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

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

Abstract

Abstract:

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

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.

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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

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

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

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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