糖皮质激素对骨髓微血管内皮细胞一氧化氮合酶磷酸化的影响

Effect of glucocorticoid on enos phosphorylation in bone microvascular endothelial cells

目的 观察经糖皮质激素处理后的骨髓微血管内皮细胞一氧化氮（NO）的合成变化,探讨激素性股骨头缺血性坏死的发病机制.方法 取行人工全髋关节置换术患者自愿捐献的股骨头内松质骨,酶消化法分离培养骨髓微血管内皮细胞,取第3代细胞.利用氢化可的松诱导建立激素性股骨头缺血性坏死模型.分别与不同浓度（0、0.10、0.50、1.00、2.00g/L）氢化可的松培养,孵育8h;不同时间（0、4、8、16h）,终质量浓度为1 g/L氢化可的松培养,随后对骨髓微血管内皮细胞中NO浓度进行检测,Western blot检测蛋白激酶B（Akt）、磷酸化Akt（p-Akt）、内皮型一氧化氮合酶（eNOS）和磷酸化eNOS （p-eNOS）蛋白表达.Pull-down检测eNOS与Akt蛋白质之间的相互作用力.结果 氢化可的松对骨髓微血管内皮细胞NO的合成有抑制作用,表现出浓度依赖性和时间依赖性（P＜0.05）.eNOS Thr 495位点的磷酸化状态未发生改变,而Ser633位点发生了去磷酸化.同时检测到Akt Thr308/Ser473位点发生了去磷酸化,eNOS与Akt蛋白质之间的相互作用力减弱.结论 糖皮质激素抑制骨髓微血管内皮细胞NO合成,其机制可能与抑制磷酸肌醇3激酶（PI3K）-Akt-eNOS信号转导通路有关.

Objective Glucocorticoid is the main cause of non-traumatic avascular necrosis of femoral head.To explore the production of nitric oxide （NO） in the bone microvascular endothelial cells treated with glucocorticoid so as to investigate the pathogenesis of steroid-induced avascular necrosis offemoral head.Methods The cancellous bone of femoral head was harvested from voluntary donators undergoing total hip arthroplasty,and then the bone microvascular endothel ial cells were isolated by enzyme digestion.The cells at passage 3 were cocultured with different concentrations of hydrocortisone （0,0.10,0.50,1.00 and 2.00 g/L） for 8 hours.With the final concentration of 1 g/L was added,the cells were incubated for 0,8,16 and 24 h respectively.NO level in the cell culture medium was assayed by NO assay kit.Intracellular signal molecules such as protein kinase B （Akt）,phosphorylated Akt,endothelial nitric oxide synthase （eNOS） and phosphorylated eNOS protein expressions were detected by Western blotting.Protein and protein interaction between eNOS and Akt was detected by Pull-down assay.Results eNOS dephosphorylation was selective to Ser-635 but not to Thr-497 in concentration-and time-dependent manner,which was parallel to Akt Thr308/Ser473 dephosphorylation.Glucocorticoid inhibited eNOS from binding to Akt in vitro.Conclusion NO production in bone microvascular endothelial cells can be down-regulated by glucocorticoid,and the mechanism may be related to the activation of the phosphatidylinositol 3 kinase （PI3K）-Akt-eNOS signal transduction pathway.