脑脉通联合骨髓干细胞动员对脑缺血大鼠神经细胞凋亡相关蛋白Fas、FasL及caspase-3表达的影响

Effects of Naomaitong combined with mobilization of bone marrow mesenchymal stem cells on neuron apoptosis and expressions of Fas,FasL and caspase-3 proteins in rats with cerebral ischemia

目的:观察脑脉通联合骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)动员对脑缺血大鼠神经细胞凋亡和凋亡相关蛋白Fas、FasL及半胱氨酸天冬氨酸特异性蛋白酶3(caspase-3)表达的影响。方法:202只SD大鼠分为假手术组、模型组、人重组粒细胞集落刺激因子(recombinant granulocyte colonystimulatingfactor,rG-CSF)组、脑脉通组和脑脉通联合rG-CSF组(联合组)。采用改良线栓法阻塞大鼠大脑中动脉制各局灶性脑缺血动物模型。rG-CSF组和联合组大鼠分别于术前3 d和术后2 d皮下注射10μg/(kg·d)rG-CSF,脑脉通组和联合组灌胃脑脉通。术后早期(2 d和3 d)和后期(7 d和14 d)观察大鼠神经功能变化;腹主动脉取血,流式细胞仪测定外周血CD34阳性细胞数量;原位末端标记法观察神经细胞凋亡情况;免疫组织化学法检测脑组织CD34、caspase-3、Fas和FasL蛋白表达情况。结果:模型组大鼠外周血和脑组织CD34阳性细胞增加,分别于3 d和7 d时达到峰值;各治疗组尤其联合组脑组织CD34表达增强明显。各模型组大鼠神经功能评分降低,rG-CSF组7 d和14 d时的神经功能评分增加,联合组较rG-CSF组的变化显著。模型组脑组织2、3和7 d时的Fas、FasL及caspase-3蛋白表达增强;rG-CSF组2 d时FasL,各联合组Fas、FasL及caspase-3表达均减弱;联合组7 d和14 d时的Fas、FasL及caspase-3表达较rG-CSF组减弱;各实验组14 d时Fas、FasL及caspase-3表达较3 d时减弱。结论:脑脉通和BMSC动员在改善大鼠脑缺血中、后期神经功能和抑制细胞凋亡方面存在交互效应.其作用可能与脑脉通使BMSC动员后早期的Fas和FasL表达下调,以阻抑caspase-3引起的凋亡瀑布反应,进一步抑制促凋亡基因Fas和FasL的表达有关。

Objective： To observe the effects of Naomaitong, a compound traditional Chinese herbal medicine, combined with mobilization of bone marrow mesenchymal stem cells （BMSCs） on neuron apoptosis in rats with cerebral ischemia, and to explore the possible mechanism by detecting the expressions of Fas, FasL and caspase-3 proteins. Methods： Two hundred and two SD rats were divided into sham-operated group, untreated group, recombinant granulocyte colony-stimulating factor （rG-CSF） group, Naomaitong group and Naomaitong plus rG-CSF group （combination group）. Focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion using a nylon thread with some modification. Rats in the rG-CSF group and the untreated group were administered with rG-CSF 10 μg/（kg·d） by subcutaneous injection 3 d before and 2 d after the operation respectively, once a day, and rats in the Naomaitong group and the combination group were intragastrically administered Naomaitong before and after the operation until sacrificed. Two, three, seven and fourteen days after operation, count of CD34-positive cells in peripheral blood and CD34 expression in brain tissue were determined. General neural function score （GNFS） was evaluated. Neuron apoptosis, expressions of Fas, FasL and caspase-3 in rat’s brain were all measured. Results： Count of CD34-positive cells in peripheral blood and CD34 expression in brain tissue were high in the untreated group, and reached the peak at 3 d and 7 d respectively. CD34 expression in brain tissue was increased in each treated group, especially in the combination group. GNFS was increased at 3 d and 7 d in the untreated group, 7 d and 14 d in the rG-CSF group and the combination group. Expressions of Fas, FasL and caspase-3 were increased 2, 3 and 7 d after operation, while expression of FasL at 2 d in the rG-CSF group, expressions of Fas, FasL and caspase-3 in the combination group were decreased. Expressions of Fas, FasL and caspase-3 at 7 d and 14 d in the combination group were lower than those in the rG-CSF group. Meanwhile, expressions of Fas, FasL and caspase-3 were decreased in each group at 14 d as compared with those at 3 d. Conclusion： There exists interaction between Naomaitong and BMSC mobilization in the effect of improving nerve function and inhibiting neuron apoptosis in rats after cerebral ischemia. It is implied that Naomaitong combined with BMSC mobilization down-regulates the expressions of Fas and FasL in early phase and then inhibits the apoptosis cascade reaction caused by caspase-3, which causes further inhibition of Fas and FasL expression after cerebral ischemia.