Apelin/APJ axis improves angiotensin II-induced endothelial cell senescence through AMPK/SIRT1 signaling pathway

Figure 1. Apelin improved AngII-induced senescence and suppressed ROS production. A – Cells were pre-treated with apelin (10–8 M) for 1 h followed by exposure to AngII (10–6 M) for an additional 24 h. Cell senescence was evaluated by SA-β-gal activity. Representative photo micrographs showed SA-β-gal-positive cells (blue) in HUVECs. B – P21 expression was detected by western blot. The bar graphs show the quantification of the indicated proteins. C – Cells were pre-treated with apelin (10–8 M) for 1 h followed by exposure to AngII (10–6 M) for an additional 24 h. Cellular ROS was evaluated by DCFH-DA. Representative photo micrographs show ROS-positive cells (green) in HUVECs The values are expressed as means ± SD of at least 3 independent experiments or 5 random fields of vision.*P < 0.05 vs. control; #P < 0.05 vs. AngII.

Figure 2. Apelin/APJ inhibited AngII-induced senescence through AMPK/SIRT1 signal pathway. A – SIRT1, AMPK, APJ were respectively knocked down by siRNA transfection as described in the Material and methods section. APJ, AMPK and SIRT1 expression was detected by western blot. B – As shown in the left panel, cells were pre-treated with apelin (10–8 M) for 1 h followed by exposure to AngII (10–6 M) for an additional 24 h. Western blot analysis was performed. As shown in the right panel, APJ was knocked down. At 48 h after transfection, the cells were pre-treated with apelin (10–8 M) for 1 h followed by exposure to AngII (10–6 M) for an additional 24 h. Western blot analysis was performed C – As shown in the left panel, AMPK was knocked down. At 48 h after transfection, the cells were pre-treated with apelin (10–8 M) for 1 h followed by exposure to AngII (10–6 M) for an additional 24 h. Western blot analysis was performed. As shown in the right panel, SIRT1 was knocked down. At 48 h after transfection, the cells were pre-treated with apelin (10–8 M) for 1 h followed by exposure to AngII (10–6 M) for an additional 24 h. Western blot analysis was performed. The bar graphs show the quantification…

Figure 3. Apelin improved AngII-induced ageing and reduced ROS production via AMPK/SIRT1 signaling pathway. A – SIRT1, AMPK, APJ were knocked down by siRNA transfection. At 24 h after transfection, the cells were treated with apelin (10–8 M) for 1 h followed by exposure to AngII (10–6 M) for an additional 24 h. Cell senescence was evaluated by SA-β-gal activity. Representative photomicrographs show SA-β-gal-positive cells (blue) in HUVECs. B – The level of intracellular ROS was assessed. Representative photomicrographs show ROS-positive cells (green) in HUVECs The values are expressed as means ± SD of at least 3 independent experiments or 5 random fields of vision.*P < 0.05 vs. control; #p < 0.05 vs. AngII; Δp < 0.05 vs. apelin + AngII

Figure 4. Apelin promoted telomerase activity in AngII-treated HUVEC. A – SIRT1, AMPK, APJ were knocked down by siRNA transfection. At 24 h after transfection, the cells were treated with apelin (10–8 M) for 1 h followed by exposure to AngII (10–6 M) for an additional 24 h. Telomerase activity was investigated by RQ-TRAP. Telomerase activity in the control group was considered as 100% compared to the other groups. B – Cell viability was detected by CCK-8 assay. The bar charts show the cell viability of the indicated group The values are expressed as means ± SD of at least 3 independent experiments.*P < 0.05 vs. control; #p < 0.05, vs. AngII; Δp < 0.05, vs. apelin + AngII.

Figure 5. Pathway involved in the protective effect of apelin/APJ axis in AngII induced HUVECs ageing. Apelin/ APJ axis induces the phosphorylation of AMPK, causing SIRT1 activation. This process inhibits AngII-induced ROS generation and increases telomerase and finally improves AngII-induced HUVECs ageing