Fig 1.
Diagrammatic representation of the device used for cyclic compressive stress.
Fig 2.
Effect of different levels of cyclic compressive stress on apoptosis in osteoblasts.
Osteoblasts were treated with different levels of mechanical stress (0.15 Hz×4cm,0.15Hz×8cm, 0.3Hz×8cm, 0.6Hz×8cm, 0.6Hz×16cm)(0 as control) for 30min and then cultured for another 24h. Cells were harvested and measured for apoptosis and caspase-3 activity. A. The apoptotic cells were determined by TUNEL staining. Scale bar = 50μm. B. Quantitative analysis of apoptotic cells in each group. *P<0.05 versus control group; #P<0.05 versus 0.3Hz×8cm group; &P<0.05 versus 0.6Hz×8cm group. C. Quantitative analysis of caspase-3 activity in each group. *P<0.05 versus control group; #P<0.05 versus 0.15Hz×4cm group; &P<0.05 versus 0.3Hz×8cm group; $P<0.05 versus 0.6Hz×8cm group. Each value is presented as mean ± SD of three independent experiments and data of the treatment group was expressed as fold change vs. that of control group (labeled as “1.00”).
Fig 3.
Activation of MAPKs and Akt in response to different levels of mechanical stress.
Osteoblasts were stimulated with different levels of mechanical stress for 30 min. The kinase activity of MAPKs and PI3K/Akt were detected by western bolt using p-ERK, p-JNK, p-p38 and p-Akt antibodies. (A) The expression levels of MAPKs and Akt examined by western blot. (B) Quantitative analysis of activations of MAPKs and Akt. *P<0.05 versus control group; #P<0.05 versus 0.15Hz×4cm group; &P<0.05 versus 0.15Hz×8cm group. Each value is presented as mean ± SD of three independent experiments and data of the treatment group was expressed as fold change vs. that of control group (labeled as “1.00”).
Fig 4.
Effect of mechanical stress and specific inhibitors on phosphorylation of MAPKs and Akt.
Osteoblasts were treated with mechanical stress of 0.6Hz×8cm(0 as control) for 30min in the presence or absence of SP600125, SB203580, PD98059 and LY294002. (A) Western blot analysis of the protein levels of p-ERK, p-JNK, p-p38 and p-Akt. (B) Quantitative analysis of the ratio of p-ERK/t-ERK, p-JNK /t-JNK, p-p38/t-p38 and p-Akt/t-Akt. *P<0.05 versus control group; #P<0.05 versus loaded group. Each value is presented as mean ± SD of three independent experiments and data of the treatment group was expressed as fold change vs. that of control group (labeled as “1.00”).
Fig 5.
Effect of large-magnitude mechanical stress-induced cell apoptosis in osteoblasts pretreated with or without specific inhibitors.
Osteoblasts were pretreated with or without specific inhibitors for 1 h and then treated with mechanical stress of 0.6Hz×8cm (0 as control) for 30min and then cultured for a further 24h. (A) Apoptosis assayed by flow cytometry using annexin-V/PI double staining. (B) Quantitative analysis of apoptotic osteoblasts in each group. *P<0.05 versus control group; #P<0.05 versus loaded group. Data are presented as mean ± SD of three independent experiments.
Fig 6.
Effect of large-magnitude mechanical stress on apoptosis-related proteins expression in osteoblasts.
Osteoblasts were treated with large-magnitude mechanical stress (0.6Hz×8cm) for 30min and then cultured for a further 24h. (A) Cell lysates were analyzed by western blot. (B) Quantitative analysis of expression of p-Bad, Bax, Bcl-2 and caspase-3. *P<0.05 versus control group. Each value is presented as mean ± SD of three independent experiments and data of the treatment group was expressed as fold change vs. that of control group (labeled as “1.00”) after normalized to β-actin.
Fig 7.
Apoptosis-related proteins expression in osteoblasts pretreated with or without JNK and Akt inhibitors respectively.
Osteoblasts were pretreated with or without specific inhibitors for 1 h and then treated with mechanical stress of 0.6Hz×8cm (0 as control) for 30min and then cultured for a further 24h. (A) Cell lysates were analyzed by western blot. (B) Quantitative analysis of expression of p-Bad, Bax, Bcl-2 and caspase-3. *P<0.05 versus stress group; #P<0.05 versus stress+LY294002 group. Each value is presented as mean ± SD of three independent experiments and data of the treatment group was expressed as fold change vs. that of control group (labeled as “1.00”) after normalized to β-actin.
Fig 8.
Signaling pathways in regulating the osteoblasts apoptosis.
The fine balance of apoptotic and anti-apoptotic effect of mechanical stress on osteoblasts is achieved by the actions of critical signaling pathways and key signaling proteins such as JNK and Akt.