银杏达莫注射液不同成分对亚急性期卒中小鼠神经功能及G-CSF、MAC-1的影响及作用机制

Effects and mechanism of different components of Ginkgo damol injection on neurological function and G-CSF and MAC-1 in subacute stroke mice

目的探讨银杏达莫注射液中的不同成分(银杏总黄酮、双嘧达莫、银杏总内酯)对亚急性期卒中小鼠神经功能及粒细胞-集落刺激因子(Granu1ocyte colony-stimu1ating factor,G-CSF)、巨噬细胞分化抗原1(Macrophage differentiation antigen 1,MAC-1)的影响及作用机制。方法选取无特定病原体(specific pathogen free,SPF)小鼠134只建立脑卒中亚急性期模型和脑缺血再灌注损伤模型,根据脑缺血再灌注损伤模型的实现情况(舍弃血流再灌注失败的14只小鼠)分组,假手术组、模型组、银杏总黄酮组(3 ml·kg^(-1))、双嘧达莫组(3 ml·kg^(-1))和银杏总内酯组(3 ml·kg^(-1)),各24只。前两组只予以等量(3 ml·kg^(-1))的生理盐水干预,后三组在给药且造模成功后,立即在小鼠尾静脉注射相应药物各1次,连续注射1周(尾静脉:每天早晚各注射1次)。取各组小鼠腹主动脉血进行生化处理,用酶联免疫吸附试验法(enzyme linked immunosorbent assay,ELISA)检测G-CSF蛋白含量,用免疫印迹法检测MAC-1蛋白含量。采用改良神经系统严重程度评分表(modified neurologic severity scores,mNSS)记录各组小鼠脑缺血再灌注后1、3、5、7天时的各项指标。根据上述指标的具体表达情况分析银杏总黄酮、双嘧达莫、银杏总内酯对小鼠神经功能及G-CSF和MAC-1的影响及其与亚急性期卒中的作用机制。结果(1)生存率:模型组死亡率较假手术组更高(P<0.05)。与模型组相比,使用银杏总黄酮、双嘧达莫、银杏总内酯治疗后银杏总黄酮组、双嘧达莫组和银杏总内酯组整体的生存率均显著提升,三者相比双嘧达莫组略低于银杏总黄酮组、银杏总内酯组(P<0.001)。(2)体重:模型组体重下降最明显,显著低于假手术组(P<0.001),从治疗后第3天开始,银杏总黄酮组、双嘧达莫组和银杏总内酯组的体重与模型组相比逐渐升高(P<0.001),双嘧达莫组体重升高程度依次高于银杏总黄酮组、银杏总内酯组(P<0.001)。(3)mNSS:与假手术组比,模型组mNSS评分最高,神经功能缺损最严重(P<0.001),从术后第3天开始,银杏总内酯组mNSS评分下降程度依次高于双嘧达莫组、银杏总黄酮组(P<0.001)。(4)假手术组G-CSF、MAC-1表达均低于模型组、银杏总黄酮组、双嘧达莫组、银杏总内酯组(P<0.001),模型组G-CSF表达依次高于银杏总黄酮组、银杏总内酯组、双嘧达莫组,模型组MAC-1表达依次高于双嘧达莫组、银杏总内酯组和银杏总黄酮组(P<0.001)。银杏达莫注射液全方和银杏总内酯能显著降低G-CSF、MAC-1表达,效果显著优于双嘧达莫和银杏总黄酮(P<0.001)。结论银杏达莫注射液的不同有效成分可改善亚急性期卒中小鼠的神经功能损伤,可能通过银杏总内酯调节G-CSF、MAC-1介导的相关病变机制来抑制机体的粒细胞黏附与浸润通路。

Objective To investigate the effects of different components(total flavonoids,dipyridamole)on nerve function and granulocyte-colony-stimulating factor(Granu1ocyte colony-stimu1ating factor,G-CSF)and macrophage differentiation antigen 1(Macrophage differentiation antigen 1,MAC-1)in subacute stroke mice.Methods 134 mice without specific pathogens(specific pathogen free,SPF)subacute stroke model and cerebral ischemia and reperfusion injury model,according to the implementation of cerebral ischemia and reperfusion injury model(14 mice with failed reperfusion),24 each in sham group,model group,total flavonoids group(3mL·kg^(-1)),dipyridamole group(3 mL·kg^(-1))and total ginkgo group(3 mL·kg^(-1)).The first two groups received only equal saline intervention(3 mL·kg^(-1)),and in the last three groups,the corresponding drugs were immediately injected in the tail vein of the mouse for 1 week(tail vein:1 injection in the morning and evening).Main abdominal artery blood from each group was collected for biochemical treatment,G-CSF protein content was measured by enzyme-linked immunosorbent test(enzyme linked immunosorbent assay,ELISA),and MAC-1 protein content was measured by immunoblotting.The modified Neurological Severity Scale(modified neurologic severity scores,mNSS)was used to record the indicators at 1,3,5,and 7 days after cerebral ischemia reperfusion.According to the specific expression of the above indexes,the effects of total flavonoids,dipyridamole and total olide in ginkgo on the neural function,G-CSF and MAC-1 and the mechanism of subacute stroke were analyzed.Results(1)Survival:higher mortality in the model group than in the sham group(P<0.05).Compared with the model group,the overall survival rate of the total flavonoids group,dipyridamole group and intracolide group was significantly improved compared with the dipyridamole group,and the dipyridamole group was slightly lower than the total flavonoids group and intracolide group(P<0.001).(2)Body weight:the weight loss in the model group,significantly lower than the sham group(P<0.001),from the third day after treatment,dipyrimidole group and ginkgo group(P<0.001),dipyrimidole group than ginkgo total flavonoids and ginkgo group(P<0.001).(3)mNSS:Compared with the sham group,the mNSS score was the highest and the most severe neurological deficit(P<0.001).From the third postoperative day,the decline of the mNSS score in total olide group was higher than that in the dipyridamole group and total flavonoids group(P<0.001).(4)The expressions of G-CSF and MAC-1 were lower than that of model group,ginkgo total flavonoids group,dipyrimidole group and ginkgo total inside group(P<0.001).The expressions of G-CSF expression in model group was successively higher than that of ginkgo total flavonoids group,ginkgo total inside group and dipyrimidole group,and MAC-1 expression was successively higher than dipyrimidole group,ginkgo total inside group and ginkgo total flavonoids group(P<0.001).Ginkgo ol injection and total ginkgo lactone could significantly reduce G-CSF and MAC-1 expression,which was significantly better than dipyridamole and ginkgo total flavonoids(P<0.001).Conclusion The different active components of ginkgo damol injection can improve the neurological damage in subacute stroke mice,which may inhibit the granulocyte adhesion and infiltration pathway through the mechanism of G-CSF mediated by MAC-1.