Effects And Mechanism Of Intermittent Fasting In Epilepsy-induced Cognitive Impairment

Epilepsy is a common neurological disease that often causes impairment of learning and memory in patients.Clinical studies have shown that high-fat and low-carbohydrate ketogenic diets can effectively reduce seizures in patients with early-stage epilepsy.However,this treatment has a negative impact on patients with low muscle mass and low body weight,and most patients cannot adhere to this diet for a long time.Intermittent fasting(IF)is a dietary intervention by changing the time and frequency of eating.Compared with the ketogenic diet IF,it can be more effectively controlled without affecting the overall energy intake and nutritional changes.Blood glucose,thus being more widely used in clinical research.There is evidence that IF can attenuate epilepsy-induced hippocampal damage in rats,and that intermittent fasting can enhance various forms of memory,including spatial memory,associative memory,and working memory.However,the specific molecular mechanism is still unclear,and further research is urgently needed.A large number of studies have shown that mitochondrial dysfunction may be the main mechanism of epilepsy,in which the production of free radicals and metabolic imbalance play a key role in the occurrence and development of epilepsy.SIRT3 is the main deacetylase in mitochondria,targeting superoxide dismutase SOD2 in mitochondria to reduce the level of oxidative stress in mitochondria.Previous research results suggest that intermittent fasting can activate SIRT3 protein by activating the SENP1-SIRT3 pathway,which may be closely related to the occurrence and development of epilepsy.This topic intends to explore the role of fasting in epileptogenesis through intermittent fasting mice and acute fasting cell models,and to study its mechanism of action from the pathway that SIRT3 can be activated by intermittent fasting.OBJECTIVE: After Kainic acid(KA)induced epilepsy,the impairment of learning and memory was compared between intermittent fasting pretreatment and free feeding mice after epilepsy,and SIRT3 protein was activated by IF to induce oxidative stress,mitochondria Injury and apoptosis to explore its possible mechanism.METHODS: Animal level: Taking C57BL/6 wild-type mice as objects,the epilepsy model was established by localized injection in the hippocampus,and the highest level of seizures and the time of the first seizure were recorded.After modeling,the following research methods were used to study related issues: 1)After 4 weeks of modeling,behavioral methods were used to detect the learning and memory ability of mice;2)Neu N and PI staining were used to observe the death of neurons in the hippocampal region of the mouse brain;3)WB detection 4)SOD,ATP,and MDA kits were used to detect the level of oxidative stress in the hippocampus.Cellular level: N2 A cells were treated with KA to establish an excitotoxicity model,and treated with low-glucose medium to establish an acute fasting model,and then the following four experiments were performed to detect the internal mechanism of the protective effect of IF on epilepsy injury 1)DCFH-DA and The Mito-Sox TM probe detects the activity level of ROS in whole cells and the ROS activity level in mitochondria;2)immunofluorescence co-localization and co-immunoprecipitation experiments detect the position changes of SENP1 and SUMO1 and the co-localization of SUMO1 and SIRT3.3)Si-RNA was transfected into cells using Poly Jet TM transfection reagent;4)Western blotting was used to detect the expression of related proteins,JC-1 assay was used to detect the membrane potential of mitochondria,and CCK8 method and Calcein/PI were used to detect cell activity.RESULTS: In the animal epilepsy model,there was no significant change in the body weight and seizure level of intermittent fasting mice compared with free-feeding mice,but the behavioral results showed that IF can alleviate the spatial memory dysfunction caused by epilepsy;activity Cells(Neu N)and dead cells(PI)immunofluorescence results showed that a large number of neurons in the hippocampus of mice injected with KA in free-feeding mice died in a large number,and the number of neurons in the hippocampus of IF mice injected with KA was compared with Neuronal mortality was significantly reduced in freely fasted mice injected with KA.At the same time,after IF mice were injected with KA,the deacetylation levels of ATP and mitochondrial SOD2 were significantly increased compared with those of free-feeding mice given KA.Hoechst33342 and WB experiments showed that IF can inhibit the apoptosis of neurons to reduce neuronal death in the hippocampus.The above results show that intermittent fasting can improve cognitive impairment and neuron death in the hippocampus after seizures in mice,and this protective effect may be related to the enhanced mitochondrial function and decreased oxidative stress levels,thereby inhibiting neuronal apoptosis.In order to further explore the possible mechanism of the protective effect brought about by intermittent fasting,we used mouse-derived neuroblastoma(N2A)cells to construct a KA-induced excitotoxicity model and simulated intermittent fasting with low-glucose medium for 6 h.fasting.In cell experiments,it was also shown that IF can inhibit cell apoptosis by increasing mitochondrial function and inhibiting oxidative stress levels.The results of immunofluorescence co-localization and co-immunoprecipitation showed that the protective effect brought by IF may be related to the de-SUMO1 of mitochondrial SIRT3 by the de-SUMO1 protein SENP1 that enters the mitochondria,thereby activating SIRT3.CONCLUSIONS: Intermittent fasting has a protective effect on spatial memory impairment caused by kainic acid-induced epilepsy in mice;its protective effect may be through regulating the transport of SENP1 into mitochondria,removing the SUMO1 modification on SIRT3 protein,and activating the antioxidant effect of SIRT3,and finally achieve the effect of protecting neurons.