牡荆苷调控Nrf2/ARE通路减轻急性脑缺血再灌注大鼠氧化应激反应研究

Effect of vitexin on Nrf2/ARE pathway in alleviating oxidative stress in rats with acute cerebral ischemia-reperfusion

目的探讨牡荆苷通过调控核转录因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)通路对急性脑缺血再灌注大鼠氧化应激反应的作用。方法54只SD大鼠随机分为假手术组、模型组、阳性对照依达拉奉(0.56 mg/kg)组和牡荆苷低、中、高剂量(10、20、40mg/kg)组,除假手术组外均制备急性脑缺血大鼠模型,再灌注后假手术组、模型组大鼠ip生理盐水,依达拉奉组和牡荆苷各剂量组按照相应剂量ip给药,8 h给药1次,共3次。对比干预前后大鼠神经行为学评分;HE染色检测大鼠大脑皮质病理变化;试剂盒检测大鼠大脑皮质组织氧化应激指标丙二醛(MDA)、一氧化氮(NO)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)水平;实时荧光定量PCR(qRT-PCR)和Western blotting检测大鼠大脑皮层Nrf2/ARE通路相关基因mRNA与蛋白表达水平。结果干预前后假手术组大鼠神经行为学评分无明显变化,模型组较干预前增高(P<0.01),依达拉奉组、牡荆苷各剂量组均较干预前降低(P<0.01),且干预后各组间神经行为学评分比较差异显著(P<0.01)。假手术组大鼠大脑皮质组织神经细胞分布均匀、密集,神经细胞的胞体、胞核形态正常;模型组、依达拉奉组和牡荆苷各剂量组大鼠脑组织神经细胞排列均有紊乱、疏松,其中模型组可见液化性坏死、细胞结构消失等表现;牡荆苷低剂量组可见细胞排列严重紊乱、疏松,部分液化性坏死、细胞结构消失;牡荆苷中剂量组和依达拉奉组液化性坏死部分面积小,大多细胞结构正常;牡荆苷高剂量组仅可见极少液化性坏死,细胞结构基本正常,细胞排列轻微紊乱。与假手术组比较,模型组大鼠大脑皮质MDA和NO水平显著升高(P<0.01),SOD和GSH水平显著降低(P<0.01),Nrf2、γ-GCSmRNA表达水平显著升高(P<0.01),细胞质Nrf2、γ-GCS蛋白表达水平均显著升高(P<0.01),细胞核Nrf2、HO-1蛋白表达水平均显著降低(P<0.01)。与模型组比较,依达拉奉组和牡荆苷低、中、高剂量组大鼠大脑皮质MDA和NO水平均显著降低(P<0.01),SOD和GSH水平显著升高(P<0.01),Nrf2、γ-GCS mRNA表达水平显著降低(P<0.01),细胞质Nrf2、γ-GCS蛋白表达水平均显著降低(P<0.01),细胞核Nrf2、HO-1蛋白表达水平均显著升高(P<0.01)。且牡荆苷各剂量组大鼠大脑皮质MDA、NO、SOD、GSH水平及Nrf2、γ-GCS、HO-1m RNA和蛋白表达水平均呈剂量依赖性,组间差异显著(P<0.01)。结论牡荆苷可减轻急性脑缺血再灌注大鼠氧化应激反应,推测与调控Nrf2/ARE信号通路,上调Nrf2基因与蛋白表达,促进其由细胞质向细胞核移动,上调HO-1表达,抑制γ-GCS表达,增强机体的抗氧化应激反应能力有关。

Objective To investigate the effect of vitexin on oxidative stress in rats with acute cerebral ischemia-reperfusion by regulating the pathway of nuclear factor E2-related factor 2(Nrf2)/antioxidant response element(ARE).Methods A total of 54 SD rats were randomly divided into Sham group,model group,positive control(edaravone 0.56 mg/kg)group and vitexin low,medium,and high dose(10,20,40 mg/kg)groups.The rat models with acute cerebral ischemia were established except the Sham group.After reperfusion,rats in Sham group and model group were received ip saline.The edaravone group and vitexin groups were administered according to the corresponding dose,once every 8 h for a total of three times.The neurobehavioral scores of rats before and after intervention were compared.HE staining was used to detect the pathological changes of cerebral cortex.The levels of MDA,NO,SOD,and GSH in cerebral cortex of rats were detected by the kit.The mRNA and protein expression levels of Nrf2/ARE pathway related gene in rat cerebral cortex were detected by real-time fluorescent quantitative PCR(qRT-PCR)and Western blotting.Results Before and after intervention,there was no significant change in the neurobehavioral score of rats in the Sham group,the model group was higher than before intervention(P<0.01),the edaravone group and the vitexin groups were lower than before intervention(P<0.01),and there was a significant difference in the neurobehavioral score between the groups after intervention(P<0.01).In the Sham group,the distribution of nerve cells was uniform and dense,and the morphology of cell body and nucleus was normal.In the model group,edaravone group and vitexin group,the arrangement of brain tissue was disordered and loose,in which liquefying necrosis and disappearance of cell structure were observed in the model group;in the low-dose group,the arrangement of cells was seriously disordered and loose.In the vitexin medium dose group and edaravone group,the area of liquefying necrosis was small,and most of the cells were normal;In the vitexin high dose group,only a few liquefying necrosis were found,the cell structure was basically normal,and the cell arrangement was slightly disordered.Compared with the Sham group,the levels of MDA and NO in the cerebral cortex of the model group were significantly increased(P<0.01),the levels of SOD and GSH were significantly reduced(P<0.01),and the expression levels of Nrf2 andγ-GCS mRNA were significantly increased(P<0.01),the expression of cytoplasmic Nrf2 andγ-GCS proteins were significantly increased(P<0.01),and the expression levels of nuclear Nrf2 and HO-1 proteins were significantly decreased(P<0.01).Compared with the model group,the levels of MDA and NO in the cerebral cortex of rats in the edaravone group and low,medium and high dose groups of vitexin were significantly reduced(P<0.01),and the levels of SOD and GSH were significantly increased(P<0.01),Nrf2 andγ-GCS m RNA expression levels were significantly reduced(P<0.01),cytoplasmic Nrf2,γ-GCS protein expressions were significantly reduced(P<0.01),and nuclear Nrf2 and HO-1 protein expression levels were significantly increased(P<0.01).And the levels of MDA,NO,SOD,GSH and Nrf2,γ-GCS,HO-1 mRNA and protein expression in rat cerebral cortex in each dose group were dose-dependent,with significant differences between groups(P<0.01).Conclusion Vitexin can alleviate oxidative stress in rats with acute cerebral ischemia-reperfusion.It is speculated that it is related to the regulation of Nrf2/ARE signaling pathway,the up-regulation of Nrf2 gene and protein expression,the promotion of its movement from cytoplasm to nucleus,the up-regulation of HO-1 expression,the inhibition ofγ-GCS expression,and the enhancement of the body’s ability to response to oxidative stress.