淫羊藿苷经PI3K/Akt/mTOR通路调控激素对骨微血内皮细胞自噬的影响

Icariin Regulates Glucocorticoid-induced Autophagy of Bone Microvascular Endothelial Cells Through PI3K/Akt/mTOR Pathway

目的:探讨淫羊藿苷(ICA)介导磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/m TOR)信号通路对激素性骨微血管内皮细胞(BMECs)自噬的影响。方法:从人工全髋关节置换术中获得的股骨头分离培养BMECs,并通过免疫荧光染色法鉴定细胞种类。实验细胞分为空白组、激素组(100 mg·L^(-1)氢化可的松)、ICA组(100 mg·L^(-1)氢化可的松+6.7×10^(-3)mg·L^(-1)ICA)及雷帕霉素组(100 mg·L^(-1)氢化可的松+6.7×10^(-3)mg·L^(-1)ICA+1 mg·L^(-1)雷帕霉素),使用100 mg·L^(-1)的氢化可的松诱导BMECs自噬,运用荧光染色观察不同时间节点下BMECs自噬出现的峰值,蛋白免疫印迹法(Western blot)分析各组自噬及PI3K/Akt/m TOR通路相关蛋白表达,以电镜观察各组细胞自噬小体及自噬溶酶体的产生。结果:100 mg·L^(-1)氢化可的松可诱导BMECs自噬并在5 h左右出现自噬峰值,但随着干预时间的进一步增加,自噬峰值下降。与空白组比较,激素组细胞膜破坏,细胞器排列紊乱,可见大量自噬小体及自噬溶酶体;与激素组比较,ICA组细胞膜较为完整,细胞器排列较为稀疏,自噬小体数量及自噬溶酶体数量低于激素组;与ICA组比较,雷帕霉素组细胞膜破坏,细胞器排列紊乱,有较多自噬小体及自噬溶酶体。与空白组比较,激素组自噬相关蛋白4B(Atg4B)、人微管相关蛋白/轻链3B(LC3B)、p62表达显著提高(P<0.01);与激素组比较,ICA组LC3B、Atg4B、p62、自噬效应蛋白-1(Beclin-1)表达显著下降(P<0.01);与ICA组比较,雷帕霉素组Atg4B、p62表达显著升高(P<0.01);与空白组比较,激素组磷酸化(p)-PI3K/PI3K、p-Akt/Akt、p-m TOR/m TOR表达显著降低(P<0.01);与激素组比较,ICA组p-PI3K/PI3K、p-Akt/Akt、p-m TOR/m TOR显著提高(P<0.01);与ICA组比较,雷帕霉素组p-PI3K/PI3K、p-Akt/Akt、p-m TOR/m TOR表达显著降低(P<0.01);与空白组比较,激素组泛素化水平显著降低(P<0.01);与激素组比较,ICA组泛素化表达显著提高(P<0.01);与ICA组比较,雷帕霉素组泛素化水平显著下降(P<0.01)。结论:激素所诱导的BMECs自噬具有时间依赖性,ICA对激素诱导的BMECs的自噬具有抑制作用,且该作用可能与调控PI3K/Akt/m TOR通路有关。

Objective:To investigate the impact of icariin(ICA)on autophagy in glucocorticoid-induced bone microvascular endothelial cells(BMECs)mediated by the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.Method:BMECs were isolated and cultured from femoral heads obtained during total hip arthroplasty and identified using immunofluorescence staining.The experimental cells were divided into four groups:A control group,a glucocorticoid group(100 mg·L^(-1) hydrocortisone),an ICA group(100 mg·L^(-1) hydrocortisone+6.7×10^(-3) mg·L^(-1) ICA),and a Rapamycin group(100 mg·L^(-1) hydrocortisone+6.7×10^(-3) mg·L^(-1) ICA+1 mg·L^(-1) rapamycin).Autophagy in BMECs was induced using 100 mg·L^(-1) hydrocortisone.LC3 fluorescence staining was used to observe the peak of autophagy at different time points.Western blot analysis was employed to analyze the expression of autophagy-related proteins and PI3K/Akt/mTOR pathway proteins in each group.Electron microscopy was used to observe autophagosomes and autolysosomes in the cells.Result:Hydrocortisone at 100 mg·L^(-1) induced autophagy in BMECs,reaching a peak at around 5 hours,which then declined with further intervention.Compared to the control group,the glucocorticoid group showed cell membrane damage,disordered organelle arrangement,and a large number of autophagosomes and autolysosomes.Compared to the glucocorticoid group,the ICA group had more intact cell membranes,sparser organelle arrangement,and fewer autophagosomes and autolysosomes.Compared to the ICA group,the Rapamycin group showed cell membrane damage,disordered organelle arrangement,and more autophagosomes and autolysosomes.Compared to the control group,the glucocorticoid group had significantly increased expression of light chain 3B(LC3B),Atg4B,and p62(P<0.01).Compared to the glucocorticoid group,the ICA group showed significantly decreased expression of LC3B,Atg4B,p62,and Beclin-1(P<0.01).Compared to the ICA group,the Rapamycin group had significantly increased expression of Atg4B and p62(P<0.01).Compared to the control group,the glucocorticoid group had significantly decreased expression of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR(P<0.01).Compared to the glucocorticoid group,the ICA group showed significantly increased expression of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR(P<0.01).Compared to the ICA group,the Rapamycin group had significantly decreased expression of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR(P<0.01).Ubiquitination levels were significantly decreased in the glucocorticoid group compared to the control group(P<0.01).Compared to the glucocorticoid group,ubiquitination levels were significantly increased in the ICA group(P<0.01),and significantly decreased in the Rapamycin group compared to the ICA group(P<0.01).Conclusion:The glucocorticoid-induced autophagy in BMECs is time-dependent.ICA inhibits glucocorticoidinduced autophagy in BMECs,and this effect may be related to the regulation of the PI3K/Akt/mTOR pathway.