| Objective:To explore the intervention effect of CathepsinB Inhibitor(CBI) and its signaling pathways following recurrent neonatal seizure induced growth and cognitive changes in rats.Methods: 6-day-old (P6) SD rats (quantity: 90) were randomly divided into three groups(n=30): recurrent neonatal seizure group (RS group), CBI-treated seizure group (CBI group) and the control group. Rats in RS group were subjected to 5 seizures with flurothyl during the first 14 days of life. In CBI group, CBI was injected each day before seizures were induced. Then examined development indexes such as the physical growth(weight, ear separation, incisor eruptio, etal),neural reflex(plane righting test, cliff avoidance test, the air righting reflex test, etal),neural behavior and cognitive emotional competence(Morris water maze test,open-field behavior test). Western blot was employed to determine LC3, beclin-1, CathepsinB, bcl-2, PRG-1 expression at different time points (1.5h,3h,6h,24h and P35) after the last convulsion. All the date was analyzed by the software SPSS 16.0.Results:1. behavior analysis:(1) The weights of rats in RS group were lighter than CONT and CBI group from p7 to p14. (2) The time of cliff avoidance in RS was longer than CBI. It showed statistically significant (P<0.05) in p12. (3) In the Open-field behavior test: the activities of RS group were markedly reduced than CONT group in the horizontal movements, the RS group were significantly decreased than the CONT group and CBI group in the vertical motions (P<0.05). (4) 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 RS group than that in CONT and CBI group at D5 (P<0.05). 〈2〉As far as search strategy is concerned, rats of RS group performed worse than those in CONT and CBI groups〈.3〉The frequency of passing through the platform quadrant in rats had no significantly difference among the three groups (P>0.05).2. Western blot analysis: Protein expression is consistent in hippocampus and cortex. (1)LC3-II/LC3-I,beclin-1,CathepsinB expression in RS group (1.5h,3h,6h and 24h) was significantly higher than that at the same time in CONT group (P<0.05), but bcl-2 opposite; LC3-II/LC3-I,beclin-1,CathepsinB expression in the time point (1.5h,3h,6h and 24h) of CBI group was significantly decreased compared with that in RS group (p<0.05), but except bcl-2. There were no significant differences among three groups at P35 (P>0.05). (2)However, there was no significant difference of PRG-1 among three groups in the time point (1.5h,3h,6h and 24h). In the long-term time point of P35 up-regulated expression of PRG-1 was seen in RS group compared with that in CONT and CBI groups (P<0.05).Conclusions:1.Autophagy/lysosomal and apotosis pathway were activated immediately after recurrent neonatal seizures as indicated by the elevated expression of LC3,beclin-1,CathepsinB and down-regulated of bcl-2 in hippocampus and cerebral cortex. Recurrent neonatal seizures could damage long-term learning ability which may be related to the upregulation of PRG-1.2. CBI used before seizures could significantly down-regulated the acute phase expression of LC3,beclin-1,CathepsinB and long-term PRG-1 expression in hippocampus and cerebral cortex. Additionally, CBI could increase acute phase expression of bcl-2 and improve long-term neurobehavioral injury.3. The protective effects of CBI on recurrent neonatal seizure-induced brain damage might be through the autophagy/lysosomal and apotosis pathway. |
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