| Objective:To explore the expression characteristics and neuronal apoptosis changes of sodium-hydrogen exchanger-1(NHE-1)in the hippocampus of lithium chloride-pilocarpine epileptic model rats and effect of the regulation of NHE-1 on the apoptosis of hippocampal neurons and epileptic seizures in rats.Methods:1.64 healthy adult male SD rats were selected and divided into three groups randomly,that is,control group(n=12),and lithium chloride-pilocarpine epileptic models were established for other rats.After the successful establishment of models,phenobarbital and phenytoin sodium were administered to screen out drug-resistant epileptic rats(n=17)and drug-sensitive epileptic rats(n=35).The epileptic seizures in rats were recorded by 24-hour continuous video monitoring;immunohistochemistry,immunofluorescence,RT-PCR and western blot were adopted to detect the expressions of NHE-1 and calpain1 in the hippocampus of rats,and neuronal apoptosis was detected by HE staining and TUNEL staining.2.59 healthy adult male SD rats were divided into the control group(n=12),the epileptic model group(n=12),the lentivirus empty vector model group(n=17)and the lentivirus model group(n=18)randomly.The hippocampus of the epileptic model(EP)group was injected with normal saline in the hippocampus,the lentivirus model(LV-NHE-1+EP)was injected with lentivirus interfering with the NHE-1 gene in the hippocampus and the lentivirus empty vector model(LV-empty+EP)was injected with lentivirus empty vector and the kindling of rats was recorded.The epileptic seizures in rats were recorded by 24-hour continuous video monitoring.Immunohistochemistry,RT-PCR and western blot were adopted to detect the expressions of NHE-1 and calpain1 in the hippocampus of rats,and neuronal apoptosis was detected by TUNEL staining.Results:1.The results of seizures in rats showed that during the chronic spontaneous phase,the number of seizures was significantly higher in the drug-resistant group compared with the sensitive group(P<0.05),and the duration of seizures was prolonged(P<0.05).HE staining showed the degeneration,necrosis and loss of neurons and the proliferation of glial cells in the hippocampus of epileptic rats in the drug-resistant group.The results of immunohistochemistry showed that Compared with the control group and the drug-re88sistant group,NHE-1 was over-expressed on the hippocampal neuronal membrane in the drug-resistant group,and the optical density was higher than those of the control group and the drug-sensitive group(P<0.05);compared with the control group and the drug-sensitive group,calpain1 was over-expressed in the hippocampal neuronal cytoplasm of the drug-resistant group,and its optical density was higher than those of the control group and the drug-sensitive group(P<0.01).The results of immunofluorescence showed that the immunofluorescence intensity of NHE-1 in the hippocampus of the drug-resistant group was higher than those of the control group and the drug-sensitive group,and its optical density was higher than those of the control group and the drug-sensitive group(P<0.01);compared with the control group and the drug-sensitive group,the immunofluorescence intensity of calpain1 in the hippocampus of the drug-resistant group was higher than those of the control group and the drug-sensitive group,and its optical density was higher than those of the control group and the drug-sensitive group(P<0.01).The results of RT-PCR indicated that the expression of NHE-11mRNA in the hippocampus of the drug-resistant group was higher than that of the drug-sensitive group(P<0.05),the expression of NHE-1mRNA in the hippocampus of the drug-resistant group was higher than that of the control group(P<0.01).The expression of calpain1 mRNA in the hippocampus of the drug-resistant group was higher than that of the drug-sensitive group(P<0.05),the expression of calpain1 mRNA in the hippocampus of the drug-resistant group was higher than that of the control group(P<0.01).The results of western blot showed that the expression of NHE-1 protein in the hippocampus of the drug-resistant group was higher than that of the drug-sensitive group(P<0.05),compared with the control group,the expression of NHE-1 protein in the hippocampus of the drug-resistant group significantly grew(P<0.01).The expression of calpain1 protein in the hippocampus of the drug-resistant group was higher than that of the drug-sensitive group(P<0.05),the expression of calpain1 protein in the hippocampus of the drug-resistant group was significantly higher than that of the control group(P<0.01).The results of TUNEL staining showed that the hippocampal neuronal apoptosis in the drug-resistant group was more prominent than those in the drug-sensitive group and the control group(P<0.01).2.After lentivirus was transfected into the hippocampus of rats,epileptic rat models were established.Compared with the EP group and the LV-empty+EP group,the successful kindling time of the LV-NHE-1+EP group was prolonged,and the seizure level fell within 1 hour after successful kindling,and the chronic spontaneous seizure and seizure duration decreased.The results of immunohistochemistry showed that compared with the EP group and the LV-empty+EP group,the expression of NHE-1 in the hippocampal neuronal membrane of the LV-NHE-1+EP group fell,the expression of calpain1 in the hippocampal neuronal cytoplasm of the LV-NHE-1+EP group fell.The results of RT-PCR showed that compared with the EP group and the LV-empty+EP group,the expression of NHE-1 mRNA in the hippocampus of the LV-NHE-1+EP group fell(P<0.01,P<0.05),the expression of calpain1 mRNA in the hippocampus of the LV-NHE-1+EP group fell(P<0.01,P<0.05).The results of western blot showed that compared with the EP group and the LV-empty+EP group,the expression of NHE-1 protein in the hippocampus of the LV-NHE-1+EP group fell(P<0.01),the expression of calpain1 protein in the hippocampus of the LV-NHE-1+EP group fell(P<0.01).TUNEL staining showed that compared with the EP group and the LV-empty+EP group,the hippocampal neuronal apoptosis in the LV-NHE-1+EP group was relieved(P<0.01).Conclusions:The up-regulated NHE-1 expression in the hippocampus of epileptic model rats may be involved in the occurrence and development of epilepsy by aggravating neuronal apoptosis.NHE-1/calpain signaling pathway may be an important mechanism leading to neuronal apoptosis. |
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