The Effect Of α-lipoic Acid On The Expression Of P38MAPK In Hippocampus And Behavior Of Amygdala Electrical Kindling Seizures In Rats

Objective: Epilepsy is a common syndrome of nervous system affectingpeople of all ages, epidemiological investigations show that approximately50million people are affected worldwide, and the morbidity is3.5‰-4.8‰inChina. It is characterized by recurring seizures, the principalpathophysiological basis of epilepsy is abnormally synchronized neuronaldischarges which disrupt the function of the brain regions. Repeated seizuresover time or single prolonged seizures may result in neuronal apoptosisseverely, particularly within the limbic system such as hippocampus. Thestudies show that hippocampal sclerosis results from neuronal apoptosis andgliosis, in turn, hippocampal sclerosis contributes to seizure with frequent,develops into intractable epilepsy eventually. Therefore, research deeply themolecular mechanism of neuronal apoptosis after epilepsy, and explor newand effective drugs that anti-apoptosis of neurons may have a profoundsignificance.Those in our study were chronic epileptic rats kindled by amygdalaelectrical stimulation, and α-lipoic acid (α-LA)was administrated.Pre-kindling and post-kindling afterdischarge threshold (ADT) wasdetermined, the number of cumulative stimulations to reach the respectiveseizure stage and of the cumulative duration of afterdischarge (ADD) to reachthe respective seizure stage were recorded, the expression of p38MAPK andp-p38MAPK protein in hippocampus were tested by the method of WesternBlot, apoptosis rate of hippocampal neuron was detected by Flow cytometricanalysis Propidium Iodide（PI) dyeing technology. For exploring the effects ofα-LA on the behavior and the expression of p38MAPK in hippocampus ofamygdala electrical kindling rats, and discussing the molecular mechanism of brain protection function of α-LA.Methods:36clean grade adult male Wistar rats which weight250-300gwere choosed, the rats were randomly distributed to the following six groupsconsisting of6animals each. Normal control group：the rats without anyprocessing, α-LA group：α-LA （40mg/kg）was administered intraperitonealinjection once daily, sham-operation group：after implantating electrode intoamygdala without any processing, electrical stimulation model group：animalswere stimulated by120%pre-kindling ADT once daily, electrical stimulationplus low-dose α-LA group: animals were stimulated by120%pre-kindlingADT once daily30min after α-LA (20mg/kg) was administeredintraperitoneal injection, electrical stimulation plus high-dose α-LA group:animals were stimulated by120%pre-kindling ADT once daily30min afterα-LA(40mg/kg) was administered intraperitoneal injection, the stimulationwas continued for15days. Racine scale was used to evaluate the behaviouralseizure during the kindling process, and the respective seizure stage andduration of afterdischarge (ADD) were recorded. At the end of observations,the rats were killed by decapitation and their brains were dissected on ice toremove hippocampus, the expression of the p38MAPK and p-p38MAPKprotein in hippocampus were tested by the method of Western Blot, apoptosisrate of hippocampal neuron was detected by Flow cytometric analysis PIdyeing technology.Results:1.Results of behavior①The number of cumulative stimulations to reachthe first seizure stage5: electrical stimulation plus high-dose α-LA group（11.50±0.43）was increased obviously compared to model group（10.17±0.31)(P<0.05), but there was no significant difference when electrical stimulationplus low-dose α-LA group (11.17±0.31) compared to both model group andelectrical stimulation plus high-dose α-LA（P>0.05）.②The number ofcumulative ADD to reach the first seizure stage5: electrical stimulation pluslow-dose α-LA group（191.17±5.82) and electrical stimulation plus high-dose α-LA group （184.83±7.60）was shorted obviously compared to model group（235.83±5.00）（P<0.05）, however, there was no significant differencebetween electrical stimulation plus low-dose α-LA group and electricalstimulation plus high-dose α-LA group（P>0.05）.③Pre-kindling andpost-kindling ADT: post-kindling ADT （133.33±12.29) was decreasedobviously compared to pre-kindling ADT（273.33±24.59）of model group（P<0.05）, post-kindling ADT（200.00±17.13）was decreased obviouslycompared to pre-kindling ADT（286.67±22.31）of electrical stimulation plushigh-dose α-LA group（P<0.05）, post-kindling ADT（153.33±19.78）wasdecreased obviously compared to pre-kindling ADT （280.00±30.55） ofelectrical stimulation plus low-dose α-LA group（P<0.05）.④Post-kindlingADT: electrical stimulation plus high-dose α-LA group was increasedobviously compared to model group （P<0.05）, there was no significantdifference of electrical stimulation plus low-dose α-LA group compared toboth model group and electrical stimulation plus high-dose α-LA（P>0.05）.No seizures were found in the normal control group, sham-operation groupand α-LA group.2. Result of Western Blot The expression of p-p38MAPK protein inhippocampus of the normal control group, sham-operation group and α-LAgroup were0.55±0.06,0.62±0.05,0.64±0.05, but there was no significantdifference among them（P>0.05）. The expression of p-p38MAPK protein inhippocampus of model group (3.39±0.09) was increased obviously comparedboth to electrical stimulation plus low-dose α-LA group (2.20±0.04) andelectrical stimulation plus high-dose α-LA group (1.26±0.93)（P<0.05）,electrical stimulation plus low-dose α-LA group was increased obviouslycompared to electrical stimulation plus high-dose α-LA group（P<0.05）, at thesame time, the expression of p-p38MAPK protein in hippocampus of themodel group, electrical stimulation plus low-dose α-LA group and electricalstimulation plus high-dose α-LA group were increased obviously compared tonormal control group, sham-operation group and α-LA group（P<0.05）.3. Results of PI dyeing The apoptosis rate of hippocampal neuron of the normal control group, sham-operation group and α-LA group were1.91±0.60,1.72±0.46，1.53±0.37, but there was no significant differenceamong them（P>0.05）. The apoptosis rate of hippocampal neuron of modelgroup（14.83±0.60）was increased obviously compared both to electricalstimulation plus low-dose α-LA group(11.71±0.52) and electrical stimulationplus high-dose α-LA group (8.65±0.38)（P<0.05）, and electrical stimulationplus low-dose α-LA group was increased obviously compared to electricalstimulation plus high-dose α-LA group（P<0.05）, at the same time, theapoptosis rate of hippocampal neuron of the model group, electricalstimulation plus low-dose α-LA group and electrical stimulation plushigh-dose α-LA group were increased obviously compared to normal controlgroup, sham-operation group and α-LA group（P<0.05）.Conclusions:1.This study successfully established the chronic epileptic rat modelkindled by amygdala electrical stimulation.2.The expression of p-p38MAPK protein in hippocampu was up-regulated and the apoptosis of hippocampal neuron was increased afterepilepsy.3.The seizure stage was decreased, the duration of afterdischarge wasshorten, the level of p-p38MAPK protein in hippocampus was down-regulatedand the apoptosis rate of hippocampal neuron was decreased afteradministering α-lipoic acid. Thus, we can speculate that α-lipoic acid probablyplay a neuroprotective role in epilepsy by inhibiting apoptosis, and thendown-regulate the susceptibility of electrical stimulation.