AMPK α 1 Regulate Mitochondrial Metabolic Function And Participate Study On The Pathogenesis Of Epilepsy In Mice

Objective: In this study,lithium chloride pilocarpine was used to induce seizures in male C57BL/6J mice at the age of 6 weeks.The changes in the activity of mitochondrial respiratory chain complexes Ⅰ,Ⅲ,Ⅳ(Complex Ⅰ,Ⅲ,Ⅳ)in the hippocampus of mice at different stages(0 day,2 weeks,6 weeks)after status epilepticus(SE)were detected compared with corresponding normal control mice Adenosine triphosphate(ATP)concentration changes,mitochondrial membrane potential,ΔΨ m)Injury and Adenosine 5 ’monophosphokinase(AMP)-activated protein(AMPK)α Different subunits(AMPK α、 AMPK α 1、AMPK α 2)Changes in protein level expression and exploration of AMPK in epilepsy α The mechanism of the effect of mitochondrial metabolic function.Hematoxylin eosin staining(HE)was used to display the number and morphological changes of neurons in the CA1,CA3,and DG regions of the hippocampus(HP),and to detect the changes of hippocampal neurons in mice after 6 weeks of epilepsy.Behavioral related experiments were used to examine the motor,social,learning and memory abilities of epileptic model mice.The ultrastructural changes of mitochondria in hippocampal CA1 region were observed by transmission electron microscopy.Conditional overexpression of chronic epilepsy model mice increases AMPK α To observe whether mitochondrial dysfunction and impaired learning and memory abilities in chronic epileptic mice can be improved.Methods:(1)54 healthy 6-week-old male C57 mice were divided into 6 groups according to different periods after SE(0 day,2 weeks,6 weeks).Nine mice in each group were divided into normal control group(0 day,2 weeks,6 weeks)and epilepsy model group(0 day,2 weeks,6 weeks),respectively.The weight of the experimental mice was about 20g;(2)A classical SE model of temporal lobe epilepsy was established by intraperitoneal injection of lithium chloride – pilocarpine(Li-Pilo),while the normal control group mice were injected with corresponding doses of lithium chloride and diazepam,while the rest of the drugs were replaced with the same volume of physiological saline.Epileptic seizures of grade IV or above(lasting for 30 minutes)can be included in the study;(3)After successful modeling of epileptic mice,hippocampal tissue samples were collected at the same time period at 0,2,and 6 weeks,and then tested;(4)Detection of AMPK in hippocampus by Western blot(WB)αProtein expression levels of different subunits;(5)Enzyme-linked immunosorbent assay(ELISA)was used to detect the activity of mitochondrial respiratory chain complexes Ⅰ,Ⅲ,and Ⅳ in the hippocampus of mice,as well as the changes in the concentration of Complex Ⅰ and ATP;(6)The tissue mitochondrial isolation kit extracts and purifies mitochondria,and the mitochondrial membrane potential detection kit(JC-1)detects the trend of changes in mitochondrial membrane potential levels;(7)HE staining was used to detect the morphological and quantitative changes of hippocampal cells;(8)The ultrastructural changes of mitochondria were observed by transmission electron microscopy;(9)Using SPSS26.0 statistical software to analyze experimental data;(10)36 healthy 6-week-old male mice(20g ± 1.5g)were divided into normal control group,epilepsy model group,and AMPK α1 Conditional overexpression group,AMPK α1 In the conditional overexpression epilepsy model group,a classical temporal lobe epilepsy SE model was established by intraperitoneal injection of lithium chloride pilocarpine(Li-Pilo),and the normal control group was replaced by saline;(11)Breeding AMPKα1 using Flox(+/-)and Cre tool mice Conditional overexpression mice,with their ears cut and labeled 2 weeks after birth,and their tails cut(about 1 cm);(12)The kit extracts deoxyribonucleic acid(DNA)from rat tail tissue,amplifies it by polymerase chain reaction(PCR),and uses agarose gel electrophoresis to carry out preliminary genotype verification;(13)After 6 weeks of successful modeling,behavioral related experiments were conducted to detect motor ability,cognitive function,learning and memory ability,and social ability in gene mice.After that,the brain was decapitated and tissue samples were collected.(11)Sixteen healthy 6-week old male mice(20g ± 1.5g)were divided into normal control group,epilepsy 6-week model group,normal control group+A484954,epilepsy 6-week model group+A484954.A classical temporal lobe epilepsy SE model was established by intraperitoneal injection of lithium chloride pilocarpine(Li Pilo),and the normal control group was replaced by saline;Normal control group and epilepsy model mice were given intraperitoneal injection of A484954 for 14 days after modeling for 6 weeks,while the other groups were injected with 5% DMSO of the same concentration,volume,and course of treatment.At the end of the treatment period,hippocampal tissue was extracted and AMPK was detected by protein immunoblotting assay α 1 Expression changes.Results:(1)AMPKα in hippocampal tissue of chronic epileptic mice The expression of different subunit proteins decreased;(2)The mitochondrial function of hippocampus in chronic epileptic mice was severely damaged;(3)The damage of mitochondrial ultrastructure in hippocampus of chronic epileptic mice was severe;(4)AMPK α1 The expression level of mitochondrial respiratory chain complexes I,III,and IV is closely related to their activity;(5)AMPK α1 The expression level of mitochondrial respiratory chain complexes I and III is closely related to the activity of mitochondrial respiratory chain complexes,but not related to the activity of mitochondrial respiratory chain complexes IV;(6)The morphological and structural damage of brain cells in the hippocampus of chronic epileptic mice was severe,and the number of brain cells significantly decreased;(7)Type in AMPK α 1After gene therapy,the motor ability of chronic epileptic mice returned to normal;(8)AMPK α1 The social ability of chronic epileptic mice with conditional overexpression returned to normal;(9)AMPK α1 After conditional overexpression,the cognitive function of chronic epileptic mice returned to normal;(10)AMPK α1 The learning and memory abilities of chronic epileptic mice with conditional overexpression were improved.(10)AMPKα1 in hippocampus of chronic epileptic mice after injection of eukaryotic elongation factor 2 kinase(e EF2K)inhibitor A484954 The expression level of returned to normal.Conclusion:(1)AMPKα1 in hippocampus after SE The decreased expression of may be closely related to mitochondrial dysfunction,providing a new perspective for the development of alleviating mitochondrial functional damage and neuroprotection after epilepsy;(2)Increase AMPKα1 After the expression level was increased,the mitochondrial function and learning,memory,cognitive,and social abilities of epileptic mice were improved.(3)AMPKα1 The recovery of memory ability in chronic epileptic mice by overexpression of e EF2 K may be related to the e EF2 K signaling pathway.