Figure 5.

Role of SIRT1 in the inhibitory effect of resveratrol on insulin-induced VSMC proliferation. (a) Effect of resveratrol on SIRT1 expression. VSMCs were incubated with 100 μM resveratrol for 24 h. SIRT1 protein levels were determined by Western blotting. ^∗P < 0.05 vs. control, n = 5/group. (b) Cell proliferation after resveratrol treatment was quantified by the CCK-8 assay in insulin-treated cells that had been treated with or without the SIRT1 inhibitor EX-527. Results are expressed as optical density. ^∗P < 0.05 vs. control; ^#P < 0.05 vs. insulin group, n = 9/group. (c) and (d) EdU assays were performed to assess the effect of SIRT1 inhibitor EX527 on VSMC proliferation. (c) DNA synthesis in VSMCs was determined by EdU incorporation assay. (d) The ratio of EdU-positive cells to total cells. ^∗P < 0.05 vs. control; ^#P < 0.05 vs. insulin group, n = 6/group. (e) and (f) A10 cells were treated with the blank, insulin (100 nM), resveratrol (100 μM), EX527 (25 μM), or both insulin (100 nM) and resveratrol (100 μM). Western blotting was performed to analyze PI3K and Akt phosphorylation levels. (e) Representative images and relative protein expression of p-PI3K. (f) Representative images and the relative protein expression of p-Akt. ^∗P < 0.05 vs. control; ^#P < 0.05 vs. insulin group, n = 5/group. (g) and (h) Wound healing assay was performed to measure the regulation of SIRT1 inhibitor EX527 in the effect of resveratrol on insulin-induced VSMC migration. (g) Images were taken at regular intervals. (h) Relative migration rate was calculated. ^∗P < 0.05 vs. control; ^#P < 0.05 vs. insulin group, n = 6/group. (i) and (j) Transwell assay was performed to measure the regulation of SIRT1 inhibitor EX527 in the effect of resveratrol on insulin-induced VSMC migration. (i) Representative pictures of cells that migrated through the filter and were stained with crystal violet. (j) Bar graph showing the number of migrated VSMCs. ^∗P < 0.05 vs. control; ^#P < 0.05 vs. insulin group, n = 6/group.