扎里奴思方干预骨髓间充质干细胞移植对脑缺血再灌注损伤大鼠神经轴突再生的影响

目的:观察扎里奴思方(扎方)干预骨髓间充质干细胞(BMSCs)移植对大脑中动脉阻塞(MCAO)大鼠神经轴突再生的影响,并探讨其机制。方法:SD大鼠随机分为假手术组、模型组、扎方组、移植组和联合组;线栓法制备大鼠MCAO模型,体外全骨髓贴壁筛选法培养及扩增BMSCs;大鼠灌胃给药(14.6 g·kg~(-1)·d~(-1)),BMSCs经颈内动脉移植入脑;移植后1,3,7,14 d取材,蛋白质免疫印迹(Western blot)检测生长相关蛋白(GAP-43),神经微丝蛋白(NF~(-2)00),髓磷脂相关糖蛋白(MAG)和勿动蛋白-A(Nogo-A)蛋白表达。结果:模型组GAP-43,MAG及Nogo-A蛋白表达较假手术组明显增高(P0.01),NF~(-2)00降低(P0.01);与模型组比较,扎方3,14 d组、移植3,7,14 d组及联合各组GAP-43表达增高(P0.05,P0.01),扎方、移植及联合各组NF~(-2)00表达增高(P0.01),MAG及Nogo-A降低(P0.01);与移植组比较,扎方14 d组GAP-43,7 d组NF~(-2)00及1,3,14 d组MAG表达降低(P0.05,P0.01),3 d组Nogo-A降低(P0.01),7 d组增高(P0.05),联合各组GAP-43及1,14 d组NF~(-2)00表达增高(P0.01),MAG,Nogo-A及7 d组NF~(-2)00表达降低(P0.01);扎方与联合组比较,联合3,7,14 d组GAP-43及14 d组NF~(-2)00表达增高(P0.01),各组MAG及Nogo-A表达降低(P0.01);同组间比较,模型7 d组GAP-43及MAG表达增高(P0.05,P0.01),3 d组NF~(-2)00表达较1 d组增高(P0.01),3 d组Nogo-A达高峰(P0.01),扎方、移植及联合各14 d组GAP-43表达较7 d组增高(P0.01),扎方及移植各7 d组NF~(-2)00表达较3,14 d组增高(P0.01,P0.05),联合3 d组NF~(-2)00表达较低(P0.01),后逐渐增高(P0.05),扎方、移植及联合各组MAG及Nogo-A表达呈先增后减趋势。结论:扎方可显著促进脑缺血再灌注损伤BMSCs移植后神经轴突再生,以二者联合作用显著,其机制可能与干预损伤后GAP-43,NF~(-2)00及MAG,Nogo-A的动态表达有关。

Effect of Zhali Nusi Fang in Intervening Impact of Bone Marrow Mesenchymal Stem Cells Transplantation on Neuronal Axons Regeneration in Rats After Cerebral Ischemia Reperfusion Injury

Objective: To observe the effect of Hui medicine Zhali Nusi Fang (ZLNF) in intervening the impact of bone marrow mesenchymal stem cells (BMSCs) transplantation on neuronal axons regeneration in rats after cerebral ischemia reperfusion injury (CIRI). Method: SD rats were divided randomly into Sham-operated group, model group, ZLNF group, BMSCs transplantation group and ZLNF-BMSC combination group; middle cerebral artery occlusion (MCAO) model was duplicated with nylon thread, BMSCs were cultured and amplified by the whole bone marrow adherence method; drugs were given to the rats by intragastric administration (14.6 g·kg^-1·d^-1), BMSCs were transplanted into rat brains through carotid artery; rat brains were taken out at 1, 3, 7, 14 days after transplantation; growth associated protein-43 (GAP-43), neurofilament protein-200 (NF-200), myelin associated glycoprotein (MAG) and Nogo-A protein expression were detected by Western blot. Result: The GAP-43, MAG, Nogo-A in the model groups were obviously increased than the sham-operated groups (P<0.01), and with significant decrease in NF-200 (P<0.01); compared with the model groups, the GAP-43 at 3, 14 days in the ZLNF group, at 3, 7, 14 days in the transplantation group and the combination group were increased (P<0.01, P<0.05), the NF-200 in the ZLNF group, the transplantation group and the combination group were increased (P<0.01), but with decreases in MAG and Nogo-A (P<0.05); compared with the transplantation groups, the GAP-43 at 14 day, the NF-200 at 7 day and MAG at 1, 3, 14 days in the ZLNF group were decreased (P<0.01, P<0.05), and the Nogo-A at 3 day in the ZLNF group was decreased (P<0.01) but increased at 7 day (P<0.05), the GAP-43 at 1, 14 days and the NF-200 in the combination group were increased (P<0.01), the NF-200 at 7 day and MAG, Nogo-A in the combination group were decreased (P<0.01); compared with the ZLNF group, the GAP-43 at 3, 7, 14 days and the NF-200 at 14 day in the combination group were increased(P<0.01), the MAG, Nogo-A in all of the groups were decreased (P<0.01); in the intra-group comparison, the GAP-43 and MAG at 7 day in the model group were increased compared with that at 3, 14 days (P<0.01, P<0.05), the NF-200 at 3 day in the model group was increased compared with that at 1 day, the Nogo-A at 3 day reached peak (P<0.01), the GAP-43 at 14 day group in ZLNF, transplantation and combination groups were higher than that at 7 day (P<0.01), the NF-200 at 7 day in ZLNF and transplantation groups were higher than that at 3, 14 days (P<0.01, P<0.05), the NF-200 at 3 day in the combination group was relatively low (P<0.01) and later gradually higher (P<0.05), the MAG, Nogo-A in ZLNF, transplantation and combination groups were increased first and then decreased. Conclusion: Hui medicine ZLNF can obviously promote the neuronal axons regeneration after the transplantation of BMSCs for cerebral ischemia reperfusion injury, with a significant effect in their combination, and its mechanism may be related to dynamic expressions of GAP-43, NF-200, MAG and Nogo-A after the intervention.