Bergapten prevents lipopolysaccharide mediated osteoclast formation, bone resorption and osteoclast survival

Abstract

Purpose: This study was designed to investigate the potential effect of bergapten on lipopolysaccharide (LPS)-mediated osteoclast formation, bone resorption and osteoclast survival in vitro.

Methods: After osteoclast precursor RAW264.7 cells were treated with bergapten (5, 20, 40 μmol/L) for 72 hours in the presence of LPS (100 ng/ml), osteoclastogenesis was identified by tartrate-resistant acid phosphatase (TRAP) staining, and the number of TRAP-positive multinucleated cells [TRAP(+)MNCs] per well were counted. To investigate the effect of bergapten on osteoclastic bone resorption, RAW264.7 cells were treated with bergapten for six days in the presence of LPS, and the area of bone resorption was analyzed with Image Pro-Plus. Next, we examined apoptosis of RAW264.7 cells after bergapten incubation for 48 hours by flow cytometer using annexin V/propidium iodide (PI) double labeling. Finally, osteoclast survival was observed by Hoechst 33342 labeling and Western blotting after bergapten treatment for 24 hours.

Results: Data showed that bergapten (5-40 μmol/L) dose-dependently inhibited LPS-induced osteoclast formation and bone resorption. Treatment with bergapten triggered apoptotic death of osteoclast precursor RAW264.7 cells in a dose-dependent manner. Furthermore, bergapten significantly reduced the survival of mature osteoclast, as demonstrated by emergence of apoptotic nuclei and activation of apoptotic protein caspase 3/9.

Conclusions: These findings suggest that bergapten effectively prevents LPS-induced osteoclastogenesis, bone resorption and survival via apoptotic response of osteoclasts and their precursors. The study identifies bergapten as an inhibitor of osteoclast formation and bone resorption and provides evidence that bergapten might be beneficial as an alternative for prevention and treatment of inflammatory bone loss.

Fig. 1

Chemical structure of bergapten (5-methoxypsoralen, C₁₂H₈O₄, M = 216.19)

Fig. 2

Bergapten inhibited lipopolysaccharide (LPS)-induced osteoclast formation (n = 4). A RAW264.7 cells were treated with or without bergapten (1–100 μmol/L) for 24 h, 48 h and 72 h, and cell viability was measured by thiazolyl blue tetrazolium bromide (MTT) assay. B After RAW264.7 cells were treated with or without bergapten (5–40 μmol/L) in the presence of LPS (100 ng/ml) for 72 h, cells were stained for tartrate-resistant acid phosphatase (TRAP), and TRAP(+) multinucleated cells (MNCs) containing more than three nuclei were counted as osteoclasts under I X70 microscope. C Data are expressed as the percentage of TRAP(+)MNCs/well (means ± standard deviation). a p < 0.05 compared with control group; b p < 0.05 compared with LPS-treated group. Bar = 5 μm

Fig. 3

Bergapten prevented lipopolysaccharide (LPS)-induced bone resorption (n = 4). RAW264.7 cells cultured on bone slices were treated with or without bergapten (5–40 μmol/L) for 6 days in the presence or absence of LPS (100 ng/ml). A The slices were stained with tartrate-resistant acid phosphatase (TRAP), and resorption pits were observed under an Olympus I × 70 light microscope. B Total areas of resorption pits were analysed with Image Pro-Plus version 4.0, and data are expressed as means ± standard deviation. a p < 0.05 compared with control group; b p < 0.05 compared with LPS-treated group. Red arrows resorption pits. Bar = 10 μm

Fig. 4

Bergapten triggered apoptotic death of osteoclast precursor RAW264.7 cells by flow cytometric analysis using annexin V/propidium iodide (PI) double staining (n = 3). A RAW264.7 cells were treated with or without bergapten (5–40 μmol/L) in the presence or absence of lipopolysaccharide (LPS) (100 ng/ml) for 48 h and stained with fluorescein isothiocyanate (FITC)-conjugated annexin V (5 μg/ml) and PI (10 μg/ml). Cell apoptosis or necrosis was analysed by flow cytometry. The units of the Y and X axes are fluorescence intensity. The assay identifies normal cells as PI negative and annexin V (FITC) negative, apoptotic cells as PI negative and annexin V positive and necrotic cells as PI positive and annexin V positive. Cells in the lower left (LL) region were PI negative and annexin V negative, in the lower right (LR) region PI negative and annexin V positive and in upper right (UR) region PI positive and annexin V positive. B Data obtained from a are expressed as apoptosis (%) or necrosis (%). a p < 0.05 compared with control group; b p < 0.05 compared with LPS-treated group

Fig. 5

Bergapten reduced the survival rate of mature osteoclasts (n = 5). RAW264.7 cells were cultured on glass coverslips in 35-mm dishes in the presence of lipopolysaccharide (LPS) (100 ng/ml) for 72 h to obtain mature osteoclasts. Then, osteoclasts were treated with or without bergapten for 24 h and A stained with Hoechst 33342. B Data from A are shown as apoptosis of mature osteoclasts (means ± standard deviation). C After mature osteoclasts were treated with bergapten (5, 20, 40 μmol/L) for 24 h, total protein was extracted and analysed by Western blots with specific antibodies to detect the level of procaspase-3, cleaved caspase-3, procaspase-9 and cleaved caspase-9. a p < 0.05 compared with LPS-treated group