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Abstract：
BACKGROUND: It is now understood that ferroptosis plays a significant role in the progression of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke extract (CSE). However, the mechanisms underlying this relationship remain largely unclear.
METHODS: In this study, we established a COPD mouse model through exposure to cigarette smoke particulates, followed by H&E staining, analysis of bronchoalveolar lavage fluid, and immunohistochemistry assay. A549 cells were exposed to increasing concentrations of CSE, with the addition of the ferroptosis activator erastin or the inhibitor Fer-1. Cell viability, LDH (lactate dehydrogenase) release, inflammatory cytokines, total ROS (reactive oxygen species), and lipid ROS were measured using the corresponding assay kits. The acetylation level of GNPAT was determined through immunoprecipitation. We assessed the expression levels of molecules involved in plasmalogen biosynthesis (FAR1, AGPS, and GNPAT), GPX4, and SIRT4 using quantitative real-time PCR, western blot analysis, and immunofluorescence staining.
RESULTS: CSE-induced lung tissue damage was initially observed, accompanied by oxidative stress, ferroptosis, and increased plasmalogen biosynthesis molecules (FAR1, AGPS, and GNPAT). CSE also induced ferroptosis in A549 cells, resulting in reduced cell viability, GSH, and GPX4 levels, along with increased LDH, ROS, MDA (malondialdehyde) levels, oxidized lipids, and elevated FAR1, AGPS, and GNPAT expression. Knockdown of GNPAT mitigated CSE-induced ferroptosis. Furthermore, we found that CSE regulated the acetylation and protein levels of GNPAT by modulating SIRT4 expression. Importantly, the overexpression of GNPAT countered the inhibitory effects of SIRT4 on ferroptosis.
CONCLUSIONS: Our study revealed GNPAT could be deacetylated by SIRT4, providing novel insights into the mechanisms underlying the relationship between CSE-induced ferroptosis and COPD.
METHODS: In this study, we established a COPD mouse model through exposure to cigarette smoke particulates, followed by H&E staining, analysis of bronchoalveolar lavage fluid, and immunohistochemistry assay. A549 cells were exposed to increasing concentrations of CSE, with the addition of the ferroptosis activator erastin or the inhibitor Fer-1. Cell viability, LDH (lactate dehydrogenase) release, inflammatory cytokines, total ROS (reactive oxygen species), and lipid ROS were measured using the corresponding assay kits. The acetylation level of GNPAT was determined through immunoprecipitation. We assessed the expression levels of molecules involved in plasmalogen biosynthesis (FAR1, AGPS, and GNPAT), GPX4, and SIRT4 using quantitative real-time PCR, western blot analysis, and immunofluorescence staining.
RESULTS: CSE-induced lung tissue damage was initially observed, accompanied by oxidative stress, ferroptosis, and increased plasmalogen biosynthesis molecules (FAR1, AGPS, and GNPAT). CSE also induced ferroptosis in A549 cells, resulting in reduced cell viability, GSH, and GPX4 levels, along with increased LDH, ROS, MDA (malondialdehyde) levels, oxidized lipids, and elevated FAR1, AGPS, and GNPAT expression. Knockdown of GNPAT mitigated CSE-induced ferroptosis. Furthermore, we found that CSE regulated the acetylation and protein levels of GNPAT by modulating SIRT4 expression. Importantly, the overexpression of GNPAT countered the inhibitory effects of SIRT4 on ferroptosis.
CONCLUSIONS: Our study revealed GNPAT could be deacetylated by SIRT4, providing novel insights into the mechanisms underlying the relationship between CSE-induced ferroptosis and COPD.
摘要：
背景：现已了解，铁凋亡在香烟烟雾提取物（CSE）诱导的慢性阻塞性肺疾病（COPD）的进展中起着重要作用。然而,这种关系的潜在机制仍不清楚.
方法：在本研究中，我们通过暴露于香烟烟雾颗粒建立了COPD小鼠模型，其次是H&E染色，支气管肺泡灌洗液分析，和免疫组织化学检测。A549细胞暴露于浓度增加的CSE，添加铁凋亡激活剂擦除素或抑制剂Fer-1。细胞活力，LDH（乳酸脱氢酶）释放，炎性细胞因子，总ROS（活性氧），和脂质ROS使用相应的测定试剂盒进行测量。通过免疫沉淀测定GNPAT的乙酰化水平。我们评估了参与缩醛磷脂生物合成的分子的表达水平（FAR1，AGPS，和GNPAT)，GPX4和SIRT4使用定量实时PCR，蛋白质印迹分析,和免疫荧光染色。
结果：最初观察到CSE诱导的肺组织损伤，伴随着氧化应激，铁性凋亡,和增加的缩醛磷脂生物合成分子(FAR1，AGPS，和GNPAT）。CSE还在A549细胞中诱导铁凋亡，导致细胞活力降低，GSH,和GPX4级别，随着LDH的增加，ROS,MDA（丙二醛）水平，氧化脂质，和升高的FAR1，AGPS，和GNPAT表达式。GNPAT的敲低减轻了CSE诱导的铁细胞凋亡。此外，我们发现CSE通过调节SIRT4的表达来调节GNPAT的乙酰化和蛋白质水平。重要的是,GNPAT的过表达抵消了SIRT4对铁凋亡的抑制作用。
结论：我们的研究显示GNPAT可以被SIRT4去乙酰化，为CSE诱导的铁死亡与COPD之间关系的潜在机制提供了新的见解。
方法：在本研究中，我们通过暴露于香烟烟雾颗粒建立了COPD小鼠模型，其次是H&E染色，支气管肺泡灌洗液分析，和免疫组织化学检测。A549细胞暴露于浓度增加的CSE，添加铁凋亡激活剂擦除素或抑制剂Fer-1。细胞活力，LDH（乳酸脱氢酶）释放，炎性细胞因子，总ROS（活性氧），和脂质ROS使用相应的测定试剂盒进行测量。通过免疫沉淀测定GNPAT的乙酰化水平。我们评估了参与缩醛磷脂生物合成的分子的表达水平（FAR1，AGPS，和GNPAT)，GPX4和SIRT4使用定量实时PCR，蛋白质印迹分析,和免疫荧光染色。
结果：最初观察到CSE诱导的肺组织损伤，伴随着氧化应激，铁性凋亡,和增加的缩醛磷脂生物合成分子(FAR1，AGPS，和GNPAT）。CSE还在A549细胞中诱导铁凋亡，导致细胞活力降低，GSH,和GPX4级别，随着LDH的增加，ROS,MDA（丙二醛）水平，氧化脂质，和升高的FAR1，AGPS，和GNPAT表达式。GNPAT的敲低减轻了CSE诱导的铁细胞凋亡。此外，我们发现CSE通过调节SIRT4的表达来调节GNPAT的乙酰化和蛋白质水平。重要的是,GNPAT的过表达抵消了SIRT4对铁凋亡的抑制作用。
结论：我们的研究显示GNPAT可以被SIRT4去乙酰化，为CSE诱导的铁死亡与COPD之间关系的潜在机制提供了新的见解。
