| Epilepsy, one of the most common diseases of the nervous system, is thecause of excessive brain neurons repeatedly synchronization discharge, and isa transient of the central nervous system dysfunction clinical syndromeinduced by a variety of chronic brain diseases. Epilepsy can lead to brainneuron selective damage even necrosis, and cause glial cell hyperplasia,reconstructing synaptic plasticity of the brain structure and function change.These brain plasticity changes and repeated seizure are the leading causes ofrefractory epilepsy. At the same time, the patients often accompaniedcognitive dysfunction with repeated seizures, its incidence is about30%-40%.The pathogenesis of epilepsy is very complex, and has not been fullyelucidated, oxidative stress injury, glutamate toxicity of excitatory, calciumoverload and other factors can induce the abnormal discharge of neuronsinducing seizures. The oxidative stress induced oxygen free radical (FR) chainreaction is the core of the damaged neurons pathological link. The resultsconfirmed that many chronic diseases are linked to oxidative stress inducedexcessive free radicals in the human body. The relationship between epilepsyand oxidative stress have become the research focus. There are oxidative stressresponse in the brain of epilepsy animal models or people with epilepsy.Seizure induce FR increased significantly and far more than the body’s abilityto remove FR. Lots of FR can directly make the macromolecular substancessuch as lipid, protein and DNA oxidative damage damaging cell membraneand other cell structures, and FR also can cause apoptosis by inhibiting themitochondrial function. Therefore, in view of the important role of the oxygenfree radicals in the pathological mechanism of epileptic brain damage, theinhibition of oxidative stress and remove oxygen free radicals may be the important strategy for the treatment of epilepsy.The nuclear factor erythroid2-related factor2(Nrf2) is the key factor incell oxidative stress reaction, Nrf2through reaction with the antioxidantresponse element (ARE), which regulates the expression of a group ofcytoprotective enzymes. Nrf2is an important transcription factor ofadjustment oxidation reaction. Nrf2is located in the cytoplasm combined withKeap1in the physiological state, and is rapidly degraded by ubiquitinproteasome to maintain the low activity. When the body was attacked by suchas oxygen free radicals and endogenous toxin, the Nrf2dissociated with Keapl,and the half-life of Nrf2extended obviously, then Nrf2transposition into thenucleus, and combined with the ARE inducing the expression of a group ofcytoprotective enzymes. So far, it was confirmedn that the Nrf2-ARE signalpathway encoded endogenous protection more than200species. Theendogenous protection can strengthen antioxidant capacity of cell, protectcells from the toxic injury, in anti-tumor, anti-inflammatory and anti-apoptosisHeme oxygenase1(HO-1) and NADPH: quinone oxidoreductase1(NQO1)are important antioxidant enzymes of the signal pathway coding. The previousstudy confirmed that activation of Nrf2-ARE signaling pathways increasesthe expression of the gene products play nerve protection function in nervoussystem diseases such as Parkinson’s disease, cerebral hemorrhage, cerebralinfarction and cerebral trauma and so on. However, to our knowledge, theNrf2-ARE signal pathway and two Nrf2-regulated gene products (HO-1andNQO1) have not been studied after seizure.Sulforaphane (SF) is isothiocyanates of a widely exists in cruciferousvegetables which have the biological characteristics of anti-oxidation,anti-tumor and immune regulation and so on. SF as a chemical preventiondrug has aroused the attention of the researchers. The previous study confirmsthat SF is a important activator of Nrf2-ARE signal pathway, can induce Nrf2phosphorylation transposition into the nucleus, and combine ARE, regulatingthe expression of antioxidants and detoxifying enzyme, scavenging freeradicals to protect the body’s cells. However, to our knowledge, the neuroprotective effect and the molecular mechanism of SF have not beenstudied after seizure.The present study was aimed to examine oxidative stress parameters(malondialdehyde and glutathione) and determine the expression of Nrf2,HO-1and NQO1at protein or gene levels in hippocampus of amygdalakindling rats. And further to evaluating if activation of Nrf2-ARE signalpathway with sulforaphane (SF) in hippocampus can suppress the progressionof chronic amygdala kindling, and ameliorate the cognitive impairment andoxidative stress induced by epileptic seizure. The present study was aimed toclarify that the pathophysiology of epilepsy, and to provide new targets forepilepsy treatment.Part Ⅰ The expression and significance of Nrf2-ARE signaling pathwayin hippocampus of rapid amygdala kindling ratsObjective: To observe oxidative stress parameters(malondialdehyde andglutathione) in hippocampus of rapid amygdala kindling rats and theexpression of Nrf2-ARE signal pathway.Methods: In the present study, Wistar rats were rapidly kindled in theamygdala. Twenty-four hours after the last seizure, the hippocampus of control,sham and kindled rats were examined for oxidative stress parameters(malondialdehyde and glutathione) by spectrophotometry, the expression ofNrf2, heme oxygenase-1(HO-1) and NAD(P)H:quinone oxidoreductase-1(NQO1) were determined using immunohistochemistry, Western blot andreal-time fluorescence quantitative polymerase chain reaction (PCR).Results:1The level of GSH in kindling group was significantly lowercompared to sham group (P <0.01) and the level of MDA was much higherthan that of sham group (P <0.01). There was no difference between controland sham groups(P>0.05).2To localize Nrf2and HO-1expression, immunohistochemical studywas performed. In kindling group, the immunoreactive intensity of Nrf2andHO-1significantly increased in hippocampus, and Nrf2expressed both atcytoplasm and nucleus. In addition, similar localization was found between Nrf2and HO-1in neurons and glial cells. The differences were shownbetween sham and kindling groups by AOD measurement in the expression ofNrf2(P <0.01) and HO-1(P <0.01). There was no difference between controland sham groups (P>0.05).3Western blot analysis was used to measure in protein level of Nrf2innuclear and HO-1in cytoplasm extracted from hippocampus. Groupdifferences were found between sham and kindling groups with the expressionof Nrf2(P <0.01) and HO-1(P <0.01). There was no difference betweencontrol and shamgroups (P>0.05).4The expression of Nrf2, HO-1and NQO1mRNA in hippocampus wasstudied by real-time fluorescence quantitative PCR. Group differences werefound between sham and kindling groups in the expression of Nrf2mRNA (P<0.01), HO-1mRNA (P <0.01) and NQO1mRNA (P <0.01). There was nodifference between control and sham groups (P>0.05).Conclusion:In conclusion, the results of the present study indicate thatthe seizure can induce oxidative stress in hippocampus of rats, which activateNrf2-ARE signal pathway to result in up-regulation of antioxidative anddetoxifying enzymes. Therefore, to activate Nrf2-ARE signal pathway may beone of the strategic targets for epilepsy therapies.Part ⅡThe brain protection of sulforaphane in chronic amygdalakindling ratsObjective: To observe the role of of sulforaphane in the kindlingprogression and the cognitive impairment of chronic amygdala kindling rats.At the same time, to observe the neuroprotective effect for hippocampalneurons.Methods: The rats were kindled by one daily electric stimulation of theamygdala, Electric stimulation was delivered15times for the kindling. Thesulforaphane (5mg/kg/day) was injected intraperitoneally at30min beforeevery electric stimulation. The mean seizure stage and the after dischargeduration in the course of kindling were detected for sulforaphane antiepilepticeffect. Morris water maze (MWM) test assessed the cognitive function of kindled rats, and detected the role of sulforaphane. Using Nissal’s staining andtransmission electron microscope detected the role of sulforaphane inhippocampal neuron of kindled rats.Results:1The repeated administration of subconvulsive electric stimulationinduced severe seizures during the15kindling stimulation. Electricstimulation was terminated on the15th day. By this time, all of the kindlinggroup rats reached the kindling criterion, i.e.,ageneralized stage4or5at leastthree times, while8out of12rats treated with SF did not reach kindlingcriterion over this time. There were significant differences in seizure stagebetween the kindling and kindling+SF groups from the9th day to the15th day(P<0.05from the9th day to the14th dayand P<0.01on the15th day).Meanwhile, pretreatment with SF markedly decreased the after dischargeduration (ADD) as compared to the kindling group from the7th day to the15th day (P<0.05from the7th day to the10th day and P<0.01from the11thday to the15th day). In the control and SF groups, there were no seizureactivities.2In the Morris water maze, all animals showed a progressive decline inthe escape latency with training. Rats in the kindling group exhibitedsignificantly prolonged escape latency as compared to the control group(P<0.01). However, the poor performance was mitigated by pretreatment withSF(P<0.01). In the probe trial, the kindling group spent significantly less timein the target quadrant than the control group (P<0.01), while pretreatment withSF significantly improved the performance (P<0.01). The number of crossingthe plat form in the kindling group obviously decreased as compared to thecontrol group(P<0.01), while pretreatment with SF markedly increased thenumber of crossing(P<0.01). Inaddition, SF per se had no significant effectoncognition.3Nissl staining showed that: neurons in hippocampus of control groupwere clear with normal nucleolus, well-distributed karyotin and rich nisslbodies in kytoplasm, there was no significantly neuron loss. While in the kindled rats, neuron loss was obviously, with shrunken plasma body andpyknotic nuclei. In the kindling+SF group, most pyramid cells were normaland only a few showed chromatin condensation. The hippocampus neurons ofSF group were no significant change as compared to the control group.4In hippocampal CA1area of the rats in NC group，there were intactneurons，distinct structure，uniform chromatin，abundant apparatus，andintegrate myelin．In PTZ group，however，there were condensed nucleus，edematous neuron，swollen perikaryon with vacuole，reduced mitochondriaand Golgi apparatus and polyribosome；there was also Myelin-splitting nervefiber．In kindling+SF group，the neuron and neuropile were less swollen,karyotheca were normally intact，chromatin were uniform，the apparatus wererich and basically normal，and the myelin was complete and integrate．Incontral group，the synapses in hippocampal CA1area were abundant withdistinct pre-and-post synaptic membranes and rich synaptic vesicles．Thesynaptic cleft was clear．In kindling group，there were reduced synapses withindistinct pre-and-post synaptic membranes．In kindling+SF group，there weremore synapses than that in kindling group. The ultrastructure of hippocampusneurons in SF group were no significant change as compared to the controlgroup.Conclusions：In summary, the results from the present study demonstratethat SF could suppress the progression of amygdala kindling, also amelioratethe cognitive impairment induced by epileptic seizure. SF also can protecthippocampal neuron damage induced by seizures.Part Ⅲ The neuroprotection mechanism research of sulforaphane in inchronic amygdala kindling ratsObjective: The present study was aimed to evaluate if activation ofNrf2-ARE signal pathway with SF in hippocampus can ameliorate oxidativestress induced by epileptic seizure, to explore the mechanism of brainprotection.Methods: The rats were kindled by one daily electric stimulation of theamygdala, Electric stimulation was delivered15times for the kindling. The sulforaphane (5mg/kg/day) was injected intraperitoneally at30min beforeevery electric stimulation. Twenty-four hours after the last seizure, thehippocampus of control, kindled, kindling+SF and SF rats were examined foroxidative stress parameters (malondialdehyde and glutathione) byspectrophotometry, the expression of Nrf2, heme oxygenase-1(HO-1) andNAD(P)H:quinone oxidoreductase-1(NQO1) were determined usingimmunohistochemistry, Western blot and real-time fluorescence quantitativepolymerase chain reaction (PCR).Results:1The level of Malondialdehyde (MDA) in the kindling group wasmuch higher as compared to the control group (P<0.01), and the level ofreduced glutathione (GSH) in the kindling group was significantly lower ascompared to the control group (P<0.01). However, pretreatment with SF ledto a noticeable decrease in the concentration of MDA (P<0.01) and asignificant increase in GSH level (P<0.01) as compared to the kindling group.Although SF treatment ameliorated theoxidative damage induced by seizures, the oxidative stress level wasstill higher than the control group (P<0.01). SF per se caused a decrease inthe oxidative stress as indicated by the significant decrease in MDA levelsand the significant increase in GSH levels as compared to the control group(P<0.05).2Western blot analysis of protein level of Nrf2in nuclear and proteinlevels of HO-1and NQO1in cytoplasm extracted from hippocampus. Theexpression of Nrf2in nuclear and the expression of HO-1and NQO1incytoplasm were not significantly different between control and kindlinggroups(P>0.05). However, pretreatment with SF led to significant increase inthe expression of Nrf2, HO-1and NQO1at protein levels (P<0.01) ascompared to the control and kindling groups. SF per se also causedsignificant increase in the expression of Nrf2, HO-1and NQO1at proteinlevels (P<0.01) as compared to the control group.3The expression of Nrf2, HO-1and NQO1mRNA in hippocampus was studied by real-time fluorescence quantitative PCR. The expressionof Nrf2, HO-1and NQO1mRNA were not significantly different betweencontrol and kindling groups(P>0.05). However, pretreatment with SF led tosignificant increase in Nrf2, HO-1and NQO1mRNA levels (P<0.01) ascompared to the control and kindling groups. SF per se also causedsignificant increase in Nrf2, HO-1and NQO1mRNA levels (P<0.01) ascompared to the control group.Conclusions： In summary, the results from the present studydemonstrate that SF could activate the Nrf2-ARE singal pathway, increasethe expression of antioxidative enzymes(HO-1and NQO1) and alsoameliorate oxidative stress induced by epileptic seizure. Therefore, toactivate Nrf2-ARE signal pathway maybe one of the strategic targets forepilepsy therapies. |
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