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  • Crocin protects against dexamethasone‑induced osteoblast apoptosis by inhibiting the ROS/Ca2+‑mediated mitochondrial pathway.

Crocin protects against dexamethasone‑induced osteoblast apoptosis by inhibiting the ROS/Ca2+‑mediated mitochondrial pathway.

Molecular medicine reports (2019-05-23)
Zhigang Nie, Shuang Deng, Lei Zhang, Sen Chen, Qiang Lu, Hao Peng
ABSTRACT

Osteoblast apoptosis has been identified as an important event in the development of glucocorticoid (GC)‑induced osteoporosis and osteonecrosis of the femoral head. Crocin, a bioactive ingredient of saffron, has been demonstrated to induce antiapoptotic effects on numerous types of cell in vitro; however, the effects of crocin on the dexamethasone (Dex)‑induced apoptosis of osteoblasts remain unclear. In the present study, the protective effects of crocin during Dex‑induced apoptosis of MC3T3‑E1 osteoblasts, and the underlying mechanisms, were investigated. MTT and Annexin V‑FITC/PI flow cytometry assays were performed to evaluate the viability and apoptosis of cells, respectively. The mitochondrial transmembrane potential, reactive oxygen species (ROS), intracellular Ca2+ levels and apoptosis‑associated protein expression were assessed via flow cytometry, fluorescence microscopy and western blotting. It was demonstrated that crocin pretreatment inhibited Dex‑induced apoptosis of osteoblasts in a dose‑dependent manner. Crocin reversed Dex‑induced decreases in the mitochondrial transmembrane potential, and increases in ROS and intracellular Ca2+ levels. Furthermore, crocin upregulated the expression levels of B‑cell lymphoma-2 (Bcl‑2) and mitochondrial cytochrome c (Cyt C), and downregulated those of cleaved caspase‑9, cleaved caspase‑3, Bcl‑2‑associated X protein and cytoplasmic Cyt C. N‑acetylcysteine, a ROS inhibitor, and 1,2‑bis(2‑aminophenoxy)ethane‑N,N,N',N'‑tetraacetic acid, a calcium chelator, attenuated Dex‑induced osteoblast apoptosis, whereas H2O2 and ionomycin, a calcium ionophore that increases intracellular calcium levels, reversed the antiapoptotic effects of crocin on Dex‑treated osteoblasts. These results indicated that crocin may protect osteoblasts from Dex‑induced apoptosis by inhibiting the ROS/Ca2+‑mediated mitochondrial pathway, thus suggesting that crocin has potential value as a treatment for GC‑induced bone diseases.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, ≥96.0% (HPLC)
Sigma-Aldrich
N-Acetyl-L-cysteine, Sigma Grade, ≥99% (TLC), powder
Sigma-Aldrich
Dimethyl sulfoxide-d6, "100%", 99.96 atom % D
Millipore
Hydrogen peroxide solution, 3%, suitable for microbiology
Sigma-Aldrich
Crocin, for microscopy
Sigma-Aldrich
Thiazolyl Blue Tetrazolium Bromide, 98%
Sigma-Aldrich
Amyloid Protein Non-Aβ Component, ≥80% (HPLC)