Study On The Mechanism Of Parthanatos In Hippocampal Neuron Death Following Epileptic Seizure

This study is divided into 3 parts:PART 1: Study on the mechanism of Parthanatos induced by glutamate in HT22 cellsObjective: Polymer(ADP-ribose)polymerase 1(PARP-1)dependent cell death,also known as Parthanatos,is a new and unique way of cell death,but rarely reported in the field of epilepsy.This study aims to investigate whether Parthanatos was involved in the mechanism of epilepsy-induced neuronal damage in vitro study,and to provide new targets for neuroprotective treatment of epilepsy.Methods: HT22 cells,cultured in vitro,were stimulated by GLU at different concentrations and at different times.CCK8 kit was used to detect mortality of HT22 cells induced by GLU to choose the best cell injury model.In addition,PARP-1 inhibitor PJ34 and si RNA silencing PARP-1 gene technology were used to intervene in GLU-induced HT22 cell injury.Then,LDH release test,Western blot,immunofluorescence,flow cytometry and other techniques were used to observe the relevant indicators to explore the function and the mechanism of PJ34 and si RNA silencing PARP-1 gene on GLU-induced HT22 cell injury.Results:(1)GLU induced the damage of HT22 cells in a concentration and time dependent manner.With the prolonged treatment time of GLU on HT22 cells,the mortality of the cell was progressively increased(p < 0.01),mitochondrial membrane potential was progressively decreased(p < 0.01),the expression of intracellular PARP-1 and the synthesis of PAR were significantly increased,and the translocation of AIF from mitochondrial to the nucleus was also increased.(2)Compared with the mortality of HT22 cells treated with GLU for 12 h and 24 h,the corresponding pretreatment of PJ34 significantly decreased the mortality of HT22 cells(12 h,P < 0.05;24 h,P < 0.01).Meanwhile,application of PJ34 significantly reduced the expression of intracellular PARP-1 and the synthesis of PAR in GLU-induced HT22 cells,and inhibited the translocation of AIF from mitochondrial to thenucleus.Also,PJ34 inhibited the decrease of mitochondrial membrane potential in GLU-induced HT22 cells(p < 0.01).(3)Silencing PARP-1 gene by si RNA technology significantly reduced the mortality of GLU-induced HT22 cells(p < 0.01)and the expression of intracellular PARP-1 and the synthesis of PAR.It also inhibited the translocation of AIF from mitochondrial to the nucleus and the decline of mitochondrial membrane potential in GLU-induced HT22 cells(p < 0.01).Conclusion:(1)GLU induces the death of HT22 cells in a concentration and time dependent manner.(2)Parthanatos is involved in the pathway of GLU-induced HT22 cell death.(3)PARP-1 can be a potential intervention target for neuroprotective therapy.PART 2: Role of oxidative stress in Parthanatos induced by glutamate in HT22 cellsObjective: This study aims to investigate the relationship between ROS produced by glutamate on HT22 and Parthanatos,and to clarify the initial role of ROS in the mechanism of Parthanatos.Methods: HT22 cells,cultured in vitro,were stimulated by GLU to simulate the cell damage model in vitro after epilepsy.LDH release test,Western blot,flow cytometry,enzyme labeling and ELISA were used to detect cell mortality,ROS,8-OHd G,glutathione(GSH)and related proteins to clarify the role of oxidative stress in Parthanatos induced by GLU in HT22 cells,and to confirm the protective effect of NAC on HT22 cells.Results:(1)With the prolonged treatment time of GLU on HT22 cells,the levels of ROS and 8-OHd G(DNA damage marker)were progressively increased(p < 0.01).Application of NAC significantly reduced the production of ROS(p < 0.01)and the expression of 8-OHd G generated from GLU-induced HT22 cells(p < 0.01).Meanwhile,NAC also increased the level of GSH(p < 0.01).(2)Pretreatment of NAC significantly reduced the mortality of HT22 cells induced by GLU(p < 0.01),decreased the expression of intracellular PARP-1 and the synthesis of PAR,and inhibited the translocation of AIF from mitochondrial to the nucleus.Conclusion:(1)ROS may be the initial factor of Parthanatos in HT22 cells induced by GLU.(2)NAC plays an antioxidant role by increasing the level of GSH in GLU-inducedHT22 cells.(3)NAC can effectively inhibit the occurrence of Parthanatos in GLU-induced HT22 cells.PART 3: Neuroprotective effects and mechanisms of NAC and PJ34 on epilepsy rats induced by kainic acidObjective: This study aims to investigate the role and mechanism of antioxidant NAC and PARP-1 inhibitor PJ34 on epilepsy model in vivo.Methods: kainic acid(KA)was injected into the right amygdala of Wistar rats to establish epilepsy model,and the behavioral characteristics and pathological changes(HE staining)were observed.NAC and PJ34 were used for pretreatment,FJB staining,Western blot,enzyme labeling and ELISA were used to determine the related indicators to explore their function and mechanism in epilepsy model induced by KA.Results:(1)The behavioral characteristics of the epilepsy rats induced by KA were similar to those of patients with epilepsy.This model had a high success rate and low mortality rate.(2)The epilepsy rats induced by KA showed neurons damage located in CA1 and CA3 areas of hippocampus,especially in CA3 area.(3)Application of PJ34 significantly reduced the damage of hippocampal neurons after epileptic seizures in rats(p < 0.01).It also reduced the expression of PARP-1 and the synthesis of PAR in hippocampus,and inhibited the translocation of AIF from mitochondrial to the nucleus.(4)Application of NAC significantly reduced the death of hippocampal neurons(p < 0.01)and DNA damage(p <0.01)after epileptic seizures in rats(p < 0.01),accompanied with the increased level of GSH in hippocampus(p < 0.01).Meanwhile,NAC also reduced the expression of PARP-1 and the synthesis of PAR in hippocampus of epilepsy rats,and inhibited the translocation of AIF from mitochondrial to the nucleus.Conclusion:(1)In KA-induced epilepsy model,pathological changes in hippocampus were the damage of neurons located in CA1 and CA3 regions(more severe in CA3 regions).(2)PJ34 has protective effect on the damage of hippocampal neurons after epilepsy in rats,and inhibits the occurrence of Parthanatos in hippocampal neurons.(3)NAC has protective effect on the damage of hippocampal neurons after epilepsy in rats,and alleviates DNAdamage of hippocampal tissues caused by epilepsy by increasing the level of GSH.In addition,NAC also inhibit the occurrence of Parthanatos in hippocampal neurons.