Fig. 5: Inhibition of Sirt1 in H/R-stimulated HBMVECs increased amyloidogenic pathway in neuronal cells. | Cell Death & Disease

Fig. 5: Inhibition of Sirt1 in H/R-stimulated HBMVECs increased amyloidogenic pathway in neuronal cells.

From: Sirtuin 1-dependent regulation of high mobility box 1 in hypoxia–reoxygenated brain microvascular endothelial cells: roles in neuronal amyloidogenesis

Fig. 5

ah HBMVECs were transfected with scrambled or Sirt1 siRNA for 24 h followed by exposure to 24-h H/R or normoxia, and CM samples were harvested. SH-SY5Y cells (a) or H4swe cells (bh) were cultured with HBMVEC–CM for 24 h. The levels of neprilysin (NEP) and IDE in SH-SY5Y cells were analysed by western blotting; β-actin was used as a loading control. Representative blots from duplicated experiment are presented (a). In H4swe cells, the expression of full-length APP (APP-N), APP-CTF (CTF-alpha, α-C83 and CTF-beta, β-C99) and BACE1 were detected by western blotting; β-actin was used as a loading control. Representative blots are presented, and bar graph indicate the semi-quantitative levels of full-length APP, APP-CTF, APP-CTFβ and BACE1, as compared to CM without H/R or Sirt1 siRNA (bf, n = 3, mean ± SD *p < 0.05 versus without treatment by one-sample t-test, #p < 0.05 versus H/R CM without treatment of Sirt1 siRNA by unpaired t-test). HBMVECs were treated with Ex527 (a Sirt1 inhibitor) at 1 μM (Ex. 1) or 5 μM (Ex. 5) for 24 h, followed by 24-h H/R or normoxia treatment. H4swe cells were cultured with the HBMVECs NC-CM or H/R-CM for 24 h. Full-length APP (APP-N) and the carboxy-terminal fragment of CTFβ were detected by western blotting. Representative blots are presented (g), and bar graph indicate the semi-quantitative levels of CTF-β/APP (h n = 5, mean ± SD; *p < 0.05, **p < 0.01 versus CM with normoxia without treatment of Ex527 by one-way ANOVA with multiple comparison). il HBMVECs were transfected with scrambled or Sirt1 siRNA (i, j) or treated with Ex527 (k, l) for 24 h followed by 24-h H/R exposure. H4swe cells were cultured with each CM for 24 h. The concentrations of Aβ40 and Aβ42 in H4swe CM were analysed using ELISA. Data are shown as means ± SD from repeated independent experiments (n = 9-15, *p < 0.05, **p < 0.01, ***p < 0.001 versus scrambled or vehicle control by unpaired t-tests).

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