丙泊酚对大鼠局灶性脑缺血-再灌注时NF-κB活化和PUMA表达的影响

Effect of propofol on activation of NF-κB and PUMA in cerebral cortex following transient focal cerebral ischemia-reperfusion in rats

目的观察丙泊酚对大鼠局灶性脑缺血-再灌注(IR)时核因子(NF)-κB活化和P53正向凋亡调控因子(PUMA)、Bcl-2和Caspase-3基因表达的影响,并探讨其脑保护的机制。方法 90只雄性大鼠建立局灶性脑IR模型,随机分为IR组、丙泊酚组(P组)和假手术组(C组)。缺血前腹腔注射丙泊酚100mg/kg,各组再灌注时间为2、3、6、12、24、72h。蛋白质印迹法检测不同时间点的NF-κB、PUMA、Bcl-2和Caspase-3表达,凝胶电泳迁移率(EMSA)检测12、24h的NF-κB和PUMA活性;检测不同时间点Caspase-3活性;DNALader观察细胞凋亡。结果与C组比较,IR组缺血侧大脑皮层于再灌注2～24hNF-κB、PUMA表达逐渐升高,72h开始下降,Bcl-2在灌注6h内表达降低,以后逐渐升高,而Caspase-3在灌注2～24h内逐渐升高,72h开始下降。与IR组比较,P组在灌注后的各个时间点NF-κB、PUMA表达明显减少,而Bcl-2表达逐渐升高,Caspase-3逐渐降低。IR组Caspase-3的活性在24h最高,72h开始降低。在P组,Caspase-3蛋白活性升高不显著。DNALader发现,IR组6、12、24、72h见明显的DNA片段,而在P组未见明显的DNA片段。结论丙泊酚抑制NF-κB活化引起的PUMA上调,后者下调Caspase-3和上调Bcl-2,从而抑制神经组织细胞凋亡。

Objective To investigate she effect of propofol on the activation of NF-κB, PUMA and the expression of bcl 2, caspase-3 gene in cerebral cortex after transient focal cerebral ischemiareperfusion（IR）. Methods Ninety male Wistar rats aged 3 4 months weighing 250 300 g were randomly divided into 3 groups： sham group, IR group and propofol group. In propofol group, propofol 100 mg/kg was given IP 10 min before middle cerebral artery occlusion. 2,3,6,12,24 and 72 h were the time point of reperfusion. NF-κB, PUMA, bcl 2 and caspase-3 protein expression and caspase-3 active units were determined by Western blotting, NF-κB and PUMA activity were determined by EMSA, and TUNEL was used for apoptosis assay. Results In IR group, NF-κB and PUMA were significantly activated in the ischemic cerebral cortex during 2-72 h of reperfusion. The expression of NF-κB, PUMA and caspase-3 were significantly higher in ischemie cortex than that in sham groups, and the expression of Bcl-2 was significantly lower in iscbemic cortex. NF-κB was highly correlated to PUMA. In propofol group the expression of NF^B, PUMA and caspase-3 decreased, bcl 2 protein expression increased as compared with IR group. Increased DNA band was seen in IR groups but not in propofol group. Conclusion PropofoI pretreatment can inhibit apoptosis of neurons induced by IR by inhibiting the activation of NF-κB, which up-regulates the PUMA and up-regulating bcl-2 gene and down-regulating caspase-3 gene.