| 【Purpose】In this study,epilepsy mice induced by Kainic Acid(KA)were used as animal models to observe the expression of Bcl3 at different time points and different brain regions and to explore the cells expressing Bcl3 in the acute phase of epileptic seizures in mice.And a KA-stimulated neuron toxicity model was also established to explore the effects of Bcl3 on neuron activity and the possible mechanisms by regulating the expression of Bcl3.【Methods】1.Group of experimental animals: Adult male C57BL/6 mice were randomly divided into a Control group(Control group,n = 6)and an epilepsy model group(SE group,n = 60).The mice of SE group were divided into 8 subgroups according to different time points(6 h,12 h,24 h,2 d,3 d,4 d,7 d,2 w)after the seizure,with 6 mice in each subgroup.KA was used to establish the status epilepticus(SE)models.According to Racine classification,those with seizures of grade IV or above and lasting 30 minutes were included in subsequent experiments.2.Quantitative Real-time PCR(q PCR)was used to detect the expression of Bcl3 in mice hippocampus at different time points after seizures.The expression of Bcl3 in olfactory bulbs,prefrontal lobes,striatum,hippocampus,temporal cortex and cerebellum of mice at this time point were detected,after obtaining time point of the highest expression.Immunofluorescence staining(IF)was used to co-stain Bcl3 with neuron-specific marker Neu N,microglial-specific marker CD68,and astrocytespecific marker GFAP on frozen sections of mice brains tissue to explore the cells expressing Bcl3.3.Establish combinations of different KA concentration and time gradients to construct a KA-stimulated neuron toxicity model,and use the CCK8 experiment to detect the results and select the optimal combination.4.Groups of cell experiment: Lentivirus transfection was used to inhibit Bcl3 expression in primary cultured neurons.According to different conditions for neurons,they were divided into control group,model group(KA group),KA toxicity model plus empty virus group(KA + KBD group),and KA toxicity model plus lentivirus group(KA + bcl3-RNAi group).5.CCK8 test was used to detect the changes of neuronal cell activity in each group,and the expression of Bcl3,Caspase3,Cleaved-caspase3,ATG7,Beclin1 and LC3bⅡ/Ⅰ were detected by WB and q PCR.【Results】1.Compared with the control group,the expressions of Bcl3 m RNA in the hippocampus of mice at all time points in the SE group were significantly increased(P < 0.01).It was showed that with the prolongation of the time after the seizure,the expressions of Bcl3 m RNA increased rapidly,reached the highest level at 24 hours after the seizure,and then gradually decreased slowly.The expression level was still higher than normal even 2 weeks after the seizure(P < 0.01).2.Compared with the control group,expressions of Bcl3 m RNA were all increased in different regions of the mice brain 24 hours after the seizure(P < 0.05).Among them,the hippocampus increased the most,followed by temporal cortex,prefrontal,striatum,cerebellum,olfactory bulb.3.Immunofluorescence staining revealed that Bcl3 expressed on mouse neuron and microglia,but not on astrocyte.4.Through comparison,with the combination of 100 u M of KA for 12 h,the relative activity of neurons was the lowest,which was(40.72±6.40)% of the blank control group,so this condition was selected for subsequent experiments.5.The results of CCK8 experiment showed that when the expression of Bcl3 in the lentiviral interference group was suppressed,the neuron activity was increased significantly(P < 0.05).Compared with the empty virus group,WB and q PCR experiments showed that Caspase3 and Cleaved-caspase3 did not changed significantly(P > 0.05),while ATG7,Beclin1 and LC3bⅡ/Ⅰ all decreased notably(P< 0.05).【Conclusions】1.The expression of Bcl3 increased in the brain of KA-induced epilepsy mice,and the increase in hippocampus and temporal cortex was the most obvious.2.Inhibiting the expression of Bcl3 shows a protective effect on neurons,which may be achieved by inhibiting autophagy... |
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