Fig 1.
Cyclic stretch changed the morphology of the human PDL cells.
(a) non-stretching control. Cells aligned multidirectionally. (b) 6 h stretched cells. (c) 24 h stretched cells. (b-c) Cells were elongated and prone to be parallel to each other, with their long axis aligned perpendicularly to the force vector. 24 h stretched cells were altered more significantly. Black arrow showed the stretching direction.
Fig 2.
Cyclic stretch for 6 and 24 h induced apoptosis of different stages.
(a-c) Dot plots of Annexin V and PI staining were obtained from flow cytometry. The lower right quadrant (Annexin V+/PI-) reflects cells at early stage of apoptosis, while the upper right quadrant (Annexin V+/PI+) indicates cells at late stage of apoptosis. (d) Apoptotic rates in each group were analyzed. (a) non-stretching control. (b) 6 h stretched cells. (c) 24 h stretched cells. (d) The apoptotic rates (including the early and the late apoptosis) increased in the 6 and 24 h stretched groups (P<0.01 VS non-stretching control). The apoptotic rates in the 6 h stretched cells increased mainly due to the up-regulation of the early apoptosis (P<0.01 VS non-stretching control), and 24 h stretch further raised the late apoptotic rate apparently (P<0.01 VS 6 h stretch). Bars represent standard deviations (n = 3). **: P<0.01 VS non-stretching control; ##: P<0.01 VS 6 h stretch.
Fig 3.
Cyclic stretch induced the expression of activated caspase-3, -7, -8 and -9.
Western Blot results for the protein level of caspase-3, -7, -8 and -9. (i-iv) Mean expression levels of the proteins normalized to GAPDH, from three independent experiments, in response to 6 and 24 h stretch loading. (a-d) Representative protein bands of Western Blot experiments. The protein level of the cleaved caspase-3 (P<0.05) and the cleaved caspase-7 (P<0.01) and up-regulation of the pro-caspase-7 (P<0.01) were detected after 24 h stretch. The up-regulation of the cleaved caspase-8 (P<0.05), the cleaved caspase-9 (6 h stretch: P<0.05; 24 h stretch: P<0.01), the pro-caspase-8 (6 h stretch: P<0.05; 24 h stretch: P<0.01) and the pro-caspase-9 (P<0.01) were detected in both 6 and 24 h stretched cells. Bars represent standard deviations (n = 3). **: P<0.01 VS non-stretching control; *: P<0.05 VS non-stretching control.
Fig 4.
Cyclic stretch increased the activity of caspase-8 and caspase-9.
The results of activity analysis of caspase-8 and -9. Compared with the non-stretching control, the caspase-8 (P<0.05) and the caspase-9 (P<0.01) activities in human PDL cells in response to 20% cyclic stretch for 6 or 24 h increased significantly. Cyclic stretch for 24 h further increased the activity of caspase-9, comparing with 6 h stretch (P<0.01). Bars represent standard deviations (n = 3). **: P<0.01 VS non-stretching control; *: P<0.05 VS non-stretching control; ##: P<0.01 VS 6 h stretch.
Fig 5.
Inhibition of caspase-8 and caspase-9 suppressed the apoptotic rate.
The results of apoptotic analysis after inhibiting either caspase-8 or caspase-9 or both of them. (a-f) Dot plots of Annexin V and PI staining. (i, ii) Apoptotic rates in each group were calculated. In 6 h stretched cells, the apoptotic rates (including the early and the late apoptosis) were identified no statistical difference by using either caspase-8 inhibitor or caspase-9 inhibitor (P>0.05). However, inhibition of both caspase-8 and caspase-9 significantly reduced the apoptotic rates in 6 h stretched cells (P<0.01). In 24 h stretched cells, the apoptotic rate was significantly down-regulated by using caspase-9 inhibitor (P<0.01), while showed no change by using caspase-8 inhibitor (P>0.05). Inhibition of both caspase-8 and caspase-9 also significantly reduced the apoptotic rate in 24 h stretched cells (P<0.01). **: P<0.01 VS non-inhibiting cells.
Fig 6.
Inhibition of caspase-8 and caspase-9 suppressed the protein level of the cleaved caspase-3.
Western Blot results after inhibiting either caspase-8 or caspase-9 or both of them. (i, ii) Representative protein bands of Western Blot experiments. (a, b) Mean expression levels of the proteins normalized to GAPDH, from three independent experiments. In 6 h stretched cells, the activation of caspase-3 were identified no statistical difference by using either caspase-8 inhibitor or caspase-9 inhibitor (P>0.05). However, inhibition of both caspase-8 and caspase-9 significantly reduced the protein level of the cleaved caspase-3 in 6 h stretched cells (P<0.01). In the 24 h stretched cells, caspase-9 inhibitor alone reduced the protein level of the cleaved caspase-3 (P<0.01), while only using caspase-8 inhibitor had no inhibitory effect on the protein level of the cleaved caspase-3 (P>0.05). Inhibition of both caspase-8 and caspase-9 also significantly reduced the protein level of the cleaved caspase-3 in 24 h stretched cells (P<0.01). Bars represent standard deviations (n = 3). **: P<0.01 VS non-inhibiting cells.
Fig 7.
Schematic of the apoptotic pathway in the 6 and 24 h stretched cells.
The red and the black arrows showed the apoptotic pathways in the 6 and 24 h stretched cells, respectively. After 6 h stretch, the extrinsic and the intrinsic pathways contributed equally to the stretch-induced apoptosis. The cleaved caspase-8 was capable of directly activating caspase-3. After 24 h stretch, the caspase-3 was activated both directly by caspase-9 through the intrinsic pathway and indirectly by caspase-8 in a mitochondrion-dependent manner via caspase-9.