Macrophage cell culture
RAW264.7 macrophage were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, USA) containing 10% fetal bovine serum (FBS; Corning, USA) and 1% penicillin/streptomycin (Invitrogen, USA), in 37°C, 5% CO2incubator.
Cell MTT test
Cell viability was tested by MTT assay, 1 x 105 cells were seeded in a 96-well plate. After incubation for 24h, treated with various concentrations of LPS (Sigma, USA) (100, 500, 1000, 2000ng/ml) and Irisin(400ng/ml) (Phoenix Pharmaceuticals, Inc) for 24h. Then, 20 μl of 5% MTT (Solarbio, USA) solution was added to each well and incubated for 4 h. The supernatant was discarded and the resulting crystals were dissolved in DMSO (Solarbio, USA). Subsequently, the cells were further incubated (37 °C, 5% Co2) for 10 min. Absorbance was measured at 490nm.
RAW264.7 was cultured in 12-well plates at 1 × 105 cells/well. After incubation for 24h, cells were cultured with medium, Irisin(400ng/ml), and LPS (1000ng/ml), respectively, or cells were pretreated with Irisin for 12 hours, and then LPS was added. Cell morphology was evaluated with light microscopy.
IL-12 and IL-23 levels
The levels of IL-12 and IL-23 in the supernatants of the four groups of cells were taken and detected by enzyme-linked immunosorbent assay (ELISA) kits (Hermes Criterion Biotechnology, Canada), respectively.
Macrophage phagocytosis test
The cell suspension concentration of log-phase RAW264.7 was adjusted to about 1 × 105/mL and added 500 μL into each well of the 24-well plate in a 5% CO2 incubator for 24h at 37℃. Zymosan particles(Invitrogen, USA) were added to the cells. Phagocytosis was allowed to proceed for 30min. Cells were fixed with 4% paraformaldehyde for 30min at 4℃, then washed with PBS. The plate was examined by light microscopy at 400magnification using a Nikon Eclipse Ti2-E microscope (Nikon, Tokyo, Japan). Phagocytosis was determined by counting a total of 100 macrophages in at least four different fields in 400 magnification photomicrographs. Phagocytosis was assessed as the percentage of phagocytic cells (PP), mean of particles per cell (MNP), and as the phagocytic index (PI) determined as PI =PP x MNP.
The total proteins in each group were extracted with RIPA (Solarbio, USA), PMSF (Solarbio, USA), Protein Phosphatase Inhibitor (Solarbio, USA). The total protein was quantified by the BCA protein assay kit (Beyotime, China), and the protein was denatured at 95℃ for 5min. After electrophoresis, proteins were transferred on to polyvinylidene difluoride (PVDF) (Immobilon, Germany), followed by 5% non-fat milk（Solarbio, USA）to block the band. The band was then incubated with primary antibody (β-actin, Proteintech, China, Mouse, 1:5000, ERK1/2, Proteintech, China, Rabbit, 1:1000; Phospho-ERK1/2, Proteintech, China, Rabbit, 1:1000; AKT, Proteintech, China, Mouse, 1:2000; Phospho-AKT, Proteintech, China, Rabbit, 1:5000; PPAR α，Proteintech, China, Mouse, 1:1000; PPAR γ，Proteintech, China, Rabbit, 1:1000) at 4°C overnight, the appropriate secondary antibody HRP-conjugated affinity purified goat anti-rabbit, or goat anti-mouse lgG was applied at 1:5000 dilution and incubated for 1 hours at room temperature. Images were taken with Bio-Rad imaging system (Bio-Rad, USA) and analyzed by image analysis system for the optical density of the protein bands.
To compare the apoptosis rate, RAW264.7 macrophages were divided into four groups, treated with a culture medium, Irisin (400 ng/ml), LPS (1000 ng/ml) alone, pretreated with Irisin, and then added with LPS. In addition, to assess the apoptotic rate of thymocytes, thymocytes and supernatants were collected after thymocytes were treated with dexamethasone (5 μM) (Yeasen, China) for 12 hours. then, centrifuged at 3000 r/min for 5 minutes, and resuspended in 500 μL Binding Buffer. Annexin V-FITC and PI (Procell, China) were added with 5 μL respectively, and flow cytometry quantitative detection was performed with FACS.
Apoptotic Cell Clearance Assay
Macrophages were seeded in 24-well plates. Apoptotic thymocytes were added to each well at a macrophage: thymocyte ratio of (1:10). Thymocytes were labeled with fluorescent cell tracking reagent (Invirtrogen, USA) before co-culture with macrophages. Phagocytosis assays were performed at 37°C for 1 hour. Macrophages were then washed thoroughly to remove non-phagocytic cells. Fix cells with 4% paraformaldehyde. Image analysis was performed by a fluorescence microscope (thymocytes, red). Quantify thymocytes phagocytosed by each macrophage using Image J by counting 50-100 macrophages in each well. Data are presented as phagocytosis index (PI). This index was defined as the total number of apoptotic cells phagocytosed by each macrophage present in the field of view.
Results were expressed as mean ± S.E.M, all data were analyzed by one-way ANOVA using Prism 9.0 software (GraphPad Software). P-values <0.05 were considered statistically significant.