[Effect of glucocorticoid on production of reactive oxygen species in bone microvascular endothelial cells]

Glucocorticoid is the main cause of non-traumatic avascular necrosis of femoral head. To explore the changes of reactive oxygen species (ROS) in the bone microvascular endothelial cells treated with glucocorticoid so as to investigate the pathogenesis of steroid-induced avascular necrosis of femoral head.

The cancellous bone of femoral head was harvested from voluntary donators undergoing total hip arthroplasty, and then the bone microvascular endothelial cells were isolated by enzyme digestion. The cells at passage 3 were cocultured with different concentrations of hydrocortisone (0, 0.03, 0.10, 0.30, and 1.00 mg/mL) for 24 hours. MTT assay was used for the inhibitory rate of cell proliferation, flow cytometry for apoptosis rate, and fluorescence probe for the production of ROS and xanthine oxidase (XOD).

At 2-3 days primary culture, the cells were spindle and arranged like cobbles and they reached confluence after 1 week. The inhibitory rates of cell proliferation in 0.03, 0.10, 0.30, and 1.00 mg/mL groups were 20.22% +/- 2.97%, 22.94% +/- 4.52%, 43.98% +/- 3.35%, and 78.29% +/- 3.85%, respectively; and 2 high-concentration groups (0.30 and 1.00 mg/mL groups) were significantly higher (P < 0.05) than 2 low-concentration groups (0.03 and 0.10 mg/mL groups). The apoptosis rates in 0, 0.03, 0.10, 0.30, and 1.00 mg/mL groups were 0.10% +/- 0.01%, 0.23% +/- 0.02%, 1.83% +/- 0.04%, 6.34% +/- 0.11%, and 15.33% +/- 0.53%, respectively; 2 high-concentration groups (0.30 and 1.00 mg/mL groups) were significantly higher (P < 0.05) than 0 mg/mL group. In 0, 0.30, and 1.00 mg/mL groups, the ROS levels were 57.35 +/- 7.11, 120.47 +/- 15.68, and 166.15 +/- 11.57, respectively, and the XOD levels were 0.017 9 +/- 0.000 9, 0.028 3 +/- 0.0017, and 0.067 7 +/- 0.0041, respectively; there were significant differences in the levels of ROS and XOD among 3 groups (P < 0.05).

Increasing of ROS production in bone microvascular endothelial cells can be induced by high concentration glucocorticoid, and it can result in cell injury.