冷缺血期氢预处理对大鼠肺微血管内皮细胞缺氧复氧时Nrf2活性的影响  

Effect of hydrogen preconditioning during cold ischemia phase on activity of Nrf2 in rat pulmonary microvascular endothelial cells subjected to hypoxia-reoxygenation

作　　者：李喆 康继宇 张广超[1] 蔡海龙 周华成[1] Li Zhe;Kang Jiyu;Zhang Guangchao;Cai Hailong;Zhou Huacheng(Department of Anesthesiology,The Fourth Affiliated Hospital of Harbin Medical University,Harbin 150001,China)

机构地区：[1]哈尔滨医科大学附属第四医院麻醉科,150001

出　　处：《中华麻醉学杂志》2019年第6期680-683,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(81570088).

摘　　要：目的评价冷缺血期氢预处理对大鼠肺微血管内皮细胞(PMVECs)缺氧复氧时核因子E2相关因子2(Nrf2)活性的影响.方法雄性清洁级SD大鼠,2~3周龄,使用组织块贴壁法,提取PMVECs,采用随机数字表法分为4组(n=25):对照组(C组)、缺氧复氧组(H/R组)、氧气组(O组)和氢气组(H组).经95%氧气-5%二氧化碳预平衡的低钾右旋糖酐液(LPD)4℃孵育4 h,模拟冷缺血期;LPD替换经95%氧气-5%二氧化碳预平衡的LPD,室温孵育1 h,模拟肺移植期;37℃M199完全培养液迅速替换LPD,在40%氧气-5%二氧化碳-55%氮气混合气体中孵育,模拟再灌注期.O组和H组于冷缺血期密闭培养箱分别通入40%氧气-60%氮气、3%氢气-40%氧气-57%氮气,每20 min置换1次.4 h后收集细胞培养液,采用ELISA法检测IL-6、IL-10、TNF-α浓度,采用硫代巴比妥酸法测定MDA浓度;提取细胞质和细胞核蛋白,采用Western blot方法测定Nrf2表达水平;分别采用流式细胞术和TUNEL法检测细胞凋亡率.结果与C组比较,H/R组IL-6、TNF-α和MDA水平升高,IL-10水平降低,细胞凋亡率升高,细胞核Nrf2表达上调(P<0.05);与H/R组比较,O组和H组IL-6、TNF-α和MDA水平降低,IL-10水平升高,细胞凋亡率降低,细胞质Nrf2表达下调,H组细胞核Nrf2表达上调(P<0.05),O组细胞核Nrf2表达差异无统计学意义(P>0.05);与O组比较,H组IL-6、TNF-α和MDA水平降低,IL-10水平升高,细胞凋亡率降低,细胞核Nrf2表达上调,细胞质Nrf2表达下调(P<0.05).结论冷缺血期氢预处理减轻大鼠PMVECs缺氧复氧损伤的机制与激活Nrf2,从而抑制氧化应激有关.Objective To evaluate the effect of hydrogen preconditioning during cold ischemia phase on the activity of nuclear factor erythroid 2-related factor 2 ( Nrf2) in rat pulmonary microvascular en-dothelial cells ( PMVECs) subjected to hypoxia-reoxygenation ( H/R). Methods PMVECs were isolated from clean-grade male Sprague-Dawley rats, aged 2-3 weeks, using the tissue block adherence method and divided into 4 groups ( n=25 each) using a random number table method: control group ( group C), H/R group, oxygen group ( O group) and hydrogen group ( H group). Cells were incubated for 4 h with 4℃ low potassium dextransolution ( LPD) pre-equilibrated with 95% oxygen and 5% carbondioxide to simulate the cold ischemia phase. LPD pre-balanced with 95% oxygen and 5% carbon dioxide was replaced with LPD, and then cells were incubated for 1 h at room temperature to simulate the lung transplantation period. LPD was rapidly replaced with 37℃ M199 complete culture solution, and cells were incubated in the mixture of 40% oxygen-5% carbondioxide-55% nitrogen to simulate the reperfusion period. In O and H groups, the cells were exposed to 40% oxygen-60% nitrogen and 3% hydrogen-40% oxygen-57% nitrogen during the cold ischemia period, respectively, and the gas mixture was replaced every 20 min. The cell culture fluid was collected 4 h later for determination of interleukin ( IL )-6, IL-10 and tumor necrosis factor-alpha ( TNF-α) concentrations ( by enzyme-linked immunosorbent assay) and malondialdehyde ( MDA) concen-trations ( by thiobarbituric acid method). The cytoplasm and nucleoproteins were extracted for measurement of Nrf2 expression ( by Western blot) and cell apoptosis ( by flow cytometry and TUNEL assay). The cell apoptosis rate was calculated. Results Compared with C group, the IL-6, TNF-α and MDA levels were significantly increased, the IL-10 level was decreased, the apoptosis rate was increased, and the expres-sion of Nrf2 in nucleus was up-regulated in H/R group ( P>0. 05). Compared with H/R group, the IL-6, TNF-α an

关 键 词：氢 冷缺血 微血管 内皮细胞 肺 NF-E2相关因子2 

分 类 号：R-332[医药卫生]