| Objective:To observe dynamic expression of rat’s Mipul which under condition in Cerebral ischemia-reperfusion, and study whether the role of brain protection of NBP by promoting the expression of Mipul.Methods:64clean-grade male SD rats are randomly divided into four groups:4rats for control group which do not do any processing directly put to death;20rats for sham-operated control group which only in the right common carotid artery into about10mm or so, incomplete embolization of cerebral artery;20rats for cerebral ischemia reperfusion group which are operated in line on the right side of the brain artery embolization;20rats for drug intervention group which are stomach filled with butyl phthalide (NBP) of80mg/kg per day after cerebral ischemia reperfusion for seven days. Each group are randomly divided into four groups, sacrificed in6h,1st day,3rd day,7th day, and the brain tissue were kept in the-70℃. According to Bedersonl evaluation method, we evaluate nerve function defect degree of rats before and after operation. We monitor the pathological changes of brain tissue with HE staining, immunofluorescence and electrophysiological, and quantify the mRNA expression by RT-PCR and protein expression by western blot of Mipul, Bcl-2, Caspase-3.Results:1. Bedersonl score:Rats in control group and sham-operated control group were not seen nerve function defect symptoms, scored of0. Obvious nerve dysfunction after operation was observed in ischemia-reperfusion group, scored between2to3. After intervention with butylphthalide NBP, nerve dysfunction in ischemia-reperfusion group was significantly alleviated, scored between1to2. Ischemia reperfusion group, the longer reperfusion time is, the heavier of the degree of nerve function defect shows. Meanwhile, the longer the drug intervents, the lighter of the degree of nerve function defect shows.2. HE staining:Brain tissues in normal control group and sham-operated control group appear normal, and there is no obvious change on the pathological or morpho-logical structure. The structures of hippocampus were visible contraction, with thick dyeing cone cells, loose hippocampus tissue, and dyeing uniformity. Pathological changes after ischemia reperfusion (2h), damage of hippocampus cell is serious. The tissues appear a large number of empty and parts of nerve cells are missing, accompanied by osteoporosis of brain. Pathological brain injury was significantly alleviated after NBP intervention compared with ischemia reperfusion group, which was statistically significant (P<0.05).3. RT-PCR:(1) Compared with control group, Mipu1were differentially expressed at mRNA level in ischemia reperfusion group. The expression of Mipul increased with the extension of perfusion time, peaked at6h and continued to24h. Mipu1expression showed a trend of gradual decline ever since. The difference of Mipul expression between control group and ischemia reperfusion group was significantly different (P<0.01).(2) With butyl phthalide intervention after cerebral ischemia reperfusion, the expression of Mipul increased. The difference of Mipul expression between un-treated group and treated group was significantly different (P<0.05).4. Western Blot:(1) Compared with control group, the protein expression level of Mipul was different after perfusion, which increased at the first few hours and reached the highest at6h. Then it declined slowly since24h, and7d to a minimum.(2) The Bcl-2protein expression was positively related to Mipul, increasing as Mipul increased and reducing as Mipul reduced. However, the expression of Caspase3was negatively related to Mipul.(3) After drug intervention, expression of Mipul and Bcl-2increased, while Caspase-3dropped. The differences of expression between ischemia reperfusion group and drug-intervention group were significantly different (P<0.05).Conclusion:1.The expression of Mipul was increased after cerebral ischemia-reperfusion in rat, and this kind of phenomenon were observed at multiple time-point, and the mechanism of Mipul was associated with apoptotic pathway.2. Butylphthalide intervention promoted the expression of Mipul, it indicated that Mipul maybe was the molecular targets of Butylphthalide. |
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