基于Nrf2/ARE信号通路研究绞股蓝皂苷ⅩⅦ抗脑缺血/再灌注机制

Mechanism of gypenosideⅩⅦagainst cerebral ischemia/reperfusion injury based on nuclear factor erythroid 2-related factor 2/antioxidant responsive element signaling pathway

目的:探讨绞股蓝皂苷ⅩⅦ调控核因子E2相关因子2/抗氧化反应元件(Nrf2/ARE)信号通路抗脑缺血/再灌注(I/R)的机制。方法:将40只SPF级SD大鼠随机分为假手术组、I/R模型组及25、50和100 mg/kg绞股蓝皂苷ⅩⅦ组,每组8只。绞股蓝皂苷ⅩⅦ组分别给予绞股蓝皂苷ⅩⅦ 25、50、100 mg/kg灌胃(灌胃量0.01 mL/g),连续给药14 d;其余两组给予相同剂量生理盐水灌胃。然后采用改良线栓法制备大鼠脑I/R模型;假手术组大鼠采用相同方法手术,但不产生实质性栓塞。再灌注后24 h,评估各组大鼠神经功能缺损评分;采集大鼠腹主动脉全血检测血清活性氧(ROS)、血红素加氧酶-1(HO-1)、γ-谷氨酰半胱氨酸合成酶(γ-GCS)、超氧化物歧化酶(SOD)、醌NADH氧化还原酶1(NQO1)、丙二醛(MDA)水平;随后取完整大脑组织并分离大脑皮质脑梗死周围半暗带组织,观察大鼠脑组织大体情况及脑梗死体积,光镜下观察脑组织病理学变化,采用实时荧光定量反转录-聚合酶链反应(RT-qPCR)检测脑缺血半暗带组织Nrf2和Keap1的mRNA表达,采用蛋白质免疫印迹试验(Western blotting)检测脑缺血半暗带组织Nrf2和Keap1的蛋白表达。结果:再灌注后24 h,与假手术组比较,I/R模型组大鼠神经功能缺损评分明显升高,脑组织梗死体积明显增大,血清NQO1、SOD和γ-GCS水平均明显降低、血清MDA、HO-1和ROS水平均明显升高,脑缺血半暗带组织Nrf2和Keap1的mRNA及蛋白表达均明显升高。与I/R模型组比较,50 mg/kg与100 mg/kg绞股蓝皂苷ⅩⅦ能明显降低脑I/R大鼠的神经功能缺损评分(分:1.50±0.53、1.37±0.52比2.75±0.46),缩小脑组织梗死体积〔(19.8±5.1)%、(21.4±6.4)%比(42.3±5.8)%〕,明显提高血清NQO1、SOD、HO-1和γ-GCS水平〔NQO1(ng/L):186.05±10.38、220.75±16.22比131.36±5.95,SOD(kU/L):63.23±5.30、72.70±8.62比36.75±6.55,HO-1(ng/L):60.57±7.93、60.35±4.72比42.72±4.95,γ-GCS(kU/L):8.81±0.53、8.72±0.69比6.80±0.56〕,明显降低血清MDA和ROS水平〔MDA(μmol/L):5.94±0.66、5.61±0.53比10.88±1.34,ROS(kU/L):69.11±4.23、67.12±4.52比104.43±7.54〕,同时可明显提高脑缺血半暗带组织Nrf2和Keap1的mRNA及蛋白表达〔Nrf2 mRNA(2^(-△△Ct)):1.90±0.13、2.13±0.18比1.48±0.11,Keap1 mRNA(2^(-△△Ct)):1.78±0.11、1.85±0.10比1.43±0.10,Nrf2/β-actin:0.73±0.04、0.79±0.03比0.60±0.03,Keap1/β-actin:0.71±0.01、0.76±0.03比0.61±0.01〕,比较差异均有统计学意义(均 P<0.01);25 mg/kg绞股蓝皂苷ⅩⅦ无明显作用。 结论:绞股蓝皂苷ⅩⅦ (50 mg/kg和100 mg/kg)可能通过Nrf2/ARE信号通路调节NQO1、SOD、HO-1、γ-GCS、ROS及MDA起到抗脑I/R损伤的作用。

Objective To explore the mechanism of gypenosideⅩⅦagainst cerebral ischemia/reperfusion(I/R)through nuclear factor erythroid 2-related factor 2/antioxidant responsive element(Nrf2/ARE)signaling pathway.Methods Forty SPF Sprague Dawley(SD)rats were randomly divided into sham operated group,I/R model group,25,50 and 100 mg/kg gypenosideⅩⅦgroups(n=8).GypenosideⅩⅦgroups were administered 25,50 or 100 mg/kg(0.01 mL/g)gypenosideⅩⅦby intragastric administration for 14 days;the other two groups received the same dose of saline.Rat cerebral I/R model was established by modified line bolt method;rats in the sham operated group underwent the same procedure without producing substantial embolization.After 24 hours of reperfusion,the neurological deficit scores of the rats in each group were assessed.Rat abdominal aortic whole blood was collected and the serum reactive oxygen species(ROS),heme oxygenase-1(HO-1),γ-glutamylcysteine synthase(γ-GCS),superoxide dismutase(SOD),quinone NADH oxidoreductase 1(NQO1),and malondialdehyde(MDA)were detected.Then whole brain tissue was harvested and penumbra tissue was isolated from cerebral cortex,the general condition of rat brain tissue and the volume of cerebral infarction were evaluated,the histopathological changes in the brain were observed under light microscopy,the mRNA expressions of Nrf2 and Keap1 were measured by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR),the protein expressions of Nrf2 and Keap1 were determined by Western blotting.Results After 24 hours of reperfusion,compared with the sham operated group,the score of neurological deficit and infarct volume were significantly increased,the NQO1,SOD andγ-GCS levels in serum were significantly decreased,MDA,HO-1 and ROS levels in serum were significantly increased,the Nrf2 and Keap1 mRNA and protein expressions in the ischemic penumbra were significantly increased in rats from I/R model group.Compared with the I/R model group,the neurological deficit scores(1.50±0.53,1.37±0.52 vs.2.75±0.46)and brain infarct volume[(19.8±5.1)%,(21.4±6.4)%vs.(42.3±5.8)%]were significantly reduced,serum NQO1,SOD,HO-1 andγ-GCS were significantly increased[NQO1(ng/L):186.05±10.38,220.75±16.22 vs.131.36±5.95,SOD(kU/L):63.23±5.30,72.70±8.62 vs.36.75±6.55,HO-1(ng/L):60.57±7.93,60.35±4.72 vs.42.72±4.95,γ-GCS(kU/L):8.81±0.53,8.72±0.69 vs.6.80±0.56],serum MDA and ROS levels were significantly reduced[MDA(μmol/L):5.94±0.66,5.61±0.53 vs.10.88±1.34,ROS(kU/L):69.11±4.23,67.12±4.52 vs.104.43±7.54],the mRNA and protein expressions of Nrf2 and Keap1 in the ischemic penumbra were significantly increased in rats from 50 mg/kg and 100 mg/kg gypenosideⅩⅦgroups[Nrf2 mRNA(2^(-△△Ct)):1.90±0.13,2.13±0.18 vs.1.48±0.11,Keap1 mRNA(2^(-△△Ct)):1.78±0.11,1.85±0.10 vs.1.43±0.10,Nrf2/β-actin:0.73±0.04,0.79±0.03 vs.0.60±0.03,Keap1/β-actin:0.71±0.01,0.76±0.03 vs.0.61±0.01],all the comparative differences were statistically significant(all P<0.01);25 mg/kg gypenosideⅩⅦhad no significant effect.Conclusion GypenosideⅩⅦ(50 mg/kg and 100 mg/kg)may play a role in anti-cerebral I/R injury by regulating NQO1,SOD,HO-1,γ-GCS,ROS and MDA through Nrf2/ARE signaling pathway.