Study On The Neuroprotective Effect Of Modified Atkins Ketolone Diet And ?-hydroxybutyrate In Antiepileptic Therapy

Epileptic seizures and antiepileptic drugs may cause many adverse effects on children,such as the tolerance,compliance and long-term maintenance of antiepileptic therapy,especially cognitive function.Current studies have shown that the ketogenic diet has no adverse cognitive or behavioral effects,and is effective and well tolerated in children with refractory epilepsy.Many studies suggest that the antiepileptic effect of the ketogenic diet may be related to the neuroprotective effect of ketone bodies.Therefore,exploring antioxidant neuroprotective compounds can bring new ideas for the treatment of epilepsy,and investigating the regulatory mechanism of the neuroprotective effect of ketone bodies will be our further research direction.Because of adverse effects of epileptic seizures and antiepileptic drugs on children's cognitive function,our clinical observation and study focuses on the protective effect of the ketogenic diet on cognitive function.78%ketone bodies are?-hydroxybutyric acid.Therefore,the antiepileptic and neuroprotective effects of BHBA on a mouse model of refractory epilepsy was investigated in this study.This study was divided into two parts.In the first part,through the observation and study of electroencephalography(EEG)changes and cognition of children with refractory epilepsy treated with a ketogenic diet,and the comparison of early EEG changes and neurobehavioral development before and after ketogenic diet(modified Atkins diet),the effects of modified Atkins diet on the clinical seizures,EEG changes and cognitive function of children with refractory epilepsy were determined.In the second part,neurobehavioral and pathological changes in mice with refractory epilepsy were detected using the Morris water maze,and the levels and activities of MDA,ROS,SOD and GSH-Px in the hippocampus were measured,to investigate the protective effect of ?-hydroxybutyric acid on epileptic seizure-mediated oxidative stress in the hippocampus.The effect of BHBA on apoptosis of hippocampal neurons was observed by detecting the expression of Bcl-2 and Bax.and to further observe the role of AMPK/PPAR a signaling pathway in BHBA repair of oxidative stress injury and protection of nervous system.Part IEffect of modified Atkins ketogenic diet on cognitive function of children with refractory epilepsyObjective:Through the analysis of the clinical efficacy of modified Atkins ketogenic diet,the number of epileptiform EEG discharges,relative EEG power and intelligence test in the treatment of refractory epilepsy in children,cognitive function,intelligence scale and EEG waveform analysis were associated.On this basis,early EEG changes and neurobehavioral development before and after ketogenic diet were compared,so as to determine the clinical and cognitive effects of modified Atkins diet in children with refractory epilepsy.Methods:The subjects were 80 children with refractory epilepsy treated in the Department of Pediatrics from 2015 to 2018.The subjects were randomly divided into two groups.32 cases in the experimental group was treated with KD+AEDs,while 28 case in the control group was treated with AEDs only.The ketogenic diet was supplemented according to modified Atkins diet.The basic data of the patients were recorded,including age,gender and the AEDs administrated orally.The 24-hour ambulatory electroencephalogram(AEEG)and Wechsler 's intelligence test for children were carried out and the number of clinical seizures was recorded in the experimental group and the control group before and 6 months after treatment.According to the reduction in the number of clinical seizures,the clinical efficacy was evaluated.The EEG was evaluated by epileptiform discharges at the interval of seizures(after ketogenic diet),Computing the relative power of each band(alpha,beta,theta,delta)in EEG monitoring,and the intelligence was graded using FIQ.Statistical analysis was conducted by SPSS 19.0.Results:1.The total effective rate of clinical treatment was 71.42%(20/28)in the control group and 96.87%(31/32)in the experimental group.The clinical total effective rate showed a difference between the 2 groups(?2=6.405,P=0.011<0.05)2.The total effective rates of EEG in the experimental group and the control group were 93.75%(30/32)and 78.57%(22/28),respectively,showing a difference between the 2 groups(x2=7.428,P=0.007<0.05),3.The difference of relative power of alpha,beta,Delta and theta bands between the control group and the experimental group before and after treatment showed that the difference between the two groups was uniform P<0.05.After ketogenic diet for 6 months,the relative power(0 bands)of the control group was higher than that of the experimental group,and the relative power(?/? band)of the control group without ketogenic diet was lower than that of the experimental group(P<0.05).Compared with before treatment,the relative power at ? band in the control group(without ketogenic diet)was lower after treatment(P<0.05).Compared with before treatment,the relative power at P band in the experimental group was higher after treatment(P<0.05).After 6 months of treatment,the relative power at a band was higher while at ? and ? bands was lower than those before treatment(P<0.05)4.The FIQ,PIQ and VIQ were higher in the experimental group after 6 months of ketogenic diet combined with clinical AEDs treatment(P<0.05).5.EEG background fast activity(alpha and beta bands)and cognitive improvement have positive effects in the experimental group6.There was no correlation between changes in fast activity(alpha and beta bands)and changes in cognitive function in the experimental groupConclusions:1.Modified Atkins ketogenic diet in children with refractory epilepsy can significantly improve the clinical symptoms of children,improve neuroelectrophysiological indicators of EEG(discharge index at inter-seizure intervals and relative power of ?-band EEG),and improves the cognitive function of children.2.Fast background activity(alpha and beta bands)of EEG is correlated with improvement of cognitive function.There was no correlation between the changes of fast activity(alpha and beta bands)and the changes of cognitive function in children the experimental group.Part ?The neuroprotective effects of ?-hydroxybutyric acid on epileptic miceObjective:KD is characterized by the oxidation of fatty acids to produce ketone bodies(KBs),and ?-hydroxybutyric acid is the main component of KBs.Current studies have shown that antioxidant stress,alleviating neuroinflammation and protecting the nerve tissue are the main mechanisms for the treatment of refractory epilepsy.Neurobehavioral test and hippocampal histopathological examination were carried out in mice with refractory epilepsy.The neuroprotective effect of ?-hydroxybutyric acid on epilepsy-mediated oxidative stress in the hippocampus was investigated by measuring MDA,ROS,SOD and GSH-Px in the hippocampus.the expressions of p-AMPK,AMPK,PPARa,Bcl-2 and Bax proteins were detected using Western blots,to investigate the effect of BHBA on Bcl-2/Bax gene expression and apoptosis,explore the protective mechanism of BHBA treatment on oxidative stress injury of neurons mediated by the AMPK signaling pathway,and study the role of peroxisome proliferator-activated receptor a(PPARa)in anticonvulsion after BHBA treatment,so as to provide a basic theory for clinical treatment and new drug development,and provide clues for the research on ketogenic diets.Methods:1.The mice were divided into three groups and animal models of epilepsy was established:A:control group,B:epileptic group and C:?-hydroxybutyric acid group,with 15 mice per group.2.Firstly,Morris water maze test was conducted,during which the mice underwent a five-day learning.In this period,whether mice could find the platform after 60 s in water was observed.On the 6th d,the platform was removed.Moreover,the number of times the mice crossed the platform and the percentage of time spent in the target quadrant within 60 s were recorded using a camera.Further,the mice were guaranteed a 30-min rest between the two experiments.3.The hippocampus of mice was isolated and prepared into sections.4.The histopathology of the hippocampus in mice was detected using Nissl and Tunel staining.The staining effect was observed under a microscope and photographed.5.The level ROS and MDA and the activities SOD and GSH-Px in the hippocampus of each group were measured.6.Samples were provided by the laboratory and stored in a refrigerator at-80 C.The expressions of p-AMPK,AMPK,PPARa,Bcl-2 and Bax proteins in the three groups(A:control group,B:epileptic group and C:?-hydroxybutyric acid group)were detected using Western blots.The films were scanned,and the optical density of the target bands was analyzed using the gel image processing system(Gel-Pro-Analyzer software).Results:1.Morris water maze test.Compared with the control mice,the escape latency of epileptic mice increased significantly at the acquisition stage,and decreased after BHBA treatment.On the 6th day,probe test showed that epileptic mice crossed the platform less frequently and spent less time in the target quadrant than the control mice.2.Nissl and Tunel staining showed significant loss and apoptosis of hippocampal neurons after epilepsy.After BHBA treatment,the number of integrative neurons in the hippocampus increased and the number of apoptotic cells decreased.3.BHBA attenuated the overproduction of ROS and MDA in the hippocampus caused by epilepsy.4.Compared with the control group,SOD and GSH-Px activities in the hippocampus of epileptic mice decreased,which was partly but significantly enhanced by BHBA.5.Compared with the control group,Western blots revealed down-regulated bcl-2 protein and up-regulated Bax protein in the hippocampus of epileptic mice.6.Compared with the control group,AMPK phosphorylation and PPARa protein expression in the hippocampus of epileptic mice decreased,and PPARa in the hippocampus recovered significantly after BHBA treatment.Conclusion:1.BHBA attenuates epilepsy-mediated spatial learning deficits.BHBA improves hippocampal injury induced by epilepsy and prevents epilepsy-mediated oxidative stress in the hippocampus.The up-regulation of Bcl-2 and the down-regulation of Bax suggest that BHBA treatment can improve apoptosis by epilepsy.2.BHBA plays a protective role by activating the AMPK signaling pathway,The regulation of PPARa may be involved in the antiepileptic effect of BHBA.