| Summary: | Background: Osteoblasts play important roles in the process of osteogenesis and prevention of osteonecrosis. Dexamethasone (Dex), a type of glucocorticoids (GCs), induces apoptosis of osteoblasts and leads to the occurrence of non-traumatic osteonecrosis. This study aimed to explore the effects of phosphatidylinositol 3-kinase/Protein kinase 3 (PI3K/AKT) and glycogen synthase kinase 3β (GSK3β) on Dex-induced osteoblasts apoptosis. Methods: Viabilities, proliferation, and apoptosis of primary osteoblasts and pre-osteoblast MC3T3-E1 cells after Dex treatment were detected using cell counting kit-8 (CCK-8) assay, 5-bromo-2′-deoxyuridine (BrdU) incorporation assay, FITC-Annexin V/PI staining and western blotting, respectively. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to measure the intracellular reactive oxygen species (ROS) levels after Dex treatment. N-acetyl-l-cysteine (NAC) was used as ROS scavenger in this research. The expressions of PI3K/AKT and GSK3β in osteoblasts and MC3T3-E1 cells after Dex treatment were analyzed using western blotting and qRT-PCR, respectively. Then the effects of GSK3β knockdown on Dex-induced apoptosis of osteoblasts were explored. Alkaline phosphatase (ALP) activity assay was used to detect the role of Dex in regulating ALP activity. Results: Dex remarkably inhibited proliferation and induced apoptosis of osteoblasts and MC3T3-E1 cells. Dex potentially attenuated the osteoblast differentiation. The intracellular ROS levels were significantly increased after Dex treatment. Dex suppressed the activation of PI3K/AKT pathway in osteoblasts and MC3T3-E1 cells by down-regulating the expressions of p-PI3K and p-AKT. The expressions of GSK3β in osteoblasts and MC3T3-E1 cells were obviously up-regulated after Dex treatment. Knockdown of GSK3β alleviated Dex-induced osteoblast and MC3T3-E1 cell apoptosis by decreasing the expressions of Bax, cleaved-caspase 3, cleaved-caspase 9 and increasing the expression of Bcl-2. Conclusion: Our research verified that Dex induced osteoblasts apoptosis by ROS-PI3K/AKT/GSK3β signaling pathway.
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