Fig. 6

Mesenchymal stem cells (MSCs) inhibit Kupffer cell apoptosis via the PGE2-TLR4-ERK1/2-Fas/FasL-caspase3 pathway. Murine-derived RAW264.7 cells were used to represent Kupffer cells; 5 × 10⁵ macrophages per well were seeded in a six-well plate, and 10⁵ MSCs were seeded in a 0.4-μm polycarbonate membrane insert Transwell in medium containing or lacking NS-398 (5 μM). After 24 h, the medium was changed once to remove non-adherent dead cells. After 2 h of incubation at 37 °C without O₂, the macrophages were transferred into co-culture with the Transwells containing MSCs, and 0, 100, 200, 400, or 800 μM hydrogen peroxide (H ₂ O ₂ ) was added to specific wells. Then, the co-cultured cells were incubated at 37 °C with 5% CO₂ for 6 h to mimic the process of IRI during NHB liver transplantation in vivo. a RAW264.7 cells were stained with F4/80 antibody, a surface marker of Kupffer cells, and analysed by flow cytometry. Representative images from one experiment are shown. Blue shading and red shading indicate immunofluorescence intensity of cells for the isotype and F4/80 antibodies, respectively. b In vitro co-culture system. After H₂O₂ stimulation for 6 hours, apoptosis of Kupffer cells was detected by Annexin V/PI staining. Representative images from one experiment out of three are shown. c Summarized results are presented as mean ± SEM (N = 3 for each group). d After H₂O₂ stimulation for 6 hours, expression of TLR2, TLR4, ERK1/2, p-ERK1/2, Fas, FasL, cleaved caspase3, and β-actin from Kupffer cells was determined by Western blotting analysis. Representative images from one experiment are shown. e Grey value of each band was calculated by ImageLab software. Representing relative expression of target proteins, the grey value ratio compared with β-actin was analysed and shown as mean ± SEM (N = 3 for each group). *P < 0.05, **P < 0.01, ***P < 0.001