2023, Vol. 27 ›› Issue (31): 5015-5021

Role of pyruvic acid in osteoclast differentiation

Liu Guanjuan1, Xia Qianxi1, Song Na1, Huo Hua1, Hong Wei2, Liao Jian1   

Abstract: BACKGROUND: Glycolysis is essential for osteoclast differentiation and bone resorption of mature osteoclasts is mainly dependent on glycolysis. Pyruvic acid, as the end product of glycolysis, plays an important role in the metabolism of the three nutrients.
OBJECTIVE: To observe the effect of pyruvic acid on osteoclast differentiation.
METHODS: The toxicity of different concentrations of pyruvic acid (0.1, 1, 10, 20, 30, 50 mmol/L) to RAW264.7 cells was detected by cell counting kit-8 assay, and the safe concentration of pyruvic acid was selected. RAW264.7 cells were grouped into interventions: blank control group was cultured with a complete medium (α-MEM medium containing volume fraction 10% fetal bovine serum and 1% double antibodies); control group was cultured with a complete medium containing nuclear factor κB receptor activating factor ligand, and experimental group was cultured with a complete medium with nuclear factor κB receptor activating factor ligand and different concentrations of pyruvic acid, followed by tartrate-resistant acid phosphatase staining, cytoskeletal F-actin staining, RT-qPCR assay, western blot assay and bone resorption lacuna assay.
RESULTS AND CONCLUSION: (1) The maximum half inhibitory concentration of pyruvic acid on RAW264.7 cells was 8.923 mmol/L, and 0.1, 1, 5 mmol/L were selected as the safe concentrations for subsequent experiments. (2) Tartrate-resistant acid phosphatase staining revealed that 1 mmol/L pyruvic acid promoted the differentiation of RAW264.7 cells into osteoclasts. (3) F-actin staining showed that 1 mmol/L pyruvic acid promoted the formation of osteoclast F-actin ring, while 5 mmol/L pyruvic acid did not interfere with the formation of osteoclast F-actin ring. (4) RT-qPCR assay indicated that pyruvic acid within the safe concentration range had no significant effect on the relative mRNA expression of osteoclast differentiation related genes nuclear factor of activator T-cells and tartrate-resistant acid phosphatase. (5) Western blot assay showed that 0.1 and 1 mmol/L pyruvic acid within the safe concentration range had no significant effect on the protein expression of nuclear factor of activator T-cells, while 5 mmol/L pyruvic acid inhibited the protein expression of nuclear factor of activator T-cells, and 0.1, 1, and 5 mmol/L pyruvic acid promoted the expression of osteoclast differentiation related protein c-Fos. (6) 1 mmol/L pyruvic acid promoted the formation of bone resorption lacunae in osteoclasts. (7) To conclude, 1 mmol/L pyruvic acid can promote the differentiation of receptor activator for nuclear factor-κB induced RAW264.7 cells into osteoclasts in vitro.

Key words: osteoclast, glycolysis, pyruvic acid, bone metabolism, bone homeostasis