[Effects of icariin on autophagy and exosome production of bone microvascular endothelial cells].
Authors of this article are:
Zhang Q, Gao F, Cheng L, Liu L, Sun W, Li Z.
A summary of the article is shown below:
in English, Chinese目的: 评估淫羊藿苷对低浓度糖皮质激素诱导的骨微血管内皮细胞（bone microvascular endothelial cells，BMECs）自噬和外泌体分泌的影响。.方法: 从行全髋关节置换术切取的股骨头中分离 BMECs，用一系列低浓度梯度氢化可的松（0、0.03、0.06、0.10 mg/mL）干预（设为 A、B、C、D 组），在此基础上再用 5×10 −5 mol/L 淫羊藿苷干预（设为 A1、B1、C1、D1 组），24 h 后采用 Western blot 检测自噬相关蛋白微管相关蛋白轻链 3B（microtubule-associated protein 1 light chain 3B，LC3B）及死骨片 1（p62）的表达。从经淫羊藿苷处理（干预组）和未经淫羊藿苷处理（未干预组）的 BMECs 中提取外泌体，纳米颗粒跟踪分析技术检测其直径和浓度，BCA 法检测外泌体总蛋白质含量，Western blot 检测外泌体 CD9、CD81、TGF-β 1 和 VEGFA 蛋白的表达。进一步将 BMECs 分为 3 组，实验组和对照组分别分离经或未经淫羊藿苷处理的 BMECs 分泌的外泌体，与 BMECs 共培养；空白对照组为单纯 BMECs。氢化可的松处理后，采用 Western blot 检测 LC3B 和 p62 表达，划痕实验检测细胞迁移能力，并观察血管生成能力。.结果: 随氢化可的松浓度升高，各组 LC3B-Ⅱ蛋白相对表达量逐渐增加，p62 蛋白相对表达量减少，各组间差异均有统计学意义（ P<0.01）；相同激素浓度下，淫羊藿苷干预后，LC3B-Ⅱ蛋白相对表达量减少，p62 蛋白相对表达量增加（ P<0.01）。干预组外泌体浓度显著高于未干预组（ t=−10.191， P=0.001）；两组外泌体直径和总蛋白质含量比较差异均无统计学意义（ P>0.05）。未干预组和干预组 CD9 和 CD81 蛋白均高度表达；干预组 VEGFA/CD9 和 TGF-β 1/CD9 蛋白相对表达量比值均显著高于未干预组（ P<0.01）。外泌体共培养后，空白对照组、对照组和实验组中 p62 蛋白相对表达量呈递增趋势，LC3B-Ⅱ蛋白相对表达量呈递减趋势，各组间比较差异均有统计学意义（ P<0.05）。氢化可的松处理 12、24 h 时，对照组和实验组划痕闭合率明显高于空白对照组（ P<0.05），实验组明显高于对照组（ P<0.05）；氢化可的松处理 4、8 h 时，实验组和对照组管腔数、出芽数和小管分支长度均显著大于空白对照组（ P<0.05）；实验组小管分支长度和管腔数显著大于对照组（ P<0.05）。.结论: 淫羊藿苷及 BMECs 产生的外泌体能改善低浓度激素诱导的 BMECs 自噬，对内皮细胞起到保护作用。.Objective: To evaluate the effects of icariin on autophagy induced by low-concentration of glucocorticoid and exosome production in bone microvascular endothelial cells (BMECs).Methods: BMECs were isolated from femoral heads resected in total hip arthroplasty and then intervened with hydrocortisone of low concentration (0, 0.03, 0.06, 0.10 mg/mL), which were set as groups A, B, C, and D, respectively. On the basis of hydrocortisone intervention, 5×10 -5 mol/L of icariin was added to each group (set as groups A1, B1, C1 and D1, respectively). Western blot was used to detect the expressions of microtubule-associated protein 1 light chain 3B (LC3B) and dead bone slice 1 (p62) after 24 hours. Exosomes were extracted from BMECs treated with icariin (intervention group) and without icariin (non-intervention group), and the diameter and concentration of exosomes were evaluated by nanoparticle tracking analysis technique. The total protein content of exosomes was detected by BCA method, and the expressions of proteins carried by exosomes including CD9, CD81, transforming growth factor β 1 (TGF-β 1), and vascular endothelial growth factor A (VEGFA) were assessed by Western blot. The BMECs were further divided into three groups: BMECs in the experimental group and the control group were co-cultured with exosomes secreted by BMECs treated with or without icariin, respectively; the blank control group was BMECs without exosome intervention. The three groups were treated with hydrocortisone and Western blot was used to detect the expressions of LC3B and p62. The scratching assay was used to detect cell migration ability; angiogenic ability of BMECs was also assessed.Results: With the increase of hydrocortisone concentration, the protein expression of LC3B-Ⅱ increased gradually, and the protein expression of p62 decreased gradually ( P<0.01). Compared with group with same concentration of hydrocortisone, the protein expression of LC3B-Ⅱ decreased and the protein expression of p62 increased after the administration of icariin ( P<0.01). The concentration of exosomes in the intervention group was significantly higher than that in the non-intervention group ( t=-10.191, P=0.001); and there was no significant difference in exosome diameter and total protein content between the two groups ( P>0.05). CD9 and CD81 proteins were highly expressed in the non-intervention group and the intervention group, and the relative expression ratios of VEGFA/CD9 and TGF-β 1/CD9 proteins in the intervention group were significantly higher than those in the non-intervention group ( P<0.01). After co-culture of exosomes, the protein expression of p62 increased in blank control group, control group, and experimental group, while the protein expression of LC3B-Ⅱ decreased. There were significant differences among groups ( P<0.05). When treated with hydrocortisone for 12 and 24 hours, the scratch closure rate of the control group and experimental group was significantly higher than that of the blank control group ( P<0.05), and the scratch closure rate of the experimental group was significantly higher than that of the control group ( P<0.05). When treated with hydrocortisone for 4 and 8 hours, the number of lumens, number of sprouting vessels, and length of tubule branches in the experimental group and the control group were significantly greater than those in the blank control group ( P<0.05); the length of tubule branches and the number of lumens in the experimental group were significantly greater than those in the control group ( P<0.05).Conclusion: Icariin and BMECs-derived exosomes can improve the autophagy of BMECs induced by low concentration of glucocorticoid.
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This article is a good source of information and a good way to become familiar with topics such as: Femoral head; autophagy; exosome; glucocorticoid; icariin; microvascular endothelial cells.
