Bilirubin regulates cell death type by alleviating macrophage mitochondrial dysfunction caused by cigarette smoke extract.

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.

Self-injury and suicidal ideation among Chinese adolescents involved in different subtypes of aggression: The role of gender.

BACKGROUND: Although aggression associated self-injury and suicidal ideation, but the specific impact of different subtypes of aggression is unclear. Therefore, the current study aims to quantify the associations between five subtypes of aggression, self-injury, and suicidal ideation in Chinese adolescents. METHOD: A total of 18,532 students were recruited in grades 7 to 12 using a stratified cluster sampling across five representative provinces in China. The Functional Assessment of Self-Mutilation (CH-FASM) and Buss and Warren's Aggression Questionnaire (BWAQ) assess self-injury and aggression, respectively. RESULTS: During the last year, the proportion of self-injury, suicidal ideation, and self-injury plus suicidal ideation (SSI) were 13.4 %, 10.0 %, 12.4 %, respectively. Multivariate logistic regression indicated that hostility was associated with self-injury-only (OR = 1.033, 95 % CI = 1.021-1.044), suicidal ideation-only (OR = 1.075, 95 % CI = 1.061-1.088), and SSI (OR = 1.100, 95 % CI = 1.087-1.114) (all P < 0.001) across five subtypes of aggression. In gender stratification, physical aggression was risk factor for self-injury-only, suicidal ideation-only, and SSI (OR = 1.028, 95 % CI = 1.018-1.037, P < 0.001) in females. While anger was association with self-injury-only and suicidal ideation-only in males (P < 0.05). LIMITATIONS: This study was a cross-sectional design and self-reported questionnaire. CONCLUSION: Hostility is a strong predictor of self-injury and suicidal ideation across five subtypes of aggression. There are gender differences in relationships. Prevention programs for adolescents' self-injury and suicidal ideation should consider different subtypes of aggression and gender differences.

[Effects of cigarette smoke extract on mitochondrial function of macrophages].

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: ①Compared with 0% CSE, cell viability was increased significantly in 1% CSE group (P＜0.01), cell viability was decreased significantly when CSE concentration was above 5% (P＜0.05); Macrophages were treated with 5% CSE, and cell viability was decreased significantly with the increase of treatment time (P＜0.01). ②Compared with 0% CSE, 5% CSE and 25% CSE mainly caused macrophage necrosis, decreased mitochondrial membrane potential, increased ROS production and decreased ATP significantly (P＜0.05 or P＜0.01), and the changes were more significant in 25% CSE treatment group(P＜0.05 or P＜0.01). Conclusion: CSE may affect mitochondrial function of macrophages, leading to decreased cell viability and necrosis.