Intervention And Mechanisms Of E-64d On Developmental Seizure-induced Cognitive Deficit

Object: To explore the effects of E-64d on developmental seizure-induced cognitive deficit and its underlying mechanism.Methods: 120 Sprague-Dawley rats(postnatal days P21)were randomly divided into three groups:the control group (n=24),the recurrent-seizure group (RS group,n=48) and the E-64d treated-seizure group ( E-64d group,n=48).The penicillin was used to induce seizure attack. Rats in RS group were injected i.p. with penicillin(4.8×106 U·kg-1·d-1)every other day in eleven consecutive days from postnatal day 21 to induce seizure; Rats from the control group were injected equal amount of normal sodium(NS) for the same time;In E-64d group, E-64d(4μl, 1μg/μl)was injected each day before seizure was induced. Selected randomly 24 rats that up to standard from each group. At 3h,12h and 24h after the last convulsion, Beclin1,LC3,Bcl-2 and PRG-1 protein level in hippocampus and cerebral cortex were detected by western blot method. Morris water maze test were performed to assess learning and memory ability during P45-P51. Western blot analysis was used to determine Beclin1,LC3,Bcl-2 and PRG-1 protein level in hippocampus and cerebral cortex after the Morris water maze test.Results: 1. Western blot analysis: (1)The level of Beclin1and LC3II/LC3I in hippocampus and cerebral cortex of RS group were significantly higher than that of control group at 3h,12h and 24h after the last convulsion(P<0.05).The level of Beclin1and LC3II/LC3I in hippocampus and cerebral cortex of E-64d group was significantly lower than that of RS group(P<0.05). (2)The level of Bcl-2 protein in hippocampus and cerebral cortex of RS group were significantly lower than that of control group at 3h,12h and 24h after the last convulsion(P<0.05).The level of Bcl-2 protein in hippocampus and cerebral cortex of E-64d group was significantly higher than that of RS group(P<0.05). (3)At 3h,12h and 24h after the last convulsion the expression of PRG-1 in hippocampus and cerebral cortex were not significantly different between RS and control group(P>0.05). The expression of PRG-1 in hippocampus and cortex were not significantly different between E-64d and RS group(P>0.05). (4) There were no significant differences among three groups about the level of Beclin1,Bcl-2 and LC3II/LC3I in hippocampus and cerebral cortex after morris test(P>0.05). (5)The level of PRG-1 in hippocampus and cerebral cortex of RS group were significantly higher than that of control group after morris test(P<0.05). The level of PRG-1 in hippocampus and cerebral cortex of E-64d group was significantly lower than that of RS group(P<0.05).2.Morris water maze test: (1) The average latencies of all rats were reduced by degree from D1 to D5. The escape latency was significantly longer in rats of RS group than that of the control at D5(P<0.05). The escape latency was significantly shorter in rats of E-64d group than that of RS group at D5(P<0.05). However, there were no significantly difference between E-64d and control groups (P>0.05). (2)As far as search strategy is concerned ,rats of RS group performed worse than those in control and E-64d groups at D5,with statistically significant difference (P<0.05). However, the performance of rats between the E-64d and control groups had no significantly difference. (3) The frequency of passing through the platform quadrant in rats had no significantly difference among the three groups(P>0.05).Conclusion: 1. The protein level of Beclin1,LC3 in hippocampus and cerebral cortex were significantly increased and the level of Bcl-2 was significantly decreased in the acute period after penicillin-induced development seizures, which suggest that autophagy and apoptosis pathway is activated. Moreover, there were long-term cognitive deficit and up-regulated expression of PRG-1 in the forebrain following developmental seizures.2. Pretreatment with E-64d could significantly inhibit the abnormal expression of LC3,Beclin1, Bcl-2, PRG-1 in in hippocampus and cerebral cortex and attenuate cognitive dysfunction following penicillin-induced recurrent developmental seizures. 3. These results showed that E-64d improved learning capacity through autophagy/apoptosis signal pathways and by modulating the long-term expression of PRG-1 in hippocampus and cerebral cortex.