大黄苷元对骨髓间充质干细胞移植脑缺血大鼠血脑屏障的作用与机制

Influence of Rhubarb Aglycone on fibrinolysin of BMSCs transplanting in rats with cerebral ischemia

目的观察脑缺血/再灌注损伤大鼠血脑屏障通透性(BBB)改变,并从对纤溶酶系统调节的影响方面探讨大黄苷元、BMSCs移植及其联合保护微血管损伤的作用机制。方法体外培养扩增BMSCs;大鼠随机分组;线栓法制备MCAO模型;术后3h再灌注24h,将BMSCs由同侧颈内动脉移植入脑,大黄苷元灌胃用药;观察脑微血管病理改变,免疫组织化学法测定BMSCs移植后d7(早期)、d14(中期)和d28(后期)IgG和LN、tPA和PAI表达变化。结果模型组大鼠微血管残缺、破损,脑组织IgG和tPA表达增强,随再灌注时间延长呈增加趋势;LN和PAI表达减弱,其中PAI随再灌注时间延长呈减弱趋势。与模型组比较,大黄苷元组d7、移植组d14和d28、联合组各时间点大鼠微血管结构改善明显;大黄苷元组各时间点的IgG和tPA表达均减弱,LN增强,d14的PAI表达增强;移植组d14和d28的IgG和tPA表达减弱,LN增强;联合组各时间点IgG和d14的tPA表达减弱,d7的LN和各时间点PAI表达增强。联合组d7的微血管结构较移植组完整,其d28的tPA表达分别较大黄苷元和移植组降低,d14和d28的LN、d7和d28的PAI-1均较大黄苷元和移植组增强。结论脑缺血/再灌注损伤后微血管基底膜层连蛋白降解明显,BBB通透性随再灌注时间延长而增加;BMSCs移植后早期保护微血管受损的作用不明显,此后随移植时间的延长而增强;大黄苷元可使BMSCs移植早期的作用提前及中、后期作用增强,其作用机制可能与增强PAI-1表达以调节纤溶酶平衡,进而减轻微血管基底膜胶原降解有关。

Aim To observe the changes of permeability of BBB in rats with cerebral ischemia reperfusion injury, and then explore the mechanisms of protecting rats against brain micrangium injury of Rhubarb Aglycone, BMSCs transplantation and their combination from the influence on regulation of fibrinolysin system. Methods Rat whole bone marrow was cultivated and BMSCs were purified and increased by methods of adherence and selection in vitro. 190 rats were randomly divided into different groups. Middle cerebral artery occlusion （MCAO） model was duplicated with nylon thread. Rats from transplantation and combination groups were transplanted with BMSCs via carotid artery at 24 h after reperfusion. Rhubarb Aglycone was used in intragastric administration. Brain micrangium pathological changes were observed by light microscope and immunohistochemical method was used to determine the expression of IgG, LN, tPA and PAI-1. Results Rat brain micrangium in model groups was mutilated and damaged the expression of IgG and tPA increased, and the progressively enhanced tendency appeared following prolongation of reperfusion, while the expression of LN and PAI decreased, and PAI showed the attenuated tendency following the prolongation of reperfusion. Compared with model groups, improvements of brain micrangium structure in d 7 Rhubarb Aglycone group, d 14 and d 28 transplantation groups, and all combination groups were significant, and IgG and tPA expression decreased and LN increased in each Rhubarb Aglycone group, while PAI expression decreased in d 14 Rhubarb Aglyeone group. IgG and tPA expression decreased and LN increased in d 14 and d 28 transplantation groups. IgG in all combination groups and tPA in d 14 combination group was weakened, and expression of LN in d 7 and PAl in each combination group increased. Integrity of rat brain micrangium was significant in d 7 combination group than that in transplantation group, and the expression of tPA was lower than that in Rhubarb Aglycon and transplantation group respectively, while LN expression in d 14 and d 28 groups and PAI-1 in d 7and d 28 were more obvious than that in Rhubarb Aglycon and transplantation group respectively. Conclusions Degradation of laminin of brain micrangium in rats with cerebral ischemia reperfusion injury was obvious, and the permeability increase of BBB aggravated followed the prolongation of reperfusion. BMSCs transplantation showed no significant effects on brain micrangium in earlier period, and the protection appeared to be enhanced following the prolongation of transplantation. Rhubarb Aglycone could make the effect-of BMSCs transplantation ahead of schedule and enhance the protection in metaphase and anaphase, and the mechanisms might be related to softening the degradation of basal lamina collagen of brain micrangium by regulating the balance of fibrinolysin, especially by increasing the expression of PAI-1.