Role of Teriparatide in Glucocorticoid-induced Osteoporosis through Regulating Cellular Reactive Oxygen Species

Abstract

Objective: To determine the signaling pathways mediated by teriparatide in MLO-Y4 cell lines based on the evaluation of reactive oxygen species (ROS) through AKT pathways, which regulate apoptosis of bone cells.

Methods: We performed the DCFH-DA assay to investigate the role of ROS in MLO-Y4 cells caused by dexamethasone (Dex). Four groups were included: Dex group, Dex+NAC, Dex+ teriparatide group and control group (without any dispose). Real-time reverse transcriptase polymerase chain reaction was used to test the SOD2 and Cat mRNA expression. Western blot (WB) was used to investigate the AKT and caspase-3 protein expression. A Cell Counting Kit-8 (CCK-8) assay test was conducted to explore the cell viability, and we also studied the apoptosis through western blot assay. A glucocorticoid-induced osteoporosis (GIOP) model was used to confirm the anti-ROS and anti-apoptosis ability of teriparatide.

Results: The CCK-8 assay revealed that Dex reduced the proliferative capability of cells significantly, whereas incubation with teriparatide resulted in a remarkable increase in the proliferation of osteocytes. In addition, teriparatide can rescue the effect of inhibiting cell proliferation due to Dex treatment. Immunofluorescence analysis showed that ROS levels increased in Dex-treated MLO-Y4 cells when compared with control groups. However, the Dex+Teriparatide group showed less ROS when compared with the Dex group. The expression of Sod2 and Cat, two antioxidant enzymes crucial for ROS elimination, was decreased in the Dex group, indicating a defect of the enzymatic antioxidant system. Compared to the Dex group, incubation with teriparatide resulted in a significant decrease in caspase-3 level; when compared with the control group, the caspase-3 level was not significantly different, indicating that teriparatide can rescue apoptosis during Dex exposure. Moreover, teriparatide promotes the expression of AKT, and rescues the apoptosis effect caused by Dex. The results of immunofluorescence also showed that Akt was highly expressed in the teriparatide group when compared with the Dex group. The microstructural parameters Tb.Th, BV/TV, and Tb.N in the methylprednisolone (MPS) group were markedly reduced compared with the control group, but additional treatment with teriparatide could remarkably reverse the methylprednisolone-induced reduction of these parameters. Moreover, the parameter Tb.Sp was significantly increased in the methylprednisolone group compared to the control group, and this increase could be inhibited by teriparatide.

Conclusions: Teriparatide can reduce the cellular ROS level caused by glucocorticoids to facilitate the proliferation of osteocytes through activating the AKT pathway. Meanwhile, the activated AKT can inhibit the activity of proteolytic enzyme caspase-3 and prevent the activation of apoptosis cascade.

Keywords: AKT pathway; Apoptosis; Glucocorticoid-induced osteoporosis; Teriparatide.