The Effect And Potential Mechanism Of The Deubiquitinating Enzyme USP15 In Epilepsy

Purpose: Epilepsy is a chronic disease in central nervous system.Although antiepileptic drugs have made great progress over the past few decades.Nevertheless,about 30% of patients of epilepsy are resistant to drugs treatment.Therefore,it is urgent to further investigate the pathogenesis of epilepsy,providing a novel therapeutic strategy for epilepsy.Research content: In the present study,we conducted experiments in vivo and in vitro.Firstly,we detected the expression level of USP15 in the pentylenetetrazole(PTZ)kindling rat model of epilepsy to investigate the correlation between USP15 and epilepsy.Secondly,in the light of the significant role of glutamate-mediated oxidative toxicity in epilepsy,we established the oxidative stress model in HT22 cells treated with glutamate.To explore the role and the potential mechanism of USP15 in oxidative stress,we detected the expression changes of USP15 and the effects of USP15 knockout on cell viability and morphology,apoptosis,ROS activity,SOD activity and Nrf2 / HO-1 signaling pathway under the condition of glutamate treatment.Methods:(1)We intraperitoneally injected a sub-convulsive dose of PTZ in every other day to establish the chronic-kindling epileptic model.35 male SD rats were randomly divided into two groups,with 20 in the epilepsy group and 15 in the control group-epilepsy group: intraperitoneal injection of PTZ(35mg/kg)in every other day,control group:intraperitoneal injection of the same amount of saline as PTZ.The injection time was 13:00-18:00pm.After each injection,rats were put into separate cages to observe their behaviors for at least 30 minutes,and the seizure scale was recorded.The rats successfully kindled were included in the later experiments.At 24 hours,7 days and 30 days after kindling,the expression level of USP15 in the hippocampus of epileptic rats was detected by Western blot and immunohistochemical staining.(2)In addition,we established glutamate-induced oxidative stress model in vitro.Firstly,Western blot was used to detect the expression of USP15 at 0,8,16 and 24 hours after glutamate treatment.USP15 knockout cell was generated by CRISPR / cas9 technology.Then,CCK-8 was applied to detect cell viability;TUNEL staining was used to detect apoptosis in HT22 cells;Western blot was used to analyze the expression of apoptosis related proteins Cleaved caspase-3 and Bcl-2;ROS activity and SOD activity were investigate through ROS test kit and SOD test kit respectively.Finally,to clarify the effect of USP15 on Nrf2 nuclear expression,we analyzed Nrf2/HO-1 expression by Western blot and explored Nrf2 nuclear localization by immunofluorescence staining.Results:(1)In the epilepsy group: 6 rats died in the experiment,with a mortality rate of 30%;3 rats failed to be kindled;11 rats met the standard of full kindling and were included in the later experiment,with a success rate of 55%.In the control group,no rats had a seizure or died.Compared with the control group,the expression level of USP15 significantly increased at 24 hours,7 days and 30 days after kindling,suggesting the correlation between USP15 and epilepsy.(2)USP15 expression decreased at 8 hours but increased at 16 and 24 hours after glutamate treatment,initially implying the correlation between USP15 and oxidative stress at the cell level.Western blot was used to detect the expression of USP15 in HT22 cells after viral infection.The result showed that USP15 expression was deficient,indicating that the USP15 knockout cell line was successfully constructed.Moreover,under the condition of glutamateinduced oxidative stress,USP15 knockout could increase cell viability,maintain the normal morphology of HT22 cells,reduce apoptosis,inhibit Cleaved caspase-3 expression but promote Bcl-2 expression,demonstrating that USP15 inhibition could enhance the antagonism of cells against oxidative toxicity.Knockout of USP15 also reduced ROS activity but enhanced SOD activity.Further studies showed that USP15 knockout enhanced Nrf2 nuclear expression in HT22 cell,leading to increased expression of HO-1 as one of its downstream antioxidant genes.Conclusion: The increased expression level of USP15 in the hippocampus of kindled rats suggested the correlation between USP15 and epilepsy.Additionally,in the glutamate-induced oxidative stress model,USP15 inhibition could promote the endogenous Nrf2/ARE antioxidant pathway and thus antagonize the oxidative toxicity induced by glutamate,indicating that USP15 participated in the regulation of oxidative stress.In the light of the important role of glutamate-induced oxidative toxicity in the pathogenesis of epilepsy and the regulatory effect of USP15 on oxidative damage,we preliminarily speculated that USP15 may be involved in the occurrence and development of epilepsy.In the future,we will carry out further research at the animal level to provide a novel strategy for the treatment of epilepsy.