Lipopolysaccharides (lps)

RAW264.7 cells, a macrophage line of mouse, was obtained from Cell Bank of Academy of Sciences (Shanghai, Chain). After detection of mycoplasma-free contamination, the cells were seeded in DMEM medium (Gibco, Carlsbad, CA, United States) with 10% fetal bovine serum, 200 μg/ml L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and cultured at 37°C, 5% CO₂.
M1 macrophages were generated by stimulation with 100 ng/mL LPS (Escherichia coli, Sigma-Aldrich, United States) and 10 ng/mL IFN-γ (Sino Biological, Shanghai, China) for 24 h. RAW264.7 cells were induced with 10 ng/mL IL-4 and 10 ng/mL IL-13 (both purchased from Sino Biological) for 24 h to generate of M2 macrophages. LBP and LPS/IFN-γ treated simultaneously RAW264.7 cells for 24 h, or LBP alone treated RAW264.7 cells for 24 h. Fludarabine and AS1517499 (both purchased from TargetMol, Shanghai, China) were employed to validate the STAT1and STAT6 signaling pathways.

The immortalized BV-2 microglial cells (the Institute of Basic Medicine Chinese Academy of Medical Sciences, Beijing, China) were maintained in high glucose DMEM (Sigma, Saint Louis, MO, USA, Catalog No. D6429) supplemented with 10% FBS (Sigma, Saint Louis, MO, USA, Catalog No.12103C) at 37 °C in a humidified atmosphere of 5% CO₂ in air.
Cells were seeded in 96 well plates at 1 × 10⁵ cells per well, then cultured for 24 h. These cells’ group distribution and administration were divided into the following groups (n = 3 per group): control group; LPS (5 μg/mL) group; LPS + TAK-242 (1 μM) group (TAK-242, a TLR4 inhibitor which can prevent LPS-induce inflammatory response, was used as positive control [32 (link),33 (link)]); and LPS + dutasteride (10 nM–20 μM) group. Cells were incubated with TAK-242 (TargetMol, Washington, USA, Catalog No. TQ0181) and dutasteride (TargetMol, Washington, DC, USA, Catalog No. T1499) for 1 h, and subsequently stimulated with LPS (TargetMol, Washington, DC, USA, Catalog No. T11855) for 24 h, then stored for later tests.

The electrospun nanofiber membranes were first placed in 6-well plates, and RAW264.7 cells were seeded on their surfaces at a density of 2 × 10⁵ cells per well. Following the incubation, the culture medium was discarded, and the cells were washed three times with PBS to remove debris and non-adherent cells. Lipopolysaccharide (LPS, TargetMol) was then added to the culture to activate macrophages. After another 24 h, cells were collected for analysis.
The cell pellets were resuspended in 100 μL of PBS containing 1.25 μg of PE-conjugated anti-mouse CD86 antibody and 0.25 μg of FITC-conjugated anti-mouse CD206 antibody (Biolegend). This step was performed on ice for 30 min to preserve cell viability and prevent nonspecific antibody binding. The expression of CD86 and CD206 was subsequently analyzed using the CytoFLEX flow cytometry system (Beckman Coulter). Additionally, the intracellular ROS level was detected using 2,7dichlorodihydrofluorescein diacetate (DCFH-DA; Yeasen), following the same procedure as described above (n = 3).