Tetrandrine inhibits osteoclast differentiation by inducing autophagy

doi:10.12307/2023.531

Abstract

Abstract: BACKGROUND: Previous studies have shown that tetrandrine can inhibit the proliferation of tumor cells by activating AMPK signaling pathway to induce autophagy. However, there are few studies on tetrandrine induced autophagy and differentiation of osteoclasts.
OBJECTIVE: To explore the effect of tetrandrine at different concentrations on autophagy during osteoclast differentiation, and analyze whether inducing autophagy of osteoclasts can inhibit their differentiation.
METHODS: Part one: Recombinant mouse macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand cytokines were used to induce mouse RAW264.7 macrophages into osteoclasts. Mouse RAW264.7 macrophages were divided into five groups: blank group (complete medium), positive control group (treated by two cytokines), and low-, medium-, and high-concentration drug groups (treated by two cytokines and added 0.1, 0.5, and 1.0 μmol/L tetrandrine, respectively). Tartrate resistant acid phosphatase staining method was used to detect the number of mature osteoclasts. Western blot assay and RT-PCR were utilized to detect the relative expression levels of proteins of Cathepsin K, autophagy microtubule-associated protein 1 light chain 3-II, and autophagy substrate ubiquitin-binding protein (p62) at protein and mRNA levels, respectively. Monodansyl cadaverine staining was used to detect the number of autophagosomes in osteoclasts. Part two: The concentration of tetrandrine (1.0 μmol/L) with the best inhibitory effect on osteoclast differentiation was selected for regression verification. Cells were divided into positive control group (treated by two cytokines), autophagy inhibitor group (bafloamycin), drug intervention group (tetrandrine), and drug intervention+authophage inhibitor group. Tartrate resistant acid phosphatase staining was used to detect the number of mature osteoclasts. Western blot assay was utilized to detect the relative protein expression level of Cathepsin K.
RESULTS AND CONCLUSION: Tetrandrine at a certain concentration inhibited the differentiation of osteoclasts and the inhibitory effect increased with the increase of drug concentration. Tetrandrine at a certain concentration could stimulate the expression of autophagy markers, reduce the accumulation of autophagy specific substrates during osteoclast differentiation, and stimulate the formation of autophagosomes during osteoclast differentiation. After using autophagy inhibitor, the effect of tetrandrine on inhibiting osteoclast differentiation was significantly affected. In conclusion, tetrandrine at a certain concentration can inhibit the differentiation of osteoclasts by activating autophagy.

Key words: tetrandrine, osteoclast, autophagy, osteolysis, microtubule-associated protein 1 light chain 3-II, CTSK

CLC Number:

Zhang Bowen, Li Mei, Zhang Jinning, Yang Tianxiang, Cheng Mengqi, Chen Desheng. Tetrandrine inhibits osteoclast differentiation by inducing autophagy[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(28): 4473-4479.

Figures/Tables 9

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