Exosomes derived from Wharton's jelly of human umbilical cord mesenchymal stem cells reduce osteocyte apoptosis in glucocorticoid-induced osteonecrosis of the femoral head in rats via the miR-21-PTEN-AKT signalling pathway
Exosomes derived from Wharton's jelly of human umbilical cord mesenchymal stem cells reduce osteocyte apoptosis in glucocorticoid-induced osteonecrosis of the femoral head in rats via the miR-21-PTEN-AKT signalling pathway
复制标题
人脐带间充质干细胞沃顿胶来源的外泌体通过 miR-21-PTEN-AKT 信号通路减少糖皮质激素诱导的大鼠股骨头坏死中的骨细胞凋亡
DOI：
10.7150/ijbs.32262
复制
发表时间：
2019-01-01
影响因子：
9.2
通讯作者：
Ma, Xin-long
中科院分区：
文献类型：
--
作者：
Kuang, Ming-jie;Huang, Ying;Ma, Xin-long
目的：糖皮质激素性股骨头坏死（GIONFH）是长期或大剂量使用糖皮质激素后的常见疾病。GIONFH的发病机制目前仍存在争议，糖皮质激素引起的骨代谢异常可能是重要因素之一。外泌体由于其对骨修复的积极作用，对骨相关疾病显示出有希望的治疗效果。在这项研究中，我们假设外泌体通过miR-21-PTEN-AKT信号通路减少大鼠GIONFH中的骨细胞凋亡。研究方法：为了评价exosomes在GONFH中的作用，建立了地塞米松处理或exosomes处理的体外细胞模型和甲基强的松龙处理的体内大鼠模型。体外实验采用CCK-8法和5-乙炔基-2 ′-脱氧尿苷（5-ethynyl-2′-deoxyuridine，5-EDU-U）染色法检测成骨细胞的增殖情况。此外，末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）法，膜联蛋白V-异硫氰酸荧光素-碘化丙啶染色，和蛋白质印迹进行评估骨细胞的凋亡。在体内，我们使用显微计算机断层扫描和组织学和免疫组织化学分析来评估外泌体的影响。此外，外泌体通过miR-21-PTEN-AKT途径对骨细胞凋亡的作用机制通过高通量RNA测序、荧光原位杂交、荧光素酶报告基因测定和蛋白质印迹进行了研究。结果：高通量测序结果显示，AKT信号通路在exosome组中表达上调。定量PCR和Western blotting证实AKT通路基因的相对表达量上调。Western blotting结果显示，外泌体激活的AKT可抑制骨细胞凋亡。进行RNA荧光原位杂交和荧光素酶报告基因测定以确认miR-21和PTEN之间的相互作用。根据体内实验，如通过显微计算机断层扫描和组织学和免疫组织化学分析所证明的，外泌体在大鼠模型中预防GIONFH。结论：外泌体可有效抑制骨细胞凋亡（在MLO-Y 4细胞中）并预防大鼠GIONFH。这些有益作用是由miR-21-PTEN-AKT信号通路介导的。
Purpose: Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a common disease after long-term or high-dose glucocorticoid use. The pathogenesis of GIONFH is still controversial, and abnormal bone metabolism caused by glucocorticoids may be one of the important factors. Exosomes, owing to their positive effect on bone repair, show promising therapeutic effects on bone-related diseases. In this study, we hypothesised that exosomes reduce osteocyte apoptosis in rat GIONFH via the miR-21-PTEN-AKT signalling pathway. Methods: To evaluate the effects of exosomes in GIONFH, a dexamethasone-treated or exosome-treated in vitro cell model and a methylprednisolone-treated in vivo rat model were set up. In vitro, a CCK-8 assay and 5-ethynyl-2′-deoxyuridine staining were performed to evaluate the proliferation of osteocytes. Further, a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, annexin V-fluorescein isothiocyanate-propidium iodide staining, and western blotting were conducted to evaluate the apoptosis of osteocytes. In vivo, we used micro-computed tomography and histological and immunohistochemical analyses to assess the effects of exosomes. Moreover, the mechanism of exosome action on osteocyte apoptosis through the miR-21-PTEN-AKT pathway was investigated by high-throughput RNA sequencing, fluorescence in situ hybridisation, luciferase reporter assays, and western blotting. Results: High-throughput RNA sequencing results showed that the AKT signalling pathway was up-regulated in the exosome group. Quantitative PCR and western blotting confirmed that the relative expression of genes in the AKT pathway was up-regulated. Western blotting revealed that AKT activated by exosomes inhibited osteocyte apoptosis. RNA fluorescence in situ hybridisation and luciferase reporter assays were performed to confirm the interaction between miR-21 and PTEN. According to the experiment in vivo, exosomes prevented GIONFH in a rat model as evidenced by micro-computed tomography scanning and histological and immunohistochemical analyses. Conclusions: Exosomes are effective at inhibiting osteocyte apoptosis (in MLO-Y4 cells) and at preventing rat GIONFH. These beneficial effects are mediated by the miR-21-PTEN-AKT signalling pathway.