| Objective To investigate the effect of Purα on lithium pilocarpine kindling rat hippocampal neurons apoptosis and cognitive function of learning and memory.Material and Methods The choice of laboratory animal: Approved by the ethics committee of Ningxia Medical University, in accordance with the operating standards of animal experiments. Selection of male rats. 6-8 week-old male SD rats, weighing between 220~250g. Feeding conditions: avoiding bright light, suitable temperature and humidity 24 hours a day and night cycle.90 SD rats were randomly divided into Purα over-expressed group, Purα SiRNA group and control group, n = 30 in each groupThe rats in each group were randomly selected, with 40mg/kg standard weight intraperitoneal injection of 1% pentobarbital sodium solution for anesthesia. According to the rat brain stereotaxic atlas, determine the CA1 region of hippocampus in rats.(coordinates: anterior fontanelle after 3.6mm, next to the open 2.2mm, depth 2.5mm), By using a micro injection device slow bolus 4μL lentiviron suspension(5 x 108 TU virus particles). The control group according to the same method and coordinate, injected with the same volume of saline to complete the operation process. After the surgery was finished,the rats were individually housed and received intraperitoneal injection of penicillin 50000 U/d for 3 consecutive days to prevent infection. The injection of the virus after two weeks, 10 rats in each group were randomly selected and decapitated, stripping out the hippocampus by Western Blotting confirmed that Purα lentivirus was expressed in the hippocampus.The other 20 rats in each group were observed with pilocarpine induced epilepsy,according to the Racine score standard as the basis to determine the seizure grade, if seizures reach III ~ V level is regarded as successful. If was seizure or seizures did not reach the level III is according to the required every 30 min increase the injection time of pilocarpine, increasing doses of 10 mg / kg, supplement the total injection times up to no more than three times. One hours after seizures observed behavioral changes in rats, seizures after three days in each group were randomly selected 10 rats by water maze test, the experimental design five days and can be divided into the search space and cruise positioning experiment. The cruise record for four days(4 times/day), observe the rat spatial learning ability. The fifth day was giving the space search experiment, recording the number of rats through the platform quadrant within the specified time and residence time, as judged by the spatial memory of rats reference index. The remaining rats in each group were dissected from the brain and frozen in the hippocampal CA1 region. the other rats were perfused and the hippocampus samples were used for paraffin embedding and the histological examination. Immunofluorencent assay and western blotting to examine Caspase-3 expression. The statistical software SPSS20 was used for data analysis, the experimental data were presented as mean standard ± deviation(judges S), Using single factor variance analysis(One-Way ANOVA) data for statistical comparison, comparison between groups using LSD method, while the group of homogeneity of variance with Dunnett method, Experimental results for P < 0.05 was statistically significant.Results 1. Morris water maze: Compared with the other two groups,the silent group escape latency time is long, the difference was statistically significant(P<0.05).Compared with overexpression group and epilepsy model group, the difference was not statistically significant(P>0.05).With the increase of experimental days, the escape latency of each group was gradually shortened. Overexpression group compared with the other two groups, the trend is not obvious. Comparison between the platform quadrant time of three groups, the differences were statistically significant(P<0.05). The number of crossing platform quadrant, overexpression group compared with silent group,the difference was statistically significant, compared with the epilepsy model group no significant difference.2. The expression of Purα in the hippocampal: Two weeks after injection of lentiviral particles in hippocampal CA1 region, west-blot results show Purα protein expression in overexpression group was significantly increased and silent group decreased significantly; among the three groups comparisons, the difference is statistically significant, The results demonstrated that the infection with Purα lentivirus was successful.3. The results of HE staining in the hippocampal: Epilepsy was induced successfully after three days, the morphological and pathological changes of hippocampus. The control group cell arrangement rules, cell structure is not complete. The overexpression group, cells arranged closely, chromatin structure is relatively complete and clear, uniform distribution. Silence group, cell structure disorder, degeneration and necrosis of neurons, increase the spacing, there are obvious neuronal apoptosis.4.Immunohistochemical Staining:Three days after pilocarpine induced seizures, Compared with the control group and the silence group,the overexpression group occasionally saw the Brown stained cells and stained,epileptic model group positive cells than the overexpression group;Silence group showed a large number of positive cells, the cytoplasm and nuclei were brown yellow or brown granules. The results demonstrated that Purα has protective effect on hippocampal neurons in the seizures rats. Silencing of Purα gene, neuronal apoptosis induced by epilepsy can make worse.5. Western-Blot: The expression of Caspase-3 in hippocampus of rats in each group, compared with the epilepsy model group and overexpression group,the silence group of Caspase-3 was significantly increased. The epilepsy model group compared with over- expression group(P < 0.05). Overexpression group compared with silent group(P < 0.01). The results with immune tissue chemical observation is consistent, indicating that Purα can reduce the apoptosis of epilepsy.Conclusions: Purα can repair the DNA damage caused by seizures, and reduce apoptosis, thereby improving the cognitive and learning ability of rats. |
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