Effect And Mechanism Of DRP1 In Epileptic Seizures Of Rats Through The Regulation Of ENT1

Objective:To explore the inhibited DRP1 signaling pathway and observe the behavior change of lithium chloride-pilocarpine epilepsy rat model and DRP1 and ENT1 expression change within the brain.Methods: The healthy adult male SD rats were selected and injected intraperitoneally with lithium chloride-pilocarpine to establish the animal model of epilepsy.The rats were randomly divided into four experimental groups: epilepsy group(6h,24 h,72h,1wk,four time points,n = 5 × 4),mdivi-1 group(specific inhibitor of mitochondrial isolated protein)(n = 5),DMSO group(n = 5)and normal control group(n = 5).Racine scoring criteria was used to observe the latent period of epileptic seizures and the number of epileptic seizures in 1 hour,and to assess the extent of epileptic seizures in rats.Ultrastructure changes of mitochondria in hippocampus were observed by transmission electron microscopy.The expression of DRP1 in hippocampus and temporal cortex of rats were detected by immunohistochemistry,immunofluorescence and Western Blot.Western blot was used to detect the expression of ENT1 in hippocampus of each group.Results:1.Behavioral assessment results showed: Normal rats didn't have epileptic seizures.The latency of epilepsy group and solvent control group were 24.4 ± 5.12 min and 23.6 ±4.67 min respectively.mdivi-1 group was38.8±1.30 min.The number of first epileptic seizures in the experimental group was 9 + 0.52,the control group was 9 + 0.37,and the mdivi-1 inhibitor group was 4.5 + 0.33.The first epileptic seizure latency difference and the number of epileptic seizures difference between the epilepsy group and the solvent control group was not statistically significant(P> 0.05).The latency of seizure was significantly prolonged in the mdivi-1 group in comparison to the above two groups,the number of episodes of seizures decreased within 1 hour,and has statistically significant difference in comparison to the epilepsy group(P ?0.05).2.Observation of mitochondrial ultrastructure in CA1 region of hippocampus by transmission electron microscope results: Normal group have a complete mitochondrial structure of the hippocampus;The neuronal mitochondria in the epileptic group and the mdivi-1 group were damaged in different degrees.Neuronal mitochondriain epileptic group were obviously swollen,it's structure was obviously destroyed and some of the inner and outer membrane were disintegrated.Neuronal mitochondria in mdivi-1 group was slightly swollen,partial ridge was divided,density of the matrix was reduced.3.Using Western Blot,immunohistochemical and immunofluorescent to test the expression of DRP1 in different groups of brain tissues.3.1 Western Blot results: The expression of DRP1 detected in the normal group of rat hippocampus and new temporal lobe cortex had a basic level.The expression of DRP1 began to rise at 6h after epileptic seizures,and reached the peak at 24 h,72h still has a high level of expression,with a significant drop in it.The results have significant difference in comparison to control group(P<0.05).Western Blot showed that the expression of DRP1 was significantly lower in group mdivi-1 than that of the epileptic group(24h)after the successful modeling of 24 h,two groups of expression of DRP1 compared statistically significant difference(P<0.05).3.2 Immunohistochemical results:DRP1 immunoreactive cells were mainly expressed in the neuronal cell membrane,the number of positive cells of DRP1 were observed in epilepsy group increased significantly and have statistically significant difference in comparison to the normal group(P < 0.05);the number of DRP1 positive cells decreased in mdivi-1 group have statistically significant difference in comparison to the epilepsy group(P < 0.05).3.3 Immunofluorescence results: in hippocampal CA1,CA3 and DG regions,DRP1 was co expressed with neuron specific dendrite specific MAP2,and mainly expressed in the membrane of positive cells,but not co expressed with GFAP,a specific marker of astrocytes.4.The expression of ENT1 in hippocampus of rats in each group was detected by Western Blot.Results: the expression of ENT1 in hippocampus tissue of mdivi-1 group(24h)was significantly lower than that of epilepsy group(24h),two groups of expression of ENT1 compared statistically significant difference(P<0.05).Conclusion: DRP1 mediated the imbalance of mitochondrial dynamics and caused the disorder of synthesis of ATP and adenosine production,and effected the ENT1 expression and function changes,resulting in excitatory glutamate neurotransmitter changes,which may be one of the mechanism of epilepsy.