Study On Mechanism Of Amentoflavone On Epilepsy And Cognition

ObjectiveObservation Amentoflavone of pilocarpine induced epileptic rats on learning and memory function, to explore the mechanism of action; study amentoflavone on rat insular neurons epileptiform discharge effect and of GABAR currents regulatory role. Material and Methods1. To improve the effect of amentoflavone on memory impairment in epileptic rats: 36 clean grade healthy male SD rats were randomly divided into two groups(group AF), epilepsy group(group EP) and control group(group Control), 12 rats in each group. The control group(group 220~250g) was divided into two groups. In the intervention group, the intervention group was injected with AF(25mg/kg), and the other two groups were injected intraperitoneally with the same volume of normal saline, and the other groups were injected with the same volume of normal saline. The 5 day by intraperitoneal injection of lithium chloride(127mg / kg). After 18 h biflavonoids intervention group and epilepsy group were treated with intraperitoneal injection of pilocarpine goods(30mg / kg). Seizure behavior according to Racine standard is divided into 6, rats appeared three times in a row grade IV, V grade convulsion is believed to successfully ignited. Morris water maze test in rats with learning and memory, to observe the effect of drug intervention on epileptic rats memory impairment by finding the hidden platform in the water maze escape latency and by number of the location of the original platform to assess rats learning and memory function.2. Effect of amentoflavone on oxidative stress and cholinergic function within the hippocampus: Morris water maze experiment after the end of line of the rats were decapitated, taking the brain acetylcholine content in hippocampal tissue was detected by immunohistochemical staining and enzyme-linked immunosorbent adsorption determination of acetylcholinesterase activity and MDA was measured by thiobarbituric acid method, with dithio bis Nitrobenzoic Acid Method Determination of GSH. To observe and compare the manto o biflavonoids intervention effects on epileptic rats oxidative stress and cholinergic function, to explore the improvement of the mechanism of memory impairment.3. Effect of rat brain island arnentobiflavone induced by 4- aminopyridine leaves neurons epileptiform discharge: Choose to be born 4-5 weeks of clean grade healthy male SD rats, intraperitoneal injection of chloral hydrate anesthesia, brains were removed quickly put on the back burner vibration slicing machine of water bath, removed 400 um thick insular brain slices, to 37°C artificial cerebrospinal fluid incubation for 1 hour, stabilize neuronal to contain 4-amino pyridine concentration 100 um artificial cerebrospinal fluid continuous perfusion 30-40 min, evoked Island lobe neurons epileptiform discharges. Using whole cell patch clamp recording and current clamp mode recorded induced by 4-aminopyridine typical epileptiform discharges, and intervention in different doses(5ug / ml, 10 ug / ml, 20 ug / ml, respectively) of amentoflavone, O manto biflavonoids stem prognosis of neuronal firing frequency and duration changes were observed and compared. Evaluation amentoflavone of epileptiform discharge effect.4. Regulation effect of amentoflavone on GABAR channel current: Brain slice preparation stability, selecting good shape of insular neurons, were randomly divided into GABA group?control group and O manto biflavonoids group, using whole cell patch clamp recording mode, to contain tetrodotoxin concentration degree 1um artificial cerebrospinal fluid blocking sodium ion current with GABA concentration to 100 um artificial cerebrospinal fluid through drug delivery system for rapid administration, neuronal GABAR currents, and then different concentrations of amentoflavone intervention, relatively dry changes in GABAR current prognosis and evaluation o mentor biflavone of GABAR current regulatory role.5. Statistical analysis SPSS21.0 statistical software was used to analyze the data, the results of the experiment with S, the comparison between the groups using one factor analysis of variance, with P<0.05 as the difference was statistically significant Results1. Morris water maze: Find the hidden platform in the water maze escape latency and the number of times through the original platform where the number of times, respectively, said the spatial learning ability and spatial memory ability of rats is stronger, and vice versa. Comparison between groups of the escape latency and epilepsy group than the control group significantly increased(P < 0.01); double flavonoid intervention group than in the epilepsy group significantly shortened(P < 0.01); through original platform location number comparison, epilepsy group compared with the blank control group decreased significantly(P < 0.01); double flavonoid intervention group than in the epilepsy group increased significantly(P < 0.01).2. Expression of Ach E positive neurons in the hippocampus: The background of immunohistochemistry was light yellow, Ach E immunoreactive neurons showed different levels of brown yellow. Blank control group, epilepsy group, the two flavonoids intervention group CA3 area Ach E immunoreactive neurons were 40.6 + 10.2, 7.1 + 2.2, 32.8 + 8.4. Epilepsy CA3 region of ache immunoreactive positive cell number compared with the blank control group significantly reduced, pale staining(P < 0.01); biflavone intervention significantly increased the number of CA3 region of ache immunoreactive positive cells than in the epilepsy group and deep staining(P < 0.01).3. Hippocampal Ach E activity concentration: The Ach E activity concentration(U/L) of the blank control group, epilepsy group and the double flavonoid intervention group were 38.61 + 4.90, 7.54 + 2.34, 30.66 + 5.79, respectively. Compared with the control group, the activity of Ach E in the hippocampus of epileptic group was significantly lower(P<0.01), and the Ach E activity was significantly increased in the intervention group(P<0.01).4. MDA and GSH content: The contents of MDA(nmol/mgprot) in the blank control group, epilepsy group and the double flavonoid intervention group were 4.10 + 0.42, 9.50 + 1.11, 5.25 + 0.98, GSH(mg/gprot) 5.20 + 0.53, 2.81 + 0.31, 6.08 + 0.54, respectively. Compared with the blank control group, MDA level of epileptic rats was significantly increased(P < 0.01) and GSH decreased significantly(P < 0.01); relative to the epilepsy group, biflavone intervention group, the level of MDA significantly reduced(P < 0.01) and GSH increased significantly(P < 0.01).5. Effect of rat brain island arnentobiflavone induced by 4- aminopyridine leaves neurons epileptiform discharge: Under whole cell current clamp recording mode, control group rat insular lobe neurons showing a single incident of depolarization issued, which is electrophysiological activity of rat insular area neurons, by containing 4-amino pyridine concentration 100 um artificial cerebrospinal fluid continuous perfusion flow incubation of rat island lobe 30-40 min, insular lobe neurons gradually appeared stable inducing epileptiform discharge. Records show that after 5ug / ml, 10 ug / ml and 20 ug / ml of different concentrations of amentoflavone intervention, insular lobe neurons promote epileptiform power duration and epileptic discharges issued intermittent period had no obvious change. Using normal artificial cerebrospinal fluid for the elution of the insular lobe of the brain slices, the number of epileptic discharges of neurons decreased gradually, and the intermittent period was prolonged, and finally recovered to the state of the Control group. Statistical results show that manto the concentration biflavonoids intervention group were not significantly reduced insular lobe epileptiform discharge duration and frequency of discharge.6. Regulation effect of amentoflavone on GABAR channel current :With a concentration of 20 ug / ml o manto biflavonoids extracellular fluid of rat insular cortex brain slices were perfused and induce a GABAR currents and irrigation for GABA outflow liquid compared, current amplitude without significant changes(Figure 2.1 AF group). The experimental results show that o mentor biflavone of GABAR currents do not produce effect. Conclusions1. Amentoflavone can improve induced epileptic rats learning and memory function, by increasing the activity of acetylcholinesterase(ACh E) in hippocampus, inhibition of oxidative stress. Epileptic discharge.2. Amentoflavone cannot inhibit insular neurons Epileptic discharge.. Insular neurons GABAR channels.3. Amentoflavone no moderating effect on Insular neurons GABAR channels. induced by GABA.