2Department of Biology, Changzhi Medical College, 046000 Changzhi, Shanxi, China
3Changzhi Medical College, 046000 Changzhi, Shanxi, China
4Department of Pharmacology, Changzhi Medical College, 046000 Changzhi, Shanxi, China
# These authors contributed equally to this study.
* To whom correspondence should be addressed.
Received March 15, 2019; Revised April 8, 2019; Accepted April 8, 2019
Resveratrol has been shown to stimulate differentiation of osteoblastic MC3T3-E1 cells in vitro; however, the mechanisms underlying the anabolic effect of resveratrol on osteoblasts remain largely unknown. Our study was aimed to investigate the molecular mechanism of resveratrol-induced differentiation of MC3T3-E1 cells. MC3T3-E1 cells were treated for 8 days with different concentrations of resveratrol (10–8-10–6 M) and 10–6 M cyclosporine A (CsA), a specific inhibitor of the calcineurin/NFAT pathway. According to the results of pilot studies of cell proliferation and alkaline phosphatase activity, 10–7 M concentration of resveratrol was used in subsequent experiments. The levels of mRNA expression of the osteosis-related genes CaN, NFATc1, and Runx2 were analyzed by real-time RT-PCR; the levels of the corresponding proteins were estimated by Western blot analysis. Resveratrol upregulated expression of the CaN, NFATc1, and Runx2 genes at both mRNA and protein levels compared to the control group (p < 0.05), while CsA reduced the effects of resveratrol (p < 0.05). Using immunohistochemical staining, we showed that resveratrol induced NFATc1 accumulation in the cell nuclei, and treatment with CsA inhibited resveratrol-mediated induction of NFATc1, suggesting that the calcineurin/NFATc1 signaling pathway plays an important role in the regulatory effect of resveratrol on osteoblasts.
KEY WORDS: resveratrol, cyclosporine A, calcineurin/NFATc1, osteoblast differentiation