Resveratrol Synergistically Promotes BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

Figure 5

Resveratrol promotes BMP9-induced bone formation by activating the AMPK/autophagy signaling pathway. (a) Cell lysates were collected after infection with Ad-GFP or Ad-BMP9 with or without resveratrol (50 μM) for 48 h. The protein levels of p-AMPK and t-AMPK were measured by WB analysis. (b) Autophagy was induced by adding 50 μM resveratrol to Ad-GFP or Ad-BMP9. The LC3, Beclin1, and p62 autophagy markers were assessed by WB analysis. (d, e) siAMPKα inhibited the ALP of BMP9 and BMP9+resveratrol-induced osteogenic differentiation of C3H10T1/2 cells. After pretreatment of C3H10T1/2, cells with siAMPKα followed by osteogenic differentiation with BMP9 or BMP9+resveratrol for 7 d, ALP staining (c) and ALP activity assays (d) were performed to measure the osteogenic ability. (e, f) The SP-1 autophagy inhibitor suppressed the osteogenic differentiation in the BMP9 and BMP9+resveratrol groups. Cells were treated with the combination of SP-1 and Ad-BMP9 or Ad-BMP9+resveratrol for 7 d, and ALP staining (e) and ALP activity assays (f) were performed to measure the osteogenic induction in C3H10T1/2 cells. (g) WB analysis showed that pretreatment with siAMPKα decreased the AMPK level. C3H10T1/2 cells were transfected with siAMPKα 24 h prior to treatment with Ad-BMP9 or Ad-BMP9+resveratrol. After another 48 h, protein lysates were collected to measure AMPK levels by WB analysis. (h) WB analysis showed that autophagy was inhibited by SP-1. After treatment of C3H10T1/2 cells with SP-1 combined with Ad-BMP9 or Ad-BMP9+resveratrol for 48 h, cell lysates were collected to measure the protein levels of LC3, Beclin1, and p62 by WB analysis. , , and compared to the control group. ^#, ^##, and ^### compared with the BMP9 group. ^&, ^&&, and ^&&& compared to the BMP9+resveratrol group.

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