The Department of Sports Medicine, the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China.
Acta Chir Orthop Traumatol Cech. 2023;90(4):267-276.
PURPOSE OF THE STUDY Articular cartilage injury is a common disease in daily life, with a high incidence. The aim of this study was to investigate the effect and mechanism of miRNA-140-3p in bone mesenchymal stem cells (BMSCs)-derived exosomes under hypoxia on inflammatory articular chondrocytes. MATERIAL AND METHODS To simulate the pathological status of arthritis, rat chondrocytes were used to establish the osteoarthritis (OA) model by IL-1β (10 μg/ml) as a modulating in vitro, and exosomes were isolated by differential ultra-high speed centrifugation. The cell counting kit-8, wound healing and flow cytometry assays were utilized to assess proliferation, migration and apoptosis of chondrocytes, respectively. Lipogenic and chondrogenic differentiation of chondrocytes were detected by oil red O staining and toluidine blue staining individually. The expressions of miR-140-3p and chondrocyte-specific gene mRNA were investigated using qRT-PCR. Western blot was applied to assess chondrocyte associated proteins and BMSC-Exo surface protein markers, and immunohistochemistry was adopted to detect the staining of collagen I and II. RESULTS Under scanning electronic microscope, the shape of exosomes was almost round. Exosome treatment prominently impaired the inhibition of chondrocytes' proliferative and migrative ability by IL-1β. It was found hypoxia had a more marked impact on proliferation, expression of collagen II and apoptosis in OA chondrocytes than normoxia, as well as a stronger effect on weakening adipose differentiation and enhancing chondrogenic differentiation in inflammatory chondrocytes. Furthermore, incubation with BMSC-Exo overexpressing miR-140-3p can remarkably increase the survival rate and migration in inflammatory chondrocytes. In addition, overexpression of miR-140-3p was found to enhance the chondrogenic differentiation of inflammatory chondrocytes. Furthermore, we found that the healing effect of exosomes on inflammatory chondrocytes under hypoxic conditions was produced by a rise in miR-140-3p expression within them and that hypoxia-mediated upregulation of miR-140-3p expression occurred through HIF-1α. CONCLUSIONS Under hypoxia, BMSC-Exo enhanced the chondrogenic phenotype, increased the viability of inflammatory chondrocytes. The overexpression of miR-140-3p in BMSC-Exo is beneficial to protect joints and delaying the pathogenesis in OA. Key words: HIF-1α, apoptosis, lipogenic differentiation, chondrogenic differentiation.
关节软骨损伤是日常生活中的常见病，发病率较高。本研究旨在探讨缺氧条件下骨髓间充质干细胞（BMSC）衍生的外泌体中 miRNA-140-3p 对炎性关节软骨细胞的作用及机制。
为模拟关节炎的病理状态，采用白细胞介素-1β（10μg/ml）作为调节剂，在体外建立大鼠软骨细胞骨关节炎（OA）模型，并通过差速超速离心分离外泌体。分别采用细胞计数试剂盒-8（CCK-8）、划痕愈合和流式细胞术检测软骨细胞的增殖、迁移和凋亡，油红 O 染色和甲苯胺蓝染色分别检测软骨细胞的成脂和成软骨分化。采用 qRT-PCR 检测 miR-140-3p 和软骨细胞特异性基因 mRNA 的表达。采用 Western blot 检测软骨细胞相关蛋白和 BMSC-Exo 表面蛋白标志物，采用免疫组织化学检测胶原 I 和 II 的染色。
扫描电子显微镜下，外泌体的形状几乎呈圆形。外泌体处理显著削弱了 IL-1β对软骨细胞增殖和迁移能力的抑制作用。结果发现，与常氧相比，缺氧对 OA 软骨细胞的增殖、Ⅱ型胶原表达和凋亡的影响更为显著，对炎性软骨细胞的弱脂分化和强软骨分化的影响也更强。此外，用过表达 miR-140-3p 的 BMSC-Exo 孵育可显著提高炎性软骨细胞的存活率和迁移率。此外，过表达 miR-140-3p 可增强炎性软骨细胞的软骨分化。此外，我们发现，外泌体在低氧条件下对炎性软骨细胞的修复作用是通过其内 miR-140-3p 表达的增加产生的，而低氧介导的 miR-140-3p 表达上调是通过 HIF-1α 发生的。
在缺氧条件下，BMSC-Exo 增强了软骨细胞的软骨表型，提高了炎性软骨细胞的活力。BMSC-Exo 中 miR-140-3p 的过表达有利于保护关节，延缓 OA 的发病机制。
HIF-1α；细胞凋亡；成脂分化；软骨分化。
