PDF(Pubmed)
Abstract：
UNASSIGNED: To explore the effects and mechanisms of bilirubin on mitochondrial function and type of macrophage cell death after exposure to cigarette smoke extract (CSE).
UNASSIGNED: RAW264.7 macrophages were treated with different concentrations of CSE and bilirubin solutions and divided into four groups: control, CSE, bilirubin, and bilirubin + CSE groups. The necrotic and apoptotic states of the macrophages were determined using an Annexin V-fluorescein 5-isothiocyanate/propidium iodide (FITC/PI) staining kit. Cytoplasmic NOD-like receptor family, pyrin domain containing 3 (NLRP3) expression in macrophages was detected by immunofluorescence and the levels of IL-1β and IL-18 in the supernatants of culture medium were detected by enzyme linked immunosorbent assay (ELISA) test. A JC-1 mitochondrial membrane potential detection kit was used to assess mitochondrial membrane damage and the adenosine triphosphate (ATP) assay kit was used to determine intracellular ATP levels. After the macrophages were stained with reactive oxygen species (ROS) specific dye, 2\',7\'-Dichlorodihydrofluorescein diacetate (DCFH-DA), the fluorescence intensity and proportion of ROS-positive macrophages were measured using flow cytometry.
UNASSIGNED: We observed that compared with those of 0 μM (control group), concentrations of 5, 10, or 20 μΜ bilirubin significantly decreased cell viability, which was increased by bilirubin exposure below 1 μM. The effect of CSE on macrophage viability was concentration- and time-dependent. Bilirubin of 0.2 μM could alleviate the inhibition of macrophage viability caused by 5% CSE. In addition, bilirubin intervention could reduce the occurrence of necrosis and pyroptosis to a certain extent.
UNASSIGNED: CSE could cause mitochondrial dysfunction in macrophages, as demonstrated by a decrease in mitochondrial membrane potential and intracellular ATP levels and an increase in ROS production, while bilirubin could relieve mitochondrial dysfunction caused by CSE.
UNASSIGNED: RAW264.7 macrophages were treated with different concentrations of CSE and bilirubin solutions and divided into four groups: control, CSE, bilirubin, and bilirubin + CSE groups. The necrotic and apoptotic states of the macrophages were determined using an Annexin V-fluorescein 5-isothiocyanate/propidium iodide (FITC/PI) staining kit. Cytoplasmic NOD-like receptor family, pyrin domain containing 3 (NLRP3) expression in macrophages was detected by immunofluorescence and the levels of IL-1β and IL-18 in the supernatants of culture medium were detected by enzyme linked immunosorbent assay (ELISA) test. A JC-1 mitochondrial membrane potential detection kit was used to assess mitochondrial membrane damage and the adenosine triphosphate (ATP) assay kit was used to determine intracellular ATP levels. After the macrophages were stained with reactive oxygen species (ROS) specific dye, 2\',7\'-Dichlorodihydrofluorescein diacetate (DCFH-DA), the fluorescence intensity and proportion of ROS-positive macrophages were measured using flow cytometry.
UNASSIGNED: We observed that compared with those of 0 μM (control group), concentrations of 5, 10, or 20 μΜ bilirubin significantly decreased cell viability, which was increased by bilirubin exposure below 1 μM. The effect of CSE on macrophage viability was concentration- and time-dependent. Bilirubin of 0.2 μM could alleviate the inhibition of macrophage viability caused by 5% CSE. In addition, bilirubin intervention could reduce the occurrence of necrosis and pyroptosis to a certain extent.
UNASSIGNED: CSE could cause mitochondrial dysfunction in macrophages, as demonstrated by a decrease in mitochondrial membrane potential and intracellular ATP levels and an increase in ROS production, while bilirubin could relieve mitochondrial dysfunction caused by CSE.
摘要：
■探讨胆红素对暴露于香烟烟雾提取物（CSE）后线粒体功能和巨噬细胞死亡类型的影响和机制。
■用不同浓度的CSE和胆红素溶液处理RAW264.7巨噬细胞，并分为四组：对照组，CSE,胆红素,和胆红素+CSE组。使用膜联蛋白V-荧光素5-异硫氰酸酯/碘化丙啶(FITC/PI)染色试剂盒测定巨噬细胞的坏死和凋亡状态。细胞质NOD样受体家族,免疫荧光法检测巨噬细胞中pyrin结构域3（NLRP3）的表达，酶联免疫吸附试验（ELISA）法检测培养基上清液中IL-1β和IL-18的水平。使用JC-1线粒体膜电位检测试剂盒来评估线粒体膜损伤,并且使用三磷酸腺苷(ATP)测定试剂盒来确定细胞内ATP水平。巨噬细胞用活性氧（ROS）特异性染料染色后，2\',7'-二氯二氢荧光素二乙酸酯（DCFH-DA），采用流式细胞术检测ROS阳性巨噬细胞的荧光强度和比例.
■我们观察到，与0μM（对照组）相比，浓度为5、10或20μM的胆红素显着降低了细胞活力，胆红素暴露低于1μM会增加。CSE对巨噬细胞活力的影响是浓度和时间依赖性的。0.2μM的胆红素可以减轻5%CSE对巨噬细胞活力的抑制作用。此外,胆红素干预可在一定程度上减少坏死和焦亡的发生。
■CSE可引起巨噬细胞线粒体功能障碍，线粒体膜电位和细胞内ATP水平降低，ROS产生增加，胆红素可以缓解CSE引起的线粒体功能障碍。
■用不同浓度的CSE和胆红素溶液处理RAW264.7巨噬细胞，并分为四组：对照组，CSE,胆红素,和胆红素+CSE组。使用膜联蛋白V-荧光素5-异硫氰酸酯/碘化丙啶(FITC/PI)染色试剂盒测定巨噬细胞的坏死和凋亡状态。细胞质NOD样受体家族,免疫荧光法检测巨噬细胞中pyrin结构域3（NLRP3）的表达，酶联免疫吸附试验（ELISA）法检测培养基上清液中IL-1β和IL-18的水平。使用JC-1线粒体膜电位检测试剂盒来评估线粒体膜损伤,并且使用三磷酸腺苷(ATP)测定试剂盒来确定细胞内ATP水平。巨噬细胞用活性氧（ROS）特异性染料染色后，2\',7'-二氯二氢荧光素二乙酸酯（DCFH-DA），采用流式细胞术检测ROS阳性巨噬细胞的荧光强度和比例.
■我们观察到，与0μM（对照组）相比，浓度为5、10或20μM的胆红素显着降低了细胞活力，胆红素暴露低于1μM会增加。CSE对巨噬细胞活力的影响是浓度和时间依赖性的。0.2μM的胆红素可以减轻5%CSE对巨噬细胞活力的抑制作用。此外,胆红素干预可在一定程度上减少坏死和焦亡的发生。
■CSE可引起巨噬细胞线粒体功能障碍，线粒体膜电位和细胞内ATP水平降低，ROS产生增加，胆红素可以缓解CSE引起的线粒体功能障碍。
