Resveratrol Protects Osteoblasts Against Dexamethasone-Induced Cytotoxicity Through Activation of AMP-Activated Protein Kinase

Abstract

Purpose

Glucocorticoids are used for the treatment of inflammatory diseases, but glucocorticoid treatment is associated with bone damage. Resveratrol is a phytoalexin found in many plants, and we investigated its protective role on dexamethasone-induced dysfunction in MC3T3-E1 cells and primary osteoblasts.

Materials and Methods

MC3T3-E1 cells and primary osteoblasts were treated with dexamethasone in the presence/absence of different doses of resveratrol for 24 or 48 h. Then, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium (MTT) and lactate dehydrogenase (LDH) assays were used to evaluate cell viability. Apoptosis was analyzed by a flow cytometry. An alkaline phosphatase (ALP) activity assay and Alizarin Red S staining were used to study osteoblast differentiation. Expression of osteoblast-related genes was measured by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The AMP-activated protein kinase (AMPK) signaling pathway and mitochondrial expression of superoxide dismutase were evaluated by Western blotting. Intracellular reactive oxygen species (ROS), adenosine triphosphate (ATP) content, mitochondrial-complex activity, and mitochondrial DNA content were measured to evaluate mitochondrial function.

Results

Resveratrol induced the proliferation and inhibited apoptosis of osteoblasts in the presence of dexamethasone. Resveratrol increased the ALP activity and mineralization of osteoblasts. Resveratrol also attenuated dexamethasone-induced inhibition of mRNA expression of osteogenesis maker genes, including bone morphogenetic protein-2, osteoprotegerin, runt-related transcription factor-2, and bone Gla protein. Resveratrol alleviated dexamethasone-induced mitochondrial dysfunction. Resveratrol strongly stimulated expression of peroxisome proliferator–activated receptor-γ coactivator 1α and sirtuin-3 genes, as well as their downstream target gene superoxide dismutase-2. Resveratrol induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Blockade of AMPK signaling using compound C reversed the protective effects of resveratrol against dexamethasone.

Conclusion

Resveratrol showed protective effects against dexamethasone-induced dysfunction of osteoblasts by activating AMPK signaling.

Keywords:

• resveratrol
• dexamethasone
• osteoblasts
• AMPK
• mitochondrial function

Acknowledgments

This work was supported by the Jinan Science and Technology Project (grant 201805042) and the Program of Linyi Science and Technology Innovation Development (grant 201919058).

Abbreviations

LDH, lactate dehydrogenase; ALP, alkaline phosphatase; AMPK, AMP-activated protein kinase; ROS, reactive oxygen species; BMP-2, bone morphogenetic protein 2; OPG, osteoprotegerin; Runx2, runt related transcription factor 2; BGP, bone Gla protein; PGC-1ɑ, peroxisome proliferator–activated receptor γ coactivator 1α; SIRT3, sirtuin 3; SOD2, superoxide dismutase 2; SOD1, superoxide dismutase 1; ACC, acetyl-CoA carboxylase; SIRT1, sirtuin 1; PBS, phosphate buffered saline; COX I, cytochrome c oxidase subunit I; RANKL, receptor activator of nuclear factor-κB ligand; NRF2, nuclear factor erythroid 2-related factor 2; HO1, heme oxygenase-1.

Disclosure

The authors report no conflicts of interest in this work.