4种中药单体对小鼠脑缺血再灌注损伤的神经保护作用及机制研究

Study on the neuroprotective effects and action mechanisms of four Chinese herbal ;monomer on cerebral ischemia reperfusion mice

目的：观察中药单体葛根素、川芎嗪、人参皂苷Rb1、羟基红花黄色素A对脑缺血再灌注模型小鼠神经行为学、脑梗死体积及脑血流量的影响，探讨其作用机制。方法按随机数字表法将小鼠分为假手术组、模型组、葛根素组、川芎嗪组、人参皂苷Rb1组、羟基红花黄色素A组，每组24只。除假手术组外，其余各组小鼠采用大脑中动脉栓塞法制备脑缺血1 h再灌注24 h模型。脑缺血后1 h，葛根素组小鼠尾静脉注射3μmol/kg葛根素溶液、川芎嗪组尾静脉注射3μmol/kg川芎嗪溶液、人参皂苷Rb1组尾静脉注射3μmol/kg人参皂苷Rb1溶液、羟基红花黄色素A组尾静脉注射3μmol/kg羟基红花黄色素A溶液。各组小鼠于脑缺血1 h再灌注后24 h进行神经行为学评分，采用2,3,5-氯化三苯基四氮唑染色观察小鼠脑梗死体积，采用多普勒激光血流检测仪测定小鼠脑皮层血流量，采用化学比色法检测脑组织NO含量，采用蛋白免疫印迹法检测活化半胱氨酸天冬氨酸蛋白酶-3（cleaved-caspase-3）、NF-κB p-p65蛋白表达。结果与模型组比较，葛根素组、川芎嗪组、人参皂苷Rb1组、羟基红花黄色素A组小鼠脑梗死体积[（15.83±1.83）%、（22.00±2.53）%、（22.83±1.83）%、（17.83±1.72）%比（34.67±2.66）%]降低（P＜0.01或 P＜0.05），脑皮层血流量[（598.81±9.90）μl/（kg？min）、（614.78±9.20）μl/（kg？min）、（577.83±5.55）μl/（kg？min）、（583.54±7.98）μl/（kg？min）比（548.43±1.97）μl/（kg？min）]升高（P＜0.01或P＜0.05），脑组织NO水平[（17.09±1.18）μmol/L、（18.54±0.54）μmol/L、（18.17±0.49）μmol/L、（15.10±0.73）μmol/L比（20.63±0.73）μmol/L]、cleaved-caspase-3[（1.02±0.08）、（1.12±0.04）、（0.87±0.08）、（1.07±0.08）比（1.30±0.06）]、NF-κB p-p65/NF-κB p65蛋白[（1.03±0.19）、（1.15±0.05）、（1.12±0.08）、（0.72±0.08）比（1.45±0.08）]表达降低（P＜0.01或 P＜0.05）。结论4种中药单体均可不同程度改善脑缺血再灌注小鼠的神经功能和脑组织功能，缓解缺血症状，增加血流量，抑制细胞凋亡和炎症因子的释放。

Objective To compare the therapeutical effect of puerarin, ligustrazine, ginsenoside Rb1, Hydroxysafflor yellow A on cerebral ischemia reperfusion mice. Methods The mice were randomly assigned for sham group, model group, puerarin group, ligustrazine group, ginsenoside Rb1 group, and Hydroxysafflor yellow A group, 24 mice for each group. All the groups were subjected to middle cerebral artery occlusion （MCAO） by 1 h ischemia and 24 h of reperfusion except the sham group. The puerarin, ligustrazine, ginsenoside Rb1, Hydroxysafflor yellow A were administrated by tail vein injection with 3μmol/kg at the onset of 1 h of ischemia. The neurologic deficit score, infarct area calculated by TTC staining, cerebral cortex blood flow monitored by laser doppler flowmetry, NO content measured by chemical colorimetry and western blot＆amp;nbsp;were applied to determine the expression for cleaved-caspase-3 and nuclear transcription factor NF-κB for each group. Results Compared with the model group, the infarct area （15.83%± 1.83%, 22.00%± 2.53%, 22.83%± 1.83%, 17.83%± 1.72%vs. 34.67%± 2.66%） in the puerarin group, ligustrazine group, ginsenoside Rb1 group, Hydroxysafflor yellow A group was significantly decreased （P〈0.01 or P〈0.05）;the cerebral cortex blood flow （598.81 ± 9.90 μl/kg？min-1, 614.78 ± 9.20 μl/kg？min-1, 577.83 ± 5.55 μl/kg？min-1, 583.54 ± 7.98 μl/kg？min-1 vs. 548.43 ± 1.97 μl/kg？min-1） significantly increased （P〈0.01 or P〈0.05）;the NO content （17.09 ± 1.18μmol/L, 18.54 ± 0.54μmol/L, 18.17 ± 0.49μmol/L, 15.10 ± 0.73μmol/L vs. 20.63 ± 0.73μmol/L） ignificantly decreased （P〈0.01 or P〈0.05）;the expression of cleaved-caspase-3 （1.02 ± 0.08, 1.12 ± 0.04, 0.87 ± 0.08, 1.07 ± 0.08 vs. 1.30 ± 0.06） and NF-κB p-p65/NF-κB p65 （1.03 ± 0.19, 1.15 ± 0.05, 1.12 ± 0.08, 0.72 ± 0.08 vs. 1.45 ± 0.08） ignificantly decreased （P〈0.01 or P〈0.05） Conclusions Four Chinese herbal monomers could improve nerve and cerebral dysfunctions and ameliorate ischemia symptoms with varying degrees. The mechanisms were involved with the enhancement of cerebral cortex blood flow and inhibition of cell apoptosis and the activation of inflammatory signaling pathways.