Effects Of Regulatory Synaptic Vesicle Protein 2A On Parthanatos In Hippocampal Neurons Of Pharmaco-resistant Temporal Lobe Epilepsy Rats

Objective:To explore effects of regulatory expression of synaptic vesicle protein 2A on Parthanatos in hippocampal neurons of pharmaco-resistant temporal lobe epilepsy rats.Methods:150 healthy adult male Sprague-Dawley rats,six rats were randomly selected as normal control group,the remaining rats were used to prepare epilepsy models.Lithium chloride-pilocarpine combined medication was used to induce status epilepticus in rats,and a lithium chloride-pilocarpine acute epilepsy model was prepared.Rats with spontaneous recurrent seizures is the chronic epileptic rat.Two kinds of antiepileptic drugs,phenobarbital and phenytoin sodium,were given intraperitoneal injection for 14 days.By comparing the reduction of epileptic seizures in rats before and after treatment,pharmaco-sensitive epileptic rats and pharmaco-resistant epileptic rats were screened out,and they were respectively included in the pharmaco-sensitive epileptic group(PSE)and the pharmaco-resistant epileptic group(PRE).SV2 A up-regulation lentivirus was used for over-expression regulation of the selected pharmaco-resistant epileptic rats,and they were included in the SV2 A up-regulation group(UPRE);SV2A down-regulation lentivirus was injected to interfere and included the rats in the SV2 A down-regulation group(DPRE);the empty virus was injected as a negative control,and were included in the SV2 A up-regulation control group(UPRC)and SV2 A down-regulation control group(DPRC).The brain tissues of each group were taken for Western blot and immunofluorescence experiments.By detecting the expression of PARP-1 and AIF,the changes of Parthanatos in hippocampal neurons of pharmaco-resistant rats were observed.Results:(1)Compared with PSE group,the frequency of spontaneous recurrent seizures in PRE group increased(T=-7.869,P<0.05),the racine degree was higher than PSE group(T=-3.154,P=0.010),and the duration of seizures was longer(T=-2.816,P=0.018).Compared with UPRC group,the frequency of spontaneous recurrent seizures in UPRE group was reduced(T=3.461,P=0.026),and the duration of seizures was significantly shortened(T=2.848,P=0.047),the level of seizures was not significantly different(T=1.555,P=0.195).Compared with DPRC group,the frequency of spontaneous recurrent seizures in the DPRE group increased after lentiviral knockdown interference regulation(T=-2.940,P=0.042)and there was no significant difference in the level and duration of seizure(T=-0.288,P=0.788;T=-0.204,P=0.848).(2)Immunofluorescence results showed that PARP-1 mainly exists in the nucleus of granular cells in the hippocampal dentate gyrus.The mean fluorescence intensity of PARP-1 among the three groups is not completely consistent,and the difference is statistically significant(F=11.273,P=0.001).After LSD-t test,it was found that the mean fluorescence intensity of PARP-1 in PRE group and PSE group was significantly higher than normal control group(P<0.05),and the mean fluorescence intensity of PRE group was stronger than PSE group(P <0.05).WB results showed that the expression of PARP-1 in the three groups of rats was significantly different(F=40.839,P<0.05),and the expression of PARP-1 in the PSE group and PRE group was significantly higher than normal control group(P< 0.05),and the PRE group had higher expression than the PSE group(P<0.05).(3)Immunofluorescence results showed that in the normal control group,AIF was mainly expressed in the cytoplasm of granular cells in the dentate gyrus.In PSE group,part of AIF was expressed in the cytoplasm,and a small part was expressed in the nucleus.In PRE group,the AIF expression site changed from the cytoplasm to the nucleus,and a large amount of expression was in the nucleus.The difference in mean fluorescence intensity among three groups was statistically significant(F=4.313,P=0.037).The PRE group was significantly enhanced compared with the normal control group(P<0.05),and there was no significant difference between the PRE group and the PSE group and the PSE group and the normal control group.Western blotting also obtained similar results: there were differences among the three groups(F=13.448,P=0.002),The expression of AIF in PRE group was significantly higher than the normal control group and the PSE group(P<0.05).There was no significant difference between the PSE group and the normal control group.(4)After SV2 A lentivirus treatment,the fluorescence intensity of PARP-1 in each group is not completely the same(F=5.089,P=0.011).The mean fluorescence intensity of UPRC group,DPRC group,and DPRE group was higher than the normal control group(P<0.05).Compared with the UPRC group,the mean fluorescence intensity of PARP-1 in the hippocampal DG area of UPRE group was significantly reduced(P<0.05).There were also significant differences in the expression of PARP-1 in the hippocampus among the groups(F=18.592,P<0.05).In UPRC group,DPRC group and DPRE group,the expression of hippocampal PARP-1 was higher than normal control group(P<0.05).Compared with UPRC group,the expression of PARP-1 was reduced in the UPRE group(P<0.05).(5)The results of AIF immunofluorescence showed(F=4.406,P=0.018),In UPRC group,DPRC group and DPRE group,the mean fluorescence intensity of AIF in the hippocampal DG area was significantly higher than normal control group(P<0.05).However,there were no statistically significant differences between the UPRE group and the UPRC group,and between the DPRC group and the DPRE group.The WB result showed(F=8.509,P=0.003),the expression of AIF in UPRC group,DPRC group and DPRE group was significantly higher than normal control group(P<0.05).Compared with the DPRE group,the UPRE group had significantly lower AIF expression in the hippocampus(P<0.05).The difference between the remaining groups was not significant.Conclusions:(1)Chronic temporal lobe seizures may induce Parthanatos in hippocampal neurons of epileptic rats.(2)Pharmaco-resistant temporal lobe epilepsy may aggravate Parthanatos death of hippocampal neurons in epileptic rats.(3)By up-regulating SV2 A,it may have a partial inhibitory effect on Parthanatos of hippocampal neurons in pharmaco-resistant temporal lobe epilepsy rats.