| Objective The aim of this study is to establish a pentylenetetrazol(PTZ)-induced epileptic seizure model in immature mice,observe and analyze the behavioral changes,neuronal apoptosis and the expression of PI3 K /AKT/m TOR signaling pathway in brain tissue of immature mice with PTZ induced epileptic seizures and after everolimus treatment.To further explore whether everolimus can exert neuroprotective effect on brain injury after epilepsy by inhibiting neuronal apoptosis and PI3 K /AKT/m TOR signaling pathway,and whether it is expected to become a new antiepileptic drug.Research Methods(1)Healthy,male,SPF balb/c immature mice(2-3 weeks old,weight 13-20 g,average about 17g)were chosen and randomly divided into 3 groups of 12 mice,which consisted of the Control+vehicle group,PTZ+vehicle group,PTZ+Eve+vehicle group.The Control+vehicle group received 834.88 u L/kg.d of organic solubilizing agent orally,and the mice were intraperitoneally injected with normal saline 7ml/kg.d.The PTZ+vehicle group received 834.88 u L/kg.d of organic solubilizing agent orally.The PTZ+Eve+vehicle group was treated with 4mg/kg.d(834.88 u L/kg.d)of everolimus orally and 7ml/kg.d of pentylenetetrazol intraperitoneally.All oral drugs were given orally 2 hours before intraperitoneal injection.According to the Racine classification standard,the occurrence of grade IV-V seizures was considered as successful modeling.During the experiment,the behavioral changes(seizure latency,seizure time,seizure grade)were observed and recorded.(2)TUNEL staining was used to observe the apoptosis rate of neurons.(3)Western blot was used to detect the protein expression of PI3 K,AKT and m TOR involved in the brain tissue of immature mice.(4)Quantitative PCR was used to detect the expression levels of PI3 K,AKT and m TOR in brain tissue of immature mice.Results(1)Behavioral changes of immature mice in each group The study found that the immature mice in the PTZ epileptic group showed obvious seizure behavior.A Compared with epileptic group,the latent period of seizure was prolonged and the seizure grade decreased obviously after everolimus treatment.(2)The apoptosis of hippocampus CA1 and CA3 neurons was observed by TUNEL staining.The results showed that the apoptosis of the PTZ and everolimus group was obviously higher than that of the control group,and the apoptosis of everolimus was obviously decreased in comparison with PTZ.(3)Western Blot was used to detect the expression of PI3 K,AKT and m TOR proteins in the brain tissue of immature mice The results showed that the expression of PI3 K,AKT and m TOR in PTZ with epilepsy group and everolimus treatment group were obviously higher than that in control group.Compared with the PTZ-induced epilepsy group,the expression of the three proteins was obviously decreased in the mice treated with everolimus.(4)Quantitative PCR was applied to detect PI3 K,AKT and m TOR expression in brain tissue The results showed that the expression levels of PI3 K,AKT and m TOR in PTZ group and everolimus treatment group were significantly higher than those in control group.The expressions of the three proteins in the everolimus treatment group were significantly lower than those in the pentylenetetrazol epileptic group.Conclusion(1)Pentylenetetrazol successfully induced epilepsy model in immature mice,and the immature mice showed obvious seizure susceptibility;Everolimus can reduce the susceptibility and intensity of seizures.(2)Everolimus significantly inhibited PTZ-induced neuronal apoptosis in immature mice.(3)Everolimus significantly inhibited the expression of PI3 K,AKT and m TOR proteins in the brain tissue of immature mice with PTZ induced epilepsy.(4)Everolimus can inhibit the expression of PI3K/AKT/m TOR signaling pathway,inhibit neuronal apoptosis,reduce the susceptibility and intensity of seizures,and play a neuroprotective role to achieve the purpose of treating epilepsy,which is expected to become a new Anti-seizure medication(ASM). |
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