Synthetic resveratrol aliphatic acid inhibits TLR2-mediated apoptosis and an involvement of Akt/GSK3beta pathway

Abstract

As resveratrol derivatives, resveratrol aliphatic acids were synthesized in our laboratory. Previously, we reported the improved pharmaceutical properties of the compounds compared to resveratrol, including better solubility in water and much tighter binding with human serum albumin. Here, we investigate the role of resveratrol aliphatic acids in Toll-like receptor 2 (TLR2)-mediated apoptosis. We showed that resveratrol aliphatic acid (R6A) significantly inhibits the expression of TLR2. In addition, overexpression of TLR2 in HEK293 cells caused a significant decrease in apoptosis after R6A treatment. Moreover, inhibition of TLR2 by R6A decreases serum deprivation-reduced the levels of phosphorylated Akt and phosphorylated glycogen synthase kinase 3beta (GSK3beta). Our study thus demonstrates that the resveratrol aliphatic acid inhibits cell apoptosis through TLR2 by the involvement of Akt/GSK3beta pathway.

Figure 1

Structural formula of resveratrol (Res).

Figure 2

Structural formulae of resveratrol derivatives.

Figure 3

R6A decreases the expression of TLR2. HEK293 and TLR2/HEK293 cells were treated with R6A at 0, 10μM, or 100μM for 24 h and then subjected to serum deprivation (SD) for 0, 12, and 24 h, respectively. Cell lysates were probed for TLR2 expression by Western blot. Representative results of TLR2 immunobloting are shown at the top of each pane. Results represent mean ± s.e.m. of three independent experiments. * p < 0.01 compared with TLR2/HEK293 cells for SD 12 h without R6A treatment group. ** p < 0.01 compared with TLR2/HEK293 cells for SD 12 h with 10 μM R6A treatment group. # p < 0.01 compared with TLR2/HEK293 cells for SD 24 h without R6A treatment group. ## p < 0.01 compared with TLR2/HEK293 cells for SD 24 h with 10 μM R6A treatment group.

Figure 4

Role of R6A in TLR2-mediated apoptosis. We treated HEK293 and TLR2/HEK293 cells with or without 100μM of R6A. After 24 h treatment, the cells were subjected to serum deprivation (SD) for 0, 12, and 24 h, respectively. Apoptotic cells (dark cells) were determined by TUNEL assay. Photographs of representative TUNEL-stained cells are shown at the top. Magnification 200 ×. The bar graph at the bottom shows the percentage of apoptotic cells. Results represent mean ± s.e.m. of three independent experiments. * p < 0.01 compared with TLR2/HEK293 cells for SD 12 h without R6A treatment group. ** p < 0.01 compared with TLR2/HEK293 cells for SD 24 h without R6A treatment group.

Figure 5

Effect of R6A on the levels of phospho-Akt in TLR2/HEK293 cells following serum deprivation. HEK293 and TLR2/HEK293 cells were treated with R6A at 0, 10μM, or 100μM. At 24 h after treatment, the cells were employed to serum deprivation (SD) for 0, 12, and 24 h, respectively. The levels of phospho-Akt and total Akt were determined by Western blot with specific antibodies. Representative results of phospho-Akt and total Akt immunobloting are shown at the top of each pane. * p < 0.05 compared with TLR2/HEK293 cells for SD 12 h without R6A treatment group. ** p < 0.05 compared with TLR2/HEK293 cells for 12 SD with 10 μM R6A treatment group. # p < 0.01 compared with TLR2/HEK293 cells for SD 24 h without R6A treatment group. ## p < 0.01 compared with TLR2/HEK293 cells for SD 24 h with 10 μM R6A treatment group.

Figure 6

Inhibition of TLR2 by R6A inhibits serum deprivation-reduced the levels of phospho-GSK3β. We treated HEK293 and TLR2/HEK293 cells with R6A at 0, 10μM, or 100μM. After 24 h treatment, the cells were subjected to serum deprivation (SD) for 0, 12, and 24 h, respectively. The cells were harvested and the levels of phospho-GSK3β and total GSK3β were determined by Western blot with specific antibodies. Representative results of phospho-GSK3β and total GSK3β immunobloting are shown at the top of each pane. Results represent mean ± s.e.m. of three independent experiments. * p < 0.05 compared with TLR2/HEK293 cells for SD 12 h without R6A treatment group. ** p < 0.05 compared with TLR2/HEK293 cells for 12 h with 10 μM R6A treatment group. # p < 0.05 compared with TLR2/HEK293 cells for SD 24 h in control group (without R6A treatment group). ## p < 0.05 compared with TLR2/HEK293 cells for 24 h with 10 μM R6A treatment group.