丹龙醒脑方对脑缺血再灌注损伤大鼠SVZ区Frizzled3、Dsh1及β-catenin表达的影响与促进神经干细胞增殖的机制

Effects of Danlong Xingnao Formula on the expression of Frizzled3, Dsh1 and β-catenin in SVZ of rat model of cerebral ischemia-reperfusion and its mechanism underlying promoting proliferation of neural stem cells

目的:探讨丹龙醒脑方对局灶性脑缺血再灌注大鼠侧脑室室管膜下区(SVZ)神经干细胞(NSCs)增殖与Frizzled3、Dsh1、β-catenin表达的关系。方法:将120只雄性SD大鼠随机分为假手术组、模型组、依达组、丹龙组。后3组用线栓法制备局灶性脑缺血再灌注(MCAO/R)模型,再灌注7d后取缺血侧SVZ脑组织。采用Brdu免疫荧光法检测SVZ区NSCs增殖,RT-q PCR法、Western Blot法分别检测Frizzled3、Dsh1、β-catenin m RNA和蛋白的表达。结果:与假手术组比较,其余各组Brdu阳性细胞数明显增多,Frizzled3、Dsh1、β-catenin蛋白及m RNA的表达明显升高(P<0.05,P<0.01);与模型组比较,依达组、丹龙组Brdu阳性细胞数明显增多,Dsh1、β-catenin m RNA及蛋白的表达明显增强(P<0.05,P<0.01),丹龙组Frizzled3表达明显增强(P<0.05,P<0.01),但依达组不明显。依达组、丹龙组组间比较,除Frizzled3蛋白表达丹龙组要优于依达组(P<0.01),其余指标均无明显差异。结论:丹龙醒脑方能促进脑缺血后SVZ区NSCs增殖,其机制可能与通过上调Frizzled3和Dsh1的水平,进而促进Wnt信号的传递和增强关键因子β-catenin的表达有关。

Objective： To investigate whether the effects of Danlong Xingnao Formula on promoting proliferation of neural stem cells（NSCs） in subventricular zone（SVZ） were related to the expression of Frizzled3, Dsh1 and β-catenin in hippocampus of rat model of cerebral ischemia-reperfusion（MCAO/R）. Methods： One hundred and twenty male SD rats were randomly divided into the sham group, MCAO/R（model） group, edaravone group and Danlong Xingnao Formula（DLXNF） group. The rats in all groups except for the sham group were subjected to a middle cerebral artery occlusion to establish the MCAO/R model. The brain tissues in SVZ of focal cerebral ischemia were harvested after 7 days of reperfusion. The NSCs proliferation in SVZ was determined by Brdu immunofluorescence. The expressions of hippocampal Frizzled3, Dsh1 and β-catenin m RNA and protein were determined respectively by RT-q PCR and Western Blot. Results： Brdu positive cells and the expression of Frizzled3, Dsh1 and β-catenin protein and m RNA in other groups were improved obviously than those in the sham group（P〈0.05, P〈0.01）; Compared with the model group, Brdu positive cells and the expression of Dsh1 and β-catenin protein and m RNA in edaravone group and DLXNF group improved obviously（P〈0.05, P〈0.01）; the expression of Frizzled3 protein and m RNA in the DLXNF group also improved obviously（P〈0.05, P〈0.01）, but the expression in edaravone group had no difference.Comparison between edaravone group and DLXNF group showed that the differences of all indicators were not statistically significant except the expression of Frizzled3 protein（P〈0.01）. Conclusion： Danlong Xingnao Formula can stimulate the proliferation of NSCs by activating the expression of Frizzled3 and Dsh1, then promoting the transduction of Wnt signal channel and enhancing the expression of β-catenin.