Lipopolysaccharides (lps)

RAW 264.7 macrophages were divided into three groups according to treatment: macrophages cultured in normal medium for 48 hours were defined as M0; macrophages undergoing a 2 hour incubation with lipopolysaccharide (LPS, Sigma-Aldrich, Castle Hill, Australia) (2.5 mg/mL) and then cultured in normal medium for another 46 hours were defined as M1 [24 (link)]; and macrophages co-cultured with MSCs for 72 hours were defined as M2. For transwell co-cultures, a 0.4-μm pore size insert (Corning, Lowell, MA, USA) was placed into the six-well plate with LPS-stimulated macrophages on the bottom well, while 2 × 10⁵ MSCs were seeded onto inserts and cultured for another 72 hours [23 (link)].

Interferon (IFN)-γ/lipopolysaccharide (LPS) and Interleukin (IL)-4 were applied to generate the M1 and M2 phenotypes of BV2 cell lines in vitro, respectively. LPS and IL-4 were from Abcam (Cambridge, MA, USA). BV2 cells (1 × 10⁶ cells/mL) were seeded into the upper insert of a six-well Transwell plate (Corning Inc., Corning, MA, USA) and cultured at 37°C for 6 h, followed by incubation with either LPS (20 ng/mL) or IL-4 (20 ng/mL) for another 24 h at 37°C. LPS-polarized M1 and IL4-polarized M2 microglia were then washed with phosphate-buffered saline (PBS) and co-cultured with GL261 cells (2 × 10⁵ cells per well) without direct contact for 48 h at 37°C. The co–cultured GL261 cells were then washed and harvested for subsequent experiments.

Freshly isolated monocytes (CD14+) were cultured at a concentration of 4.0 × 10⁵ cells/well in 12-well plates containing 1 ml RPMI (Invitrogen Corp., Paisley, UK) with 100 U/ml penicillin and streptomycin (P/S; Invitrogen Corp), L-glutamine (Invitrogen Corp., Paisley, UK), 10% FBS and the growth factors IL4 (50 ng/ml, PeproTech, USA) and GM-CSF (50 ng/ml, PeproTech, USA) for 5 days, resulting in the generation of immature DCs (iDCs; CD14-/CD1a+). After the 5-day incubation, to gain mature DCs (mDCs), LPS (100 ng/ml, Sigma-Aldrich, USA) was added to stimulate the cells for 48 h.
To examine the effect of UC-MSCs on monocyte differentiation into mDCs, UC-MSCs treated with mitomycin were cultured at a concentration of 2.0 × 10⁵ cells/well in 12-well plates containing 1 ml RPMI for 24 h, and then monocytes, IL4 and GM-CSF were added with direct cell-cell contact or in a transwell system (pore size 0.4 μM; Corning Inc., Lowell, MA, USA) at a MSC-monocyte ratio of 1:10 for 5 days and then LPS was added to stimulate the cells for 48 h. After 7-day incubation, monocytes were the separated from the UC-MSCs by spinning the cells in suspension and then washing them.
To examine the effect of some soluble factors, IL10 (50 ng/ml), IL6 (50 ng/ml), HGF (50 ng/ml), anti-IL10 (5 μg/ml), anti-HGF or anti-IL6 (5 μg/ml) (R&D Systems Europe Ltd., Abingdon, UK) was added every 3 days.

BV2 and HT22 cells were purchased from Procell and cultured in six-well plates with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at a density of 2 × 10⁵ cells per well. For control group, BV2 or HT22 cells were cultured with 2 ml complete medium. BV2 or HT22 cells were induced by incubation with LPS (Sigma, China) or L-Glutamic acid (GLU, MCE, China) at a corresponding concentration. Pre-incubate with LY294002 for 2 h prior to LPS or GLU treatment. In the treatment group, a co-culture transwell chamber (0.4 μm pore size; Corning) was used to assess the effects of hDPSCs on LPS-induced BV2 or GLU-induced HT22 cells. hDPSCs were seeded in the upper compartment at a 1 × 10⁵ cells per well in 1 mL of α-MEM complete medium with 24 h.

RAW264.7 and Caco-2 cells were bought from ATCC. Cultures were performed respectively in RPMI1640 medium or DMEM in a 6 well plate (NEST Biotechnology, Wuxi, China), and both media were supplemented with 10–20% heat-inactivated FBS, 50 U/ml penicillin and 50 U/ml streptomycin (Invitrogen, USA) in a 37 °C incubator containing 5% CO₂. RAW264.7 cells were trypsinized and resuspended at 1 × 10⁶ cells/ml for the subsequent determination of cytokine gene expression. For the lipopolysaccharide (LPS) experiments, cells were treated with 1.0 µg/ml LPS (Sigma, USA) from Escherichia coli 0:55:B55 diluted in ddH₂O for 12 h. In the NZ9000 and NZ9000SHD-5 groups, RAW264.7 cells were treated with LPS and supernatants from NZ9000 or NZ9000SHD-5 cells for 12 h. Caco-2 cells were plated at a density of 1 × 10⁶ cells/ml on collagen-coated permeable polycarbonate membrane Transwell supports with 0.3 µm pores (Corning, USA) and were grown as monolayers for subsequent measurement of TJ gene and protein expression.

The murine RAW264.7 macrophage line was purchased from the Cell Culture Center, Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in high-glucose DMEM (Servicebio) with 10% foetal bovine serum (Zeta Life, Menlo Park, CA, USA) and 1% penicillin‒streptomycin (Biosharp, Anhui, China) at 37℃ under 5% CO₂. RAW264.7 cells (1×10⁶) were treated with 1 μg/ml lipopolysaccharide (LPS, Corning, Wilmington, NC, USA) to mimic the septic environment in vitro (6, 12, and 24 h). For cell transfection, RAW264.7 cells were seeded on 60 mm-cell dishes and cultured with serum-free Opti-MEM (Gibco, Brooklyn, NY, USA) 1 h before transfection when cell confluence reached 70-80%. Then, the cells were transfected with ADAR1-overexpression adenovirus (GenePharma), ADRA1-siRNA (RiboBio, Guangzhou, China), A20-siRNA (RiboBio), miR-21 mimic, miR-21 inhibitor (RiboBio) and their negative controls through Zeta transfection reagents (Zeta Life). Further LPS treatment was performed 24 h after transfection. The cells were collected for further experiments 48 h after transfection.

To investigate the effects of UCMSCs on macrophages, Raw264.7 cells were suspended in a culture medium and stimulated with 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich). After 5–10 min, 1 × 10⁵ LPS-induced macrophages were transferred to 6-well culture plates and treated as follows: (1) cocultured with 1 × 10⁵ UCMSCs, which were seeded onto the 0.4-μm-pore-size Corning Transwell inserts; (2) cultured in concentrated UCMSC-CM diluted 1:4 in fresh medium; and (3) cultured in fresh medium as a control. After 3 days, supernatants were collected, centrifuged to remove possible cell contamination (500×g for 10 min), and stored at − 20 °C until the levels of cytokines were examined by enzyme-linked immunosorbent assay (ELISA).