Neutrophil Elastase Induces Chondrocyte Apoptosis and Facilitates the Occurrence of Osteoarthritis via Caspase Signaling Pathway

Abstract

Osteoarthritis (OA) is the most common and prevalent chronic joint disorders in the elderly population across the globe, resulting in severe disability and impairment of quality of life. Existing treatment can only alleviate the symptoms and delay the progression of OA. Therefore, novel and effective therapeutics strategies for OA need to be developed. Our present study first found that neutrophil elastase (NE) was significantly increased in OA patients' synovial fluid. Next, we examined the effect of neutrophil elastase (NE) on chondrocytes in vitro and in vivo. The results showed that NE suppressed cell proliferation, induced apoptosis and prevented cell migration in chondrocytes in vitro, accompanied by the elevation of intracellular ROS and calcium level. Moreover, NE enhanced the cleaved caspase-3 levels and disrupted the mitochondrial transmembrane potential balance. Meanwhile, chondrocytes apoptosis induced by NE can be alleviated by caspase inhibitor, zVAD-FMK and antioxidants, GSH. Besides, treatment of sivelestat, the inhibitor of NE, significantly reduced the pathological processes in OA model rats in vivo. The results of our study suggested that NE is an important factor in OA, which induces chondrocyte apoptosis and facilitates the occurrence of OA via caspase signaling pathway, and targeting the crucial signal centering around NE may be the potential therapies for OA.

Keywords: apoptosis; caspase-3; chondrocyte; neutrophil elastase; osteoarthritis.

FIGURE 1

Neutrophil elastase is overexpressed in OA patients. (A) X ray images and macroscopic views (arthroscopic image and intraoperative image) of knee joint from healthy donors and OA patients. (B) NE relative activity in synovial fluid of healthy donors and OA patients. Data are mean ± SEM; n = 10 (healthy donors), n = 45 (OA patients). ***p < 0.001.

FIGURE 2

Neutrophil elastase shows potent growth inhibition in chondrocytes in vitro. (A) Representative images of chondrocytes treated with NE for 48 h. The representative fields were photographed at ×40 and ×100 magnification. (B) Inhibitory rate histogram of chondrocytes treated with NE for 24 h, 48 h, and 72 h. (C) The fluorescence pattern of annexin V-FITC/PI staining chondrocytes. (D) Percentages of annexin V positive cells for chondrocytes treated with different doses of NE. Data are mean ± SEM; n = 3. **p < 0.01, ***p < 0.001.

FIGURE 3

Neutrophil elastase induces chondrocytes apoptosis through cleaved caspase-3. (A) Representative histograms of cleaved caspase-3 levels of chondrocytes were shown. (B) Cell associated mean relative fluorescence intensities. Data are mean ± SEM; n = 3. ***p < 0.001. (C) The fluorescence pattern of annexin V-FITC/PI staining chondrocytes. (D) Percentages of annexin V positive cells for chondrocytes treated with NE and Z-VAD-FMK. Data are mean ± SEM; n = 3. ***p < 0.001. (E) Western blot of ERK1/2 phosphorylation, total ERK1/2, caspase-3 and cleaved caspase-3 levels of chondrocytes treated with NE.

FIGURE 4

Neutrophil elastase induces DNA fragmentation, mitochondrial disruption and intracellular calcium release of chondrocytes. (A,B) TUNEL levels of chondrocytes were measured by flow cytometry. (A) Representative histograms were shown. (B) Cell associated mean relative fluorescence intensities. Data are mean ± SEM; n = 3. ***p < 0.001. (C,D) Intracellular free Ca²⁺ of chondrocytes were measured by flow cytometry. (C) Representative histograms were shown. (D) Cell associated mean relative fluorescence intensities. Data are mean ± SEM; n = 3. ***p < 0.001. (E,F) JC-1 staining of chondrocytes were measured by flow cytometry. (E) The fluorescence pattern of JC-1 staining chondrocytes. (F) Red/Green fluorescence intensities. Data are mean ± SEM; n = 3. ***p < 0.001.

FIGURE 5

Neutrophil elastase induces ROS production of chondrocytes. (A,B) ROS levels of chondrocytes were measured by flow cytometry. (A) Representative histograms were shown. (B) Cell associated mean relative fluorescence intensities. Data are mean ± SEM; n = 3. ***p < 0.001. (C,D) Chondrocytes were treated with NE and GSH for 48 h and then stained with annexin V-FITC/PI followed by flow cytometry analysis. (C) The fluorescence pattern of annexin V-FITC/PI staining chondrocytes. (D) Percentages of annexin V positive cells for chondrocytes treated with NE and GSH. Data are mean ± SEM; n = 3. ***p < 0.001.

FIGURE 6

Neutrophil elastase inhibits the motility of chondrocytes. (A,B) Migration of chondrocytes was evaluated using the in vitro wound-healing scratch assay. (A) Representative photomicrographs of chondrocytes. (B) For quantitative analysis, percentage of wound closure was determined. Data are mean ± SEM; n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Neutrophil elastase induces retraction of chondrocytes and disrupts actin cytoskeleton. Representative confocal photomicrographs of cytoskeleton on chondrocytes treated with different dose of NE. Cells were fixed and stained with TRITC-phalloidin (red) and nuclei were stained with DAPI (blue). Original magnification, ×630.

FIGURE 8

Sivelestat decreased apoptosis of articular chondrocytes and impeded the loss of cartilage in the knee joint from neutrophil elastase induced rat osteoarthritic model. (A) Macroscopic view, micro-CT image and 3D image of the specimens from sham group, OA model group and treatment groups. (B) Representative safranin O-fast green staining of the knee joint in sham group, OA model group and treatment groups. (C) OARSI scores based on staining results. Data are mean ± SEM; n = 3. **p < 0.01. (D) Western blot of ERK1/2 phosphorylation, total ERK1/2, caspase-3 and cleaved caspase-3 levels of articular cartilage from rat model. (E) Western blot of ERK1/2 phosphorylation, total ERK1/2, caspase-3 and cleaved caspase-3 levels of articular cartilage from healthy donors and OA patients.