脱嘌呤/脱嘧啶核酸内切酶1在氧化应激介导大鼠肺微血管内皮细胞损伤中的调控作用

doi:10.3877/cma.j.issn.1674-6880.2016.05.005

中华危重症医学杂志(电子版) ›› 2016, Vol. 09 ›› Issue (05) : 309 -314. doi: 10.3877/cma.j.issn.1674-6880.2016.05.005

所属专题：文献；

论著

脱嘌呤/脱嘧啶核酸内切酶1在氧化应激介导大鼠肺微血管内皮细胞损伤中的调控作用

翁杰¹, 侯金珍¹, 汤鲁明¹, 谢慧¹, 陈大庆¹, 孙来芳¹, 应斌宇², 龚裕强²^,^†(

)

^1. 325000　浙江温州，温州医科大学附属第二医院急诊科
^2. 325000　浙江温州，温州医科大学附属第二医院重症医学科

收稿日期:2016-07-17 出版日期:2016-10-01
通信作者: 龚裕强
基金资助:
浙江省医药卫生科技计划项目(2012KYB129); 浙江省自然科学基金资助项目(LY13H150003)

Modulation of apurinic/apyrimidinic endonuclease-1 on oxidative stress induced pulmonary microvascular endothelial cells injury in rats

Jie Weng¹, Jinzhen Hou¹, Luming Tang¹, Hui Xie¹, Daqing Chen¹, Laifang Sun¹, Binyu Ying², Yuqiang Gong²^,^†()

^1. Departmnet of Emergency, Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China
^2. Department of Intensive Medicine, Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China

Received:2016-07-17 Published:2016-10-01
Corresponding author: Yuqiang Gong
About author:
Corresponding author: Gong Yuqiang, Email: gyq12120@163.com

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引用本文：

翁杰, 侯金珍, 汤鲁明, 谢慧, 陈大庆, 孙来芳, 应斌宇, 龚裕强. 脱嘌呤/脱嘧啶核酸内切酶1在氧化应激介导大鼠肺微血管内皮细胞损伤中的调控作用[J]. 中华危重症医学杂志(电子版), 2016, 09(05): 309-314.

Jie Weng, Jinzhen Hou, Luming Tang, Hui Xie, Daqing Chen, Laifang Sun, Binyu Ying, Yuqiang Gong. Modulation of apurinic/apyrimidinic endonuclease-1 on oxidative stress induced pulmonary microvascular endothelial cells injury in rats[J]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2016, 09(05): 309-314.

目的

探讨脱嘌呤/脱嘧啶核酸内切酶1（APE1）在过氧化氢（H₂O₂）介导的大鼠肺微血管内皮细胞（PMVECs）氧化应激中的调控作用。

方法

分离培养大鼠PMVECs并分成4组：对照组（未加H₂O₂）及各剂量H₂O₂组（终浓度分别为50、100、200 μmol/L的H₂O₂）。在H₂O₂刺激细胞24 h后检测细胞增殖活性、细胞内活性氧水平及APE1蛋白表达，同时检测100 μmol/ml的H₂O₂刺激细胞24、48、72 h后APE1蛋白表达并比较。另外利用慢病毒载体转染构建APE1过表达PMVECs，将其分成对照组（未加H₂O₂），H₂O₂组（终浓度为100 μmol/L的H₂O₂），H₂O₂+空载体转染组（100 μmol/L的H₂O₂刺激慢病毒空载体转染的细胞）及H₂O₂+APE1转染组（终浓度为100 μmol/L的H₂O₂刺激APE1过表达细胞），并在24 h检测细胞活性氧水平及细胞增值情况。

结果

不同剂量组H₂O₂刺激细胞24 h后，四组间细胞增殖活性、活性氧的水平和APE1蛋白表达相对值差异均有统计学意义（F = 80.238、445.608、547.566，P均< 0.001）。在100 μmol/L的H₂O₂刺激细胞0、24、48和72 h后，APE1蛋白表达相对值的比较有统计学意义（F = 422.324，P < 0.001），且随时间增加APE1表达也逐渐下降[（0.781 ± 0.043）、（0.611 ± 0.026）、（0.407 ± 0.014）、（0.272 ± 0.013），P均< 0.05]。在转染慢病毒载体后，H₂O₂组活性氧水平明显高于对照组[（86.4 ± 1.2）vs.（56.4 ± 0.9），P < 0.05]；而H₂O₂ + APE1转染组细胞活性氧水平较H₂O₂组显著下降[（66.8 ± 1.0）vs.（86.4 ± 1.2），P < 0.05]，细胞增殖活性明显增加[（0.209 ± 0.001）vs.（0.153 ± 0.001），P < 0.05]。

结论

H₂O₂刺激可导致APE1蛋白表达下降，而上调APE1表达可增加细胞增殖活性，并抑制活性氧的产生。因此，APE1可能具有减轻H₂O₂介导的大鼠PMVECs氧化应激反应的作用。

关键词: 过氧化氢, 氧化性应激, 活性氧类, 脱嘌呤/脱嘧啶核酸内切酶1, 肺微血管内皮细胞

Objective

To investigate the modulation of apurinic/apyrimidinic endonuclease-1 (APE1) on oxidative stress reaction of pulmonary microvascular endothelial cells (PMVECs) in rats induced by H₂O₂.

Methods

PMVECs were cultured and randomly divided into 4 groups: control group (no H₂O₂), H₂O₂ groups (the final concentrations of H₂O₂ in cultured cells were 50, 100, 200 μmol/L, respectively). The levels of proliferative activity, reactive oxygen species (ROS) and APE1 were detected at 24 h after the different concentrations of H₂O₂ stimulation. Meanwhile, APE1 protein expression was checked at 24, 48, 72 h after 100 μmol/mL H₂O₂ stimulation. Later, the PMVECs were transfected with lentiviruses and randomly divided into 4 groups: control group (no H₂O₂), H₂O₂ group (100 μmol/L H₂O₂), H₂O₂ + empty vector group (lentivirus vector transfected empty cells with 100 μmol/L H₂O₂) and H₂O₂+ APE1 group (APE1 gene transfected lentivirus cells with 100 μmol/L H₂O₂). The levels of proliferative activity and ROS after 24 h culture were detected and compared.

Results

Compared with the control group, the levels of proliferative activity, ROS and APE1 in the other three groups increased obviously after being induced by different doses of H₂O₂ for 24 h (F = 80.238, 445.608, 547.566; all P < 0.05). In the 100 μmol/L H₂O₂ group, APE1 expression decreased obviously after 0, 24, 48 and 72 h stimulation [(0.781 ± 0.043), (0.611 ± 0.026), (0.407 ± 0.014), (0.272 ± 0.013); all P < 0.05]. After transfection, the level of ROS in the H₂O₂ group was much higher than that in the control group [(86.4 ± 1.2) vs. (56.4 ± 0.9), P < 0.05]. The level of ROS decreased [(66.8 ± 1.0) vs. (86.4 ± 1.2), P < 0.05] and the level of proliferative activity increased [(0.209 ± 0.001) vs. (0.153 ± 0.001), P < 0.05] obviously in the H₂O₂ + APE1 group, comparing with the H₂O₂ group.

Conclusions

H₂O₂ stimulation can downregulate the APE1 protein expression; conversely, upregulating the APE1 expression increases the level of cell proliferative activity, also inhibit the level of ROS. Thus, APE1 can relieve the oxidative stress reaction of PMVECs in rats induced by H₂O₂.

Key words: Hydrogen peroxide, Oxidative stress, Reactive oxygen species, Apurinic/apyrimidinic endonuclease-1, Pulmonary microvascular endothelial cells

表1 不同浓度H₂O₂刺激大鼠PMVECs24 h后对细胞增殖活性、细胞内活性氧以及APE1表达的影响（±s）

组别	细胞数（个/孔）	H₂O₂终浓度(μmol/L)	A₄₉₀值	二氯荧光黄相对荧光强度	APE1蛋白表达相对值
对照组	1 × 10⁵	0	0.255 ± 0.001	58.0 ± 1.0	0.757 ± 0.044
低浓度组	1 × 10⁵	50	0.211 ± 0.002	68.1 ± 1.4	0.694 ± 0.013
中浓度组	1 × 10⁵	100	0.158 ± 0.002^a	87.5 ± 1.9^a	0.509 ± 0.018^a
高浓度组	1 × 10⁵	200	0.099 ± 0.001^a	101.8 ± 1.6^a	0.309 ± 0.015^a
F值	?	?	80.238	445.608	547.566
P值	?	?	< 0.001	< 0.001	< 0.001

表1 不同浓度H₂O₂刺激大鼠PMVECs24 h后对细胞增殖活性、细胞内活性氧以及APE1表达的影响（±s）

组别	细胞数（个/孔）	H₂O₂终浓度(μmol/L)	A₄₉₀值	二氯荧光黄相对荧光强度	APE1蛋白表达相对值
对照组	1 × 10⁵	0	0.255 ± 0.001	58.0 ± 1.0	0.757 ± 0.044
低浓度组	1 × 10⁵	50	0.211 ± 0.002	68.1 ± 1.4	0.694 ± 0.013
中浓度组	1 × 10⁵	100	0.158 ± 0.002^a	87.5 ± 1.9^a	0.509 ± 0.018^a
高浓度组	1 × 10⁵	200	0.099 ± 0.001^a	101.8 ± 1.6^a	0.309 ± 0.015^a
F值	?	?	80.238	445.608	547.566
P值	?	?	< 0.001	< 0.001	< 0.001

表2 APE1过表达后H2O2对各组PMVECs细胞增殖活性、细胞内活性氧的影响（±s）

分组	细胞数（个/孔）	A₄₉₀值	二氯荧光黄相对荧光强
对照组	1 × 10⁵	0.256 ± 0.007	56.4 ± 0.9
H₂O₂组	1 × 10⁵	0.153 ± 0.001^a	86.4 ± 1.2^a
H₂O₂ +空载体转染组	1 × 10⁵	0.154 ± 0.002^a	83.2 ± 1.0^a
H₂O₂ + APE1转染组	1 × 10⁵	0.209 ± 0.001^ab	66.8 ± 1.0^ab

表2 APE1过表达后H2O2对各组PMVECs细胞增殖活性、细胞内活性氧的影响（±s）

分组	细胞数（个/孔）	A₄₉₀值	二氯荧光黄相对荧光强
对照组	1 × 10⁵	0.256 ± 0.007	56.4 ± 0.9
H₂O₂组	1 × 10⁵	0.153 ± 0.001^a	86.4 ± 1.2^a
H₂O₂ +空载体转染组	1 × 10⁵	0.154 ± 0.002^a	83.2 ± 1.0^a
H₂O₂ + APE1转染组	1 × 10⁵	0.209 ± 0.001^ab	66.8 ± 1.0^ab

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