Prdx1通过维持线粒体稳态调节巨噬细胞的极化

Prdx1 regulates macrophage polarization by maintaining mitochondrial homeostasis

摘要:

为了研究过氧化物还原酶1 (peroxiredoxin 1, Prdx1)在巨噬细胞极化过程中的作用，使用脂多糖(lipopolysaccharides, LPS)联合干扰素γ (interferon gamma, IFNγ)、白细胞介素-4 (interleukin-4, IL-4)处理小鼠白血病单核巨噬细胞(mouse leukemia cells of monocyte macrophage, RAW264.7)，敲除Prdx1标记为Prdx1^-/-组，采用流式细胞术检测巨噬细胞分化标志物，采用酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)法检测细胞因子水平，检测诱导型一氧化氮合酶(inducible nitric-oxide synthase, iNOS)和精氨酸酶-1 (arginase-1, Arg-1)活性反应以及Prdx1^-/-细胞氧化损伤情况，通过能量分析仪检测细胞胞外酸化率和氧消耗速率，线粒体膜电位染料(mitochondrial membrane potential dye, JC-1)荧光探针法检测线粒体膜电位，荧光染色法检测线粒体超氧化物水平，并用线粒体活性氧(reactive oxygen species, ROS)清除剂处理RAW264.7细胞来评估细胞极化情况。结果表明，敲除Prdx1导致巨噬细胞ROS、过氧化氢和8-羟基-2-脱氧鸟苷(8-hydroxy-2 deoxyguanosine, 8-OHDG)水平升高，线粒体拷贝数降低，线粒体内膜转位酶23 (translocase of inner mitochondrial membrane 23, TIM23)蛋白和热休克蛋白60 (heat shock protein 60, HSP60)表达减少，线粒体膜电位下降，超氧化物增多，三磷酸腺苷(adenosine-triphosphate, ATP)水平降低；在1型巨噬细胞(type 1 macrophage, M1)中敲除Prdx1，细胞外酸化率(extra cellular acidification rate, ECAR)增加，在2型巨噬细胞(type 2 macrophage, M2)中敲除Prdx1，耗氧率(oxygen consumption rate, OCR)降低。这表明敲除Prdx1能够通过损伤巨噬细胞线粒体来降低其氧化磷酸化功能，导致巨噬细胞倾向M1型极化而抑制其向M2型极化，本研究可为巨噬细胞介导的免疫治疗提供新的治疗策略。

Abstract:

In order to investigate the role of Prdx1 in macrophage polarization, mouse leukemia cells of monocyte macrophage (RAW264.7) were treated with lipopolysaccharides (LPS)+ interferon gamma (IFNγ) or IL-4 to induce type 1 macrophage (M1) and type 1 macrophage (M2) macrophages, respectively. The Prdx1 gene knockout cells (Prdx1^-/-;) were used for the study. Flow cytometry was conducted to detect M1/M2 macrophage markers, and ELISA kits were used to measure M1/M2 cytokine levels. Inducible nitric-oxide synthase (iNOS) activity, arginase-1 (Arg-1) activity, and oxidative damage were also assessed. The Seahorse XFe24 Extracellular Flux Analyzer was employed to measure extracellular acidification rate and oxygen consumption rate. The mitochondrial membrane potential was analyzed using the mitochondrial membrane potential dye (JC-1) fluorescent probe, and mitochondrial superoxide was detected through fluorescence staining. Additionally, the impact of adding a mitochondrial reactive oxygen species (ROS) scavenger on RAW264.7 macrophage polarization was examined. The results demonstrated an increase in ROS, hydrogen peroxide, and 8-hydroxy-2 deoxyguanosine (8-OHDG). Cytotoxicity and mitochondrial toxic effects, including mitochondrial superoxide accumulation, decreased adenosine-triphosphate (ATP) production, reduced mitochondrial membrane potential, and decreased mitochondrial DNA copy number, were observed. Furthermore, down-regulation of translocase of inner mitochondrial membrane 23 (TIM23) mitochondrial protein and mitochondrial stress protein heat shock protein 60 (HSP60) was noted. The extra cellular acidification rate (ECAR) in M1 macrophage polarization in RAW264.7 cells was increased, while oxygen consumption rate (OCR) in M2 macrophages was reduced. These findings indicate that Prdx1 knockout in RAW264.7 cells can inhibit M2 macrophage polarization but promote M1 macrophage polarization by impairing mitochondrial function and reducing oxidative phosphorylation.