The Mechanism Of Circular RNA CircIgf1r Regulates Neuron Damage After Epilepsy

Objective:Epilepsy,characterized by persistent spontaneous seizures,is one of the most common brain disorders.It affects more than 70 million of the population worldwide.At present,the main treatment for epilepsy is to control the symptoms of seizures by taking anti-seizure medications.However,there are still 30% of the patients who respond poorly to antiepileptic drugs and have difficulty controlling seizures.Studies have shown that neuronal damage or even loss,proliferation of glial cells,recombination of synaptic circuits and formation of abnormal neural networks are often observed after seizures,which may become the pathological basis for the next seizure or even recurrent seizures.Therefore,reducing neuronal damage after seizures is of great significance for treating recurrent seizures.Circular RNA is a type of RNA with a closed circular structure,and the increasing number of evidences show that circular RNA is an important participant in neurological diseases such as epilepsy and plays a key role in the development of the disease.However,it is not clear that the mechanism of circular RNA in neuronal damage after epilepsy.In our study,through high-throughput sequencing of a mouse epilepsy model,the differentially expressed circular RNA circIgf1r was identified.We mainly explored the mechanism of circIgf1r regulating neuronal damage in the development of epilepsy.Methods:1.Eight-week-old wild-type C57BL/6 mice were taken to construct status epilepticus(SE)model by intraperitoneally injecting 4-Aminopyridine(4-AP).Seizures were detected by electroencephalogram(EEG)recording and epilepsy behavioral scoring.High-throughput sequencing was used to detect circ RNA changes after seizures in mice.2.Circ Igf1 r si RNA was injected into the lateral ventricle through a stereotaxic instrument.EEG monitoring,behavioral observation and pathological section were used to detect seizure status and the pathological damage after the epilepsy.3.One-day-old wild-type C57BL/6 neonatal mice were used to culture primary astrocytes.Q-PCR was used to detect the effect of 4-AP,si RNA of circIgf1r and overexpression vectors of circIgf1r on GFAP expression,related markers of polarized phenotypes and circular RNA in astrocyte.Immunofluorescence was used to detect the effect of astrocytic circIgf1r on neuronal damage.4.Western blot was used to detect the effects of 4-AP,si RNA of circIgf1r and overexpression vectors of circIgf1r on the expression of autophagy-associated proteins LC3 and P62 in astrocytes.Pm Cherry-EGFP-LC3 B plasmid was transfected into primary astrocytes for detection of autophagy flow.Results:1.Four up-regulated circ RNAs were screened from the high-throughput sequencing results,and the expression of candidate circ RNAs was confirmed by q-pcr.These results showed that circIgf1r was up-regulated in SE mice.2.The EEG and behavioral observations showed that injecting mice with circIgf1r si RNA markedly reduced the duration and the severity but increased the latency of seizure after SE.Tissue immunofluorescence results showed that SE promoted the phenotypic polarization of astrocytes to A1 type which was reversed by knocking down circIgf1r.Nissl staining showed that knocking down circIgf1r alleviated neuronal loss after SE.3.Q-PCR results showed that circIgf1r was up-regulated in 4-AP-treated primary cultured astrocytes.In addition,4-AP induced primary astrocyte activation and promoted astrocytes to polarize towards A1.Knocking down astrocytic circIgf1r reversed the phenotypic polarization of astrocytes.Immunofluorescence results showed that 4-AP-treated astrocytes promoted neuronal damage,while knocking down astrocytic circIgf1r alleviated neuronal damage.4.Western blot and IF results showed that 4-AP caused destruction of autophagy flux in astrocytes.Knocking down circIgf1r promoted 4-AP-treated astrocyte autophagy and alleviated the destruction of autophagy flux,which was opposite to the result of overexpression.5.Functional rescue experiments showed that autophagy inhibitors reversed the phenotypic polarization of astrocytes regulated by knocking down astrocytic circIgf1r.Conclusions:In this research,we explored the molecular mechanism that circIgf1r in astrocytes regulates neuronal damage after epilepsy.SE resulted in astrocytes activation and polarized towards A1 which had a damaging effect on neurons.In vitro,knocking down circular RNA promoted autophagy and alleviated the damage of 4-AP to astrocyte autophagy flux,and promoted the polarization of astrocytes to A2 type to protect neurons.Therefore,circIgf1r could be used as a potential target for the prevention and treatment of neuron damage after epilepsy.