糖皮质激素通过PI3K-Akt-mTOR信号通路诱导股骨头骨微血管内皮细胞凋亡的研究

目的 探讨糖皮质激素通过PI3K-Akt-mTOR信号通路诱导股骨头骨微血管内皮细胞凋亡的机制。方法分离培养人骨微血管内皮细胞，取第3代细胞，给予不同剂量的氢化可的松处理24 h，浓度分别为0.1 mg/mL（低剂量）、0.2 mg/mL（中剂量）和0.3 mg/mL（高剂量），Western blot法检测PI3K-Akt-mTOR信号通路关键蛋白磷脂酰肌醇激酶（PI3K）、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白（mTOR）及其磷酸化形式的表达，AnnexinV/PI染色的流式细胞检测细胞凋亡。结果 Western blot电泳条带灰度值分析提示高浓度氢化可的松可以使该通路关键蛋白表达下降。与对照组相比，0.1 mg/mL、0.2 mg/mL和0.3 mg/mL三组的p-Akt表达分别下降60.72%、61.91%、100.00%（P＜0.01）。p-PI3K表达分别下降12.95%、36.26%、100.00%（P＜0.01）。p-mTOR表达分别下降22.95%、41.28%、57.11%（P＜0.01）。流式细胞检测显示正常对照组的早期凋亡、晚期凋亡/坏死以及活细胞比例分别为16.08%、30.86%和52.52%；激素损伤组这一比例分别为21.67%、37.2%、39.85%。激素损伤+PI3K抑制剂组这一比例分别为19.05%、37.9%和41.26%。统计学分析显示细胞比例组间差异均具有统计学意义（P＜0.001）。结论 糖皮质激素可以显著抑制人股骨头骨微血管内皮细胞内PI3K-Akt-mTOR信号通路关键蛋白的表达，从而诱导股骨头骨微血管内皮细胞发生凋亡和坏死。

Glucocorticoids induce apoptosis of bone microvascular endothelial cells in human femoral head by inhibiting PI3K-Akt-mTOR signal pathway

Objective To investigate the mechanism of glucocorticoids on the apoptosis of bone microvascular endothelial cells through PI3K-Akt-mTOR signal pathway in human femoral head. Methods The cancellous bone of the femoral head was harvested from fractured femoral neck undergoing total hip arthroplasty. Bone microvascular endothelial cells were isolated with enzyme digestion. The cells passed to the third generation were cultured with different concentrations of hydrocortisone for 24 hours. Intracellular signal molecules such as phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, protein kinase B (Akt), phosphorylated Akt, mammalian rapamycin target protein (mTOR), and phosphorylated mTOR protein expressions were detected using Western blotting. Cell apoptosis rate was detected with flow cytometry. Results The results showed that high concentration of hydrocortisone reduced the expression of key proteins in signal pathway. Compared with that in the control group, the expression of p-Akt in 0.1 mg/mL, 0.2 mg/mL, and 0.3 mg/mL group decreased by 60.72%, 61.91%, and 100.00%, respectively (P<0.01). The expression of p-PI3K decreased by 12.95%, 36.26%, and 100.00%, respectively (P<0.01). The expression of p-mTOR decreased by 22.95%, 41.28%, and 57.11%, respectively (P<0.01). Flow cytometry results showed that the proportions of early apoptosis, late apoptosis/necrosis, and living cells in the normal control group were 16.08%, 30.86%, and 52.52%, respectively. They were 21.67%, 37.2%, and 39.85%, respectively, in the hormone injury group. They were 19.05%, 37.9%, and 41.26%, respectively, in the hormone injury+PI3K inhibitor group. Statistical analysis showed that there were significant differences in cell proportion among the groups (P<0.01). Conclusion Glucocorticoids inhibit PI3K-Akt-mTOR signal pathway in bone microvascular endothelial cells, which induce apoptosis and necrosis in the bone microvascular endothelial cells of the femoral head.