Research Based On The Neuroprotection Of ?-Hydroxybutyrate To Explore The Antiepileptic Mechanisms Of Ketogenic Diet

BACKGROUND:Epilepsy is a kind of chronic disease,which is caused by the sudden abnormal discharge of neurons.It is a common disease of the nervous system.In China,the prevalence of epilepsy is high,it is about 3.6 per thousand to 4.4 per thousand in children and teenagers under 16 years old.Although up to 30%patients treated with regular antiepileptic drugs for more than two years,repeated seizures still can not be controlled,and become intractable/refractory epilepsy[1].Childhood is an important stage of the rapid development of brain function,if the treatment effect of intractable epilepsy is not satisfactory,it is serious harmful to children's physical and mental health.Therefore,it is of great scientific significance and clinical value to carry out the research on the therapeutic aspect of intractable epilepsy in children and explore new therapeutic targets.The ketogenic diet(KD)is a high fat,low carbohydrate diet,which cuold produce ketosis metabolic process by mimicing a fasting state.The ketogenic diet is an important method of treatment for refractory epilepsy in children[2]Wilder applied KD as a clinical treatment method of epilepsy for the first time in 1921,and KD showed relatively good efficacy.A large number of clinical studies and animal experiments have confirmed that KD can effectively control the seizures of epilepsy,especially for the treatment of intractable epilepsy in children.A randomized clinical trial found a reduction of seizures in 75%children after 3 months' KD therapy[3-5].The antiepileptic mechanisms of the ketogenic diet have not been folly elucidated,the recent domestic and foreign researches suggest that direct neuroprotection and ketone bodies are closely related to the antiepileptic effect.However,the underlying mechanisms of ketone bodies' neuroprotection are not clear.This paper is divided into two parts:Part one,we established the cell model by HT22 cell line and studied the protective effect of beta hydroxybutyrate on glutamate induced damage in HT22 cells.Part two,we carried out research on the redox level and cell signaling pathways to investigate the protective mechanisms of neural regulation of beta hydroxybutyrate.Part One Beta hydroxybutyrate has a neuroprotective effect on glutamate induced injury in HT22 cellsObjectives:Refractory epilepsy is still a thorny problem in the clinical treatment of intractable epilepsy,we can choose the ketogenic diet(high fat and low carbohydrate)therapy to deal with it.Fat instead of carbohydrate as the main energy source of the body,produces a lot of acetyl coenzyme A(CoA)in the liver,and a large number of ketone bodies including beta-hydroxybutyrate(BHB),acetoacetate(ACA)and acetone are synthesized in the mitochondria of liver.When the sugar's utilization is limited,ketone bodies become the main energy source.A large number of clinical studies and animal experiments show that KD treatment of refractory epilepsy can significantly increase the content of beta-hydroxybutyrate both in serum and urine[6],which suggest that beta-hydroxybutyrate might play a key role in the antiepileptic mechanisms of KD,but the underlying mechanisms are still unknown.Therefore,in this study,we took beta hydroxybutyrate which accounts for 78%of the ketone bodies as the starting point,and explored the neuroprotection role of beta hydroxybutyrate by establishing cell model(in vitro).Methods:We carried out HT22 cell line culture,and established HT22 cell model of 5mM glutamate induced injury.We used MTT assay to detect HT22 cells survival rate of each group which was pretreated with different concentrations of BHB for 24 hours,and to detect the rate of survival of HT22 cells in all groups.We discovered whether different concentrations of BHB pretreatment have protective effect on glutamate injury cell model or not,then observed morphous of cells by Hoechst 33258 staining in each experimental group.Results:1.We used 2mM,4mM,8mM,10mM concentrations of BHB to pretreat HT22 cells for 24 hours,then the survival rate of cells in each group was detected by MTT assay,and there was no significant difference between the normal control group and the test groups(P>0.05).2.The effects of 4mM and 8mM BHB pretreatment on the survival rate of cells treated by 5mM GLU for another 24 hours were analyzed by MTT assay.The results of MTT were as follows:The survival rate of GLU treated groups was lower than that of normal control groups(P<0.05);the survival rate of 4mM BHB pretreatment groups was higher than that of GLU treated only groups,and there was statistically significant compared with GLU groups(P<0.05),the survival rate of 8mM BHB pretreatment groups was higher than that of GLU treated only groups,but there was no significant difference between 8mM BHB pretreatment groups and GLU treated only groups(P>0.05).3.Morphological Observation of cells in different experimental groups by inverted phase contrast microscope showed:In normal control group,the cells grew well,the cell density was high,and organelles were complete,and the nuclei were clear;in GLU treated only group of HT22 cells,we could see the apoptosis of cells obviously,low cell density,cell body shrinkage,cytoplasmic vacuoles,nuclear pyknosis,fragmentation,fragmentation of cell bodies and nerve cell processes,apoptotic bodies;in 4mM BHB pretreated group,the cell density was higher compared with GLU group,we found that some of the cells the body were swelling,some of cells the body showned shrinkage,some of cells showed pyknosis and apoptosis were fewer compared with GLU group.Hoechst 33258 staining was observed by fluorescence microscope:In the normal control group,the nuclei of the cells were uniform blue,the density was high,nuclei were clear,and the color was dark;in the GLU group,the color of nuclei was bright blue,the density of nuclei was low,a part of the nuclei showed white light,nuclear shrinkage,margination and broken;in the BHB pretreatment group,the color of nuclei was blue,the density was higher than that in GLU group,the number of nuclei was more than GLU groups,a part of the nuclei showed a bright blue,a small amount of nuclei showed condensation and margination.Conclusions:1.BHB has no effect on the survival rate of HT22 cells and no toxicity;2.GLU can induce damage to HT22 cells;3.BHB has protective effect on GLU induced damage in HT22 cells.Part Two The related research on neuroprotective mechanisms of beta hydroxybutyrateObjectives:Nerve injury is an important mechanism of refractory epilepsy,while antiepileptic mechanisms of ketogenic diet is not clear,we conclude that it is related to the ketone bodies.The first part of our study found that BHB has neuroprotective effect,and it may play an important role in the therapeutic mechanisms of epilepsy.However,the neuroprotective mechanisms of beta hydroxybutyrate have not been fully elucidated.In the animal model of hypoglycemia,beta hydroxybutyrate and acetoacetate inhibited lipid peroxidation and scavenged ROS to inhibit neuronal apoptosis by maintaining ATP levels in brain[7];in animal model of Parkinson's disease,beta hydroxybutyrate had the protective effects by regulating mitochondrial functionc[8].Through our experiments we know that BHB has a protective effect on oxidative damage induced by GLU,then the BHB is involved in the regulation of nerve cell injury and apoptosis induced by oxidative damage.In order to explore the possible mechanisms,this part intended to use the cell model,to detect the level of oxidation-reduction in each group,and to study the regulatory mechanisms of neuroprotective effect on cell signaling pathway.We offered experimental evidence to explain the antiepileptic mechanisms of KD,and aimed to find new targets for treatment and provide clues for the new development of therapeutic methods for refractory epilepsy.Methods:We detect ROS level in each group by DCHF fluorescence probe,and detect the MDA level by the MDA kit according to colorimetry method.Western blot was carried out to detecte protein expression level in the JNK pathway and P38 MAPK pathway.Results:1.Reactive oxygen species(ROS)were detected by DCHF fluorescent probe:Observation on the formation of ROS in each group by laser confocal microscope showed:In the Control group,the visual field was dark,and the dark green cells were not so visible;in the GLU group which was treated for 24 hours by 5mM GLU,bright green fluorescent cell bodies were seen in the field of vision,some of the cells were broken;in the BHB+GLU group which was pretreated by 4mM BHB for 12 hours,and another 24 hours treated by 5mM GLU,the visual field was dark,and the dark green or green fluorescent cells were visible.Levels of ROS by the quantitative analysis of relative fluorescence levels in each group:compared with the normal control group,level of ROS in GLU group was higher(P<0.05):compared with GLU group,the expression of ROS in BHB+GLU group was lower(P<0.05).2.Detection of the levels of malondialdehyde(MDA)in each group:1)Control group was the normal control group,GLU group was not pretreated with BHB,only treaed by 5mM GLU for 12 hours,BHB+GLU group was pretreated with 4mMBHB for 12 hours,and treated by 5mM GLU for another 12 hours:The level of MDA in GLU group was highest,compared with the normal control group,the content of MDA was increased(P<0.05);the level of MDA in BHB+GLU group was significantly decreased compared with GLU group(P<0.01).2)Control group was the normal control group,GLU group was not pretreated with BHB,only treaed by 5mM GLU for 24 hours,BHB+GLU group was pretreated with 4mMBHB for 12 hours,and treated by 5mM GLU for another 24 hours:The level of MDA in GLU group was highest,compared with the normal control group,the content of MDA was increased(P<0.05);the level of MDA in BHB+GLU group was significantly reduced compared with GLU group(P<0.05).3.Wetern blot was carried out to detect the expression of P38 and JNK,the expression of phosphorylated P38 and JNK in each groupThe phosphorylation level of P38 MAPK in GLU group was highest,the level of phosphorylated P38 was higher than the normal control group(P<0.05);the level of phosphorylated P38 in BHB+GLU group was lower than the GLU group(P<0.05);the expression level of JNK and phosphorylation were consistent with P38 MAPK pathway.Conclusions:1.BHB reduces the production of reactive oxygen species and lipid peroxides in GLU induced cell damage of HT22,and plays a neuroprotective role in the redox level;2.GLU increases the phosphorylation level of P38 and JNK in the MAPK pathway,and plays an important role in the damage of nerve cells by activating P38 and JNK pathway;3.BHB decreased the phosphorylation level of P38 and JNK MAPK pathway.It suggests that the neuroprotection of BHB may be involved in the regulation of neuronal function by inhibiting P38 MAPK and JNK pathway.