Effect And Mechanism Of Mitochondrial Calcium Uniporter On Endoplasmic Reticulum Stress In Hippocampal Neurons Induced By Magnesium-free External Fluid

Background and purposeEpilepsy（EP）is a clinical syndrome of transient brain dysfunction caused by abnormal discharge of brain neurons in a variety of ways.Seizures seriously affect the lives and work of patients,and also have a large negative impact on families and society.Although various new anti-epileptic drugs have brought hope to the majority of patients,one third of the patients have not been effectively controlled after the regular administration of the drug,and it is a drug-refractory epilepsy.The complexity and uncertainty of the pathogenesis of epilepsy are the root causes of refractory epilepsy.Therefore,it is of great significance to further clarify the pathogenesis and to seek new targets for diagnosis and treatment.Mitochondria are important organelles for bilayer membrane coating of cellular aerobic respiration,energy production,and cell death signaling,and are called"energy factories"of cells.Studies have confirmed that it not only provides energy for cell life activities through oxidative phosphorylation pathway,but also participates in cell metabolism and signal transduction,calcium homeostasis regulation,reactive oxygen species（ROS）production and apoptosis.In recent years,the role of mitochondria in epileptic nerve injury has attracted more attention.The imbalance of mitochondrial dysfunction and quality control system may play an important role in epileptic neuronal injury.Mitochondria are important intracellular calcium stores.Recent studies have found that mitochondrial calcium(Calcium,Ca²⁺)homeostasis plays an important role in seizure-induced neurological damage.Mitochondrial calcium uniporter（MCU）is a selective calcium channel for uptake of calcium ions in the mitochondrial inner membrane.Its uptake of calcium ions depends on the mitochondrial inner membrane potential,and calcium ions diffuse along the electrochemical gradient,maintaining mitochondria and even cellular calcium.It plays an important role in the steady state.At the time of seizure,the MCU exerts mitochondrial compensatory action by ingesting a large amount of Ca²⁺that flows into the cytoplasm to maintain the intracellular homeostasis;however,excessive Ca²⁺entering the mitochondrial matrix leads to an increase in ROS production,triggering mitochondrial permeability transition pores（mitochondrial）.The permeablity transition pore（mPTP）is open and proapoptotic factors such as cytochrome C（Cyt C）and activated caspase release promote cell apoptosis.At present,it is found that inhibition of MCU activity can reduce the content of free calcium ions in mitochondria of rats with cerebral ischemia,increase ATP content,and alleviate mitochondrial damage by inhibiting mitochondrial division,thereby alleviating ischemia-reperfusion brain injury.MCU has been found to play an important regulatory role in the pathogenesis of hypertension,subarachnoid hemorrhage and diabetic cardiomyopathy.Our previous study found that MCU plays an important role in hippocampal nerve injury in a rat model of epilepsy,but its specific mechanism remains to be elucidated.ObjectiveIn this study,primary rat hippocampal neurons were treated with magnesium-free extracellular fluid to establish an in vitro cell model of acute epilepsy,and by observing endoplasmic reticulum stress protein（GRP78）and CHOP（C/EBP homologous protein）,CHOP),explored the effects of MCU inhibitor Ru360 and agonist Spermine on endoplasmic reticulum stress and apoptosis in hippocampal neurons of rats with magnesium-free epileptic seizures,and further explored its mechanism through Mito Q and CHOP siRNA intervention.MethodThe healthy SD rats,which were fresh within 24 hours,were immersed in 75%alcohol,and were killed by decapitation.The bilateral brains were removed and placed in a petri dish containing pre-cooled PBS buffer to expose the hippocampus.Excessive impurities such as microvessels,the hippocampus were cut into small tissue blocks with ophthalmic scissors,trypsinized,fetal bovine serum medium was terminated for digestion,and filtered through a 200-mesh cell sieve to form a single cell suspension.The cell concentration was adjusted and planted in a cell culture plate,and the hippocampal neurons were cultured in vitro to determine the purity of hippocampal neurons.After 14 days,the culture was terminated and randomly divided into four groups:CON group,AE group,AE+Ru360 group and AE+Spermine group.After treatment with magnesium-free extracellular fluid for 3 hours,the cells were returned to normal cell culture medium.The AE+Ru360 group and the AE+Spermine group were pretreated with Ru360 and Spermine 30 min before the magnesium-free extracellular fluid treatment.Neuronal cell activity was measured by MTT colorimetry,and neuronal damage and apoptosis were detected by TUNEL method.The total protein was extracted from each group,and the expression levels of GRP78 and CHOP were detected by Western blot.The relationship between ERS and ROS was further investigated by Mito Q and CHOP siRNA.Statistical analysis was performed using SPSS 17.0,and the data obtained were expressed as mean±standard deviation.One-way analysis of variance and LSD（Least-Significant Difference）-t test were used to compare the neuronal activity,mitochondrial calcium concentration,mitochondrial ROS production and endoplasmic reticulum stress protein expression in hippocampus of each group.P<0.05 indicates that the difference is statistically significant.Results1.Neuron purity test showed that the purity of hippocampal neurons was over92%.2.MTT colorimetric assay was used to detect the activity of primary cultured hippocampal neurons.Compared with AE group,the activity of hippocampal neurons in Ru360 group was significantly increased,while the effect of Spermine group was opposite.3.TUNEL staining detection of apoptotic cells found that compared with the AE group,Ru360 significantly reduced hippocampal neuronal apoptosis,while the Spermine group had the opposite effect.4.The calcium ion fluorescent probe Rhod-2/AM and the mitochondrial fluorescent probe Mito SOX showed that compared with the AE group,the Ru360group significantly reduced the mitochondrial calcium ion concentration and mitochondrial ROS production,while the Spermine group had the opposite effect.5.Western blotting showed that Ru360 reduced the expression of GRP78 and CHOP compared with AE group,while Spermine had the opposite effect.6.Mito Q intervention significantly reduced mitochondrial ROS production in hippocampal neurons of epilepsy,and Mito Q pretreatment reduced GRP78 and CHOP expression levels.7.CHOP siRNA intervention significantly attenuated hippocampal neuronal cell damage.Conclusion1.Inhibition of MCU reduced seizure-induced ERS and apoptosis.2.ROS-mediated ERS may play an important role in the involvement of MCU in the apoptosis of hippocampal neurons in epilepsy.