长期丁苯酞预处理通过Nrf2通路减轻永久性大脑中动脉远端闭塞小鼠缺血性脑损伤

Long-term butylphthalide pretreatment attenuates ischemic brain injury in mice with permanent distal middle cerebral artery occlusion through Nrf2 pathway

目的探讨长期预防性应用丁苯酞对永久性大脑中动脉远端闭塞小鼠的神经保护作用及其与核因子E2相关因子2(Nuclear factor erythroid 2-related factor 2,Nrf2)通路的关系。方法将Nrf2^(+/+)野生型和Nrf2^(-/-)基因敲除小鼠随机分为对照组(等体积植物油)、小剂量丁苯酞组(20 mg/kg)和大剂量丁苯酞组(60 mg/kg),每组各6只。通过灌胃方式每日给药1次,持续1个月,然后通过电凝法建立永久性大脑中动脉远端闭塞模型,模型制作后继续给药,直至第10天处死。在模型制作后第3天和第10天时应用改良Longa分级量表和转棒实验评价神经功能缺损情况。在处死后通过三苯基氯化四氮唑染色检测脑梗死体积,通过干湿重法检测脑含水量,通过定量实时聚合酶链反应以及蛋白质印迹法测定缺血区脑组织Nrf2通路相关因子表达,包括Nrf2、血红素加氧酶1(heme oxygenase-1,HO-1)和NAD(P)H醌氧化还原酶1(NAD(P)H:quinone oxidoreductase 1,NQO1)。结果造模后第10天时,与Nrf2^(-/-)对照组相比,Nrf2^(+/+)对照组神经功能缺损显著较轻,脑梗死体积和脑含水量均显著较小,而且Nrf2、HO-1、NQO1 mRNA及蛋白水平显著较高,差异均有统计学意义(P均<0.05)。对于Nrf2^(+/+)小鼠,与对照组相比,大剂量丁苯酞组脑梗死体积显著缩小(P<0.05),脑含水量显著降低(P<0.05),神经功能恢复显著更好(P<0.05),而且Nrf2、HO-1、NQO1 mRNA及蛋白水平显著较高(P均<0.05)。对于Nrf2^(-/-)小鼠,各组间神经功能、脑梗死组体积、脑含水量、Nrf2、HO-1、NQO1 mRNA及蛋白水平均无显著统计学差异。结论长期丁苯酞预处理能显著改善永久性大脑中动脉远端闭塞小鼠小鼠神经功能,缩小脑梗死体积,降低脑含水量,并且提高Nrf2、HO-1、NQO1 mRNA及蛋白表达水平,提示丁苯酞可能通过上调Nrf2基因及其下游抗氧化应激因子HO-1和NQO1表达发挥神经保护作用。

Objective To investigate the neuroprotective effect of long-term prophylactic use of buphthalein on mice with permanent distal middle cerebral artery occlusion and its relationship with the nuclear factor erysid 2 related factor 2(Nrf2)pathway.Methods Nrf2^(+/+)wild-type and Nrf2^(-/-)knockout mice were randomly divided into control group(equal volume vegetable oil),low-dose butylphthalide group(20 mg/kg)and high-dose butylphthalide group(60 mg/kg),with 6 mice in each group.The drug was administered once a day by gavage for 1 month,and then a permanent middle cerebral artery occlusion model was induced by electrocoagulation.After the model was made,the drug was continued and the mice were sacrificed on the 10th day.The modified Longa grading scale and the rotating rod test were used to evaluate neurological deficits on the 3rd and 10th day after the model was made.After the mice were sacrificed,the cerebral infarct volume was measured by triphenyltetrazolium chloride staining.The brain water content was measured by dry and wet weight method.The expression of Nrf2 pathway related factors,including Nrf2,heme oxygenase 1(HO-1)and NAD(P)H quinone oxidoreductase 1(NQO1)were measured by quantitative real-time PCR and Western blotting.Results On the 10th day after modeling,compared with the Nrf2^(-/-)control group,the neurological deficit was significantly milder,the volume of cerebral infarction and brain water content were significantly smaller,and the mRNA and protein levels of Nrf2,HO-1 and NQO1 were significantly higher in the Nrf2^(+/+)control group,and the differences were statistically significant(P<0.05).For Nrf2^(+/+)mice,compared with the control group,the cerebral infarct volume was significantly reduced(P<0.05),the brain water content was significantly reduced(P<0.05),and the neurological function recovery was significantly better(P<0.05),and the levels of Nrf2,HO-1,and NQO1 mRNA and protein were significantly higher in the high-dose butylphthalide group(all P<0.05).For Nrf2^(-/-)mice,there were no significant differences in neurological function,cerebral infarction group volume,brain water content,Nrf2,HO-1,NQO1 mRNA and protein levels among the groups.Conclusion Long-term butylphthalide pretreatment can significantly improve the neurological function,reduce cerebral infarction volume,reduce brain water content,and increase Nrf2,HO-1,NQO1 mRNA and protein expression levels in mice with permanent distal middle cerebral artery occlusion,suggesting butylphthalide may play a neuroprotective effect by up-regulating the expression of Nrf2 gene and its downstream antioxidant stress factors HO-1 and NQO1.