| Aim: To investigate the effect of Tacrolimus (FK506), an immunosuppressive agent, on cell proliferation and osteoblastic differentiation of primary human bone marrow-derived mesenchymal stem cells (HBMSCs).Methods: Primary HBMSCs were cultured in osteogenic differentiation medium, which consists of phenol red-free a-MEM plus 10% FBS (dextran-coated charcoal stripped, DCS) supplemented with 10 nmol·L-1 dexamethasone, 50 mg·L-1 ascorbic acid and 10 mmol·L-1β-glycerophosphate for inducing osteoblastic differentiation. The cellsd were treated with FK506 (0.012.5μmol·L-1) alone or in combination with estradiol (E2, 0.01μmol-L-1) or resveratrol (RSVL, 1μmol-L-1) together. The cell proliferation was measured by BrdU incorporation. The osteoblastic differentiation in HBMSCs culture was assessed by culture duration-dependent increments in cellular alkaline phosphatase (ALP) activity and calcium deposition. Nitric oxide (NO) production and calcineurin (CAN) activity level were measured by using commercial NO kit and CaN cellular assay kit, respectively. In addition, Western blotting was employed to determine core binding factor al (Cbfa1/RUNX2) gene expression after treament.Results: FK506 (0.012.5μmol·L-1) resulted in a dose-dependent decrease in cell proliferation as measured by decreased BrdU incorporation, and inhibited osteoblastic maturation as assessed by reductions in cellular alkaline phosphatase (ALP) activity and calcium deposition in HBMSC cultures. FK506 also resulted in a dose-dependent decrease in NO formation. FK506 (1μmol·L-1) markedly reduced CaN activity as well as RUNX2/Cbfa1 gene expression. However, E2 (0.01μmol·L-1) or RSVL (1μmol·L-1) fail to reverse the FK506 (1μmol·L-1)-induced inhibitions in the osteoblastic differentiation of HBMSCs cultures.Conclusion: FK506 inhibites cell proliferation and osteoblastic differentiation of HBMSC cultures through CaN/NO/RUNX2 pathway. |
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