Upregulation of osteoprotegerin inhibits tert-butyl hydroperoxide-induced apoptosis of human chondrocytes

Abstract

Necrosis of the femoral head (NFH) is an orthopedic disease characterized by a severe lack of blood supply to the femoral head and a marked increase in intraosseous pressure. NFH is associated with numerous factors, such as alcohol consumption and hormone levels. The present study focused on the expression levels of osteoprotegerin (OPG) in NFH and the effect of OPG overexpression on chondrocyte apoptosis. The results demonstrated that OPG expression was markedly decreased in the femoral head of patients with NFH compared with normal femoral heads. Lentivirus-mediated overexpression of OPG in human chondrocytes reversed the decrease in cell viability and the increase in reactive oxygen species production induced by an oxidative stress-inducing factor, tert-butyl hydroperoxide. Flow cytometry and TUNEL assays revealed that OPG overexpression inhibited the apoptosis of chondrocytes. In addition, it was revealed that OPG exerted its anti-apoptotic effect mainly by promoting Bcl-2 expression and Akt phosphorylation and inhibiting caspase-3 cleavage and Bax expression. The present study revealed that OPG may be an important regulator of NFH.

Keywords: apoptosis; chondrocytes; necrosis of the femoral head; osteoprotegerin; tert-butyl hydroperoxide.

Figure 1

Hematoxylin and eosin staining of femoral head tissue. (A) Normal cartilage. (B) Full anomaly layer. (C) Abnormal metaplasia. (D) Abnormal multinucleated giant cells. (E) Abnormal cystic lesions. (F) Abnormal cartilage hyperplasia. Scale bar, 60 µm.

Figure 2

Expression levels of OPG in patients with NFH. (A) Reverse transcription-quantitative PCR detection of OPG in femoral head tissues of normal subjects and patients with NFH (control vs. patients with NFH). (B) Receiver operating characteristic analysis of OPG expression in patients with NFH. (C) Representative western blots of OPG in tissues from controls and patients with NFH, and semi-quantitative analysis of OPG expression (n=30 for control; n=28 for alcohol type; n=22 for steroid type). ^*P<0.05, ^**P<0.01. AUC, area under the curve; NFH, necrosis of the femoral head; OPG, osteoprotegerin.

Figure 3

Effects of OPG on chondrocyte viability and ROS production. (A) Increased mRNA level of OPG in the cells transfected with the OPG plasmid compared with cells transfected with the control plasmid (pLVX-TRE3G). (B) The cells transfected with the OPG plasmid showed higher OPG protein level compared with that in cells transfected with the pLVX-TRE3G. (C) Cell viability was determined using an MTT assay in human chondrocytes transfected with OPG or the pLVX-TRE3G only. (D) ROS levels were determined via flow cytometry in human chondrocytes transfected with OPG or pLVX-TRE3G only. ^*P<0.05, ^**P<0.01. OPG, osteoprotegerin; ROS, reactive oxygen species; tBHP, tert-butyl hydroperoxide.

Figure 4

Effects of OPG on tBHP-induced apoptosis of chondrocytes transfected with the OPG plasmid or pLVX-TRE3G. (A) Flow cytometric analysis of cells transfected with OPG plasmid or pLVX-TRE3G only and stained with PI and Annexin V-FITC after incubation with tBHP for 24 h. (B) Percentage of dead cells (E1), those in late apoptosis (E2) and viable cells (E3), and early apoptosis (E4), as analyzed by flow cytometry. ^**P<0.01. OPG, osteoprotegerin; tBHP, tert-butyl hydroperoxide.

Figure 5

TUNEL assay showing the effects of tBHP on chondrocytes transfected with the OPG plasmid or vector only. (A) Representative microphotographs of TUNEL staining in human chondrocytes transfected with the OPG plasmid or pLVX-TRE3G only. TUNEL-positive cells are green and nuclei labeled with DAPI are blue. (B) Percentage of TUNEL-positive cells. DAPI-stained cells were counted in four random regions. Scale bar, 50 µm. ^**P<0.01. OPG, osteoprotegerin; tBHP, tert-butyl hydroperoxide.

Figure 6

Western blot analysis of apoptosis-related proteins in OPG plasmid-transfected chondrocytes. (A) Western blots of Bax, Bcl-2, caspase-3 and β-actin in human chondrocytes transfected with the OPG plasmid or pLVX-TRE3G only and treated with tBHP or vehicle. (B) Relative levels of p-Akt, Akt and β-actin in human chondrocytes transfected with OPG or vector only, and then treated with vehicle or tBHP for 24 h. (C) Relative levels of Bax, Bcl-2, caspase-3 and p-Akt in human chondrocytes. ^**P<0.01. OPG, osteoprotegerin; tBHP, tert-butyl hydroperoxide; p-, phosphorylated.