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. 2013 Jul 19;8(7):e69403.
doi: 10.1371/journal.pone.0069403. Print 2013.

"VSports手机版" Static mechanical stress induces apoptosis in rat endplate chondrocytes through MAPK and mitochondria-dependent caspase activation signaling pathways

Dechao Kong  1 , Tiansheng Zheng, Ming Zhang, Daode Wang, Shihao Du, Xiang Li, Jiahu Fang, Xiaojian Cao
Affiliations

Static mechanical stress induces apoptosis in rat endplate chondrocytes through MAPK and mitochondria-dependent caspase activation signaling pathways (VSports)

Dechao Kong et al. PLoS One. .

Abstract

Mechanical stress has detrimental effects on cartilaginous endplate chondrocytes due to apoptosis in vivo and in vitro. In this study, we investigated the possible apoptosis signaling pathways induced by mechanical stress in cultured rat cervical endplate chondrocytes. Static mechanical load significantly reduced cell viability in a time- and load-dependent manner, as demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Chondrocyte apoptosis induced by mechanical stress was confirmed by annexin V/propidium iodide (PI) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Western blot analysis revealed that static load-induced chondrocyte apoptosis was accompanied by increased phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). The loss of mitochondrial membrane potential (ΔΨm), increased Cytochrome c release, and activated Caspase-9 and Caspase-3, indicating that the mitochondrial pathway is involved in mechanical stress-induced chondrocyte apoptosis VSports手机版. Treatment with inhibitors of JNK (SP600125), p38 MAPK (SB203580), and ERK (PD98059) prior to mechanical stimulation reversed both the static load-induced chondrocyte apoptosis and the activation of JNK, p38 MAPK, and ERK. Taken together, the data presented in this study demonstrate that mechanical stress induces apoptosis in rat cervical endplate chondrocytes through the MAPK-mediated mitochondrial apoptotic pathway. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures (VSports手机版)

Figure 1
Figure 1. Static mechanical load-induced cell death in endplate chondrocytes.
Chondrocytes were exposed to various static loads for 1, 6, 12, 24, 36, and 48 h. Cell viability was detected by using the CCK-8 assay. Each value represents the mean ± SD, n = 3. *p<0.05; **p<0.01; ***p<0.001 compared with the control group.
Figure 2
Figure 2. Static compression-induced chondrocyte apoptosis as assessed by TUNEL staining.
(A) TUNEL-positive cells (green fluorescence) were observed under a fluorescence microscope. (B) The frequency of apoptotic cells was expressed as a percentage of the total cells. Data are presented as the mean ± SD from three independent experiments. ***p<0.001 versus unloaded cells; ###p<0.001 versus loaded cells. Scale bar = 50 µm.
Figure 3
Figure 3. Effect of mechanical stress-induced cell apoptosis in endplate chondrocytes.
Chondrocytes were pretreated with or without specific inhibitors for 1 h and then incubated under 0.5 MPa for a further 24 h. (A) Apoptosis was assayed by flow cytometry using annexin-V/PI double staining. (B) The percentage of apoptotic chondrocytes loaded for 24 h. Data are presented as the mean ± SD of three independent experiments. ***p<0.001 versus unloaded cells; ###p<0.001 versus loaded cells.
Figure 4
Figure 4. Static compression-induced mitochondrial dysfunction in endplate chondrocytes.
Cells were exposed to 0.5 MPa in the presence or absence of specific inhibitors for 24 h, after which the mitochondrial membrane potential was determined by staining with the mitochondrial dye JC-1. Red fluorescence represents JC-1 aggregates formed in healthy cells with high mitochondrial membrane potential, whereas green fluorescence highlights JC-1 monomers in cells with low ΔΨm. Scale bar = 100 µm.
Figure 5
Figure 5. Effect of mechanical stress on the expression of apoptosis-related proteins.
Chondrocytes were loaded under 0.5 MPa for 24 h in the presence or absence of specific MAPK inhibitors, and the protein levels of Bax, Bcl-2, and Cytochrome c were assessed by western blot analysis. Expression of β-actin was used as an internal control. Data are presented as the mean ± SD of three independent experiments. *p<0.05; **p<0.01 versus unloaded cells, #p<0.05; ##p<0.01; ###p<0.001 versus loaded cells.
Figure 6
Figure 6. Effect of mechanical stress on the activation of caspases in endplate chondrocytes.
(A) Chondrocytes were pre-treated with or without 20 µM Ac-DEVD-CHO for 1 h and then loaded under 0.5 MPa for 24 h. The expression levels of cleaved Caspase-9 and cleaved Caspase-3 were examined by western blotting. (B) Quantitative analysis of cleaved Caspase-9 and cleaved Caspse-3. The results are expressed as the mean ± SD of three separate experiments. *p<0.05; ***p<0.001 versus control cells, ###p<0.001 versus loaded cells.
Figure 7
Figure 7. Effect of mechanical stress on phosphorylation of MAPKs.
Endplate chondrocytes were loaded (0.5 MPa) for 24 h in the presence or absence of 10 µM SP600125, 10 µM SB203580, and 20 µM PD98059. (A) Western blot analysis of the protein levels of p-JNK, p-p38 MAPK, and p-ERK. (B) Relative intensities are represented as the fold changes of the phosphorylated MAPK protein levels normalized to total MAPK protein levels. Data are presented as the mean ± SD of three independent experiments. **p<0.01; ***p<0.001 versus unloaded cells.
Figure 8
Figure 8. Apparatus used for the static mechanical stress.
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