| ObjectiveIntractable epilepsy(IE)is causing serious impact on the patient's health and daily life.New therapeutic targets become one of the mainstream in the field of epilepsy research.The pathogenesis of epilepsy has not been fully understood,but hippocampal neurons loss after epilepsy has been confimed.Abnormal activation of apoptosis signaling pathways in hippocampal neuron was thought to be the most important mechanisim of hippocampal neuron death.Inhibition of apoptosis can be benefit for epilepsy patients and will be the target of epilepsy therapy.MicroRNA(miRNA),as a transcriptional regulatory factor for gene expression,may play a key role in seizures by regulating signaling pathways.It has been found that the expression of miR-124 in the hippocampal tissues of chronic epilepsy mouse models and patients with temporal lobe epilepsy was decreased,and the excitability of neurons was enhanced through the downstream regulation signaling pathway,and participated in the seizure process.Therefore,the study of whether miR-124 affects seizures by regulating Sphingosine kinase 1(SPHK1)and its downstream signaling pathways can provide a theoretical basis for finding new effective therapeutic targets.The aim of this study is to investigate the contribution of miR-124 regulates SPHK1,activiting Akt involved downstream signaling pathways in hippocampal neurons apoptosis in epilepsy pathway,inducing apoptosis of hippocampal neurons using status epilepticus(SE)mouse model by lithium chloride-pilocarpine treatment.The further study is to investigate the position of machanism of Akt and downstream signaling pathways in hippocampal neurons apoptosis in epilepsy.Methods1.Animals and treatments.Healthy adult male C57BL/6 mouse were used as experimental subjects.The experimental group was divided randomly into the normal control group and the experimental groups.Mouse in the experimental group were treated by lithium chloride and pilocarpine.Control animals were given saline by intraperitoneal injection(n=6).2.Mir-124 regulated of SPHK1 expression after status epilepticus.(1)The experimental groups(n=18)were divided randomly into three subgroups accroding to duration of status epilepticus(SE),0h(n=6),0.5h(n=6)and 4h after onset of seizures(n=6).(2)Real-time fluorescent quantitative PCR(qRT-PCR)was used to examine the change of miR-124 expression in the hippocampus after seizures.(3)To detect the expression of SPHK1,qRT-PCR was used to examine the change of SPHK1 mRNA expression,western blotting was used to explore the activity of SPHK1 in the hippocampus after seizures.3.The activation of major downstream signaling pathway regulated by SPHK1,Akt/NF-?B signaling pathway,were explored using Western Blotting.4.Study on apoptosis mechanism of hippocampal neurons induced by Akt after seizures: the representative signaling molecules of apoptotic pathways including Bim and Bcl-2.The changes of protein levels of Akt,Bim and Bcl-2 in the hippocampus after seizures were detected by Western Blotting.Results1.Reduced the expression of miR-124 induced decreased expression of SPHK1,activating Akt/NF-?B signaling pathway to induce apoptosis of hippocampal neurons:(1)The expression of miR-124 in epilepsy was down-regulated: compared with the Control group,the expression of miR-124 in the hippocampal tissue of SE mice was significantly reduced(F=23.761,p<0.001).Compared with the Control group,the longer duration of epileptic seizure,the lower the expression of miR-124 and the lowest expression in 0.5h(LSD test,p<0.001).Compared with the Control group,the expression of miR-124 was significantly reduced in 4h after SE(LSD test,p=0.002),but the expression of miR-124 was higher than that in group 0h(LSD test,p=0.041)and 0.5h(LSD test,p<0.001).(2)There was no significant change in the expression of SPHK1 mRNA between the hippocampal tissue of acute epileptic mouse model and control mice: compared with the Control group,there was no significant difference in SPHK1 mRNA in the hippocampal tissue of SE mice(F=4.734,p=0.328).There was no statistically significant difference between the expression of SPHK1 mRNA and the duration of seizure(F=1.965,p=0.175).Compared with Control group,the grey value SPHK1 of experimental group reduced significantly(F=50.316,p<0.001).Compared with the Control group,the longer duration of seizure,the lower the expression of SPHK1(F=32.964,p<0.001)and the lowest expression in 0.5h(LSD test,p<0.001).Compared with the Control group,the expression of SPHK1 was significantl reduced in 4h after SE(LSD test,p<0.001),but the expression of miR-124 was higher than that in group 0h(LSD test,p<0.001)and 0.5h(LSD test,p<0.05).(3)The expression of miR-124 in the model of acute epileptic mouse model was positively correlated with SPHK1 activity: using SPSS Pearson correlation analysis method to analyze it.The results showed that miR-124 was significantly positively correlated with SPHK1(r=0.803,p=0.011).(4)Akt/NF-?B singnal pathway regulated by SPHK1 inducing hippocampal neuron apoptosis in the mouse model of acute epileptic was activated:the p-akt /Akt ratio in experimental group was significantly higher than Control group(t=5.232,p=0.001).In the cell nucleus,the p-NF-?B /NF-?B ratio of the experimental group was significantly higher than Control group(t=2.473,p=0.039).There was no statistically significant difference between p-NF-?B /NF-?B in the experimental group(t=0.842,p=0.424).2.Activation of the Akt signaling pathway after epileptic seizure leads to apoptosis of hippocampal neurons:(1)Change of Akt protein expression: compared with the Control group,the expression of Akt in the experimental group was significantly altered(F=3.954,P=0.003).The expression level of Akt was increased from 0h to 12 h after SE,and the expression level decreased from 16 h to 24 h after SE,and the most obvious timepoint after SE was 24h(LSD test,p<0.05).(2)Change of Bim protein expression: compared with the Control group,the expression level of Bim in the experimental group increased significantly after SE(t=2.572,P=0.021).(3)Change in Bcl-2 protein expression: the expression level of Bcl-2 was significantly decreased in 8h,12 h,16h and 24 h after SE(F=2.67,P=0.043)in the experimental group,and the most obvious decrease was 24h(LSD test,P=0.011).Conclusion1.The expression of miR-124 was down-regulated in status epilepitus mouse model,and inhibited the expression of SPHK1 protein.2.The expression of SPHK1 in status epilepticus mouse model activated the Akt/NF-?B signaling pathway was suggested.3.Seizures changed Akt protein activity,activating the downstream apoptosis signaling pathway to induced apoptosis of hippocampal neurons. |
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