Fig. 7

Stimulation of SIRT1 by melatonin was closely linked to ROS level and oxidative stress. In order to investigate the mechanisms by which melatonin stimulate SIRT1, ROS inhibitor NAC (N-Acetylcysteine) was used to pre-treat TM3 cells prior to melatonin treatment in high glucose condition. A The protein levels of antioxidant enzymes GPX4, GPX5 and SOD2 in TM3 cells detected by Western blot and statistical analysis of gray values. B ROS levels in TM3 cells were detected by DCFH-DA. The fluorescence intensity was analyzed statistically. C Expression levels of SIRT1, AMPK, autophagy marker LC3 as well as mitochondrial synthesis transcription factor mtTFA in TM3 cells of each group were detected by Western blot and gray values of protein bands were analyzed statistically. NG, normal glucose control group; HG, high glucose treatment group; HG + MT, high glucose and melatonin treatment group; siSirt1, HG + MT + siSirt1 group; HG + MT + NAC, HG + MT + NAC group. A and C, Data are expressed as fold change relative to NG. *, P < 0.05; #, P < 0.01. Bar: B, 100 μm