The effects of Clomiphene on the proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells / 中国药理学通报

ABSTRACT

Aim: To investigate the effects of Clomiphene (CLO) on proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. Methods: Mouse BMSCs, cultured in phenol red-free, α-MEM supplemented with 10% charcoal-stripped fetal bovine serum, were added β-glycerolphosphate and Vitamin C for in-ducing osteoblastic differentiation, and treated with 0.1 nmol·L-1 to 10 nmol·L-1 CLO. The cell proliferation was measured by cell number count. The temporal sequence of osteoblastic differentiation in BMSCs was assessed by measuring alkaline phosphatase (ALP) activity and calcium deposition. Nitric oxide (NO) production was determined by measuring the stable end product of NO, nitrite, in the culture medium using commercial NO kit. Results: CLO (0.1-10 nmol·L-1) resulted in a dose-dependent increase in cell number, ALP activity and calcium deposition in the BMSCs cultures. CLO treatment was associated with elevated nitrite formation in conditioned media of BMCSs cultures. Concurrent treatment with ICI 182, 780 (0.1 μmol·L-1), an estrogen receptor antagonist, or N ω-nitro-L-arginine methylester (L-NAME, 6 mmol·L -1), a NOS inhibitor eliminated the stimulation of CLO on the proliferation and osteoblastic differentiation of BMSCs cultures, and abolished the CLO (1 nmol·L-1)-induced increase in NO production. Conclusion: Our results indicated that CLO at the range of 0.1-10 nmol·L-1 might be a estrogen-like receptor (ER) agonist, and stimulate the proliferation and osteoblastic differentiation of mouse BMSCs through ER/NO pathway.