| Objective: Wnt signaling pathway by observing two key signaling moleculeβ-catenin (β-catenin), glycogen synthase kinase 3β(GSK-3β) and their respective phosphorylation (p-β-catenin, p-GSK-3β) and cell Apoptosis and apoptosis factor caspase-3, caspase-10 in patients with acute cerebral ischemia reperfusion rat hippocampal CA1 area of the expression changes of the signal pathway and ischemic reperfusion brain damage in cell apoptosis, the brain Infarction provide the main basis for clinical treatment.Methord: 1.Selected weight 200 ~ 250g of clean grade healthy male Sprague-Dawley (SD) 72 rats were randomly divided into sham operation group (12) and ischemia-reperfusion model group (60), model group was divided into 5 time points, namely, 3h, 6h, 24h, 48h, 72h, 6 rats at each time point.2.Longa method using modified middle cerebral artery occlusion (MCAO) and reperfusion model.3.Animals awake after the zen-longa modified rate scale: score of 1 to 3 points into this study. Score of 0 points and 4 points were removed were randomly added to ensure that each group 6.4.By HE staining of the cells in hippocampal CA1 area of the morphological changes.5.By TUNEL assay apoptosis in hippocampus CA1 area.6.By immunohistochemistry and Western-blotting Western blot hippocampal CA1 area were detected by caspase-3, caspase-10,β-catenin, GSK-3β, p-β-catenin, p-GSK-3βprotein.7.The data presented as mean±standard deviation, after finishing the database to Excel with the results of statistical software SPSS17.0 ANOVA (Analysis of single variance), q test (Student-Newman-Kueuls), with p <0.05 was statistically significant; statistical non-linear regression analysis using pearson correlation coefficient, p <0.05 indicated statistical significance.Results :1.HE staining: sham cells in hippocampal CA1 area neat and compact structure of the normal; model in the hippocampal CA1 region cell disorder, there nuclear pyknosis, cell number decreased.2.TUNEL staining: sham hippocampal CA1 area occasionally apoptotic cells; cerebral ischemia and reperfusion in the hippocampal CA1 area of apoptotic cells appears in 3h, 6h increased significantly, 24h continue to increase, 48h peak , 72h begun to reduce, over time the difference was significant (P <0.05), compared with the sham group was significantly (P <0.05).3.Caspase-3, caspase-10 results3.1 The results of immunohistochemistry: ischemia-reperfusion in hippocampal CA1 area 3h shows caspase-3, caspase-10 expression, 24h began to increase, 48h peak, 72h began to decline, over time the difference was statistically significant (P <0.05), compared with the sham group was significantly (P <0.05). Apoptosis was detected with the TUNEL method consistent with the changes.3.2 Western-blotting results: ischemia group Caspase-3, caspase-10 expression at all time points significantly enhanced, 48h peak, 72h begun to decline. Over time the difference was significant (P <0.05), compared with the sham group was significantly (P<0.05). And immunohistochemistry consistent with the results.4.β-catenin, p-β-catenin, GSK-3β, p-GSK-3βresults4.1β-catenin, p-β-catenin immunohistochemical staining results: ischemia group the expression ofβ-catenin in the nucleus over time the difference was not statistically significant (P> 0.05), compared with the sham group showed no statistical significance (P> 0.05); p-β-catenin expression in the cytoplasm was significantly increased compared with the sham group was significantly (P <0.05), but the model group showed no change over time statistically significant (P> 0.05).4.2 GSK-3β, p-GSK-3βresults of immunohistochemistry: ischemia group GSK-3βin the nucleus and / (or) the expression level of pulp and significantly increased compared with the sham group, the difference was statistically significant ( P <0.05), but change over time within the model group was no significant difference (P> 0.05); model group, p-GSK-3βexpression in the cytoplasm over time the difference was not statistically significant (P> 0.05), and false operated group had no statistically significant difference (P> 0.05).4.3β-catenin, p-β-catenin Western-blotting results: ischemia groupβ-catenin expression at all time points no significant change in the difference was not statistically significant (P> 0.05), compared with the sham group There was no significant difference compared (P> 0.05); model group, the p-β-catenin expression at each time point was significantly increased compared with the sham group was significantly (P <0.05), model group at all time point difference was not statistically significant (P> 0.05).4.4 GSK-3β, p-GSK-3βWestern-blotting results: ischemia group GSK-3βcompared with the sham group was significantly increased, the difference was statistically significant (P <0.05), while the model in each group time no significant difference (P> 0.05); model group, p-GSK-3βexpression at all time points no significant difference (P> 0.05), compared with the sham group differences no statistical significance (P> 0.05).Conclusion: 1. acute cerebral ischemia induced apoptosis in hippocampal CA1 occurred, the number of apoptotic cells in ischemic reperfusion time with a certain phase difference, ie, apoptotic cells began to appear at 3h, 6h Increased significantly, 24h continue to increase, 48h to reach the peak, 72h begun to decrease.2. acute cerebral ischemia induced apoptosis in rat hippocampal CA1 area at the same time, accompanied by a key factor in the Wnt signaling pathway p-β-catenin, GSK-3βincreased expression andβ-catenin, p-GSK -3βno significant difference, indicating that Wnt signaling pathway may be involved in ischemic brain injury in the development of cerebral ischemia can promote apoptosis. |
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