| Objective: The effects of ERK1/2 inhibitor(PD98059)on epileptic seizures and Drp1 expression in experimental rats were investigated,so as to explore the possible mechanism of ERK1/2’s involvement in epileptic seizures by regulating mitochondrial division.Methods: Healthy adult male SD rats were divided into the following groups:Normal group;Epilepsy group(6 time points: 0.5h,2h,6h,12 h,24h);PD98059 group(ERK1/2inhibitor);Mdivi-1 inhibitor group(Drp1 inhibitor group);Dimethyl sulfoxide control group(DMSO group)(Each group or time point n=5);The epilepsy model was established by intraperitoneal injection of lithium chloride and pilokapine.After the successful modeling,the behavioral assessment of the latency period of the first seizure and the number of grand mal seizures in rats within 1 hour(Racine score at grade IV and above is considered as grand mal)was conducted.Western bloting was used to detect and analyze the expression levels of ERK1/2 and Drp1 total proteins and phosphorylated proteins in rat hippocampus,and the localization of ERK1/2 and Drp1 in nerve cells was observed by immunohistochemistry.Results:1.Behavioral results:There was no seizure in the normal group,the latency of the first seizure in the epilepsy group was 15.4±1.14 min,and the latency of the first seizure in the PD98059 group was 32.8±.54min;The first seizure in the Mdivi-1 group was28.6±1.94min;The first seizure in the solvent control group was 16.0±1.58min;The number of grand mal seizures was 9.8±0.83 in the epilepsy group and 5.2±0.84 min in the PD98059 inhibitor group;The number of grand mal seizures was 5.6±1.14 min in the Mdivi-1 inhibitor group;The number of grand mal seizures was 9.4±1.14 min in the solvent control group;There was no significant difference in the latency period of the first seizure and the number of grand mal seizures within 1 hour between the epilepsy group and the solvent control group(P > 0.05);There was no significant difference between PD98059 group and Mdivi-1 group(p > 0.05);PD98059 group and Mdivi-1 group were compared with the above two groups,the latency period of the first attack in PD98059 group and Mdivi-1 group was prolonged,and the number of seizures within 1 hour was reduced,the difference was statistically significant(P < 0.05).2.HE coloration results:The structure of hippocampal nerve cells in the normal group was complete and clear,with neat arrangement,purplish-blue nuclei,and red cytoplasm and extracellular tissues.Some nerve cells in the epilepsy group and the solvent control group showed nuclear condensation and irregular cell arrangement.Some cells in the mdivi-1 group combined with PD98059 still showed nuclear condensation and irregular arrangement,but to a less severe extent than those in the epilepsy group.3.Western bloting results:P--ERK1/2,t--ERK1/2,t-Drp1 and p-Drp1 in the hippocampus of rats in the normal group were basically expressed;Compared with the normal group and the DMSO group,the differences of t-ERK1/2 and t-Drp1 in the epilepsy group and the inhibitor group were not statistically significant(P > 0.05);Compared with the normal group,the expression of p-ERK1/2 in the hippocampus of the epileptic group gradually increased after 0.5h,reached a peak at 12 h,and remained high at 24 h,with statistically significant differences(p < 0.05),the difference between the epilepsy group(12h)and the DMSO group(12h)was not statistically significant(p > 0.05),after the intervention of PD98059 and Mdivi-1,the protein content of p-erk1/2 in PD98059 group was significantly reduced compared with that in DMSO group at the 12 th hour after successful modeling,the difference was statistically significant(p < 0.05);The protein content of p-ERK1/2 in Mdivi-1 group was not significantly different from that in DMSO group;Compared with the normal group,the protein content ratio of p-drp1 to t-drp1 was significantly increased in the hippocampus of epileptic rats,with statistically significant difference(p < 0.05),but no statistically significant difference compared with DMSO group(p > 0.05);Compared with the DMSO group,the protein content ratio of p-drp1 to t-drp1 decreased,and the difference was statistically significant(p < 0.05).4.Immunohistochemical results: T-ERK1/2 and p-ERK1/2 are expressed in hippocampal nerve cells CA1 and CA3,the positive cells are brown-yellow,t-ERK1/2 is expressed inthe membrane of nerve cells,and p-ERK1/2 is expressed in the cytoplasm and nucleus of nerve cells.Drp1 was expressed in the CA1 and CA3 regions of nerve cells,and the positive cells were light yellow and expressed in the membrane of nerve cells.Conclusion: Inhibiting the over-activation of ERK1/2 signaling pathway can regulate epileptic seizures by affecting the phosphorylation of Drp1 and mitochondrial division. |
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