Fig. 3

The beneficial effects of mMuSCs on acute liver injury require iNOS. (A) Splenocytes stained with CFSE were co-cultured with mMuSCs (2.5 × 10⁴ cells per well in 48-well plates) at a ratio of 1 : 20 (mMuSC : splenocyte) for 72 h in the presence of anti-mouse CD3 antibody (1 µg/ml) with or without the iNOS inhibitor L-NMMA (1 mM). CFSE fluorescence intensity reduction of splenocytes was detected by flow cytometry to measure the proliferation of splenocytes (n = 4). (B) The protein expression of iNOS and β-actin (loading control) were determined by immunoblotting in WT mMuSCs and iNOS^−/− mMuSCs (n = 3). The original images of blots are in the Supplementary Information. (C) The supernatants of WT mMuSCs and iNOS^−/− mMuSCs were assayed for nitrate by a modified Griess reagent (n = 3). (D) Splenocytes stained with CFSE were co-cultured with WT mMuSCs or iNOS^−/− mMuSCs (2.5 × 10⁴ cells per well in 48-well plates) at graded ratios (mMuSC : splenocyte) for 72 h in the presence of anti-mouse CD3 antibody (1 µg/ml). CFSE fluorescence intensity reduction of splenocytes was detected by flow cytometry to measure the proliferation of splenocytes (n = 4). (E) Mice were intravenously injected with ConA (15 mg/kg) to induce acute liver injury; 0.5 h later, WT mMuSCs or iNOS^−/− mMuSCs (1 × 10⁶) were transfused to suffering mice in the same way. After 12 h, serum and liver tissues were sampled. (F) Serum levels of AST and ALT were measured. (G) IFN-γ and IL-12 levels in the serum were assayed by ELISA. (H) Representative H&E-stained liver sections and percentages of tissue necrosis (Control: n = 4, ConA + PBS: n = 5, ConA + WT mMuSC: n = 5, ConA + iNOS^−/− mMuSC: n = 5). Scale bar, 250 μm. (I) Absolute numbers of MNCs in liver tissues were calculated. Absolute numbers of CD3⁺ T cells were determined by flow cytometry (Control: n = 4, ConA + PBS: n = 5, ConA + WT mMuSC: n = 4, ConA + iNOS^−/− mMuSC: n = 5). Values are presented as mean ± SEM. Statistical analysis was performed by one-way analysis of variance test (A),two-tailed unpaired t test (C) or Mann-Whitney test (F-I).