Triptolide inhibits proliferation, differentiation and induces apoptosis of osteoblastic MC3T3‑E1 cells

Abstract

Ankylosing spondylitis (AS) is characterized by the formation of bony spurs. Treatment of the resulting ankylosis, excessive bone formation and associated functional impairment, remain the primary therapeutic aims in research regarding this condition. Triptolide is the primary active component of the perennial vine Tripterygium wilfordii Hook. f., and has previously been demonstrated to exert anti‑tumor activities including inhibition of cell growth and the induction of apoptosis, however, the effect of triptolide on osteoblasts remains to be elucidated. In the present study, the MC3T3‑E1 mouse osteoblast cell line was treated with differing concentrations of triptolide for various intervals. Cell proliferation was detected using the bromodeoxyuridine assay, cell cycle and apoptosis were measured by flow cytometry, nuclear apoptosis was observed by Hoechst staining and associated proteins were determined via western blot analysis. The cells were then further incubated with osteogenic induction medium supplemented with triptolide for 7 or 12 days and the differentiation to osteoblasts was examined by picrosirius staining, observation of alkaline phosphatase activity and a calcium deposition assay. It was demonstrated that treatment with triptolide significantly inhibited osteoblast proliferation and induced cell cycle arrest and apoptosis of the osteoblasts. Furthermore, treatment with triptolide reduced collagen formation, alkaline phosphatase activity and calcium deposition. The present study demonstrated an inhibitory effect of triptolide on osteoblast proliferation and differentiation, and therefore suggests a potential therapeutic agent for the treatment of AS in the future.

Figure 1.

Triptolide inhibits proliferation of MC3T3-E1 cells. (A) Cell proliferation was detected by the Bromodeoxyuridine assay; n=6. (B) The expression of PCNA was detected by western blotting at 72 h of triptolide treatment; n=3, representative protein bands are presented. Data are expressed as the mean ± standard deviation. *P<0.05; **P<0.01 vs. 0 nM triptolide treatment group. OD, optical density; PCNA, proliferating cell nuclear antigen.

Figure 2.

Triptolide induces cell cycle arrest of MC3T3-E1 cells. (A) Cells were incubated with different concentrations of triptolide for 72 h. Cell cycle was measured by flow cytometry; n=3. The protein expression levels of (B) cyclin D1 and (C) cyclin E were detected by western blotting; n=3. Data are expressed as the mean ± standard deviation. *P<0.05; **P<0.01 vs. 0 nM triptolide treatment group.

Figure 3.

Triptolide induces apoptosis of MC3T3-E1 cells. (A) Cells were incubated with increasing concentrations of triptolide for 72 h and apoptotic nuclei were observed via Hoechst staining. The proportion of apoptotic cells was detected by flow cytometry and presented as (B) a representative image and (C) quantification of results. The expression of (D) Bcl-2, (E) Bax and (F) cleaved caspase 3 was determined by western blotting. All experiments were repeated in triplicate. Data are presented as the mean ± standard deviation. *P<0.05; **P<0.01 vs. 0 nM triptolide treatment group. Bcl-2, B cell lymphoma; Bax, Bcl-2 associated X protein.

Figure 4.

Triptolide suppresses differentiation of MC3T3-E1 cells. Cells were incubated in osteogenic induction medium supplemented with different concentrations of triptolide. (A) Collagen formation at day 7 was examined by picrosirius staining. (B) Alkaline phosphatase activity and (C) calcium deposition were detected at day 12. Experiments were repeated at least three times. Data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 vs. 0 nM triptolide treatment group.