iNOS Overexpression Promotes the Recovery of Immunosuppressive Function in AdSirt1-MSCs
(A) AdSirt1-MSCs were transfected with control empty pCMV vector (vector-AdSirt1-MSCs) or pCMV plasmid containing cDNA encoding mouse iNOS (iNOS-AdSirt1-MSCs), and then the AdSirt1-MSCs, vector-AdSirt1-MSCs, or iNOS-AdSirt1-MSCs were stimulated respectively with IFN-γ and TNF-α (IT, 10 ng/mL each) for 24 h, and iNOS expression was measured by immunoblotting analysis. (B) Under the same treatment conditions as in (A), the nitrite level in cell culture supernatant was detected using Griess reagent. ***p < 0.001 versus IT + AdSirt1-MSC. p > 0.05 versus IT + AdSirt1-MSC; NS, not significant (p > 0.05). (C) MSCs, AdSirt1-MSCs, vector-AdSirt1-MSCs, or iNOS-AdSirt1-MSCs were co-cultured respectively with CFSE-labeled splenocytes at a ratio of 1:10 for 72 h in the presence of Con A (5 μg/mL). The splenocytes were collected for proliferation analysis and assessed by the decrease in CFSE fluorescence intensity for cell division via flow cytometry after 72 h of co-culture. (D) Quantitation of splenocyte proliferation was determined by the percentage of CFSE-diluted splenocytes among total CFSE-labeled splenocytes in each group. ***p < 0.001 versus splenocytes (spl) + Con A. p > 0.05 versus spl + ConA; NS, not significant (p > 0.05).