Fig. 4

miR-146a reduces the effect of metformin on SIRT1 activity. a–d miR-146a inhibits the up-regulative effect of metformin on SIRT1 activity in H₂O₂-induced senescent NIH3T3 cells. NIH3T3 cells were transfected with miR-NC or miR-146a mimics, followed by H₂O₂ and metformin treatment. a Enzymatic activity of SIRT1 in cell lysates was measured by the spectroscopic method (n = 4). b Immunoprecipitation of the SIRT1 target FOXO1 from cells, followed by the analysis of total and acetylated FOXO1 levels and the ratio of acetylated to FOXO1 levels is shown (n = 3). c SIRT1 protein expression was tested and representative immunoblots are shown (n = 4). d Intracellular NAD⁺ level was assayed by spectroscopic method (n = 4). e, f miR-146a agomir inhibits the up-regulative effect of metformin on SIRT1 activity and NAD⁺ levels, whereas the miR-146a antagomir shows comparable effects to those of metformin on D-gal-induced aged mice. SIRT1 activity and NAD⁺ levels in lungs from mice receiving D-gal, metformin, and the miR-146a agomir (e) or antagomir (f) were determined (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001. ns means no significance