Changes In Production Of Reactive Oxygen Species Following Steroid Treated In Bone Microvascular Endothelial Cells

Objective To explore the changes of reactive oxygen species in the contribution of vascular endothelial cell injury induced with steroid treatment, and the effect of vitaminC on production of ROS. ROS level and mutations of mtDNA in osteonecrosis tissue were measured. Methods By enzyme digestion, human bone vascular endothelial cells were obtained, cultured, and treated with steroid.24 hours after MTT reduction assay was used to observe the inhibition of endothelial cells by steroid in different concentrations, and fluorescence light probe was used to detect the production of reactive oxygen species. After each group was treated with vitamin, MTT was used to detect cell proliferation and the production of ROS. The D-Loop region were amplified by PCR and sequenced. Results The typical endothelial cell morphology can be seen by microscope. The cells are arranged like flagstone. The proliferation inhibition rate of endothelial cell in group 0.03 mg/ml,0.1 mg/ml,0.3 mg/ml,1.0 mg/ml was (20.22%±2.97%),(22.94%±4.52%),(43.98%±3.35%),(78.29%±3.85%) respectively. The proliferation inhibition rate of endothelial cell in high-concentration group was significantly higher than that in low-concentration group(P<0.05). The apoptosis rate of endothelial cells in group 0 mg/ml,0.03mg/ml,0.1mg/ml,0.3mg/ml,1.0mg/ml was(0.10%±0.01%),(0.23%±0.02%),(1.83%±0.04%),(6.34%±0.11%),(15.33%±0.53%) respectively. The apoptosis rate of endothelial cells in high-concentration group was significantly higher than that in low-concentration group (P<0.05). ROS in group Omg/ml,0.3 mg/ml,1.0mg/ml was (57.35±7.11),(120.47±15.68),(166.15±11.57) respectively. The value of ROS increased after cells were treated with glucocorticoid and that in high-concentration group was significantly higher than that in low-concentration group (P＜0.05). XOD in group 0 mg/ml,0.3 mg/ml,1.0 mg/ml was (0.0179±0.0009),(0.0283±0.0017),(0.0677±0.0041) respectively. The value of XOD increased after cells were treated with glucocorticoid and that in high-concentration group was significantly higher than that in low-concentration group (P<0.05). when treated with vitamin C, the proliferation inhibition rate of endothelial cell and production of ROS were both decreased. ROS level and mutations in osteonecrosis tissue were significantly higher than the control groups. Conclusions Increasing of ROS production in endothelial cell can be induced by high concentration steroid, and it can result in cells injury. Vitamin C can efficiently inhibit the injury of endothelial cell induced by steroid and has a protective ability.The mutation and ROS level play a role in the development of osteonecrosis.