SIRT1 exerts protective effects against paraquat-induced injury in mouse type II alveolar epithelial cells by deacetylating NRF2 in vitro
SIRT1 exerts protective effects against paraquat-induced injury in mouse type II alveolar epithelial cells by deacetylating NRF2 in vitro
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DOI：
10.3892/ijmm.2016.2503
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发表时间：
2016-04-01
影响因子：
5.4
通讯作者：
Lu, Zhong-Qiu
中科院分区：
文献类型：
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作者：
Ding, Ying-Wei;Zhao, Guang-Ju;Lu, Zhong-Qiu
沉默信息调节因子2相关酶1 （SIRT1）是一种蛋白质去乙酰化酶，已知可强烈保护细胞免受氧化应激诱导的损伤。核因子e2相关因子2 (NRF2)-抗氧化反应元件（ARE）抗氧化途径在百草枯（PQ）中毒的抗氧化治疗中起重要调控作用。在本研究中，我们研究SIRT1/NRF2/ARE信号通路是否在PQ诱导的肺损伤中起重要作用。为此，小鼠II型肺泡上皮细胞（aec -II）暴露于不同浓度的PQ。分别用SIRT1过表达载体或靶向SIRT1的shRNA转染细胞诱导SIRT1过表达或沉默。western blot检测SIRT1和NRF2蛋白表达水平。超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性以及谷胱甘肽（GSH）和丙二醛（MDA）水平均采用相应的试剂盒进行测定。ELISA法测定血红素加氧酶-1 （HO-1）活性。流式细胞术检测细胞凋亡情况。用环己亚胺分析NRF2的蛋白稳定性，并测定其在细胞中的乙酰化程度。结果表明：1)SIRT1过表达显著提高NRF2蛋白表达；ii) SIRT1可促进NRF2的转录活性，上调NRF2下游基因SOD、CAT、GSH、HO-1的表达，从而抑制aec - ii的凋亡；iii) SIRT1活性的抑制进一步诱导丙二醛（MDA）的产生，导致氧化损伤增加；iv) SIRT1通过调控NRF2/ARE抗氧化通路的去乙酰化和激活，促进NRF2的稳定性。本研究结果表明SIRT1在PQ中毒致肺损伤中的保护作用与NRF2/ are抗氧化通路的激活有关。
Silent information regulator 2-related enzyme 1 (SIRT1), a protein deacetylase, is known to strongly protect cells against oxidative stress-induced injury. The nuclear factor E2-related factor 2 (NRF2)-antioxidant response element (ARE) antioxidant pathway plays important regulatory roles in the antioxidant therapy of paraquat (PQ) poisoning. In the present study, we investigated whether the SIRT1/NRF2/ARE signaling pathway plays an important role in lung injury induced by PQ. For this purpose, mouse type II alveolar epithelial cells (AECs-II) were exposed to various concentrations of PQ. The overexpression or silencing of SIRT1 was induced by transfecting the cells with a SIRT1 overexpression vector or shRNA targeting SIRT1, respectively. The protein expression levels of SIRT1 and NRF2 were measured by western blot analysis. The superoxide dismutase (SOD) and catalase (CAT) activities, as well as the glutathione (GSH) and malondialdehyde (MDA) levels were measured using respective kits. Heme oxygenase-1 (HO-1) activity was also determined by ELISA. In addition, cell apoptosis was determined by flow cytometry. The protein stability of NRF2 was analyzed using cycloheximide and its acetylation in the cells was also determined. The following findings were obtained: i) SIRT1 overexpression markedly increased NRF2 protein expression; ii) SIRT1 promoted the transcriptional activity of NRF2 and upregulated the expression of the NRF2 downstream genes, SOD, CAT, GSH and HO-1, thus inhibiting the apoptosis of AECs-II; iii) the inhibition of SIRT1 activity further induced the production of malondialdehyde (MDA), which resulted in increased oxidative damage; iv) SIRT1 promoted the stability of NRF2 by regulating the deacetylation and activation of the NRF2/ARE antioxidant pathway. The findings of this study demonstrate that the protective effects of SIRT1 are associated with the activation of the NRF2/ARE antioxidant pathway in lung injury induced by PQ poisoning.