group.We detected the level of superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide respectively by pyrogallol autoxidation method, thiobarbituric method,in order to compare changes of three different groups. At the same time,we detected the level of amino acid of different groups by high performance liquid chromatography in order to reaserch comprehensively the pathogenesis of epilepsy.3. The components of Bupleuri and Ramuli Cinnamomi Decoction by gas chromatography - mass spectrometWe produced Bupleuri and Ramuli Cinnamomi Decoction by Supercritical Fluid Ext ration CO2 .Then we analyze and identify Bupleuri and Ramuli Cinnamomi Decoction by Gas chromatography-mass spectrometry. Results1. The results of Morris water mazeIn the hiddern-platform experiment , the escape latency of KO mice is longer than WT mice ,included four-week mice, six-week mice, eight-week mice, which have statistical significance.Otherwise, in the probe trial testing, numbers through plat and elapsed time in the platform of KO mice are less than WT mice, included four-week mice, six-week mice, eight-week mice.2. The results of EphA2 expression in brain of mice EphA2 protein were abundantly found in newborn mice in hippocampus and cortex, especially in CA3 of hippocampus. And both positive cell and positive fibers were expressed in cortex. The positive cells of KO mice are more than WT mice in hippocampus and cortex, included 0-week mice, two-week mice, four-week mice.3. The results of open fieldThe average speed, trajectory and numbers pass through the central field of KO and WT mice were decreased, with increasing concentration of Bupleuri and Ramuli Cinnamomi Decoction.4. The results of level of SOD,MDA,NOThe level of SOD,MDA,NO in WT blank group was higher than WT control group. The level of WT treated group was higher than WT blank group, while the level of MDA and NO in WT treated group was lower than WT blank group.(P<0.05)The level of SOD,MDA,NO in KO blank group was higher than KO control group. The level of KO treated group was higher than KO blank group, while the level of MDA and NO in KO treated group was lower than KO blank group.(P<0.05)5. The results of level of amino acidThe level ofγ-aminobutyric acid and Aspartate in WT blank group was obviously lower than WT control mice, while the level of Glutamate in WT blank group was higher than WT control group.Otherwise, the level of Glutamate in WT blank group was higher than WT treated group. (P<0.05) The level ofγ-aminobutyric acid in KO blank group was obviously lower than KO control mice, while the level of Glutamate in KO blank group was higher than KO treated group. (P<0.05)6. The results of GC-MSWe identified 28 kinds of components by GC-MS. In Bupleuri and Ramuli Cinnamomi Decoction, the most important ingredient gingerol accounted 31.94%(C15H24 accounted 19.75%, C15H26O accounted 7.36%, C17H24O3accounted 4.83%),Eugenolaccounted18.01%, Monoterpenes accounted 12.34%(C15H24accounted 7.98%, C15H24 accounted 3.90%, C15H24 accounted for 0.46%), NBP accounted 9.34% and cinnamon substances accounted 8.03%.Conclusions1. FMR1 knockout mice was an ideal animal mode of epilepsy ,because its seizures was similar to human beings and it had impairment of study and memory.The protein EphA2 , a kind of Axon guidance molecules, may be took part in the process of Axonal sprouting and synaptic remodeling,which supplied a new thought.2. Audiogenic epilepsy of FMR1 knockout mice may be related to imbalance between excitatory amino acid and inhibitory amino acid.It was a large number of free radicals, peroxide generation and NO neurotoxicity that may be play a role in epileptogenic process.3. The antiepileptic of Bupleuri and Ramuli Cinnamomi Decoction may be related to reduced ratio of excitatory amino acid / inhibitory amino acids.It can effectively eliminated free radicals, prevented peroxide formation and reduced NO neurotoxic effects. To some extent ,it can reduce the number of seizures and protect brain cells.4. The main components in Bupleuri and Ramuli Cinnamomi Decoction ,such as Gingerol, Monoterpenes, NBP and eugenol, played a role in the process of Antioxidant and anti-epileptic.