L name

Representative nitric oxide synthase (NOS) inhibitors (Beyotime, Jiangsu, China) including l-NMMA (Pan NOS inhibitor, 200 µM), 1,400 W (iNOS inhibitor, 100 µM), and L-NAME (eNOS inhibitor, 100 µM) were used in in vitro T cell suppression experiments to inhibit the generation of NO.

Before the experiment, the cell culture medium was replaced by basic medium (M199 + 5% FBS). Then EPCs were treated with different concentrations (2.5 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml) of LPS (Solarbio, Beijing, China) at different time (24, 48, 72 h) to establish a damaged model. The cell damaged model was treated with H₂ in different concentrations (20%, 40%, 60%) at different time (24, 48, 72 h) to explore the suitable conditions of H₂. The concentration of CO₂ in the H₂ incubator is 5%, the concentration of O₂ is 21%, and the concentration of H₂ is adjusted by N₂. Finally, EPCs were treated with PI3K inhibitor LY294002 (Sigma-Aldrich, St Louis, MO, United States) (10 μM, 20 μM, 30 μM) or eNOS inhibitor L-NAME (Beyotime, Shanghai, China) (100 μM, 200 μM) to find out the suitable concentration of inhibitors.

Purified CD19⁺ B cells were stimulated with 4 μg/ml CpG ODN 2006 and 1 μg/ml CD40L in the presence or absence of hMSCs, and the frequencies of IL-10 producing B cells or CD23⁺CD43⁺ B cells were evaluated. For CD23^-CD43^- B cell conversion experiments, purified CD19⁺CD23^-CD43^- B cells were stimulated with 4 μg/ml CpG ODN 2006 and 1 μg/ml CD40L in the presence or absence of hMSCs, and the increase in CD23⁺CD43⁺ B cells was evaluated. To explore whether the cell-cell contacts were involved in this process, B cells and hMSCs were cocultured separately using the Transwell assay. To explore which factor participated in this process, 1 μM indomethacin (a non-specific inhibitor for COX-2/ PGE2, Sigma), 1 μM NS398 (a specific inhibitor for COX-2/PGE2, Sigma), 1 mM 1-MT (a specific inhibitor for IDO, Sigma), 10ug/ml anti-HGF antibody (R&D Systems), 10ug/ml anti-IL-6 antibody (R&D Systems), 10ug/ml anti-IL-10 antibody (R&D Systems), and 1 mM L-NAME (a nonselective inhibitor of NOS, including human eNOS and murine iNOS; purchased from Beyotime Biotechnology) were added to the B/hMSCs cocultures.

Identification and isolation of hAMSCs were conducted according to a previously described method 13 . Briefly, culture of hAMSCs was performed in α Minimum Essential Medium (α-MEM; Gibco, Grand Island NY, USA) containing 10% fetal bovine serum (FBS, Gibco), 100 U/L penicillin, and 100 mg/L streptomycin. hAMSCs at passages 3-5 were employed in all experiments. HUVECs purchased from China Infrastructure of Cell Line Resources (Beijing, China) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 10% FBS, 100 U/L penicillin, and 100 mg/L streptomycin. Three donors were enrolled in this study for isolation of hAMSCs.
L-NAME (Beyotime Biotechnology, Shanghai, China) was used as a specific inhibitor of eNOS in HUVECs by pre-treating with 50 nM overnight. AMG706 (Beyotime Biotechnology) was employed as an inhibitor of VEGFRs in HUVECs by pre-treating with 10 nM for 2 hours.