| Objective 1.Ferrous chloride induced Post-traumatic epilepsy(PTE)rat model,to observe the effect of everolimus on epileptic seizure in PTE rats,and to find a new treatment method for the prevention and treatment of PTE.2.To observe the changes of autophagy and m TOR pathway in Everolimus intervention PTE rats,and explore the mechanism of everolimus treatment on PTE ratsMethod 1.Model preparation and grouping: PTE rat model was induced by injecting 5μL(0.2mmol/L)of ferrous chloride into the right frontal cortex with anterior fontanelle as the origin,3.0mm to the right side,and then 2.0mm forward as the injection target.PTE rats with Racine score of grade Ⅲ were randomly divided into PTE group and everolimus group,and Sham group was set as Sham group,with 10 rats in each group.The everolimus group was treated with everolimus(5mg/kg.d)within 24 hours after successful modeling,and was continuously administered for 14 days.PTE group and Sham group were treated with equal volume normal saline intragastric administration.2.Each group observed epileptic seizures in rats,staining and protein detection of brain pathological sections: Racine score was used to record epileptic seizures in rats;The grasping force of left forelimb was determined by grasping force analyzer.EEG changes of each group were detected by EEG.HE staining was used to detect the lesions in the prefrontal lobe and hippocampus of brain tissue.The changes of hippocampal neurons were detected by Nissl staining.The autophagy related white changes in the prefrontal lobe were detected byimmunohistochemical experiments.The changes of autophagy related gene expression were detected by q PCR.p-m TOR protein,inflammatory factors(IL-1β,IL-4,TNF-α)and autophagy related protein(Beclin-1,P62)were detected by western blotting.Result 1.Everolimus alleviated epileptic seizure in PTE rats:(1)Racine score: Compared with Sham group,EEG score of PTE model rats was higher than grade Ⅲ;After 7 days and 14 days,compared with PTE group,everolimus group,the scores of rats decreased(P<0.05),and with the increase of time,the farthing scores of rats in both groups decreased.(2)Grasping power measurement: compared with Sham group,the left forelimb grasping power of rats in PTE group was significantly decreased,while the left forelimb grasping power of rats in everolimus group was significantly increased compared with PTE group.(3)electroencephalogram changes: compared with Sham group,the electroencephalogram of PTE rats presented higher spikelets.After 14 days,the electroencephalogram amplitude-higher spikelets of everolimus rats decreased compared with PTE rats.(4)Brain samples of rats after perfusion showed that the lesion area was reduced in everolimus group compared with PTE group.(5)HE staining observation: compared with Sham group,PTE group showed disordered brain morphology and structure,reduced and destroyed nerve cells,and a large number of vacuole formation and liquefaction necrosis.Compared with PTE group,the number of neurons in everolimus group was significantly higher,most of the nuclei were normal,vacuole and liquefaction necrosis were rare.(6)Nissl staining: Compared with Sham group,the number of neurons in hippocampus of rats in PTE group was decreased,the arrangement was loose and disordered,and the number of Nissl bodies in the cytoplasm was decreased(P<0.001).Compared with PTE group,the number density of cells in everolimus group was increased and the number of Nissl bodies in the permutation cytoplasm was increased(P<0.01).(7)Immunohistochemistry: Compared with Sham group,Beclin-1 expression area of PTE group was decreased(P < 0.001),while Beclin-1 expression area of evolimus group was increased compared with PTE group(P < 0.001).2.Everolimus inhibited the inflammation level of brain tissue in PTE rats: Compared with Sham group,the expressions of inflammatory cytokines IL-1β,IL-4 and TNF-α in brain tissue of rats in PTE group were increased(P<0.001),while the expressions of inflammatory cytokines IL-1β,IL-4 and TNF-α in brain tissue of rats in everolimus group were decreased(P<0.05).3.Everolimus regulates the m TOR pathway to activate autophagy:(1)Western blotting test: Compared with Sham group,brain tissue P-Mtor protein of PTE group was increased(P<0.001),autophagy protein Beclin-1 was decreased(P<0.05),P62 protein was increased(P<0.001),and P-Mtor protein of epholimus group was decreased(P<0.05),compared with PTE group.Beclin-1 autophagy increased(P< 0.05),and P62 protein decreased(P<0.01).(2)q PCR experiment: Compared with Sham group,beclin-1 m RNA expression in PTE group was decreased(P < 0.01),P62 m RNA expression was increased(P < 0.001),and beclin-1 m RNA expression was increased in everolimus group compared with PTE group(P < 0.001).P62 m RNA expression increased(P < 0.001).Conclusion 1.Everolimus can alleviate epileptic seizure in PTE rats.2.Everolimus may reduce epileptic seizures in PTE rats by regulating the m TOR pathway and activating autophagy. |
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