Figure 4.

Sirt1 Impairs the Immunosuppressive Ability of MSCs and Inhibits Inflammatory Cytokine-Induced iNOS Production in MSCs

(A) MSCs, AdEGFP-MSCs, or AdSirt1-MSCs were respectively co-cultured with CFSE-labeled splenocytes at a ratio of 1:10 in the presence of Con A (5 μg/mL). The CFSE-diluted splenocytes were detected by flow cytometry after 72 h of incubation. A representative staining of three independent experiments is shown. (B) Quantitation data were determined by the percentage of proliferating CFSE-labeled splenocytes in each group. **p < 0.01 versus splenocytes (spl) + Con A. p > 0.05 versus spl + ConA; NS, not significant (p > 0.05). (C) Under the same treatment conditions as in (A), the splenocyte proliferation clones at 72 h were inspected by microscopy. Microphotographs show a representative image of splenocyte proliferation clones in each group. Scale bars, 100 μm. (D) The mRNA levels of Il6, Il10, Tnfaip6, Hgf, and Nos2 were detected by real-time PCR in MSCs, AdEGFP-MSCs, or AdSirt1-MSCs treated with or without IFN-γ and TNF-α (IT, 10 ng/mL each) for 8 h. ***p < 0.001, **p < 0.01, p > 0.05 versus MSC; **p < 0.01, p > 0.05 versus IT-MSC; NS, not significant (p > 0.05). (E) The protein levels of Sirt1 and iNOS were determined by western blotting in MSCs, AdEGFP-MSCs, or AdSirt1-MSCs treated with IFN-γ and TNF-α (IT, 10 ng/mL each) for 24 h. MSCs without IFN-γ and TNF-α treatment were used as the control. (F) The level of nitrate representing the iNOS activity was measured using Griess reagent in the supernatant of different groups of MSCs exposed to the treatments as described in (E). ***p < 0.001 versus MSC. p > 0.05 versus MSC. NS: not significant (p > 0.05).