国际麻醉学与复苏杂志   2017, Issue (10): 0-0
    
核因子E2相关因子/血红素加氧合酶1通路在硫化氢减轻心搏骤停大鼠血脑屏障损伤中的作用
蔡慎权, 李茜, 仲浩, 范婧婧, 操良斌, 段满林, 徐建国1()
1.南京军区南京总医院
Role of nuclear factor E2 related factor/heme oxygenase-1 pathway in hydrogen sulfide-induced reduction of blood brain barrier damage in rats with cardiac arrest
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摘要:

目的 探讨核因子E2相关因子(nuclear factor E2 related factor, Nrf2)/血红素加氧合酶1(heme oxygenase-1, HO-1)通路是否参与硫化氢(hydrogen sulfide, H2S)减轻心搏骤停大鼠血脑屏障(blood-brain barrier, BBB)损伤及参与该效应的可能机制。 方法 采用经食管电刺激法建立大鼠心搏骤停心肺复苏(cardiopulmonary resuscitation, CPR)模型,缺血时间4 min。140只成年雄性SD大鼠按随机数字表法分为5组:假手术组(Sham组,20只)、心搏骤停模型组(CPR组,30只)、硫氢化钠(sodium hydrogen sulfide, NaHS)组(30只)、NaHS+溶剂[二甲基亚砜(dimethyl sulfoxide, DMSO)]对照组(NaHS+DMSO组,30只)、NaHS+Nrf2抑制剂维甲酸(retinoic acid, RA)组(NaHS+RA组,30只)。Sham组仅进行麻醉,经口气管插管,经股动静脉置管操作,其他4组均行电刺激诱发心搏骤停;NaHS组与NaHS+RA组于自主循环恢复即刻经股静脉注射NaHS(0.5 mg/kg),此后持续泵入NaHS(1.5 mg·kg-1·h-1)3 h;NaHS+RA组于实验前1周每天及复苏即刻腹腔注射RA 10 mg/kg;NaHS+DMSO组于实验前1周每天及复苏即刻腹腔给予等容量溶剂DMSO,CPR组于复苏即刻静脉给予等量生理盐水。复苏成功后连续观察7 d,记录大鼠生存情况;于复苏后1、3、7 d评价神经功能;于复苏后24 h检测脑含水量、BBB通透性,Western blot法检测Nrf2、HO-1、BBB紧密连接蛋白occludin表达变化,免疫组织化学染色检测基质金属蛋白酶-9(matrix metalloproteinase-9, MMP-9)表达变化。 结果 大鼠的生存率、神经功能评分、Nrf2和HO?蛳1表达及occludin表达量比较,NaHS组均高于CPR组(P<0.05),而NaHS+RA组低于NaHS组(P<0.05);脑含水量和伊文思蓝(evans blue, EB)含量及MMP-9阳性细胞数比较,NaHS组显著低于CPR组(P<0.05),而NaHS+RA组较NaHS组增高(P<0.05)。NaHS组与NaHS+DMSO组各项结果比较,差异无统计学意义(P>0.05)。 结论 H2S可减轻心搏骤停大鼠BBB损伤,可能与H2S激活Nrf2/HO?蛳1通路、抑制MMP-9表达、减少Occludin蛋白丢失有关。

关键词: 核因子E2相关因子/血红素加氧合酶1通路; 血脑屏障; 硫化氢; 心搏骤停; 心
Abstract:

Objective To investigate whether the nuclear factor E2 related factor/heme synthase-1(Nrf2/HO-1) pathway is involved in the mechanism of hydrogen sulfide-induced reduction of blood brain barrier(BBB) injury in rats with cardiac arrest and the possible mechanism involved in the effect. Methods Cardiac arrest was induced with transoesophageal cardiac pacing followed by cardiopulmonary resuscitation to establish the cardiac arrest model and the time of ischemia was 4 min. 140 adult male SD rats were randomly divided into five groups: sham operation group(Sham group, n=20), cardiac arrest model group (CPR group, n=30), sodium hydrogen sulfide(NaHS) group(n=30), NaHS+solvent group[NaHS+dimethyl sulfoxide(DMSO) group, n=30], NaHS+Nrf2 inhibitor retinoic acid(RA) group(NaHS+RA group, n=30). In Sham group, only anesthesia was performed, and the trachea was inserted through the mouth, and the catheters were performed via femoral artery and vein. The other four groups were all induced cardiac arrest by electrical stimulation. NaHS group and NaHS+RA group, rats received NaHS (0.5 mg/kg) at the start of CPR intravenously, followed by a continuous infusion of NaHS(1.5 mg·kg-1·h-1) for 3 h. NaHS+RA group, RA was given i.p. daily at 10 mg/kg for 1 week before experiment and immediately after recovery. NaHS+DMSO group was given the same amount of solvent daily for 1 week before experiment and immediately after recovery. CPR group was given the same amount of normal saline at the start of CPR intravenously. After recovery, 7 days were observed continuously and the survival condition was recorded, and neurological function were evaluated at 1, 3 d, and 7 d after recovery, brain water content, BBB permeability and Nrf2, HO-1, BBB tight junction protein occludin expression was detected at 24 h after resuscitation, and the expression of matrix metalloproteinase-9(MMP-9) was detected by immunohistochemical staining. Results The survival rates, neurologic deficit scores(NDS), Nrf2 and HO-1 expression, and occludin expression in NaHS group were higher than CPR group(P<0.05), while the NaHS+RA group was lower than the NaHS group(P<0.05). The brain water content and evans blue(EB) content, and MMP-9 positive cells number in NaHS group were significantly lower than that in CPR group(P<0.05), and the content of brain water content and EB content, and MMP-9 positive cells number in NaHS+RA group were higher than that in NaHS group(P<0.05). There was no significant difference between the NaHS group and the NaHS+DMSO group(P>0.05). Conclusions Hydrogen sulfide could reduce the BBB damage in rats with cardiac arrest, which may be related to the activation of Nrf2/HO-1 pathway, inhibiting the expression of MMP-9 and decreasing the loss of occludin protein.

Key words: Nuclear factor E2 related factor/heme oxygenase-1 pathway; Blood-brain barrier; Hydrogen sulfide; Cardiac arrest; Cardiopulmonary resuscitation