尤瑞克林对局灶性脑缺血大鼠Bcl-2家族蛋白表达的影响

Effect of urokallikrein on the expression of Bcl-2 family protein in rats with focal cerebral ischemia

目的通过观察尤瑞克林对局灶性脑缺血大鼠Bcl-2、Bax蛋白表达的影响,探讨尤瑞克林保护缺血性脑组织的可能作用机制,为尤瑞克林治疗缺血性脑血管病在临床上的应用提供理论依据。方法选用健康SD大鼠130只,雌雄各半,随机分为假手术组、局灶性脑缺血组及同一时间尤瑞克林治疗组(3、6、12、24、48、96h);每组10只。以线栓法阻塞大鼠大脑中动脉,制备局灶性脑缺血模型,术后30min尤瑞克林组舌下静脉注射尤瑞克林8.75×10-3、PNA单位·kg-1。行神经功能缺失评分,免疫组化SABC法对凋亡相关基因bcl-2、bax蛋白进行定量分析,电镜观察大脑皮层顶叶缺血半暗带区神经细胞超微结构变化。结果尤瑞克林组与单纯缺血对照组同一时间比较:在6、12、24、48、96h五个时间段Bcl-2蛋白表达升高(P<0.05)、同时在6个时间段Bax蛋白表达均降低(P<0.05),透射电镜示:尤瑞克林组与模型组同时间段对照比较:核膜较光滑无内陷,核切迹不明显,核染色质可见少量成块。结论尤瑞克林对脑缺血保护作用机制可能与调节NO,从而促进血管新生,提高Bcl-2蛋白表达,同时抑制Bax蛋白表达对脑缺血损伤起保护作用的分子机制之一。

Objective To explore the possible action mechanism of urokallikrein in protecting ischemic brain tissue by observing the effect of urokallikrein on the expression of Bcl-2 family protein in rats with focal cerebral ischemia in order to provide theoretical basis for urokallikrein to treat ischemic cerebrovascular diseases in clinic. Methods One hundred and thirty healthy SD rats, male and female in half, were randomly divided into sham operation group,focal cerebral ischemia groups and simultaneous urokallikrein intervention groups （3h, 6h, 12h,24h, 48h, 96h）, with 10 rabbits in each group. The animal models with focal cerebral ischemia were established by ligation to block middle cerebral artery of rats. The rats in urokallikrein intervention groups were performed by sublingual vein injection of urokallikrein 8.75 × 10^ -3 ,PNA unit kg^-1 at 30 min after the operation. Nerve function defect score was recorded, and quantitative analyses for apoptosis-related gene Bcl-2, Bax protein were performed by means of immunohistochemical SABC method, and neuron uhrastructure changes of apoptosis related gene Bcl-2, Bax protein were quantitatively analyzed, and neuron uhrastructure changes of cerebral cortex in parietal lobe ischemia penumbra region were observed by electron microscopy. Results As compared with those in simple ischemic groups,the expression levels of Bcl-2 protein were significantly increased at the five time points of 6h, 12h,24h,48h,96h （ P 〈 0.05 ）, however, the expression levels of Bax protein were significantly decreased at the five time points of 6h, 12h,24h,48h,96h （ P 〈 0.05）. Transmission electron microscope examination showed that the nuclear envelope was smooth without invagination, nuclear notch was not obvious, and small amounts conglobation was visible in nuclear chromatin in urokallikrein intervention groups, as compared with those in focal cerebral ischemia groups at the same time points. Conclusion The action mechanism of protective effect of urokallikrein on cerebral ischemia maybe related to the regulation of NO, thereby, which can promote angiogenesis, enhance the expression levels of Bcl-2 protein, however,inhibit the expression of Bax protein, which maybe one of the molecular mechanisms of the protective effect of urokallikrein on cerebral ischemic injury.