活性氧通过调控过氧化物酶体增殖物激活受体-γ的表达影响泡沫巨噬细胞的形成^*

Active Oxygen Influences Foam Macrophage Formation by Regulating Peroxisome Proliferator-Activated Receptor-γ Expression

摘要

目的 探讨活性氧（reactive oxygen species, ROS）通过调控过氧化物酶体增殖物激活受体-γ（peroxisome proliferator-activated receptor-γ, PPARγ）的表达对泡沫巨噬细胞形成的影响。方法 2018年6月–2021年12月，本研究从30名健康捐赠者处获取了人体血液样本并提取外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）。将PBMCs培育成人巨噬细胞（HMDMs）后采用结核分枝杆菌H37Ra菌株感染，构建人巨噬细胞体外感染模型。采用N-乙酰半胱氨酸（N-acetylcysteine, NAC）或大黄素调节ROS生成，采用PPARγ激动剂〔罗格列酮（BRL49653）〕或PPARγ拮抗剂〔2-氯-5-硝基苯甲酰胺（GW9662）〕调节PPARγ活性。用2',7'-二氯二氢荧光素二乙酸酯（DCFH2-DA）和油红O染色检测H37Ra感染对巨噬细胞内ROS的产生和脂质形成的影响，以及ROS的生成对泡沫巨噬细胞形成的影响。通过qRT-PCR和Western blot检测ROS的生成对巨噬细胞内PPARγ表达的影响，通过调控PPARγ活性探讨ROS调控泡沫巨噬细胞的形成是否受PPARγ活性的影响。结果 油红O染色和DCFH2-DA检测结果表明，与对照组相比，H37Ra感染能促进HMDMs ROS的产生和诱导泡沫细胞的形成，差异有统计学意义（P<0.01）。采用NAC或大黄素处理HMDMs 1 h，随后感染H37Ra，结果表明，NAC处理降低了细胞内ROS水平且脂质含量减少，大黄素则增加了细胞内ROS水平且脂质含量增加，差异有统计学意义（P<0.01）。Western blot检测结果表明H37Ra感染抑制了PPARγ的产生，NAC促进PPARγ的表达，大黄素抑制了PPARγ的表达，差异有统计学意义（P<0.05）。采用BRL49653或GW9662预处理HMDMs 1 h，随后感染H37Ra，结果表明，GW9662能促进细胞脂质堆积，差异有统计学意义（P<0.01）。结论 ROS可通过下调PPARγ的表达来促进泡沫巨噬细胞的形成。

Abstract

Objective To investigate the effect of reactive oxygen species (ROS) on foam macrophage formation by regulating the expression of peroxisome proliferator-activated receptor-γ (PPARγ). Methods From June 2018 to December 2021, human blood samples were obtained from 30 healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated. The PBMCs were differentiated into human monocyte-derived macrophages (HMDMs) and subsequently infected with Mycobacterium tuberculosis H37Ra strain to establish an in vitro human macrophage infection model. ROS production was modulated using N-acetylcysteine (NAC) and emodin, while PPARγ activity was regulated using the PPARγ agonist (BRL49653) or antagonist (GW9662). The effects of H37Ra infection on intracellular ROS production and lipid formation in macrophages, as well as the impact of ROS generation on foam macrophage formation, were assessed using 2', 7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) and Oil Red O staining. The influence of ROS production on PPARγ expression in macrophages was examined by qRT-PCR and Western blot. Furthermore, the involvement of PPARγ activity in ROS-mediated regulation of foam macrophage formation was investigated by modulating PPARγ activity. Results The results of Oil Red O staining and DCFH2-DA detection showed that, compared with the control group, H37Ra infection promoted ROS production in HMDMs and induced foam cell formation, with statistically significant differences ( P < 0.01). After treating HMDMs with NAC or emodin for 1 hour and then infecting with H37Ra, NAC treatment reduced intracellular ROS levels and lipid content, while emodin increased intracellular ROS levels and lipid content, both with statistically significant differences ( P < 0.01). Western blot analysis indicated that H37Ra infection inhibited PPARγ production, NAC promoted PPARγ expression, and emodin inhibited PPARγ expression, with statistically significant differences ( P < 0.05). Pre-treating HMDMs with BRL49653 or GW9662 for 1 hour before H37Ra infection showed that GW9662 promoted lipid accumulation in the cells, with statistically significant differences ( P < 0.01). Conclusion ROS can promote the formation of foam macrophages by downregulating the expression of PPARγ.

关键词

泡沫巨噬细胞 / H37Ra / 活性氧 / 过氧化物酶体增殖物激活受体-γ

Key words

Foam macrophages / H37Ra / Reactive oxygen species / Peroxisome proliferator-activated receptor-γ

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基金资助

*国家自然科学基金(81760002)

国家自然科学基金(82160308)

江西省自然科学基金(20212BAB216028)