| Objective:Temporal lobe epilepsy is an refractory epilepsy and a common clinical neurological syndrome, whose pathogenesis has not been fully understood. At present the role of Akt in the nervous system diseases has been one of the hot spots in neurologic studies. Study found that in acute cerebral ischemia, Parkinson's disease, Alzheimer's disease and other major diseases in nervous system, Akt promos neuronal survival through anti-apoptotic mechanism. Dynamic observation of the expression of Akt 1 protein in rat hippocampus in temporal lobe epilepsy after SE to explore its role in the development of epilepsy.Method:210 healthy adult SD male rats were randomly divided into normal group (n=10), licl group (10 rats), the epilepsy group (190). Epilepsy group was induced by lithium-pilocarpine. When the rats reached seizure V grade, sulfate atropine and diazepam was given intraperitoneally to relieve seizures.1 hour later. Licl group was injected lithium chloride 3mEq/kg, while the control group was injected the same volume of saline instead of lithium chloride and pilocarpine. Total protein was extracted from hippocampus and rat brain slices were obtained at different time points (Oh, 1h,6h,12h,24h,48h,7d,10d,30d,50d) after SE in normal group and licl group. Using Western blot technique for detection of total protein in the hippocampus, conventional preparation of gel, loading samples with equal volume of protein in each channel, electrophoresis, transferring and closure, primary antibody incubation in 4℃overnight, second antibody incubation, and chemiluminescence by gel imaging system finally. Gray value was analysised by quantity one software and Aktl protein relative gray values were statistically analyzed using analysis of variance. Using immunohistochemical technique for detection of the expression of AKT 1 in the hippocampus, conventional lost-wax and water, blocking of endogenous catalase 3% H2O2, antigen retrieval by water bath heating, normal goat serum closed. AKT1 antibody were dropping resectively and then the slices were placed in 4℃wet box overnight. Dropping biotin-labeled goat anti-rabbit IgG, incubation in wet box in 37℃, dropping horseradish enzyme labeled streptavidin working solution, incubation in wet box in 37℃. color reaction using DAB in room temperature, hematoxylin staining of nuclear, hydrochloric acid alcohol differentiation, ammonia back to blue, routine dehydration, clearing, closing slices. Light microscopy shows that positive cells cytoplasm stained yellow and nuclei stained pale blue. Quantifying positive nerve cells in hippocampal region using microscopic counting method(200×), Immunohistochemistry results were analyzed statistically using analysis of variance. Test level a=0.05, data were analyzed using SPSS 17.0 statistical package.Result:1.Akt1 protein in the hippocampus was detected in normal control group licl group and epilepsy group respectively using Western blot experiments. The results showed:the gray values of Aktl protein expression in 0h,24h,30d after SE and the normal control group had significant difference,p values were 0.034,0.000,0.002. 1h,6h,12h,48h,7d, 10d,50d after SE and the control group showed no significant difference, p values were divided 0.565,0.093,0.053,0.089,0.922,0.933,0.355. The relative gray value of Aktl protein expression in 1h,6h,12h,24h,48h after SE and licl group was statistically significant difference, p values were 0.034,0.002, 0.010,0.000,0.002. The difference in 0h,7d, 10d,30d,50d after SE and licl group was not statistically significant, p values were 0.557,0.096,0.137,0.097,0.511 respectively. licl group compared with the normal group showed no significant difference, p=0.117. The results suggest that:the epilepsy group, compared with the control group, the expression of Aktl protein in the hippocampus was significantly increased, beginning in the SE and 30 days after SE, and reaching the peak (0h=0.49, 30d=0.52, p values were 0.034,0.002), the expression was significantly reduced at 24 hours after SE, quickly fell to the lowest level and lower than normal the other time points (24h=0.27, p=0.000), Aktl protein in the hippocampus in the epilepsy group shows no significant difference. In Epilepsy group and licl group, the expression began to decrease after 1 hour after SE, and in 24 hours after SE reaches to the lowest level (24h=0.27, p=0.000). The expression in 48 hour after SE begin to go upwards and increased to licl group level at 7 days after SE2.Using immunohistochemistry to study the expression of AKT1 protein in hippocampus. Aktl protein expressed mainly in the cytoplasm of hippocampal pyramidal cells and showed brown particles. The Akt1 proteins in CA3, CA2 and CA1 area in hippocampus were counted using the microscope (200×). The expression at Oh, 1h,6h,12h,24h, 10d,30d,50d after SE in CA3 group shows significant difference with the control group, and p values were 0.000,0.000,0.000, 0.000,0.000,0.000,0.000,0.000. The expression at 48h,7d after SE in CA3 area compared with the control group showed no significant difference, p values were 0.368 points,0.064. The expression at Oh, 1h,6h,12h,24h,48h,7d,30d,50d after SE in CA3 area group and licl treatment group difference was significant, p values were 0.000,0.000,0.000,0.000,0.000,0.000,0.000,0.000,0.000. CA3 area lOd and licl after SE treatment group difference was not statistically significant, p=0.822. CA3 area licl treatment group compared with the control group there was significant difference, p=0.000. The difference in CA1 and CA2 area epilepsy group, licl treatment group and control group was not statistically significant (p>0.05). The results suggest that:①Aktl protein in the epileptic hippocampal CA3 area cells immediately began to increase, peaked at 12 hours (46.7±2.9),48h back to normal controls (16.2±2.5),10 days again begin to increase (25.0±2.3),30d reached second peak (44.1±1.8),50 day began to recover (22.3±2.6) in rats after SE.②In licl group, Aktl protein expression in CA3 area than in normal group and epilepsy increased after the 0 hour group, the difference was statistically significant (p<0.05).③In the control group, licl group and epilepsy group, the Aktl protein in CA1 and CA2 areas shows no significant difference, the difference was not statistically significant (p> 0.05).Conclusion:1.Compared with normal controls, Aktl protein expression was significantly increased in the hippocampus in the SE and 30 day after SE. The expression of Aktl protein was significantly reduced in the hippocampus at 1 day after SE and its expression in the hippocampus shows dynamic changes, which increased at first and then decreased again and increase finally.2.Aktl protein was mainly expressed in the cytoplasm of hippocampa pyramidal cells in CA3, CA1, CA2 areas also have a small amount of expression.3.Akt1 protein expression increased and then decreased, then increased and then decreased in CA3 area in hippocampal after SE, resulting inferred Aktl protein may play a protective role on neurons.
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