Role And Mechanism Of Mitochondrial Calcium Uniporter In Hippocampal Neuronal Damage In Pilocarpine-induced Seizures

BackgroundEpilepsy is a clinical syndrome with sudden,transient and recurrent dysfunction of the central nervous system caused by abnormal over-discharge of neurons in the brain.It is a common neurological disorder that affects about 1%of the population worldwide,and more than a third of all patients have refractory epilepsy.Repeated seizures can cause cognitive,behavioral and mental health problems that bring a heavy burden to family and society.Therefore,it is of great practical significance to identify the pathogenesis of epilepsy and to seek new therapeutic targets.Recent years,numerous studies have shown that changes in intracellular Ca²⁺concentration and dysregulation of calcium homeostasis play an important role in the occurrence of epilepsy.Mitochondrion is an important calcium store in the cell,and the calcium homeostasis it regulates is of great significance to both cell and mitochondrion.Studies have shown that mitochondrial Ca²⁺overload could lead to the seizure-induced neuronal damage,while inhibition of the mitochondrial Ca²⁺uptake could play a role in protecting neurons via decreasing mitochondrial Ca²⁺overload.Recently identified mitochondrial calcium uniporter（MCU）plays an important role in mitochondrial Ca²⁺uptake.In recent years,studies found that MCU plays an important role in myocardial and cerebral ischemia-reperfusion injury,while the role of MCU in seizure-induced neuronal damage is still unknown.ObjectiveIn order to investigate the role of mitochondrial calcium uniporter（MCU）in hippocampal neuronal damage in pilocarpine-induced seizures,we would intervene the expression of MCU via application of its agonist and inhibitor,then observe the effect of MCU on hippocampal neuronal apoptosis,detect the mitochondrial Ca²⁺concentration and the expression of apoptosis-associated proteins such as Bcl-2,Bax,Cyt C and caspase-3 in rat hippocampus.Methods1.Grouping and establishment of epileptic rat model84 male SD rats were randomly divided into 4 groups:control（0.9%Nacl）group,PILO（PILO）group（divided into 4 subgroups:2h,8h,24h and 72h）,Ru360（Ru360+PILO）group and Spermine（Spermine+PILO）group.2.Behavioral observationObserve the behaviors in each group according to Racine seizure classification standards,and record the latency of seizure.3.Detection of cell apoptosisHippocampal neuronal apoptosis of each group was detected by TUNEL staining.4.Detection of motichondrial Ca²⁺concentrationThe hippocampal motichondrial Ca²⁺concentration was detected by fluorescent staining.5.Detection of apoptosis-associated proteinsThe expression of apoptosis-associated proteins such as Bax,Bcl-2,Cyt C and caspase-3 was detected by western blotting.Results1.Behavioral observationRats in control group had no seizures,while rats in PILO group,Ru360 group and Spermine group all had seizures.And the latencies of seizure in each group have no significant difference（P>0.05）.2.Detection of cell apoptosisCompared with control group,apoptotic neurons in PILO group increased（P<0.05）.Compared with PILO group,Ru360 reduced the apoptotic neurons while Spermine increased the apoptotic neurons（P<0.05）.3.Detection of motichondrial Ca²⁺concentrationCompared with control group,mitochondrial Ca²⁺concentration in PILO group increased（P<0.05）.Compared with PILO group,Ru360 reduced the mitochondrial Ca²⁺concentration while Spermine increased the mitochondrial Ca²⁺concentration（P<0.05）.4.Detection of apoptosis-associated proteinsCompared with control group,expression of Bax,caspase-3 activation and Cyt C release in PILO group increased,while expression of Bcl-2 decreased（P<0.05）.Compared with PILO group,Ru360 reduced expression of Bax,caspase-3 activation and Cyt C release,while increased expression of Bcl-2（P<0.05）.Compared with PILO group,Spermine increased expression of Bax,caspase-3 activation and Cyt C release,while reduced expression of Bcl-2（P<0.05）.Conclusions1.MCU plays an important role in hippocampal neuronal damage in pilocarpine induced seizures.2.Inhibition of MCU exerts a neuroprotective effect against epileptic injury.The mechanism maybe through inhibiting the mitochondrial Ca²⁺overload to avoid increasing the mitochondrial membrane permeability,thus inhibiting Cyt C release from mitochondria to block the apoptosis pathway mitochondria mediated.