RESUMEN
OBJECTIVES: To explore the effects and mechanisms of bilirubin on mitochondrial function and type of macrophage cell death after exposure to cigarette smoke extract (CSE). METHODS: 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-1ß 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. RESULTS: 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. CONCLUSIONS: 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.
Asunto(s)
Bilirrubina , Macrófagos , Mitocondrias , Especies Reactivas de Oxígeno , Animales , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Nicotiana/efectos adversos , Nicotiana/química , Humo/efectos adversos , Apoptosis/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Objective: To investigate the effects of cigarette smoke extract (CSE) on the mitochondrial function of macrophages. Methods: RAW264.7 macrophages were used for the experiment in this study. When the cell density was about 70%, the old culture medium was abandoned, and the 100% CSE stock solution was diluted with serum-free DMEM and FBS into 1%, 5%, 15%, 25% and 90% CSE and added to the well plate. The cell activity of RAW264.7 cells treated with CSE at different concentrations for 24 h was detected by CCK-8 method. Then the optimal CSE concentration was selected to treat cells for 0 h, 24 h, 48 h or 72 h respectively, and CCK-8 assay was used to detect the cell activity of CSE treated cells at different time groups. After the cells were treated with 0%, 5% and 25% CSE for 24 hours, cell necrosis and apoptosis was detected by Annexin V-FITC /PI staining; Mitochondrial membrane damage of RAW 264.7 was detected by mitochondrial membrane potential assay kit with JC-1; Macrophages were stained with ROS-specific dye DCFH-DA, and then Flow cytometer was used to determine the fluorescence and the proportion of ROS-positive macrophages; the enhanced ATP assay kit was used to detect the intracellular ATP concentration. Results: â Compared with 0% CSE, cell viability was increased significantly in 1% CSE group (Pï¼0.01), cell viability was decreased significantly when CSE concentration was above 5% (Pï¼0.05); Macrophages were treated with 5% CSE, and cell viability was decreased significantly with the increase of treatment time (Pï¼0.01). â¡Compared with 0% CSE, 5% CSE and 25% CSE mainly caused macrophage necrosis, decreased mitochondrial membrane potential, increased ROS production and decreased ATP significantly (Pï¼0.05 or Pï¼0.01), and the changes were more significant in 25% CSE treatment group(Pï¼0.05 or Pï¼0.01). Conclusion: CSE may affect mitochondrial function of macrophages, leading to decreased cell viability and necrosis.