脑泰方对脑缺血/再灌注大鼠海马区Nrf2、HO-1和膜铁转运辅助蛋白表达的影响

Effects of Naotai formula on expression of Nrf2,HO-1 and hephaestin in hippocampus of cerebral ischemia/reperfusion rats

目的研究益气活血中药脑泰方通过Keap1-Nrf2/ARE信号通路对脑缺血/再灌注后大鼠海马区血红素加氧酶-1(heme oxygenase-1,HO-1)和膜铁转运辅助蛋白(hephaestin,Heph)表达的影响。方法将80只♂大鼠随机分为假手术组、模型组、脑泰方低剂量组(4.5 g·kg^(-1))、中剂量组(9 g·kg^(-1))和高剂量组(18 g·kg^(-1))。各组大鼠术前连续灌胃给药3 d,每日1次,再行大脑中动脉线栓法脑缺血/再灌注模型制备术,术后连续灌胃给药2 d。术后72 h采用Zea Longa神经功能学评分标准记录大鼠行为活动,TTC染色法测定脑梗死体积,real-time PCR检测大鼠海马区Nrf2、HO-1、Heph的mRNA表达,Western blot检测Nrf2、HO-1、Heph的蛋白表达。结果与模型组比较,脑泰方中、高剂量组的神经行为学评分明显下降(P<0.01),各脑泰方组脑梗死体积明显缩小(P<0.01);HO-1 mRNA表达均增加(P<0.05)。脑泰方中、高剂量组Heph mRNA表达增加(P<0.05);Nrf2和Heph蛋白表达明显增加(P<0.05,P<0.01)。各脑泰方组HO-1蛋白表达增高(P<0.01)。结论脑泰方能减轻脑缺血/再灌注损伤,其机制可能是通过激活Keap1-Nrf2/ARE信号通路,促进HO-1生成,上调Heph的表达,减少脑铁沉积,发挥脑缺血/再灌注后神经元的保护作用。

Aim To investigate the effects of Naotai formula extract（ NTE） on the expression of heme oxygenase-1（ HO-1） and hephaestin（ Heph） in hippocampus of rats after cerebral ischemia/reperfusion by Keap1-Nrf2/ARE signaling pathway. Methods Eighty rats were randomly divided into five groups as follows：sham operation group（ Sham）, cerebral ischemia/reperfusion group（ I/R）,low dose group of NTE（ 4. 5g·kg（-1））,middle dose group of NTE（ 9 g·kg（-1））and high dose group of NTE（ 18 g·kg（-1））. Rats were pretreated by intragastric administration for three consecutive days,and then subjected to middle cerebral artery occlusion（ MCAO） 2 hours before reperfusion.The rats were administered with intragastric administration for two days. After cerebral ischemia reperfusion72 hours,the behavioral activity of rats was recorded by Zea Longa neurological score,and the infarct volume was measured by TTC staining. The expressions of Nrf2,HO-1 and Heph in hippocampus of cerebral ischemia reperfusion rats were observed by real-time quantitative PCR and Western blot,respectively. Results Compared with model group,the neurobehavioral scores significantly decreased in NTE high-dose and middle-dose groups（ P 〈 0. 01）; the infarct volume of NTE groups markedly decreased（ P 〈 0. 01）;the expression of HO-1 mRNA apparently increased（ P 0. 05） in NTE groups; the expression of Heph mRNA significantly increased in NTE middle-dose and high-dose groups（ P 〈 0. 05）; the expression of Nrf2 and Heph protein evidently increased in the NTE middle and high dose groups（ P 〈 0. 05,P 〈 0. 01）; and the expression of HO-1 protein also increased in NTE groups（ P 〈 0. 01）. Conclusions Naotai formula can relieve cerebral ischemia-reperfusion injury. The mechanism might be associated with activating Keap1-Nrf2/ARE signaling pathways,promoting HO-1 generation,advancing the expression of Heph,and then reducing brain iron deposition,to achieve the protection of neurons after cerebral ischemia-reperfusion.