The Protective Effects Of Pura On DNA Damage And Repair Induced By Epilepsy

Objective To investigate the protective effects of Purα protein on rat hippocampus DNA damage induced by epilepsy and the effects of Purα protein on DNA damage and repair.Material and Methods The choice of experimental animals: 6-8 week-old male SD rats were provided by the Experimental Animal Center of Ningxia Medical University, weighing 230 ~ 250 g, avoiding bright light, circadian cycle 12/12 h, single cages, quiet, balanced feeding a week in the condition of free, to eliminate the environmental impact. 60 normal male SD rats, were randomly divided into Purα over-expressed group, Purα Si RNA group, empty vector and control group according to the random number table method, n = 15 in each group. The randomly selected rats were anesthesized by intraperitoneal injection of 1% pentobarbital sodium solution(40mg/kg body weight). The surgery was performed according to the Pacino’s rat brain stereotaxic atlas. When CA1 area in the left hippocampus(coordinates: anterior fontanelle after 3.7mm, next to the open 1.2mm, depth 3.6mm) was determined, the animals were slowly injected with 3μL of lentiviron suspension(5 x 108 TU virus particles) by micro injection device. Control group was received the same surgical operation according to the same method and coordinate refferences, without injection the viron but replaced with the same volume of N.S to complete the operation as psudosurgery group. When the surgery operation was finished, the animals will be placed back to the single cage for feeding; the animals were also received intraperitoneal injection of penicillin 50000 U/d for 3 consecutive days to prevent the infection after the surgery. Two weeks after the injection, 6 rats in each group were randomly selected and killed with cervical dislocation and the brains were removed for the experiment., the hippocampus were stripped for the preparation of slice and Western Blotting assay to evaluate the expression of Purα in the hippocampus. Epileptic model was established with intraperitoneal injection of pilocarpine. The grade of epilepsy was assessed by the criterion proposed in the “Insular epilepsy “and the Racine scoring standard when the seizures reach to the III ~ V onset, the model was regarded as successful. The epileptic behavior and manifestations were observed and recorded. The hippocampal tissues were dissected from the brain and frozen for the tissue protein extraction and the other animals were perfused with paraformaldehyde and the hippocampus samples were used for paraffin embedding and histological examination. γH2AX was examined with immunohistochemistry to evaluate the DNA damage in the hippocampus of the experimental animals. Purα overexpression and silence were determined with immunofluorencent assay and western blotting. The other DNA damage associated protein, such as Parp-1, Ku80, and XRCC4 were examined by western blotting assay. The statistical software SPSS20 was used for data analysis, the experimental data were presented as mean standard ± deviation(judges S), the single factor analysis of variance(One-Way ANOVA) was used for data analysis, LSD method was employed for many groups comparison., Dunnett method was used when variance nonhomogeneity occurred in each group., P < 0.05 was set as the statistical significance.Results 1.The behavioristics manifestations: the experimental animals demonstrated a continuous shaking beard, salivation, hemifacial spasm, repetitive chewing, subsequently appeared shaking like wet dog, bilateral forelimb lift and fall down after turning around, in some dramatic onset, the animals would appear a suddenly jump and continuous rotation.2. Instant frozen sections: A large amount of fluorescent cells were appeared around the hippocampus tissue in the instant frozen slices the 14 days after the high tittered viron was injected into the hippocampus of the experimental animals. The results demonstrated that the infection with lentivirus was successful.3.Immunohistochemistry: seizures was induced in rats 1 hour after the injection of pilocarpine, the gamma H2 AX, a symbolic protein for DNA damage, was highly expressed in the hippocampus tissues of the experimental animals. The number of gamma H2 AX positive staining cells were much lower in Purα overexpression group than that in empty vector control group and control group,(p<0.01); and the number of gamma H2 AX positive staining cells is much higher in Purα silence group than that in empty vector group and the control group(p<0.01), there are significant statistical differences between these two groups but there is no statistical difference between the empty vector and control groups(p>0.05). These results implied that Purα played an important protective effects on DNA damage induced by seizures, when Purα gene is knocked down, the DNA damage induced by epilepsy would be aggravated.4. Western-Blot: The expression level of Parp-1, Ku80 and XRCC4 were dramatically increased in the Purα silence group compared with empty vector and control groups, but they were remarkably decreased in Purα overexpression group. There were significant statistical differences between these groups(p<0.01), but there is no difference between the empty vector and control groups(p>0.05). All these results demonstrated that Purα played an important protective on the DNA damage induced by epilepsy.Conclusions: Epilepsy induced by Pilocarpine can induce dramatically DNA damage in the rat hippocampus tissue, Purα plays a remarkably protective effects on the DNA damage induced by epilepsy.